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ABSTRACT
Year : 2012  |  Volume : 8  |  Issue : 3  |  Page : 468-506

International Conference on Radiation Biology (ICRB-2012) on Cosmic Radiation to Cancer Therapeutics and 11 th Biennial Meeting of the Indian Society of Radiation Biology


Date of Web Publication17-Nov-2012

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How to cite this article:
. International Conference on Radiation Biology (ICRB-2012) on Cosmic Radiation to Cancer Therapeutics and 11 th Biennial Meeting of the Indian Society of Radiation Biology. J Can Res Ther 2012;8:468-506

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. International Conference on Radiation Biology (ICRB-2012) on Cosmic Radiation to Cancer Therapeutics and 11 th Biennial Meeting of the Indian Society of Radiation Biology. J Can Res Ther [serial online] 2012 [cited 2019 Nov 19];8:468-506. Available from: http://www.cancerjournal.net/text.asp?2012/8/3/468/103588

ACTREC, Tata Memorial Centre, Navi Mumbai, India

November 22-24, 2012

Guest Editor : Dr. Abhijit Dey -

PI, Molecular Functional Imaging Lab

ACTREC, TMC

Mumbai

Partially supported by Cancer Research & Communication. India



Radiobiology at crossroads: From explanatory to utilitarian science

The biological effect of IR and its medical potential was discovered within few years of the discovery of X-Rays and radioactivity at the turn of the last century. In its hundred years of existence, radiobiology has been pre-occupied with understanding and predicting the stochastic and non-stochastic radiation effects on cellular physiology, tumour killing, carcinogenesis, genetic effect and teratogenesis. A comprehensive understanding of the inherent variability in biological effect tor response to radiation and its determinants remains a work in progress.

Starting as a science to explain the bizarre biological effects of unknown rays called 'X' rays, radiobiology evolved to integrate various experimental and modeling approaches to quantify, predict and explain various biological effects of radiation. While the radiation associated variables were measurable and few - type, dose, dose rate and periodicity of radiation exposure; the variables associated with biological systems were far too many and not always amenable to reliable measurement. With diverse and sometimes interrelated biological variables such as normal or cancer cell and its type; oxygenation; rate, phase and kinetics of cell proliferation; series or parallel tissue architecture; direct, indirect and bystander effect; stochastic or deterministic effect; linear no threshold (LNT) or non LNT response; radiobiology had a lot to explain while maintaining internal consistency.

Starting from the law of Bergonie and Tribondeau of 1906 to the era of 4 and then 5 Rs of Radio-biology (Repair, Re-oxygenation, Re-population, Re-distribution and Radio-sensitivity) and the current era of radio-genomics, a lot has been studied, published and cherished. However, many practicing physicians who use radiation as their main professional tool perceive radiobiology as a self-obsessed explanatory science that has contributed far less in shaping their clinical practice as compared to physics, engineering and technology. Not surprisingly, modern radiotherapy is hailed as a technological approach to treat cancer rather than a biological approach when in fact it is perfect blend of both.

It is time to re-examine the issue and I would do so more from a personal perspective. In late eighties, almost all my fellow trainees and many of our trendy consultants were struck by the beauty of the mathematical modeling for bio-effect of radiation on normal tissue and tumour. Before the onslaught of high-end technology it was an era of intellectual innocence when radiobiology pervaded our minds. We admired, dissected and debated how Strandquist originally proposed the bio-effect model in 1942 and manner in which Ellis, Cohen, Orton, Kirk, Withers and many others independently looked at the same or different data and came up with their own versions of refinements. The slope of the iso-effect line, the shape of the cell survival curve, with width and meaning of the shoulder on this curve and the mysteries surrounding potentially lethal damage were not only enchanting but thought to be clinically important. Before the current Linear Quadratic model gained currency in the 90s, the NSD-TDF bio-effect model was appealing and applied in many departments including ours at the Tata Memorial Hospital. When required one could deploy the very 'objective' modeling calculation to prevail over the subjective experience of others especially as the EBM bandwagon was still at some distance. The NSD-TDF-CRE model was used for refining or justifying existing or proposed fractionation schedules and for gap corrections for missed treatments. Spinal cord was the only obstacle where out fear for the dreaded but rarely every seen myelopathy prevailed over mathematical formulae. While the numerical value of Tpot of different tumours could not be resolved in our frequent tea jaunts, we absolutely believed that each tumour has its distinct Tpot value and that it would matter. In those days before the PC and EXCEL the mathematically challenged community of radiation oncologists was served in good measure by their physicists. They would flash scientific calculators to tell us a number what came out of the NSD= D N -0.24 T -0.11 equation. The present generation of radiation oncology and physics trainees cannot imagine the bonhomie and intellectual discussions that existed between these two tribes. How I wish our physicists could go back in time and rediscover their knowledge about histological type, grade Tpot and fractionation without diluting their present focus on technology, contours, OARs, constraints and QA.

As all good things come to an end we could not live in this world of simple and apparently intelligent radiotherapy for long. The reigning mathematical model of 80s gave ways to the more elegant Linear Quadractic (LQ) model. Radiobiologists and some radio-biologically inclined oncologists and physicists immediately abandoned the sinking ship of the NSD-TDF model to come on board the newly floated LQ enterprise. Once bitten twice shy, the wider community of practicing oncologists and physicists did not trust another formulation where everything hinged upon a number called the alpha beta ratio. How could someone make a confident declaration of this ratio being the same for all tumours and acute responding normal tissue and that too a perfect 10? Had we not heard of digit preference? Perhaps as a mark of respect for severe late toxicity, a more humble number was assigned for the late responding normal tissues. Despite the indifference shown by the commoners this reverential treatment of the late responding tissues was recognized as a paradigm shift. In fact, many could not understand why it took so long for the pundits to state the obvious.

While it required the finest minds of 19 th and 20 th century from Roentgen to Einstein to explain the physical basis of ionizing radiation and its interaction with the matter, the optimal use of photoelectric effect for diagnostic radiology and of the Compton effect for radiotherapy required decades of cumulative technological development. Not as illustrious as their physics counterparts, radiobiologists had however got the 5Rs right. The intellectual insight provided was as profound but its clinical exploitation turned out to be a messy long drawn battle like the Afghan wars. Mired in the biological variables and the logistics of delivering a good enough dose of a drug, oxygen or heat to the tumour the trials and tribulations of clinical exploitation of the 5 Rs of radiobiology mostly drew blanks. Amidst the growing frustration with repetitive clinical failures, some quite spectacular and dangerous, instead of abandoning their theories, a dwindling flock of radiobiologists and their clinical partners came with renewed ideas for clinical experimentation. The real evidence for exploiting radiobiological knowledge for improving therapeutic ratio started trickling from the turn of this century. Clever prospective and adequately powered randomized trials by several groups led by Overgaard, Yarnold, Bentzen and a few others showed that repair, repopulation, reoxygenation and fraction radiosensitivity of normal and tumour cells could not only be predicted but harnessed to improve radiocurability, reduce toxicity or to improve health economics. The cautions lot of radiation oncologists though pleasantly surprised with this evidence did not take up altered fractionation and radiation modifiers very enthusiastically. However they have opened up to the idea that radiobiology is not only a science which explains their clinical observation but it can contribute to the refinement of their clinical practice. The time is ripe for radiobiology which is at this crossroad to make deeper inroads in the world of clinical radiotherapy.

This issue of JCRT has the scientific proceedings of the International Conference on Radiation Biology and the Biennial meeting of the Indian Society of Radiation Biology held at the Advanced Centre for Treatment Research and Education in Cancer in Mumbai. The meeting theme "From Cosmic Radiation to Cancer Radiotherapy" brings out the wide expanse covered by the field of radiobiology. Greater and more frequent opportunities for interaction is necessary between scientists exploring different area of radiobiology with the clinicians and physicists engaged in testing some radiobiological concepts on human subjects and radiation epidemiologists examining the stochastic effect of population exposed to low dose radiation.

Rajiv Sarin

Prof. of Radiation Oncology and I/C

Cancer Genetics Unit, ACTREC,

Tata Memorial Centre, Navi Mumbai, India

E-mail: rsarin@actrec.gov.in





BIOLOGICAL DOSIMETRY

Abstract No. 74

Dicentric chromosome analysis by giemsa staining and centromeric fluorescence in situ hybridization for biological dosimetry

Manivannan Bhavani

Sri Ramachandra University, Chennai, India. Email: bhavanigenetics@gmail.com

Changes in chromosome structure and function followed by exposure to ionsing radiation are collectively referred to as biomarkers of exposure. Of the various biomarkers in use to quantify radiation exposures, scoring of dicentric chromosomes (DC) from blood lymphocytes of exposed individuals remains, as the gold standard in radiation biodosimetry due to its specificity and sensitivity. As the DC assay is well established one and used for triage scenarios, the World Health Organisation (WHO) has taken the initiative to network laboratories involved in biological dosimetry. Besides, many laboratories are in the process of comparing DC yields, coefficients of reference dose-response curves, culture variables and scoring efficiency among scorers to obtain dose-estimations that are as close as possible. Coefficients of reference dose-response curves are a critical factor in dose-estimation and may be affected by uncertainties resulting from factors like poor metaphase preparations as well as scoring criteria, experience, etc. These coefficients are thus dependent on accurate identification of DC. As DC scoring with the fluorescent in situ hybridization (FISH) technique using centromeric specific probes results in accurate identification when compared to giemsa stained preparations, the objective of this study was to compare the DC and ring chromosome (RC) yields and resultant dose response curves by scoring DC in human peripheral blood exposed to 60 Co-γ radiation by giemsa staining and FISH. The obtained results shows that mean DC and RC frequency obtained for all the doses by both methods did not show any significant difference except at one dose (p < 0.05). Furthermore, the intercept (c), dose-coefficient and dose-squared coefficient with standard error (±SE) obtained for the reference curves also did not show any difference for both the techniques. Keywords: Ionizing radiation, Dicentric chromosome, Biological dosimetry, Dose response curve, FISH.

Abstract No. 76

Flow cytometry measurement for micronucleus frequency for biological dosimetry

G. Tamizh Selvana , M. Bhavania 1 , Safa Abdul Syed Basheerudeena 1 , J. Vijayalakshmia 1 , Solomon F.D. Paula J. S. Adhikari, N. K. Chaudhury and P. Venkatachalama 1

1 Department of Human Genetics, Sri Ramachandra University, Porur, Chennai, Tamil Nadu, India. Chemical Radioprotector and Radiation Biodosimetry research group, Institute of Nuclear Medicine and Allied Health Science, DRDO, Delhi, India. Email: tamizh096@gmail.com

Micronucleus (MN) assay, a simple and rapid measure of DNA damages, is a common methodology employed as a test of genotoxicity. Increase in number of micronuclei over and above the base line value is a very good indicator of the exposure of the test sample to radiation or chemicals. Traditional micronucleus assay is based on microscopic observation and scoring and can be measured on variety of cells, which is well established globally as an alternative to dicentric chromosome (DC) assay in radiation biological dosimetry using human peripheral blood lymphocytes (PBL). Dicentric chromosomal assay is very time consuming and therefore a large number of samples cannot be assayed in any single laboratory either for resulting or emergency situation. Of late the MN measurement by rapid means using flow cytometry is being developed to use it for triage applications. In case of nuclear emergency, the dose assessment for suspected population is mandatory towards the medical management of exposed individuals. Hence, in present study, we analyzed the MN frequency in the PBL exposed to X and gamma irradiation in-vitro. The induced MN frequency was scored with conventional microscopy and flow cytometry. The data obtained for both methodologies will be presented and discussed.

Abstract No. 163

Recent advances in physical, clinical and biological dosimetry in radiation therapy

SD Sharma

CTCRS, BARC, Anushakinagar, Mumbai, India. Email: sdsbarc@gmail.com

Beam therapy & brachytherapy are 2 common modes of therapeutic dose delivery to delineated target volume. High energy photon & electron beams are commonly used in beam therapy whereas sealed radionuclides (mainly photon emitting sources) are used in brachytherapy. Therapeutic medical radiation dosimetry includes the physical dosimetry of beams & sources, clinical dosimetry pertaining to treatment planning, dose distribution & dose verification & biological dosimetry related to treatment plan ranking & intercomparison. Physical dosimetry in beam therapy includes the dose rate calibration of beams & measurement of relative dosimetry parameters required by the external beam dose calculation algorithm. In case of brachytherapy, physical dosimetry includes the source strength calibration & measurement of relative dosimetry parameters required by the brachytherapy dose calculation formalism. Clinical dosimetry is related to generation of dose distribution, optimization of dose distribution under the defined treatment strategies & verification of dose delivered to the target volume & organs at risk (OARs). Maximizing tumour control probabilities (TCPs) while simultaneously minimizing normal tissue complication probabilities (NTCPs) are 2 main biological objectives of radiation therapy. The concepts of TCP & NTCP are used for intercomparison & ranking of treatment plans. The concepts of biological effective dose (BED) & equivalent uniform dose (EUD) are sometimes used in place of TCP & NTCP for ranking & intercomparison of treatment plans.

A number of recent advances have taken place in the technology & techniques of radiation dose delivery by external photon beams. The use of narrow conical photon beams in robotic radiosurgery & narrow fan beams in helical tomotherapy necessitated the further refinement & development of modified beam calibration methods. Similarly, the use of beamlets in intensity modulated radiotherapy necessitated the modification in the methods of relative dosimetry. Newer protocols have been proposed for dealing with such situations in external photon beam therapy. Development of absorbed dose to water standard for source strength determination is the further advancement in brachytherapy source calibration & dosimetry. This development is expected to enhance the dosimetry accuracy in brachytherapy.As far dose distribution calculation in beam & brachytherapy is concerned Model as well as Monte Carlo based dose calculation algorithms are being developed. A variety of devices & techniques have been developed for dose verification measurements in radiotherapy. Such devices & techniques include pre-treatment dose verification as well as online dosimetry during dose delivery. Refined mechanistic or phenomenological models for dose response predictions of tumour & normal structures have been proposed & are being used in radiotherapy treatment planning systems. However merits & limitations of these models need to be analyzed before using them.

Abstract No. 167

Optimizing and improving reticulocyte analysis for radiation biodosimetry application

Shwetanjali Nimkar, Minakshi Mann, Vijay Pal Singh, Raghubendra Singh Dagur, Sudhir Chandna

Natural Radiation Response Mechanisms Group, Division of Radiation Biosciences, Institute of Nuclear Medicine and Allied Sciences, Brig. S.K. Mazumdar Marg, Delhi - 110 054, India.

Biodosimetry or the measurement of biological markers that can be quantitatively related to the magnitude of the biological radiation dose received can conceivably play an important role in assessing radiation health risk in the event of an accident. Haematopoietic system is known to be most sensitive to radiation damage. The conventional haematologic parameters such as leukocyte type, numerical fraction ('differential leukocyte count') or bone-marrow examination are considered occasionally to provide such information but such analyses are time-consuming. Reticulocyte count is historically considered as a helpful tool for providing information on erythropoiesis. The reticulocytes count in peripheral blood is a reflection of bone marrow activity and responds to radiation with detectable sensitivity. This study aims to improvise and optimize this biological dosimeter tool for management of unplanned exposures to ionizing radiation. To achieve the goal manual counting and flow cytometry of reticulocytes with specific dye were performed for better precision of radiation dose assessment. Among the different staining procedures used for the reticulocyte count, we used propidium iodide for the evaluation of reticulocytes, considering that the fluorescence intensity is proportional to the amount of intracellular RNA and reflects immaturity of the cells. Manual cell counting under the microscope and flow cytometry of reticulocytes with fluorescence dyes such as pyronin-Y, SYTO-RNA, Thiazole orange and Propidium Iodide were performed for improving precision of radiation dose assessment. Validations were conducted using MGG assay and established flow cytomteric analysis. Experiments performed with various dyes and approaches that have been used thus far for reticulocyte analysis yielded encouraging results, and we also performed a detailed PI-based microscopic analysis that revealed the suitability of this new and simple assay. The reticulocyte count obtained using this analysis was slightly higher than the MMG-based analysis, which might be due to increased sensitivity of this dye for staining RNA. The kinetic analysis conducted with the peripheral blood of gamma-irradiated mice, yielded good results, and emphasized on the suitability of this assay.

ABSTRACT TOPIC: HYPERTHERMIA AND RADIATION THERAPY

Abstract No. 24

Mathematical modelling of the biological effect of hyperthermia and radiation

Hans Crezee, Petra Kok, Arjan Bel, Lukas Stalpers, Klaas Franken

Dept of Radiotherapy, Academic Medical Center, Amsterdam, The Netherlands. E-mail: h.crezee@amc.nl

Background: Hyperthermia (HT) therapy or raising the tumor temperature to 41-45΀C greatly enhances the effect of radiotherapy (RT) as demonstrated in clinical trials. The sensitizing effect of HT depends on the temperature level and the sequence of application and differs for tumor and normal tissue. Purpose is to develop biological treatment planning (BTP) to determine the biological effect of RT and HT for clinically realistic temperature and dose distributions. Materials and Methods: BTP for multi-modality treatments was developed, incorporated in our research RT planning system and applied to compare the effect of RT+HT with RT alone for 15 prostate cancer patients. The temperature dependent parameters alpha and beta of the linear-quadratic model were derived from literature and clinical experience. For prostate tumor a linear 50% increase of alpha was modelled between 37΀C and 41΀C. The value of beta was kept constant. No thermal enhancement was assumed for normal tissue. Clinical IMRT radiotherapy plans for a total dose of 70 Gy on the PTV, delivered in 35 fractions of 2 Gy, were created. Heating was achieved with the 70 MHz AMC-4 system with temperature distributions optimized to achieve a 43C tumor temperature in the PTV, normal tissue was constrained to 45΀C. Results: The average tumor temperatures T90, T50 and T10 in the PTV were 40.5΀C, 41.6΀C and 42.4΀C, respectively. The minimum, maximum and average radiotherapy doses were 62.8 Gy, 80.9 Gy and 75.9 Gy, respectively. The equivalent minimum, maximum and average dose reflecting the radio sensitization by hyperthermia were 70.1 Gy, 93.4 Gy and 86.1 Gy, respectively. Conclusion: BTP allows analysis of multi-modality treatments and is useful to predict the interaction of different cancer treatments.

Abstract No. 42

Induction of non-homologous end-joining repair by hyperthermic inhibition of homologous recombination


Przemek M. Krawczyk, Berina Eppink, Jeroen Essers, Jan Stap, Hans Rodermond, Lukas J. Stalpers, Roland Kanaar, Jacob A. Aten, Hans Crezee and Nicolaas A. P. Franken

Laboratory for Experimental Oncology and Radiobiology (LEXOR), Center for Experimental Molecular Medicine, Department of Radiation Oncology, Academic Medical Center, University of Amsterdam, P.O.Box 22700, 1100 DE Amsterdam, The Netherlands. Email: n.a.franken@amc.uva.nl

In S and G2 phase mammalian cells DNA double strand breaks (DSBs) can be repaired by homologous recombination (HR) or non-homologous end-joining (NHEJ). Results of several studies suggest that these two mechanistically distinct repair pathways can compete. Because HR and NHEJ differ with respect to error susceptibility, chromosome rearrangements, potentially carcinogenic products of DSB repair, may depend on the pathway choice. Here we investigate the influence of HR and NHEJ inhibition on the frequencies of chromosome aberrations. We utilize mild (41ΊC) hyperthermia for 1 h to disable HR and KU-57788 or cisplatin to inactivate NHEJ in SW-1573 and RKO cells and measure frequencies of chromosomal fragments (resulting from unrepaired DSBs) and translocations (products of erroneous DSB rejoining) in G2 cells by premature chromosome condensation (PCC) combined with fluorescence in situ hybridization (FISH).

Also the effect of incubation of cells for 1h at 41΀C on the accumulation of Rad51, Mre11 and Mdc1 (proteins involved in DNA DSB repair) in ionizing radiation induced foci (IRIF) is studied. The temporary inhibition of HR by hyperthermia (41΀C for 1 h) results in increased frequency of ionizing-radiation (IR)-induced chromosomal translocations and to a temporary inhibition of Rad51 accumulation in IRIF. It is also demonstrated that this effect can be counteracted by KU-57788 or cisplatin-mediated inhibition of NHEJ. Our results suggest that in G2 phase the choice of DSB repair pathway is made early after damage induction. Our results suggest that in G2 the choice of DSB repair pathway is made early after damage induction. After inhibition of HR, DSB repair might be shifted to the error-prone NHEJ pathway resulting in increasing frequencies of chromosomal translocations.

Furthermore it is shown that mild (41-42.5΀C) hyperthermia inhibits the homologous recombination (HR) pathway of DSB repair by inducing degradation of BRCA2 and that this effect can be significantly enhanced by inhibition of the heat shock protein 90 (HSP90). Mammalian tumour cells deficient in HR are sensitive to the inhibition of poly (ADP-ribose) polymerase 1 (PARP-1). We demonstrate that hyperthermia can be used to sensitize innately HR-proficient tumour cells to PARP-1 inhibition. When inhibition of PARP-1 is combined with Hyperthermia and 17 DMAG (an HSP90 inhibitor) significant tumour cell killing can be obtained both in cell culture and in tumours growing in vivo.

The results enable design of new therapeutic strategies in clinical hyperthermia.

Abstract No. 112

Clinical scale preparation and bio-evaluation of 131I rituximab for Non-Hodgkin's Lymphoma

Mythili Kameswaran, Vimalnath Nair , Rajeswari A, P V Joshi, Grace Samuel, H D. Sarma, M R A Pillai

Radiopharmaceuticals Division, BARC. Email: kmythili@hotmail.com

Radioimmunotherapy (RIT) is an attractive alternative to chemotherapy or radiotherapy in the treatment of B cell lymphoma. Most B-cell lymphomas express CD20, making it a suitable target antigen for therapeutic radioactive monoclonal antibodies. RIT with anti CD20 monoclonal antibody (MoAb) conjugated to a β-emitting radioisotope will deliver radiation to the tumor cells. Rituximab (anti CD20 MoAb), being a human murine chimeric antibody would obviate induction of HAMA response and would allow repeat administration of antibody for treatment. In order to make available an indigenous and affordable radioimmunotherapeutic agent for Non Hodgkin's Lymphoma (NHL), radioiodinated Rituximab has been prepared and evaluated at a clinical scale.

Six consecutive batches of 131I-Rituximab have been prepared in a well shielded plant under aseptic conditions. Radioiodination of Rituximab was performed by the conventional chloramine T method. Purification of the product was carried out using PD10 column. Ascorbic acid was added as a radioprotectant. The final product was rendered sterile by filtering through 0.22μ filter. The product was characterized by electrophoresis and HPLC. In vitro cell binding studies were carried out in Raji cells as specific cells and U937 as non specific cells. Biodistribution studies in normal Swiss mice were carried out to determine the clearance pattern of the product in vivo.

The overall radiochemical yield of the six batches was 83 % ± 16% (n=6) and the radiochemical purity was > 95% in all the cases. HPLC of the purified product showed a retention time of ~ 11min. The product was found to be stable when studied up to 6 days after radioiodination. In vitro cell binding studies with Raji cells showed a specific binding ranging from 23-28% in the six batches. No binding was observed with U937 cells further confirming the specificity with Raji cells. Biodistribution studies showed that the clearance was largely through the gastrointestinal and hepatobiliary route.

The favorable uptake of the in-house produced 131I-Rituximab by the CD20 expressing cells and normal biological distribution suggests the potential of this preparation as a radioimmunotherapeutic agent for NHL.

Abstract No. 140

Radiopharmaceuticals as molecular medicines for cancer management

M.R.A. Pillai

Bhabha Atomic Research Centre, Mumbai, India. Email: mrap@barc.gov.in

The use of radiation emanating from external sources for the management of cancer is a well established procedure for over a Century. External beam therapy is successful when the cancer is localized; however it is less efficacious when the tumor has already spread. Targeted delivery of the radionuclides emanating particulate radiations to the site of cancer is an efficient way of endoradiotherapy. Radiopharmaceuticals are molecular medicines that take advantage of a specific biochemical mechanism for targeting them to the site of the disease and deliver cytotoxic dose to the tumor. Radionuclide therapy (RNT) using in vivo administration of site specific radiopharmaceuticals offers the possibility to effectively treat diffused cancer as well as metastasis. Management of thyroid cancer and metastasis using radioactive iodine (Na131I) is practiced for over 50 years. As nearly one third of the ingested radioactive iodine gets accumulated in the thyroid tissues mediated through sodium iodide symporter (NIS), it is the best example of a 'magic bullet'. Advances in the understanding of tumor biology as well progress in recombinant antibody technology and synthetic peptide chemistry has led to advances in the development of many new therapeutic radiopharmaceuticals which are currently in clinical use. The Radiopharmaceuticals Division, Bhabha Atomic Research Centre has been successful in developing a few products for the management of cancer. Tc99m-Hynic-TOC, a labelled somatostatin analog peptide, developed for imaging tumors over expressing somatostatin receptors is currently functioning in many nuclear medicine departments for diagnosis of neuroendocrine tumors (NETs). Lutetium-177, I-131 or Y-90 are used as radionuclides in the above as they could be prepared in large quantities either using the Dhruva Reactor (I-131, Lu-177) or by using a generator (Y-90). Among the radiopharmaceuticals developed for therapy, Lu-177-DOTA-TATE is used for the treatment of NETs and Lu-177-EDTMP is used for pain palliation of patients suffering from bone metastasis. Rituximab, a humanized monoclonal antibody has been labeled with I-131, Lu-177 and Y-90 for the development of therapeutic agents for treating Non-Hodgkin's Lymphoma. The presentation will cover the details of the development of the above radiopharmaceuticals and the physicochemical, in vivo and in vitro biological evaluations conducted with the radiopharmaceuticals for ensuring safety and efficacy of the above products for human applications.

Abstract No. 154

Enhancement of human sodium-iodide symporter gene expression by histone deacetylase inhibitors for effective targeted radio-iodine therapy in breast cancer

Madhura Kelkar and Abhijit De

KS325, Molecular Functional Imaging Lab, ACTREC, Kharghar, Navi Mumbai, India. Email: mkelkar@actrec.gov.in

Background: Human Sodium Iodide Symporter (hNIS) mediates active iodide uptake into the thyroid gland. The hNIS mediated iodide uptake and organification is the basis for the post-operative use of targeted radio-ablation in differentiated thyroid cancer. Human NIS is also over-expressed in various sub-types of breast cancer, hence can be explored for clinical procedures of radioiodine based imaging and therapy in the breast cancer management. To enhance the higher radioiodine uptake / retention, we have initiated testing agents capable of modulating hNIS expression in breast cancer cells. Extensive screening effort to identify genetic or epigenetic modulators may help to achieve this goal. Here, we aim to identify candidate HDAC inhibitors (HDACi) to modulate hNIS expression in breast cancer cells. Materials and Methods: We tested effect of four HDACi, Trichostatin A (TSA), Sodium Butyrate (NaB), CI994, and Valproic Acid (VPA) on endogenous hNIS expression in breast cancer cell lines (MCF7 and MDA-MB 231) measured by real time RT-PCR. Further hNIS promoter driving luciferase expression vector (pNIS-Fluc2-TurboFP) was designed to evaluate the drug mediated modulation of hNIS gene transcription, which was measured by luciferase reporter assay. Engineered MCF7 and MDA-MB 231 cells were developed using the reporter plasmid and used for optimizing drug concentration. The effect of these HDAC inhibitors on endogenous protein level was determined by Immunofluorescence staining using hNIS monoclonal antibody. Besides this, the effect of HDAC inhibitors on cell viability and cell cycle were also evaluated. Results: We found that HDAC inhibitors significantly (p < 0.05) increase hNIS mRNA expression in MCF-7 and MDA-MB 231 cells. HDAC inhibitors stimulated hNIS gene transcription in both the cell lines (p < 0.0001) and their effect was found to be hNIS promoter specific. HDACi TSA, NaB, CI994 and VPA enhanced hNIS promoter activity by 2.3-, 3.1-, 2.1- and 3.5- folds respectively in estrogen receptor positive MCF-7 cells. Similarly, respective 4.5-, 4.1-, 2.2- and 2.6- folds higher expression was also observed in receptor negative MDA-MB 231 cells. HDACi also increased endogenous hNIS protein level (by 3.7-, 2.5-, 1.8- and 3.1- folds respectively) in MCF-7 cells. These HDACi were found to be anti-proliferative causing significant growth arrest. Future work will include molecular imaging guided evaluation of drug effect in breast cancer animal model. Conclusion : HDACi mediated enhancement of hNIS gene expression is a novel approach expanding the scope of 131-I radioiodine therapy for breast cancer patient groups with moderate or low level of hNIS expression. Future investigations to establish the relation between hNIS activity and histone acetylation may impact the innovative endogenous hNIS mediated radioablation in breast cancer.

LOW DOSE RADIATION BIOLOGY

Abstract No. 63

Identification of radioresistance related proteins in human oral cancer cells by fractionated ionizing radiation

Mohd Yasser 1 , S Pawar 1 , R Govekar 2 , S Zingde 2 , T Teni 1

1 Teni Lab, 2 Zingde Lab, Advanced Centre for Treatment Research and Education in Cancer (ACTREC), Navi Mumbai, India. Email: yasserashfaq@gmail.com

Tumor cell resistance to ionizing radiation and chemotherapy is a major obstacle in cancer therapy. Identification of proteins that are differentially-expressed between radiosensitive and radioresistant cancer cells is important for the ability to predict clinical effectiveness of radiotherapy.

We selected two oral cancer cell lines AW13516 and SCC29B of different sites with differing radiosensitivity. Radioresistant sublines of AW13516 and SCC29B were established by treatment with 2Gy Fractionated Ionizing Radiation using 60 Co-γ Linear Accelerator (Bhabhatron, ACTREC) up to a total dose of 70Gy. Protein lysates of parental and radioresistant AW13516, SCC29B cells were separated using 2-Dimensional gel electrophoresis in the PI range 3-10, coomassie stained and differentially expressed spots were identified. The differential spots were eluted and analyzed by MALDI-TOF. A total of 45 proteins were identified to be differentially expressed between parental and radioresistant sublines. Among them 14 proteins were found to be differentially expressed in AW 13516, 9 proteins in SCC 29B and 22 common proteins in both cell lines. The parental and radioresistant sublines were further characterized by morphological evaluation, cell cycle analysis, Annexin V/PI staining and Raman spectroscopy.

Protein analysis of radioresistant sublines may provide new insights into the mechanism underlying clinical radioresistance and help in improving the efficacy of radiotherapy of oral cancers.

Abstract No. 77

Studies on the biological effects of low dose ionizing radiation received during diagnostic radiological examination

Safa Abdul Syed Basheerudeen 1 , Bhavani M 1 , Tamizhselvan G 1 , Venkatachalam P 1 , Santosh Joseph 2 , Paneerselvam S 2 , Keerthiraj B 2 , Venkatraman P 2 , Rajendran 2 , Thayalan K 3

1 Department of Human Genetics, Sri Ramachandra Medical College and Research Institute, Porur, Chennai, 600116 India. 2 Department of Radiology and Imaging Sciences, Sri Ramachandra Medical Center, Porur, Chennai, 600116. 3 Bernard Institute of Radiology, Madras Medical College attached to Rajiv Gandhi Memorial Government Hospital, Egmore, Chennai. Email: smartsafa@gmail.com

Background: Medical applications of ionizing radiation are enormous and its uses are increasing day by day, of which, the interventional radiological procedures are minimally invasive X-ray guided procedures being in use for diagnostics and therapeutics. These procedures provide great benefits but the risk due to the low dose and dose rate of these radiation exposures is not understood clearly. Hence, the aim of the present investigation was to study the effects of very low dose and dose rate exposures received during interventional radiological procedures exposed in vivo. The outcome will provide additional insights into low dose radiation biology, whether frequent exposures to diagnostic radiation will induce any genetic damage and results in enhanced risk for the development of stochastic effects. Materials and Methods : The study consists of (n=10) patients who underwent neuro-interventional radiological procedures. The entrance skin dose (ESD) was assessed using calcium sulphate thermoluminescent dosimeter (TLD) discs. To assess the biological effects, received by the exposed patients, blood samples were collected before the procedure, 2hr and 24hr post procedure; The blood samples were cultured employing standardized methodologies and analyzed for frequency of chromosomal aberrations and micronuclei. Results: The ranges of ESD measured using TLD were between 10.20 and 700.80 mSv for head, 8.35-191.85 mSv for neck and 12.55-89.50 mSv for shoulder. The blood samples collected post procedure (2hr and 24 hr), showed an overall significant (P < 0.05) increase in chromosomal aberration frequency and micronuclei frequency when compared pre-exposure. Further, there was an inter-individual variation in the induced frequencies of DNA damages among the patients.

Abstract No. 88

Low dose gamma radiation induced differential expression of AP1 complex genes in human peripheral blood mononuclear cells

Nishad S, Anu Ghosh & JR Bandekar

Radiation Biology & Health Sciences Division, Bhabha Atomic Research Centre, Mumbai-85, India. Email: nishad@barc.gov.in

The complex molecular response to ionizing radiation is mediated by a variety of regulatory pathways, including transcriptional regulation. The transcription factors constitute important mediators of such control. Activating protein 1 (AP-1) is a dimeric transcription factor comprising of fos (c-fos, fosB, fra-1 and fra-2) and jun (c-jun, junB and junD) family proteins. The wide combinatorial possibilities provided by AP-1 proteins influences its specificity of binding DNA and subsequently, the continuum of regulated genes. In this study, the gene expression of four members of fos family and three members of jun family was studied as a function of time and radiation dose. Human peripheral blood mononuclear cells (PBMCs) were separated from venous blood collected from ten healthy male individuals (age group: 25-30 years). These were irradiated with Co60 γ-rays (Blood irradiator, 2000, BRIT) at a dose rate of 0.417 Gy/minute. Three radiation doses were given (0.3Gy, 0.6Gy and 1Gy) and RNA was isolated either immediately, 1 hour post irradiation or 4 hours post irradiation. Unirradiated PBMCs incubated for the same time at 37΀C in a humidified, 5% CO2 atmosphere were used as controls. RNA was converted into cDNA and gene expression was studied with real time quantitative PCR using SYBR green. β-actin as used as the housekeeping gene. Melting curve analysis was performed for each PCR product to confirm the specificity. For most individuals, a maximum response was seen after a dose of 1 Gy. Time course study showed that the induction for most genes was transient, reaching a maximum at 1 hour and declining to the constitutive level 4 hour after irradiation. Regression analysis confirmed the presence of significant inter-individual variation. The expression of Fos and Jun proteins is known to be temporally coordinated and the composition of AP-1 may change in the cell in a context dependent manner. All these factors make AP-1 a dynamic transcriptional complex.

Abstract No. 101

Radiation dose adaptation in human lymphocytes exposed to electron beam irradiation

Sukanya Shetty 1 , Shama Rao 2 , Madhu L.N 2

1 Dept of Biochemistry, KSHEMA, 2 Department of Allied Health Sciences, Nitte University, India.

When living organisms exposed to a variety of environmental stresses like heat, radiation exposure almost all from bacteria to humans react promptly so that they may survive by DNA repair mechanisms or it may be lethal because failure in repair mechanism, or can also lead to various kinds of mutation in the system.

In the field of oncology, radiotherapy is one of the integral parts in treatment of cancer. During radiotherapy along with cancer cells normal cells also exposed to ionizing radiation, which is one of the serious problem. Radiation adaptation is one of the methods to reduce the normal cell damage. Induction of an adaptive survival response in cultured human lymphocytes exposed to Electron beam irradiation by low dose (1Gy) then to high dose (4Gy) of Electron beam irradiation was examined. The adaptive survival response was determined by comparing the cell viability assay and DNA damage was studied at 2, 4, 6, 24 and 72 hours interval. The frequencies of DNA damage in cultured human lymphocyte was higher in cells exposed only low dose (1Gy) and high dose (4 Gy) than in cells which are pre-exposed to 1 Gy and then to 4 Gy after 24 hrs. It is also found that, adaptive response induced by exposure to low doses of radiation occurred after 6 hour.

Thus, low dose electron beam irradiation induces the adaptive response in human lymphocytes. Low dose exposure may enhance the capacity of cells to repair the damage.

Abstract No. 113

Modulatory effect of polybion against radiation and cadmium induced biochemical changes in the brain of swiss albino mice

Arindam Basu, RK Purohit, Aruna Chakrawarti, Manisha Agarwal, Pankaj Joshi and KM Bhartiya

Radiation Biology Laboratory, Department of Zoology, Govt. Dungar College, Bikaner (India) 334001. Email: dr_rajendra_purohit@yahoo.co.in

The present century has been an ever-increasing use of nuclear technologies in different fields raising the alarming problem of radiation hazards to living beings including man. An increasing body of evidence indicates that human activities are responsible for global climatic changes, which, in turn may be directly or indirectly increasing human exposure to environmental hazards. On the other hand, all forms of cadmium are poisonous leading cadmium intoxication under appropriate circumstances. The interaction between radiation and other toxicants represents a field of immense potential importance as their total environmental burden may have greater effects than expected from the sum of their individual impact.

In the present study 6 to 8 weeks old male Swiss albino mice were exposed to 2.0 and 4.0 Gy of gamma rays with or without cadmium chloride treatment. The animals of experimental groups were administered polybion for seven days prior to radiation or cadmium chloride treatment. After routine procedure of histology the histopathological changes were observed in the brain of Swiss albino mice.

The histopathological changes observed were pycnotic nuclei and crenated cells with condensation of nuclear material resulting into hyperchromatic cells. Hydrocephaly with enlarged lateral ventricles was also noted. Corpus callosum was seen malformed. Thickened meninges and venous congestion were also noticed. In the irradiated brains cytoarchitectonic layers were reduced in depth and showed some degree of intermixing of cells of various laminae. Hematoma was present between the cortex and medulla with numerous pycnotic and necrotic nuclei. Disarray of the cortical tissue with disorientation of cell processes was also evident. Damage in the cortex was noticed in the form of karyolysis, pycnosis and spongy degeneration of the connective tissue with the thickening of meninges. Dilation of blood vessels was also observed at certain places.

Quality of these changes remaining the same, but their magnitude increased with dose. With an increase in the dose, time of the onset of recovery is delayed and the time required for complete recovery is longer. After the combined exposure of gamma rays and cadmium chloride, the histological changes were similar but showed higher magnitude than the individual exposure of gamma rays and cadmium chloride.

The brain of polybion treated animals exhibited less severe damage as compared to non-drug treated animals at all the corresponding intervals. An early and fast recovery was also noticed in polybion pretreated animals.

Abstract No. 115

Fukushima Daiichi Nuclear Power Plant accident from the aspect of radiation biology

Yoshihisa Matsumoto

Tokyo Institute of Technology, Japan. E-mail: yoshim@nr.titech.ac.jp

On 11 th March, 2011, Japan, especially East coast area of Tohoku destrict, was attacked by huge earthquake of M9.0 and succeeding Tsunami. As of August 2012, 15,869 lives were lost and 2,847 people are still not found. Fukushima Daiichi Nuclear Power Plant stopped automatically in response to the earthquake but lost emergency power supply for cooling because of Tsunami, leading to hydrogen explosion and massive release of radioactivities. NISA and NSC estimate the released radioactivities of iodine 131 and cesium 137 to be 150-160 PBq and 12-15 PBq, respectively. NISA evaluated the scale of the accident to be level 7 (initially level 4), according to the international nuclear event scale (INES) by IAEA. In this occasion, I would like to overview radiation countermeasures of Japanese government and public concerns regarding the health effects of radiation from the aspect of radiation biology.

Abstract No. 135

Low dose gamma-radiation induces migration in U87 glioma cell line through a connexin-43 dependent, P38-mediated and erk-independent pathway

Soma Ghosh, Ashish Kumar & Sudhir Chandna

Natural Radiation Response Mechanisms Group, Division of Radiation Biosciences, Institute of Nuclear Medicine & Allied Sciences. Email: soma_ghosh1982@yahoo.com

Loss or down-regulation of connexins (Cx) is a common feature in various tumours and up-regulation of its expression may help reduce tumour growth. Low-dose ionizing radiation is known to stimulate MAPK pathways and also upregulates expression of Cx43 in normal as well as in tumorigenic cells. The effect could be differentially induced depending on the nature of cells and may have important implication in regulating radiation-induced biological effects.

The present study aimed at investigating intracellular MAPK signalling pathways elicited by low-dose gamma-radiation and potential relation with connexin-43 regulation.

Human malignant glioma U87 cell line was irradiated at low doses (5cGy, 10cGy, 20cGy) as well as clinically relevant dose (2Gy) of gamma-radiation and alterations in the levels or activation status of MAPK pathways and/or Cx43 were detected by western blotting or Real-Time PCR. Cell proliferation and migration was assessed by phase contrast microscopy whereas cell synchronization were analyzed by flow cytometry. Knock-down of cx43 was achieved by RNA-interference.

Low doses of gamma-radiation activated the expression of Cx43 with maximum effect observed at 10-20cGy which was found to be associated with enhanced cell migration in U87 cells. Prolonged activation of phospho-p38 was also observed particularly at doses 10cGy-20cGy which was decreased with the knockdown of Cx43, directly implicating Cx43 in the p38-mediated cell migration induced at low doses. These low doses of gamma-radiation also activated p-ERK1/2 which was associated with enhanced cell proliferation at 5cGy and further supported by the activation of downstream targets like PCNA and phospho-Histone H3. Knockdown of Cx43 did not have any effect on ERK-1/2 activation. Doses higher than 5cGy (10cGy/20cGy and 2Gy) failed to induce cell proliferation and were associated with enhanced expression of p21, thus indicating activation of cytostatic signalling.

Therefore, the present study demonstrates that low doses of gamma-radiation may activate cell proliferation and/or migration through activation of ERK1/2 and/or p38 MAPK pathways. Importantly, the p38-mediated low-dose induced U87 glioma cell migration was Cx43-dependent, which may have important implication for controlling radiotherapy-induced second malignancies.

Abstract No. 142

Exploiting sensitization windows of opportunity in hypo and hyper fractionated radiation therapy

Mansoor M Ahmed

Radiation Research Program, National Cancer Institute, National Institutes of Health. Email: ahmedmm@mail.nih.gov

Conventional fractionated radiation therapy is an effective modality for the treatment of solid tumors, however, there remains dismal response even in adjuvant settings. Hence, novel radiation dose deliveries is warranted, particularly to sensitize the adjuvant agents. This talk will focus on two novel aspects of utilizing radiation therapy to enhance the effect of adjuvant agents or vice-versa. The first focus will detail the mechanisms underlying as well as developed clinical trial on "Low-dose fractionated radiation therapy (LDFRT) as a potentiator of chemotherapy. The second part of the talk will highlight potential exploitation of factors (bystander, abscopal and immunological) induced in response to high-dose hypofractionated radiation therapy to help sensitize to specific signaling adjuvants. This will open up new avenues to design novel trials even in induction setting to understand the efficacy window before the start of definitive therapy.

Abstract No. 158

Radiation-induced transformation and hyper-radiosensitivity survival response of transformed NIH-3T3 murine fibroblast cell line

Ankit Mathur, Shalini Raik, Sudhir Chandna

Institute of Nuclear Medicine and Allied Sciences. Email: aki_mathur23@yahoo.co.in

Many human tumor cell lines are known to be hypersensitive to very low doses of ionizing radiation (below 0.5Gy), a phenomenon widely known as hyper-radiosensitivty (HRS), whereas normal cell lines display this phenomenon to much lower extent. Hence process of transformation is believed to alter sensitivity of mammalian cells against ionizing radiation, although more in-depth and detailed investigations are required to completely establish this phenomenon. In this study, we analyzed the effect of radiation induced transformation in NIH3T3 murine fibroblast cell line and its effect on the hyper-radiosentivity response. We induced transformation of NIH3T3 cells by gamma-irradiating at varying single acute doses of radiation ranging from 10cGy up to 5Gy as well as by 1.5Gy gamma-radiation repeated thrice along with 3-methyl cholanthrene (3MC) treatment and observed a significant increase in transformation. The transformation of NIH3T3 was confirmed by morphological changes and population doubling time. Since Gap Junctional Intercellular Communication (GJIC) and connexin proteins are important for survival of cells we studied the activity of GJIC using Lucifer Yellow assay in transformed NIH3T3 cells. An alteration in connexin-43 and reduced GJIC activity was observed in transformed cells than normal cells indicating significant contribution of intercellular communications in HRS of transformed cells. Very importantly, we observed highly significant increase in the hyper-radiosensitivity response of transformed cells as compared to the untransformed cells in case of radiation-transformed cells. This study is the first study that explicitly displays the altered HRS response during the transformation process and also able to provide a very important mechanistic insight into the phenomenon of HRS with respect to cellular transformation and cell-cell adhesion processes.

Abstract No. 162

A novel agarose overlay technique' for improving colony forming efficiency of primary fibroblast cells: A simpler alternative to feeder cell supplementation


Sudhir Chandna 1 , 2 , Raghubendra Singh Dagur 1, Ankit Mathur 1 , Vijaypal Singh 1 , AT Natarajan 3 , Siamak Haghdoost 2 and Mats Harms-Ringdahl 2

1 Natural Radiation Response Mechanisms Group, Division of Radiation Biosciences, Institute of Nuclear Medicine & Allied Sciences (INMAS), Brig SK Mazumdar Road, Delhi-110054, India 2 Centre for Radiation Protection Research, Department of Genetics, Microbiology and Toxicology (GMT), Stockholm University, Svante Arrhenius Vag 16E, Stockholm, Sweden 3 Department of Agrobiology and Agrochemistry, University of Tuscia, Via San Camillo de Lellis s.n.c, 01100 Viterbo, Italy. Email: raghuvendrasingh1982@gmail.com

Background: The in vitro clonogenic cell survival assay or colony formation assay has been used quite extensively for characterizing the radiation sensitivity of mammalian cells. In certain cases, the adherence-dependent cell cultures are observed to be unsuitable for clonogenicity assay due to their inability to form distinct and scorable colonies. Alternative methods such as supplementation with feeder cells, which provide them with increased amounts of growth factors required for enhancing their colony forming ability. The use of feeder cells in clonogenic assay of VH10 cells can be a limiting factor in designing large experiments. In the present study, we developed a novel and simple alternative technique involving agarose overlay that resulted in significant enhancement of cloning efficiency of VH10 cells, HDFn Fibroblasts as well as primary human fibroblasts isolated from thyroid glands of patients undergoing FNAC investigations. Response of tumour cell lines was also obtained to test the suitability and selectivity of this new assay for primary cells. Objective: Evaluating an 'agarose overlay technique' for improving colony forming efficiency of primary fibroblast cells. Materials and Methods : VH10 foreskin primary fibroblasts, HDFn fibroblasts or primary thyroid fibroblasts were irradiated and radiation response was evaluated using conventional colony forming assay, feeder layers and with the new assay with agarose overlaying, colony formation was scored. Results : In all the Petri dishes used for conventional colony forming assay, a very small number of large-sized and faintly staining colonies were detected after nearly four weeks of plating the untreated VH10 cells with only 5-7% plating efficiency. With the use of irradiated feeder cells, plating efficiency of VH10 cells was observed to be in the range 15-25%, and colony staining improved significantly over the conventional assay and observed to be more densely arranged. Using the agarose overlay assay, we could successfully generate the radiation dose-response survival curve for VH10 fibroblasts with suitable plating efficiency above 40%, and the colonies were detectable at early time-point (about 7-10 days). A total of 300 cells were seeded in all treatment groups up to the dose of 5Gy and the mean the mean SF2Gy value for VH10 cells was observed.

Abstract No. 176

Inhibition of radiation induced angiogenesis by 2-Deoxy-D-glucose

th
Saurabh Singh 1none , Amanpreet Singh Chawla 2 , Sanjay Pandey 1 , Ravi Soni 1 , Daman Saluja 2 and B S Dwarakanath 1

1 Institute of Nuclear Medicine and Allied Sciences, Delhi 110054; 2 Ambedkar Centre for Biomedical Research, University of Delhi, India. Email: saurabh_11may@yahoo.co.in

Background: Radiotherapy is an important arm of cancer treatment and ionizing radiation (IR) exerts its anti-tumor effects by killing tumor cells as well as bringing changes in the microenvironment, including the microvasculature. On the other hand, there are clinical and experimental observations demonstrating pro-angiogenic and pro-metastatic effects of IR. During radiotherapy (clinical), the tissue surrounding the tumor area receives low doses of IR compared to those delivered inside the tumor mass, as multiple radiation beams are given to tumor in order to prevent damage of organs at risk. Low-dose IR has been shown to promote angiogenesis resulting in accelerated tumor growth and metastasis in a VEGFR-dependent manner. Induced angiogenesis involves a number of energy consuming biological processes such as proliferation, migration and capillary formation by endothelial cells (ECs) accompanied by increased energy needs leading to enhanced glucose metabolism. Therefore, use of Energy Restriction Mimetic Agents (ERMA) such as 2-Deoxy-D-Glucose (2-DG) may inhibit radiation-induced angiogenesis thereby modulating the metastases and invasiveness. Objective: To investigate the effects of 2-DG on radiation induced angiogenesis. Materials and Methods: thIn-vitro studies Matrigel tube formation and scratch assay was performed in normoxic and hypoxic conditions both to assess whether 2-DG affected the tube formation and migration property of Human umbilical vein endothelial cells (HUVECs). Invasion, cell viability and cytotoxicity assays are being performed presently. th In-vivo studies Low dose radiation induced angiogenesis Swiss nu/nu mice were locally exposed to low dose (0.3 Gray) radiation at right lower back region. Twenty four hours later, matrigel was injected in the exposed region to support neo-vascularisation and 2-DG administration (0.2% and 0.4% w/v) was started next day till the termination of the study. Angiogenesis from matrigel plugs were quantified by FITC-Dextran fluorescence assay. H & E staining of the matrigel plugs was performed. Results and Conclusion: 2-DG significantly inhibited radiation-induced migration of endothelial cells (ECs), which was more prominent under hypoxic conditions as observed by the scratch assay. Also in the murine model of radiation-induced angiogenesis, the degree of neo-vascularisation was greatly reduced in the 2-DG fed mice as evidenced by a marked decrease in the capillary formation in the 2-DG group revealed by H & E staining. In conclusion, our findings provide new insights into radiation-induced angiogenesis and establish the anti-angiogenic effects of 2-DG by targeting endothelial cell metabolism thereby opening opportunities for designing more effective combinational therapies using 2-DG.

MOLECULAR IMAGING IN CANCER

Abstract No. 22

Molecular imaging of tumor angiogenesis: Peptides, proteins, and nanoparticles

Weibo Cai

Department of Radiology, Medical Physics, Biomedical Engineering, University of Wisconsin - Madison, USA. Email: wcai@uwhealth.org

The Molecular Imaging and Nanotechnology laboratory at the University of Wisconsin - Madison (http://mi.wisc.edu/) is mainly focused on three areas: 1) development of multimodality molecular imaging agents; 2) nanotechnology and its biomedical applications; and 3) molecular therapy of cancer. In this talk, I will present our recent work on molecular imaging of tumor angiogenesis in various mouse models of cancer using peptides, proteins and a variety of nanomaterials. The primary imaging techniques used in these studies are positron emission tomography [PET] and optical imaging, and the major molecular targets that we are investigating are CD105 (i.e.0 endoglin) and integrin αvβ3.

Angiogenesis (i.e. new blood vessel formation) is required for solid tumor growth/metastasis, and one of the key proteins involved in angiogenesis is CD105. Studies have shown that high CD105 expression correlates with poor prognosis in >10 solid tumor types. CD105 staining is now the accepted standard for assessing tumor microvessel density, a quantitative measure of tumor angiogenesis. Our recent efforts in the development of various CD105-targeted PET and optical imaging agents will be presented in this talk.

Since both CD105 and integrin αvβ3 are tumor vascular targets, they are particularly attractive for nanomaterial-based tumor targeting and drug delivery, since extravasation (a major obstacle for in vivo targeting with most nanomaterials) is not required to achieve tumor targeting/contrast. The nanomaterials that will be discussed in this presentation include nano-graphene oxide, zinc oxide nanowires and unimolecular micelles among others.

Abstract No. 26

Nuclear imaging and tumor characteristics: Emphasis on clinical PET

Sandip Basu

Nuclear Medicine Physician & Head, Nuclear Medicine Academic Programme, RADIATION MEDICINE CENTRE, Bhabha Atomic Research Centre, Tata Memorial Hospital Annexe, Parel, Mumbai, 400012, India. Email: drsanb@yahoo.com

The inherent strength of functional radionuclide imaging lies in characterizing the tumor biology. Rather than adjudging a modality with regard to its sensitivity in lesion detection, its role in oncological setting should be viewed from how functional imaging of tumor phenotype and heterogeneity could aid in differentiating a „good" tumor from a „bad" one and how such information can influence the clinical decision making process. This obviously forms one of the bases of „personalized cancer imaging and therapy", a concept that have been stressed over recent years. The impact can be observed almost in all therapeutic domains in oncological parlance, but at present appears most promising in the areas of radiation oncology and targeted therapy. The importance and usefulness of studying tumor biology has got a major boost with the introduction of clinical PET imaging. Though well recognized amongst peers, the utilities have been relatively underutilized in routine clinical scenario. Hence, every effort should be made in the future how the functional imaging results could be aptly translated towards this direction. This treatise will focus on examples (both clinical and preclinical levels) that demonstrate the significant advantages of functional radionuclide imaging compared to the conventional imaging modalities.

Abstract No. 44

Molecular imaging with PET for Tailored Cancer Therapy

Andreas Kjaer

Rigshospitalet, University of Copenhagen, Denmark. Email ID: akjaer@sund.ku.dk

The change in paradigm towards individualized, tailored therapy has led to a need for diagnosing at the molecular level. Most of the molecular biology methods used today need tissue sampling for in vitro analysis. In contrast, molecular imaging allows for non-invasive studies at the molecular level in living, intact organisms.

With PET it is possible to label bio-molecules with radioactive isotopes. This method can be used for non-invasive visualization of tumor specific receptors and tissue characteristics such as angiogenesis and ability to metastasize. Especially within cancer biology the technique is expected to lead to a break-through in diagnosing and treatment.

Among the different techniques for molecular imaging, the nuclear medicine based technologies have the greatest potential for translational use since methods developed in animal models may directly be transferred to humans. Furthermore, PET has a high sensitivity and allows for quantification. We will show some recent examples from our institution on how molecular imaging with PET/CT and PET/MRI may be used for tailoring and monitoring cancer therapy.

Abstract No. 134

Molecular imaging of intestinal infection, inflammation and necrosis

Rao Papineni

Carestream Health Inc., USA. Email: docpapineni@gmail.com

Inflammatory diseases such as ulcerative colitis disease recruit myeloid cells and result in inflammatory responses such as generation of reactive oxygen species (ROS). Development of non-invasive luminescence imaging approaches is needed to evaluate these inflammation responses. We developed methodologies to show that vibration induced gut inflammation can be determined using luminescence imaging approach in a non-invasive fashion (Papineni and Vizard, World Molecular Imaging Congress 2012). Citrobacter rodentium (CR) a natural bacterial pathogen infects the distal colon of mice and induces transmissible murine colonic hyperplasia (TMCH). Here we determined the early inflammation response (ROS activity) to intestinal infection by CR in a longitudinal study. We further assessed the effects of allelic variation on murine chromosome 11 (B6.CAST. 11) in the inflammatory response. The ROS activity was monitored real-time in vivo using L-012 (8-amino-5-chloro-7-phenylpyridol [3, 4-d] pyridazine-1, 4(2H, 3H) dione), a chemiluminescence reporter, using planar multimodal imaging system. 0.1 ml of 1 mg/ml L-012 probe was injected (i.p.) in control and the experimental mice that were subjected to CR infection. Necrosis was imaged (post-infection day-19) using a fluorescent probe containing a bis (zinc2+dipicolylamine, Zn-DPA) motif which binds with high affinity to apoptotic, necrotic and bacterial surfaces. Significant difference in the basal gut ROS activity was found in mice with allelic variation on chromosome 11 compared to the wild type littermates. Robust elevation in ROS activity was observed in both the wild type and B6.CAST.11mice that were infected with CR (post infection 9, 12, 19 days). The variation in the levels of ROS activity however was very distinct between the wild type and B6.CAST.11 mice indicating the contribution of molecular determinants at the chromosome 11. The potential advantages in these real-time ROS monitoring methodologies by in vivo imaging, in understanding mechanisms of infection, inflammation, and development of better intervention strategies will be discussed.

Abstract No. 149

PET/PEM imaging of breast cancer: From bench to bedside

Matthew Thakur

Thomas Jefferson University, USA. Email ID: mathew.thakur@jefferson.edu

Each year in the USA, more than thirty nine mammograms are performed. Of these, nearly 20% require histologic examinations for which invasive biopsies must be performed. Nearly 80% of these biopsies find benign pathology and are considered unnecessary. At the average rate of $5,000 US per biopsy, not only do the unnecessary biopsies consume billions of healthcare dollars, but also induce enormous anxiety and trauma to the patients.

Our long term goal is not only to image breast cancer (BC) early and accurately, but also be able to distinguish malignant lesions from benign masses noninvasively, which will decrease the number of unnecessary biopsies, minimize trauma to the patients and significantly save on the healthcare dollars.

This presentation will describe the radiolabeled design of a biomolecule specific for genomic biomarkers that are abundantly expressed on the BC cell surface at the onset of BC, irrespective of their hormonal status. Data shall be presented that evaluated the probe pre-clinically in transgenic mice that spontaneously developed BC and mimicked human pathophysiology of the disease. The research has been translated into patients for imaging BC using PET (positron emission tomography) or PEM (positron emission mammography). The early results (100% sensitivity and specificity) have been highly encouraging and will be presented.

NANOTECHNOLOGY IN CANCER

Abstract No. 47

Antibacterial effect of silver nanoparticles against skin cancer

E. Jenitta Emima packiyam 1 and R.Ragunathan 2

1 Alva's college, Moodbidri, Karnataka-574227, 2 R.Ragunathan, Department of Biotechnology Maharaja Co-education Arts & Science College Perundurai - 52, Karnataka, India. Email: jenitta_emi@yahoo.co.in


The use of nanotechnology in cancer treatment offers some exciting possibilities, including the possibility of destroying cancer tumors with minimal damage to healthy tissue and organs, as well as the detection and elimination of cancer cells before they form tumours. Cancer cells have a unique properties that can be exploited by nanoparticles Their rapid rate of growth causes them to intake an abnormal amount of nutrients (i.e., folic acid) Nanoparticles can be used to target bio-markers or antigens that are highly specific to Cancer cells (tumour specific ). A method being developed to fight skin cancer using gold nanoparticles to which RNA molecules are attached. The nanoparticles penetrate the skin and the RNA attaches to a cancer related gene, stopping the gene from generating proteins that are used in the growth of skin cancer tumors. . Nanotechnology can be used to create therapeutic agents that target specific cells and deliver toxin to kill them. The importance of bactericidal nanomaterials study is because of the increase in new resistant strains of bacteria against most potent antibiotics. This has promoted research in the well known activity of silver ions and silver-based compounds, including silver nanoparticles. This effect was size and dose dependent and was more pronounced against gram-negative bacteria than gram-positive organisms. The size of nanomaterials is similar to that of micro-organisms; for this reason silver-based compound have been used extensively in many bactericidal applications silver compounds have also been used in the medical field to treat burns and a variety of infections. Several salts of silver and their derivatives are commercially employed as antimicrobial agents most biological molecules and structures; therefore, nanomaterials can be useful for both in vivo and in vitro biomedical research and applications. Studies were carried out on both multi drug antibiotic resistant (ampicillin- resistant) and non-resistant strains of gram-negative (Escherichia coli) and a non-resistant strain of gram-positive bacteria (Staphylococcus aureus). A multi-drug resistant strain of gram-negative (Salmonella typhus, resistant to chloramphenicol, amoxycilin and trimethoprim) bacteria was also subjected to analysis to examine the antibacterial effect of the nanoparticles. The development of new resistant strains of bacteria to current antibiotics has become a serious problem in public health; therefore, there is a strong incentive to develop new bactericides. Bacteria have different membrane structures which allow a general classification of them as Gram-negative or Gram positive, Gram-positive bacteria lack the outer membrane but have a peptidoglycan layer of about 30 nm thick. We can apply several electron microscopy techniques to study the mechanism by which silver nanoparticles interact with these bacteria. We can use high angle annular dark field (HAADF) scanning transmission electron microscopy (STEM), and developed a novel sample preparation that avoids the use of heavy metal based compounds such as OsO4. High resolutions and more accurate x-ray microanalysis can be obtained.

Abstract No. 99

Synthesis of oleic acid functionalized Fe 3 O 4 magnetic nanoparticles and studying their interaction with tumor cells for potential hyperthermia applications

Neena V. Jadhav 1 ,
Amresh I. Prasad 2 , Amit Kumar 1 , Raghumani S. Ningthoujam 2 , Rajesh K. Vatsa 2 and Badri N. Pandey 1

1 Radiation Biology and Health Sciences Division, 2 Chemistry Division, Bhabha Atomic Research Centre, Mumbai 400085, India.

In the present study, oleic acid (OA) functionalized Fe3O4 magnetic nanoparticles (MN) were synthesized following modified wet method of MN synthesis. The optimum amount of OA required for capping of MN and the amount of bound and unbound/free OA was determined. Further, we have studied the effect of water molecules, associated with the surface of MN, on the variation in their induction heating ability under alternating current magnetic field. We have employed a new approach to achieve dispersion of OA functionalized MN (MN-OA) in aqueous medium using sodium carbonate, which improves their biological applicability. Intracellular localization of MN-OA was studied in mouse fibrosarcoma cells (Wehi 164) by prussian blue staining and confocal laser scanning microscopy using nile blue A as a fluorescent probe, which showed them to be interacting mainly with the cell membrane. Their hyperthermic killing ability was evaluated in Wehi 164 cells by trypan blue method. Cells treated with 0.22 and 0.44 mg of MN-OA in combination with induction heating showed decreased viability of 53% and 26%, respectively as compared to respective induction heating controls (64% and 59%). These results were supported by altered cellular morphology after MN-OA treatment followed by induction heating. Further, magnitude of apoptosis induction was found to be ~ 5 folds in cells treated with MN-OA in combination with induction heating as compared to untreated control, suggesting the efficacy of MN-OA in killing of tumor cells by cellular hyperthermia.

Abstract No. 138

Nanotechnology Platforms in Cancer: Current Status and Future Directions

Rinti Banerjee

Indian Institute of Technology Bombay, Mumbai, India. Email: rinti@iitb.ac.in

Nanomedicine is an emerging field where nanotechnology can be applied to various aspects of diagnosis and treatment of diseases. Specifically, the roles of nanotechnology platforms in cancers have shown promise. Nanoparticles can be engineered to preferentially localise in the cancerous tissues using the enhanced permeation and retention effects as well as active targeting strategies. Further, trigger responsive lipid and polymeric nanoparticles can be developed which specifically release drugs in the presence internal stimuli associated with cancers like low pH, high secretory phospholipase A2 and metalloproteinases. In addition, magnetic and gold nanoparticles show potential for response to external triggers like hyperthermia or photothermal responses. Novel combinations of imaging modalities and triggered therapeutics may also be developed in theranostic nanoparticles. Ultrasound triggered nanocomplexes are one such example where the advantages of ultrasound imaging are combined with ultrasound triggered drug delivery. Another major area where nanotechnology can contribute in cancer therapy is in the treatment of multidrug resistant tumors. Suitably designed nanoparticles can bypass the p glycoprotein efflux and be actively endocytosed within the tumors, increasing the effectiveness of the drugs. With respect to radiotherapy, nanoparticles may play a role in the regional delivery of radiosensitizers to allow a combined radio-chemotherapy with reduced systemic toxicity. Another potential application is the delivery of radioprotectants to mitigate the radiation induced injuries. The talk will give an overview of the current status of nanotechnology platforms in cancers, some of our experiences in development of novel nanocarriers for anticancer therapy, future directions and the translational challenges for further development of promising platforms.

OCCUPATIONAL AND ACCIDENTAL HEALTH HAZARDS

Abstract No. 71

Role of cell membrane proteins in thorium induced cytoproliferative effect of HepG2 cells

Manjoor Ali 1 , A. Kumar 1 , K. R. Babu 2 , V. Nataraju 2 and B. N. Pandey 1

1 Radiation Biology and Health Sciences Division, 2 Technical Physics Division, Bhabha Atomic Research Centre, Trombay, Mumbai-400 085, India. Email id: manjoor1982ali@yahoo.co.in

Our earlier studies on animals showed that liver is one of the major target organ of thorium. Therefore, in present study, HepG2 human liver cell was employed as an in-vitro model to understand the mechanism of cellular effects of thorium (Th-232). Results showed that thorium treatment (10-100 μM) enhanced the proliferation ( ~ 2.0 folds) of HepG2 liver cells, that was dependent on the concentration and incubation time, which involved MAPK and PI3K- intracellular signaling pathways. Thorium-induced proliferation of HepG2 cells was significantly inhibited by pretreatment of cell with integrin receptor and insulin growth factor receptor-1 (IGFR-1) blocking antibodies. In order to understand the cellular site of thorium action, thorium estimation was done by using Inductively Coupled Plasma-Mass Spectroscopy (ICP-MS). Our results showed that cells cultured in medium (without FCS) and treated with thorium (50μM), the cell-bound thorium was found to be ~ 100 folds higher than present in culture medium. However, when thorium treated cells were harvested with trypsin-EDTA solution (0.02% EDTA), the cell bound thorium was found ~ 25 folds lower than in cells harvested without using trypsin-EDTA solution. Increase in EDTA concentration (0.02-0.5%) of trypsin-EDTA solution enhanced the thorium removal from cells, suggesting the presence of surface bound thorium on HepG2 cells. These experiments have significant understanding the mechanism of thorium-interaction with HepG2 cells.

Abstract No. 75

Radiobiological basis of hazards from cell phones/cell towers


SC Jain

Consultant, CFFES, Delhi-110054, India. Email: scjain007@gmail.com

Radiation emitted from Cell Phones, Cell phone towers, Wi-Fi, TV and FM towers, microwave ovens, etc. causes significant health hazards (biological effects) on human, animals, birds, plants and environment. Biological effects of microwave radiation effects are classified as: (a) Thermal and (b) Non-thermal. The current exposure safety standards are mainly based on the thermal effects, which are inadequate. Non-thermal effects are several times more harmful than thermal effects. There are seventy five crores cell phones and 5 lakhs cell towers.

ICNIRP is only intended to protect the public against short term gross heating effects and not against 'biological' effects such as cancer and genetic damage from long term low level microwave exposure from mobile phones, masts and many other wireless devices.

Epidemiological studies - Sleep disruption, headache, depression, discomfort, irritability, nausea, dizziness, appetite loss, muscle spasms, numbness, tingling, altered reflexes. Subjects reported buzzing in the head, palpitations of the heart, light-headedness, heat, visual disorders, cardiovascular problems, respiratory problems, nervousness, agitation. More severe reactions include seizures, paralysis, psychosis and stroke. Blood brain barrier selectively lets nutrients pass through from blood to brain, but keeps toxins out. It is reported that 4 times incidence of Alzheimer's disease and 3 times amyotrophic lateral sclerosis (ALS).

Over-expression of (HSPs) leads to inhibition of natural programmed cell death (apoptosis). In intensive users of mobile phones leads to 30% sperm decrease in addition to damage to sperms. Children are more vulnerable due to smaller size of their skulls and thinner skin, increased rate of cell division and full development of myelin sheath. RF can pass placental barrier & continuously react with the developing embryo and increasing cells. It can also damage delicate working of the inner ear leading to hearing problem. Melatonin Reduction is reported to be decreased in cell phone users. Melatonin is a powerful antioxidant, antidepressant and immune system enhancer that regulates circadian rhythm. Children and teenagers are 5 times are more likely to get brain cancer if they use mobile phones.

More studies on biological effects of cell phones and cell towers are needed to protect the public. Special attention should be given for radiation effect on young children and teens. SAR (Specific absorption rate) - Rate at which radiation is absorbed by human body, measured in units of watts per kg (W/kg) of tissue. In USA, SAR limit for cell phones is 1.6W/kg which is actually for 6 minutes . It has a safety margin of 3 to 4, so a person should not use cell phone for more than eighteen to twenty four minutes per day. This information is not commonly known to people in India. People living within fifty to three hundred m from cell towers are more prone to ill effects of electromagnetic radiation. India has adopted ICNIRP guidelines i.e. 9.2 W/m΂ for 1800 MHz.

In addition to continuous radiation from cell towers, there is radiation from cell phones, computers, laptops, TV & FM towers, microwave ovens, etc. are additive in nature. We should promulgate the message about the adverse health effects of cell phones and cell towers.

Abstract No. 117

Non-therapeutic accidental exposure to radioactive sources: Role of a radiation oncologist

Bharath G


Amrita Institute of Medical Sciences, Kochi, Kerala, India. Email: dr.bharath9@gmail.com

The spectrum of usage of radioactive isotopes outside a hospital setting is seldom known to the medical community, their uses can range from advanced applications in space crafts to detecting simple cracks in pipelines. With such extended applications, there exists an increased risk of accidental radiation exposure, which accounts for a vast majority of radiation injuries. However this could be grossly under reported. This report is about an industrial pipeline worker who was accidentally exposed to an Iridium source while checking for a pipe leak using gamma rays. He subsequently developed cutaneous radiation syndrome, which was managed by surgical debridement. Upon visiting our department, we were able to obtain the details of the source and calculate the absorbed dose and its distribution. Cytogenetic analysis was also performed to detect any abnormality. The aim of this report is to highlight the role radiation oncologist and medical physicist in identifying the injury, calculating the absorbed dose and its distribution. This will aid in surgical management and help in predicting the radio biological late sequelae, which is essential in the management and follow up of such patients.th

Abstract No. 189

Radiation biodosimetry: Preparedness for management of nuclear radiation exposure scenario


G. Tamizh Selvan 1,2 , J S Adhikari 2 and th N. K. Chaudhury 2

1 Sri Ramachandra University, Porur Chennai 600 116, 2 Institute of Nuclear Medicine and Allied sciences, DRDO, Delhi 110 054, India. Email: nkcinmas@rediffmail.com

The consequences of radiation exposure on health depend on the nature of radiation and the absorbed dose. With increasing use of nuclear energy in industries and military weapons, the possibility of occupational or accidental exposures has increased. Further the threat from radiological exposures by terrorisms and nuclear explosions, a large number of humans are likely to be affected. Medical management of suspected persons will require first assessment for absorbed dose in individuals and most important in case of large scale incidents, the medical triage. Reference Radiation Biodosimetry laboratories mandate is to provide service for assessing the radiation absorbed dose among suspected persons. As per the recommendation of IAEA, Veinna, the dose assessment is to be performed using human peripheral blood lymphocytes and the method recommended is dicentric chromosome assay, the gold standard.

The dicentric chromosomal aberration is radiation specific and sensitive towards radiation dose. The whole process is very time consuming and require implementation of skill. No single laboratory would be able to serve the purpose in large number of suspected persons. At present in India, to the best of our knowledge, there are three Radiation Biodosimetry labs, of which two are within the Department of Atomic energy for providing biodosimetry service for radiation workers engaged in nuclear reactors and the other is at Department of Human Genetics, Sri Ramachandra University, Chennai. Our Institute has undertaken an initiative to Establish Radiation Biodosimetry lab and the current status is that we have developed skill and competence for undertaking dicentric chromosomal assay. The dose response calibration curve using new protocol suggested by BARC, Mumbai has been generated.The complete data was analysed and found to be at per with the requirement of IAEA. Further, as a part of quality control and assurance, an interlab comparison on the ability to demonstrate the skill and competence for scoring coded slides from Sri Ramachandra University was undertaken and successfully completed. Thereby now both the participating laboratories are networked, a very important step achieved for increasing the capacity of Radiation Biodosimetry for the country.

Abstract No. 190

Development of field-compatible, self-usable radio-decontamination formulations for medical management of nuclear and radiological emergencies

Rakesh Kumar Sharma, Sudha Ranaa, Vinod Kaushika, Raman Chawlaa, Rajiv Goela, Abdul Wadood Khanb, Sabna Kottab, Mita Duttac, Javed Alib, Shahid Hussain Ansarib, Sarwat Sultanad

1 Division of CBRN Defence, Institute of Nuclear Medicine and Allied Sciences, Brig. S. K. Mazumdar Road, Delhi 110 054, India. 2 Faculty of Pharmacy, Jamia Hamdard, Hamdard Nagar, New Delhi 110 062, 3 Division of Nuclear Medicine, Institute of Nuclear Medicine and Allied Sciences, Brig. S. K. Mazumdar Road, Delhi 110 054, India. Email: rksharmadrl@yahoo.com

Exposure/contamination with toxic radioactive materials (TRMs) is an occupational or accidental/intentional hazard. A contaminated individual carrying TRMs may cross-contaminate other persons. Detection and removal of radioactive contaminant from skin should be undertaken quickly both for the protection of the individual concerned and the community. 'SHUDHIKA' a skin decontamination kit has been developed as per the guidelines of Atomic Energy Regulatory Board (AERB). This kit contains chemical decontamination solutions e.g., liquid soap and water, Ethylene diamine tetraacetic acid solution (10%), Oxalic acid solution (2%), Sodium bisulphate solution (5%), Hydrochloric acid solution (pH-1), Diethylene triamine pentaacetic acid solution (5%), Potassium permanganate solution (7%), Sodium bicarbonate solution (7.8%) besides medical items. 'SHUDHIKA' kit is effective in field condition for 5-7 persons (buddy decontamination). A topical decontamination lotion formulation was developed and incorporated into fabric matrix and made self-usable device. Decontamination Efficacy (DE) was evaluated applying Tc-99m as radiological contaminants on Sprague dawley's rat's skin. Contaminant was removed from skin after 0.5-3h using decontamination agents of 'SHUDHIKA'. Wipes were used for the decontamination of Tc-99m, Iodine-131 and Thallium-201 radio-contaminant. Along with Whole body imaging and static counts were record evaluated using pharmacoscintigraphic technique. Results were compared with water and. Measured DE values analysed using one way analysis of variance (ANOVA) and students t-test, found to be statistically significant (p<0.05). DE of 'SHUDHIKA' was recorded 92%±3% (soap + water- 65±2.8%, Ethylene diamine tetraacetic acid solution-10±2), Oxalic acid solution (6.0 ±0.4%), Sodium bicarbonate solution (4±0.5), Hydrochloric acid solution (3.2±1.0), Diethylene triamine pentaacetic acid solution (3.2±1.0 %), Potassium permanganate solution and sodium bisulphite solution (4.1±2%.). DE of the wipe was found to be 85±5%, 90±5% and 87±5% after 0.5 h after contamination. Lotion wipe effectively removed the radio-contaminant applied. Minimal waste generated helps in to avoid spread and re-contamination.

Abstract No. 193

Medical countermeasures for nuclear & radiological emergencies: INMAS perspective


Aseem Bhatnagar

INMAS, DRDO, Delhi, India. Email: assem_bhatnagar@indiatimes.com

Medical countermeasures against nuclear and radiological events is a neglected area due to several contributing factors; none the least being, a) a non-commercial field unable to attract the attention of pharma industry, b) conventional phase-2 clinical trials are not possible, and c) administrative bewilderment as to store what drugs, how much, where and for how long. It may not be wrong to say that practically no new and more effective drugs have been introduced for the last several decades, and what ever is approved is not available in the massive quantity that might be required at a short notice. Ironically, even potassium iodate, the most common and popularly recognized as an anti-nuclear drug (decorporating agent for Iodine-131) is not mentioned as a radiation countermeasure drug in the Indian pharmacopoeia. Three million doses of this drug was distributed within week of the Chernobyl disaster in Poland; requirement in India will easily cross three crore. To create new research products relevant medical countermeasures from lab to the field is indeed a research and logistic challenge.

INMAS is progressing on schedule on a time-bound project to provide safe and effective drugs for nuclear emergencies. These belong to different categories: a) Chemical Radioprotectors, b) New decorporating drugs, c) off-label drugs with discovered decorporation characteristic, d) skin and decontamination approaches, and e) strategies to make available the drugs in desired quantity at desired place. As the first step, INMAS endeavors to make available 15 drugs (new and generics) in doses ranging from 50,000 to 2 lakh doses by March 2013. In the ensuing years, INMAS plans to make a comprehensive NBC kit (80 odd items) and establish a micro-GMP facility for manufacture of the NBC drugs for Ministry of Defence use. Another R&D effort is planned towards increasing the shelf life of NBC drugs by 60-100%.

The second aspect of medical management is the preparedness of the medical echelons for the disaster / event that has a minimal but cognizant probability of occurring. INMAS is planning to work upon and introduce a model for medical management of such emergencies. It is envisaged to be a 6-layered medical echelon model, and based on pre-distribution of detection equipment, drugs, SoPs and training of the identified medical personnel on a quasi-permanent basis.

Abstract No. 231

Role of radioprotectors in medical management of radiation injuries

Manju Lata Gupta

Department of Radioprotective Drug Development Research,Institute of Nuclear Medicine & Allied Sciences, DRDO, Brig S K Mazumdar Road, Delhi - 110 054, India.

Multidirectional use of nuclear energy has up surged the risk of radiation exposures. Nuclear explosion/accidents occurred at Hiroshima and Nagasaki in Japan, 1945; Kyshtym, Russia, 1957; Windscale, UK, 1957; Three Mile Island in Brazil, 1979; Chernobyl, Ukraine, 26 April 1986; Sellafield, UK, 2005 and Atucha, Argentina 2005 had shown the need for development of radiation countermeasures for defence as well as civil population. However, the two very recent nuclear accidents occurred at Mayapuri India, April 2010 and Fukushima Japan, March, 2011 have forced the scientific community to put mission oriented efforts towards the completion of unfinished task for development of safe radioprotectors.

Constant efforts for many decades have brought clear understanding about radiation mediated adverse effects on biological system. Radiation when enters in the body damages the tissues directly/ indirectly. High LET radiation mainly damages the tissue by direct effect while damage caused by Low LET radiation is predominantly by indirect effect, though direct effect also in this case cannot be ruled out fully. Radiation damages the tissue within fraction of seconds by making chemical changes followed by tissue damage, organ malfunctioning, delayed and genetic effects which may be inherited later.

Radiation induced risk management is a multidirectional phenomenon. However, due emphasis on prevention by administering prophylactic agents will certainly yield significant outcome. A potential radioprotector can minimize the effect of radiation to the level which can be managed either with the medical interventions or by mild psycho-therapies.

Radiation biologists from every corner of the world have been working on radioprotector development since last many decades, but the results are not corresponding to the efforts. Limited tissue protection and inherent toxicity are some of the prominent areas preventing the experimented drugs reaching to bed side use. Though our body has an intact endogenous defense mechanism but that is effectively operational when the radiation exposure is in the very low range. For high dose radiation exposures there is a need to administer exogenous agents which can protect and support the biological system in holistic manner. A radioprotector administered in the body before irradiation usually gets circulated to all most all the organs within few minutes, remains there for at least 2-3 hours and scavenge radiation generated free radicals maximally, besides rendering protection to the radiosensitive tissues.

Administration of safe exogenous substances will extend protection to highly radiosensitive organs such as hematopoietic system, immune system, gastrointestinal tract and endogenous defense mechanism. With the minimized damage, recovery to the biological system will be faster and may not need specialized medical intervention. Prophylactic adminstration of safe agents will protect defense forces and civil population against radiation. Disaster management teams, first responders, medical fraternity and other infrastructure support including blood and bone marrow banks, sterile conditions etc will be minimally burdened. Lesser damage will lead to faster recovery, minimum mutations, almost nil transfer of deformities from generations to generations. Considering the facts it is pertinent to accept that safe radioprotectors have immense role in medical managements of radiation injuries.

RADIATION EFFECTS ON PLANTS, MICROBES AND OTHER NON-HUMAN BIOTA

Abstract No. 30

Thioredoxin peroxidase is important regulator of radioresistance in the lepidopteran insect Sf9 cells

Shashank Hambarde, Vijaypal Singh, Sudhir Chandna

Natural Radiation Response Mechanisms Group, Institute of Nuclear Medicine and Allied Sciences, Brig SK Mazumdar Road, Delhi 11, India. Email: shambarde@gmail.com

Ionizing radiation causes formation of reactive oxygen species and free radicals which can damage various macromolecules and can cause lethality. Lepidopteran insect cells display 50-100 times higher radioresistance compared to human cells, and reportedly have more efficient antioxidant system that can significantly reduce radiation-induced oxidative stress and cell death. However, the antioxidant mechanisms that contribute substantially to this excessive resistance still need to be understood thoroughly. In this study, we characterized an antioxidant enzyme thioredoxin peroxidase (TPx) in Spodoptera frugiperda, the fall armyworm and investigated its role in high-dose gamma-radiation response of Sf9 cell line derived from Spodoptera frugiperda. In silico characterization indicated 'Spodoptera frugiperda' thioredoxin peroxidase (Sf-TPx) with primarily cytosolic and mitochondrial localization. Structural modelling data included certain motifs known in TPx's of D. melanogaster, viz., Jafrac1 and Jafrac2 that affect active site conformation and separate cysteine residues at distances not suitable for disulphide bridge formation (5.21Ε; 5.73Ε). These motifs are absent in Sf-TPxs', yielding shorter distance (2.01Ε; 2.05Ε) between the cysteine residues suitable for disulphide bridge formation. The closely juxtaposed cysteine residues of Sf-TPx's can have higher peroxidase activity than Drosophila TPx's and can impart higher resistance against radiation-induced oxidative stress. Western analysis showed that gamma-irradiation at 500Gy dose significantly up-regulated Sf-TPx, while higher doses (1000Gy-2000Gy) had no such effect. G2/M checkpoint induced following 500Gy was associated with transition of Sf-TPx decamer into enzymatically active dimer, a phenomenon also observed during G2/M block induced by 5nM okadaic acid or 10μM CDK1 inhibitor roscovitine. Therefore, Sf-TPx activity at 500Gy is likely mediated by CDKs, and accumulation of TPx dimer form during G2/M checkpoint might favour higher peroxidase activity leading to efficient survival at this dose. Further confirming this, higher lethal doses (1000Gy-2000Gy) caused significantly less accumulation of dimer form and induced dose-dependent apoptosis. A ~50% knock-down of Sf-TPx by siRNA caused remarkable increase in radiation-induced ROS as well as caspase-3 dependent radiation-induced apoptosis, clearly implying TPx role in the radioresistance of Sf9 cells. However, TPx knockdown could not induce/enhance the constitutively absent radiation-induced nitrosative stress reported by us recently. Hence our study demonstrates for the first time that thioredoxin peroxidase contributes in the radioresistance of Lepidopteran Sf9 insect cells, which is an important insight into the antioxidant mechanisms existing in this highly stress-resistant model cell system.

Abstract No. 41

Effect of electron beam irradiation on microbial load and biochemical compositions of woodfordia fruticosa, adhatoda vasica, tinospora cordifolia and rubia cordifolia

th
Prajna P.S 1none , Rama Bhat P 1 , Ganesh Sanjeev 2 , D.K. Maurya 3

1 Post Graduate Department of Biotechnology, Alva's College, Moodbidri, Karnataka 574 227, 2 Microtron Centre, Mangalore University, Mangalagangothri- 574 199. 3 Radiation Biology and Health Sciences Division, Bhabha Atomic Research Centre, Mumbai. Email: meetprajna@yahoo.co.in

Herbal medicine has been used in India for thousands of years with a greater number of people seeking remedies and health approaches free from side effects caused by synthetic chemicals. Microbial contamination of dried herbal raw materials is one among the reason for the decline of India's share in the global herbal market and is preventing India from becoming herbal giant. There may be various reasons for such concerns, mainly owing to poor quality of herbal medicines due to insufficient attention being paid to the quality assurance and quality control of raw materials especially microbiological point of view.

The study was carried out to evaluate the effect of electron beam radiation on the microbial load and biochemical characteristics of four medicinal important herbal raw materials namely Woodfordia fruticosa (flower), Adhatoda vasica (leaf), Tinospora cordifolia (stem), Rubia cordifolia (stem). Irradiation was done in three different doses viz., 8, 12 and 15 kGy with the electron beam energy 8 MeV. Irradiated and control samples were analysed for bacterial and fungal load and biochemical compositions like carbohydrates, proteins, phenolics, tannins and antioxidant properties.

Results showed that total microbial load decreased linearly with absorbed radiation dose. In Woodfordia fruticosa and Rubia cordifolia complete microbial decontamination was achieved at 8kGy. Dose of 12kGy was sufficient to completely hygenise Adhatoda vasica, whereas dose of 15kGy was needed to completely hygenise Tinospora cordifolia samples without affecting the nutritional quality of herbs to a larger extent. Minimum variation was recorded in the biochemical compositions of the herbal samples. Hence electron beam irradiation can be considered to be effective to improve the quality of herbal materials in terms of microbial decontamination.

Abstract No. 50

Gamma radiation and static magnetic field affecting seed quality under accelerated ageing of soybean

th Mahesh Kumar


National Institute of Abiotic Stress Management, India. Email: mahesagrawal@gmail.com

Soybean seeds were exposed to gamma radiation (0.5, 1, 3 and 5 kGy), static magnetic field (50, 100 and 200 mT) and a combination of gamma radiation and magnetic energy (0.5 kGy + 200 mT and 5 kGy + 50 mT). Samples were subjected to accelerated ageing treatment at 42΀C temperature and 95-100% relative humidity. Observations were recorded at 0, 10th and 20th days after accelerated aging. Soybean seed ages rapidly during storage at high temperature and high relative humidity and were compared between untreated and energized seed for various physical and biochemical characteristics. Due to accelerated aging, oil and protein quantity and quality of seed was negatively affected. Antioxidant enzymes exhibited a decline in their activity during aging and LOX activity in general, increased during aging and reflect higher rate of lipid peroxidation. The level of linoleic (l8:2) and linolenic acid (18:3) in the oil decreased during ageing. It is suggested that aging causes peroxidative changes to lipids, which could contribute to loss of oil quality. Among the irradiation treatments in the present investigation, 1-3 kGy dose is more effective for reducing the seed quality deterioration under accelerated aging.th

Abstract No. 58

Influence of Gamma radiation on scavenging enzyme activity and cell ultrastructure in groundnut (Arachis hypogaea L.)

Aparna. M. 1 , Anurag Chaturvedi 1 , Sreedhar. M. 1 , Pavan Kumar. D 1 , R. K. Singhal 2 , P. Venu-Babu 2

1 Quality Control Laboratory, Acharya N.G. Ranga Agricultural University, Rajendranagar, Hyderabad- 500 030, Andhra Pradesh, India, 2 Bhabha Atomic Research Center, Trombay, Mumbai-400085, India. Email: aparna_mothe@yahoo.com

The aim of this study was to establish changes in the activity of the scavenging enzymes, plant pigments like chlorophyll which confer sensitivity to irradiation stress, and also to assess the damage caused by ionizing radiation on cell membrane and cellular organelles on exposure to gamma radiation in Groundnut.

Groundnut (Arachis hypogaea L.) cv. Narayani seedlings, when exposed to gamma rays (0.00, 10, 20, 40, 50 and 100Gy) from a cobalt source (60Co) in a gamma chamber GC 5000 with a specific activity of 3.06 kGy/hr, a dose dependent increase in the activity of peroxidase and superoxide dismutase was observed in response to free radical generated due to radiation stress.Further, gradual decline in leaf chlorophyll content was observed with increased dose and 100Gy exposure resulted in lowest leaf chlorophyll content (0.895mg/g FW) due to maximum pigment deterioration. Alternatively, ultra thin sections of leaves of selected seedlings exposed to 100 Gy radiation stress were studied under Transmission Electron Microscope to assess the damage caused to important cell organelles. The gamma ray induced ultra structural changes included distortion of nuclear membrane, chloroplast swelling, thylakoid dilation, rupture of chloroplast outer membrane and swollen endoplasmic reticulum. Damage to ultra structure, accumulated with exposure time and led to both vesiculation in the chloroplast stroma and endoplasmic reticulum of the cells after the exposure to gamma rays at a dose of 100Gy.

Abstract No. 59

Low dose gamma irradiation improved germination, vigor and gas exchange and carbon fixation attributes of groundnut (Arachis hypogaea L.)

Sumedha Ahuja

NRL, IARI, New Delhi, India. Email: sumedhabiochem@gmail.com

An experiment was carried out to determine the effect of γ - irradiation on germination, seedling vigor and physiological and carbon fixation characteristics of groundnut variety TG-37A. Seeds were subjected to the pre-sowing treatment of gamma radiation within an very low to low dose physiological effect range i.e., 0.0, 0.0082, 0.0164. 0.0328, 0.0656, 0.1312, 5, 25, 100, 500 Gray in the Co-60 based irradiation Chamber (Model GC-5000). Observations were recorded for the radiation effect on % germination, vigour, gas exchange attributes such as photosynthetic rate, stomatal conductance, transpiration rate and chl content and the activity of carbon dioxide assimilating enzyme "Rubisco". Seed germination was increased by 10-25 % at the lower doses up to 5 Gy while the improvement in plant vigour in the same dose range was significantly high over the unirradiated by 22-84 %. For radiation exposure above 5 Gy, a dose dependent decline in germination and plant vigor was measured. Results of different gas exchange parameters revealed no effect to an improvement in photosynthesis by radiation dose up to 5 Gy which was followed by a decline in the photosynthesis rate. Stomatal conductance and Transpiration however, were only inhibited at 500 Gy. Leaf Rubisco activity was remained unaffected at all the investigated doses of gamma irradiation. Results indicate that a very low dose of gamma radiation do not pose any significant threat and infect may stimulate growth and metabolic functions. It further shows that the radiation threshold for the gas exchange traits and rubisco activity which ultimately determine the plant health and produce yield, lie well beyond 500 Gy and that the dose range above 500 Gy should be targeted to measure lethal effects of radiation on carbon assimilation attributes. A significantly lower mutation frequency was measured in Drosophila irradiated with 500 μGy than the unirradiated and those irradiated at 10 Gy (Oqura et al., 2009).

Abstract No. 61

Evaluation of genotoxic/cytotoxic potential of acute gamma radiations in some liliaceae members

George Jacinta 1 , Patel Behnaz B 1 , Venu-Babu P 2 , Singhal R K 3 , Eapen Susan 2 , Pius Jessy 1 , Narula Bhavna R 1 and Shankhadarwar S 1

1 Department of Botany, R Ruia College, Mumbai 19, 2 NABTD, BARC, Mumbai 85, 3 Analytical Spectroscopy Section, ACD, BARC, Mumbai- 85, India. Email: jaci.george@gmail.com

Inhibition/arrest/delay of cell division and chromosome breakages induced by ionizing radiations have served as sensitive biological end points to determine radiosensitivity and radiation risk of cell death and mutagenesis. With the emerging trends of non-linear dose response patterns at low doses opposing the linear no-threshold concept used in radiological protection, the need to assess the effect of low doses of gamma radiation in plants is imperative. The present study deals with the assessment of cytological dose-response to low LET gamma radiations of 2 mesophytes of the genus Allium, A. cepa Linn. and A. sativum Linn., and a xerophyte, Agave amerciana Linn. of the family Liliaceae of Bentham and Hooker. The bulbs were exposed to acute doses of gamma radiation from Co-60 sources at dose rates 0.63 cGy/min to 1.9 Gy/min. Low dose range was selected based on LD50 evaluation from acute high dose radiation response. Root meristems were scored for mitotic screening end points: spindle disturbances and metaphase arrest - aneugenic; chromosome fragmentation, bridges and laggards - clastogenic and micronuclei (aneugenic/ clastogenic). With respect to effect on mitotic division, A. cepa was more sensitive to acute low doses of gamma radiation. The non-linear trends observed in micronuclei induction over cell generations served as the best indicator of total genotoxicity and relative radiosensitivity revealing that Allium species with greater interphase chromosome volumes (ICV) ( ~ 0.2-0.23) were more radiosensitive ( LD50 , 6-8 Gy) as compared to Agave with smaller ICV ( ~ 0.036), LD50 10-15 Gy.

Abstract No. 62

Gamma radiosensitivity study on rice (Oryza sativa L.)

D. Pavan Kumar 1none , Anurag Chaturvedi 1 , M. Sreedhar 1 , M. Aparna 1 , P. Venu-Babu 2 and R. K. Singhal 2

1 Quality Control Laboratory, Acharya N.G. Ranga Agricultural University, Rajendranagar, Hyderabad- 500 030, Andhra Pradesh, India. 2 Bhabha Atomic Research Center, Trombay, Mumbai- 400085, India. Email: vrspavan@gmail.com

In the present investigation, 9 popular rice varieties cultivated in Andhra Pradesh, were examined for varietal differences in radiosensitivity to gamma radiation exposure. Dry seed of 9 rice varieties (1.Samba Mahsuri, 2.Polasaprabha, 3.Surekha, 4.Vijetha, 5.Jagityal Sannalu, 6.Nellore Mahsuri, 7.Swarna, 8.Cottondora sannalu and 9.Erramallelu) were exposed to gamma radiation dose from 0.20 to 2.00 kGy using 60Co source with a specific activity of 2.64 kGy/hr, to determine the effect on germination and to find out the Lethal dose 50 (LD50). Data on germination percentage was generated as per ISTA (1985). Germination % was measured on 7th day after germination in irradiated sample. The results revealed that increasing doses of gamma radiation had significant effect on germination % for the first 7 days under laboratory conditions. With increase in radiation above 0.80 kGy a reduction in percentage of germination was observed in irradiated seeds as compared to control. Increase in gamma ray doses from 0.20 to 0.60 kGy had little or no effect and there were no significant differences in germination % of irradiated, non-irradiated seeds for all the varieties evaluated. The LD50 values determined from Polynomial fit using Origin Pro 8 software based on germination %, ranged from 0.89 (5) to 1.88 kGy (4). The varieties 2 and 1 had highest radiosensitivity while 4 and 3 were relatively tolerant. However, the varieties 8 (1.66 kGy), 9 (1.57 kGy), 7 (1.56 kGy), 2 (1.38 kGy) and 6 (1.32 kGy) are moderately tolerant. These LD50 values determined for selected rice varieties could be useful in rice varietal improvement and maintenance.

Abstract No. 81

Performance of gamma irradiated wheat under terminal heat stress

Bhupinder Singh
and Sameer Kumar Guha

Nuclear Research Laboratory, Indian Agricultural Research Institute, New Delhi, India. Email: bhupindersinghiari@yahoo.com

Heat stress during reproductive phase is likely to cause a greater damage to the plant growth and yield than that experienced at the vegetative phase. Terminal heat stress is a major production constraint in wheat where even a single degree rise in temperature from the optimum can cause floret abortion, pollen sterility, tissue dehydration, reduced CO 2 assimilation and increased photorespiration. Grain yield reduction during stress may be due to the reduced availability of assimilates and/or the duration and rate of grain filling. Involvement of heat shock proteins (HSPs) in imparting stress tolerance by protecting cells and organisms against induced oxidative damage has been extensively studied. These proteins have a role in maintaining protein assembly and function under stressed and normal condition of growth. Gamma radiation is known to target DNA either directly or through the production of deleterious free radicals and reactive oxygen species (ROS) and it is here that the HSPs may be aiding the development of radiation adaptive responses. An experiment was conducted to assess whether the gamma radiation induced higher mobilization of stem carbohydrate into the spike has any advantage under terminal heat stress (+5 o C) in wheat. Decline in grain mass at high temperature, as compared to ambient condition, was more for unirradiated control than gamma ray treated wheat at all spikelet positions. A higher availability of flag leaf sugars and the consequently improved grain mass, but not the grain number, contributed significantly towards terminal heat tolerance advantage of gamma radiation in wheat. It would be interesting to investigate the role of Hsps, if any, in the radiation protection against heat stress.

Abstract No. 82

Forced and free choice insect infestation studies and changes in quality of gamma irradiated soybean seeds during storage

Achchhelal Yadav 1 , Chitra Srivastava 2 , Mahesh Kumar 1 , Sumedha Ahuja 1 , SK Guha 1 , SK Jha 3 and Bhupinder Singh 1

1 Nuclear Research Laboratory, 2 Division of Entomology, 3 Post Harvest Technology, Indian Agricultural Research Institute, New Delhi, India. Email: achchheyadav@yahoo.com

Experiments were conducted to ascertain the effect of electromagnetic energy on post harvest storage quality and insect damage of during storage of Soybean (SL-525). Seeds were exposed to different doses of gamma radiation at 0, 0.01, 0.05, 0.5, 1.0, 3.0 and 5.0 kGy which were evaluated for damage by soybean insect pest, Callosobruchus analis through different infestation methods (a) natural infestation and, (b) forced infestation. Insect damage to the irradiated soybean seeds in general, declined in a dose dependant manner. Irradiated seeds maintained higher seed coat hardness over unirradiated control seeds and could have caused an observed change in feeding preference of the soybean pest as evident from mass loss and number of emerged adults both of which declined with irradiation in a dose dependant manner. No insect emergence was recorded at dose above 1 kGy. Gamma irradiation did not significantly alter the oil content of the stored seeds over four months storage at RT (25-30 o C) and infact caused a significant reduction in the seed lipoxygenase activity which may provide protection against lipid peroxidation. Gamma irradiation treatments did not negatively impact the seed protein during storage. Further, an improved activity of catalase and peroxidase were recorded in seeds subjected to low doses of gamma radiation and may help in reducing the oxidative stress related inhibition of physiological and biochemical functions as compared to unirradiated soybean seeds during storage.

Abstract No. 83

Gamma radiation for improving post harvest shelf-life of tomato while maintaining the fruit quality

Sameer Kumar Guha 1th , Mahesh Kumar 1 , Sumedha Ahuja 1 , SK Jha 2 , Raj Kumar 3 and Bhupinder Singh 1

1 Nuclear Research Laboratory, 2 Post Harvest Technology, 3 Division of Vegetable Science, Indian Agricultural Research Institute, New Delhi, India. Email: bhupindersinghiari@yahoo.com

Gamma radiation has been used in the past to control of storage insects, delay fruit ripening, inhibition of tuber sprouting and sterilization of processed agri-produce besides numerous other industrial applications. The critical research inputs are, however, required to optimize doses for varied applications of gamma. Present study was conducted to investigate the role of gamma radiation for improving the shelf life of tomato without causing any deterioration in fruit quality. Two varieties of tomatoes, Pusa Ruby and Pusa Rohini, picked from the vegetable farm of IARI at green mature stage, were respectively exposed to gamma radiation at 0, 0.0025, 0.1, 0.5, 1.0, 3.0, 5.0 and 1.0 kGy and stored at 18 ±2 o C and 28 ±2 o C (75 ±5 % RH) temperature in two separate experiments. Gamma irradiation, in general, reduced accumulation of lycopene in the treated fruits and, thus, yielded radiation related delayed ripening advantage. Observations on color (Hue angle) of treated and untreated fruits at different days of storage complement and support the finding on fruit lycopene. A higher dose of 3 and 5 kGy caused stay green character but induced physical damage on the fruit. High SOD activity was measured in the irradiated fruits and may help in an efficient scavenging of ROS during storage when compared to the unirradiated control. Increased fruit firmness of irradiated fruits was found to be related to a high pulp K content of the irradiated fruits. Increased fruit firmness, maintenance of high fruit mass, significantly low ethylene production and delayed redness in the stored irradiated tomatoes suggested that 1.0 kGy dose of gamma irradiation could be effectively used to extend the shelf life of tomato with least effect on the biochemical quality of stored fruits.

Abstract No. 89

Carbon (14CO 2 ) assimilation and partitioning may effect the iron deficiency response of wheat

Rinki Khobra, Sumedha Ahuja and Bhupinder Singh

Nuclear Research Laboratory, Indian Agricultural Research Institute, New Delhi, India. Email: rinkikhobra@gmail.com

Iron is an essential plant mineral nutrient required for the chlorophyll synthesis. Genotypic difference in iron deficiency tolerance may be related to an optimized translocation of the fixed carbon between the root and the shoot. A tightly regulation of the assimilate movement between different sink organs is crucial under abiotic stress and may determine stress response of crops. An experiment was conducted to measure the relationship between iron deficiency tolerance and carbon assimilation (using 14CO 2 ) and its root to shoot translocation of ten wheat genotypes. Wheat seedlings were raised in iron sufficient (100 μM) and deficient (1 μM) nutrient solution culture under controlled condition. Seedlings at 9, 12 and 15 days after transfer were exposed to 14CO2 for a fixed duration in a plexiglass airtight chamber. The treated plants were separated into root and shoot and their biomass were recorded after drying. Iron deficiency response of the genotypes was measured in terms of their efficiency for biomass production under iron deficiency with respect to iron sufficiency. In general the carbon fixation ability increased with plant vigor. Root to shoot partitioning of assimilates was greater under iron deficiency than under iron sufficient condition which is explainable on the basis of higher carbon requirement for maintaining root growth under stressful condition. Further, a higher shoot carbon-14 under Fe deficiency than under Fe sufficiency reflects a higher carbon starvation and demand of iron deficient plants. It is, however, unclear if the gas exchange attributes get affected under iron deficiency as the deficient plants measured a higher or an almost similar level of carbon fixation when compared with the iron sufficient plants.

Abstract No. 91

An insight into gamma radiation tolerance of an ancient extremophilic insect chironomus ramosus


Rita Mukhopadhyaya 1 , K.D.Datkhile 2 , th B.B.Nath 2none

1 Molecular Biology Division, Bhabha Atomic Research Centre, Mumbai. 2 Centre for Advanced Studies, Department of Zoology, Pune University, Pune, India. Email: bbnath@unipune.ac.in

Chironomus
, commonly known as 'midge' belongs to primitive group of insects that have evolved approximately 200 million years ago. Midges exhibit wide range of tolerance to various stresses. A few European and Russian laboratories have reported examples of natural populations of midges adapted to chronic levels of radiation after Chernobyl nuclear reactor explosion in 1986. No detailed studies were carried out to elucidate the adaptive basis of radiation tolerance of midges. We have been studying the laboratory population of the Indian species of midge, Chironomus ramosus for the last several years to explore the underlying parameters of radiation tolerance. The radiation doses required to cause 100% mortality immediately after radiation exposure of egg, larva, pupa and adult stages were 1000Gy, 3000Gy, 3200Gy and 3500Gy respectively. Radiation tolerance corroborated with increased activities of antioxidant enzymes and elevated levels of Hsp70 expression in the irradiated salivary gland cells (SGCs). DNA of SGCs was extracted at various time points during recovery from radiation stress and subjected to pulse field gel electrophoresis, single cell gel electrophoresis (comet assay) and competitive ELISA. All three techniques used to detect extent of damage to DNA showed complete reconstitution of DNA of polytene chromosomes by 48 hours of post radiation exposure recovery. The findings provided evidence for the first time for existence of a possible DNA repair mechanism in any insect following gamma radiation stress and further justified the inclusion of C. ramosus in the category of radiation tolerant group of insects.

Abstract No. 120

Gamma irradiation alters the free polyamine levels in vigna radiata (l.) wilczek

Mandar Sengupta


Department of Biophysics, Molecular Biology and Bioinformatics, University of Calcutta, Kolkata, India. Email: mandar.sengupta@gmail.com


Exposure to gamma radiation incurs physical stress in living systems. Gamma radiation damages critical targets in the cell by direct ionization or indirectly through radiolysis of water to form free radicals. Ionizing radiation induced damage to the DNA lead to mutation and cell death.

Putrescine, Spermidine and Spermine are the three major polyamines found in plants. Polyamines remain in free, conjugated and bound forms. Polyamines are small aliphatic amines with uniform distribution of positive charge. Polyamines bind to DNA, RNA and protein and provide protection and stability to the cell. The polyamines play important roles in help in development, differentiation, growth and other physiological processes. Polyamines also protect plants exposed to abiotic stress.

The present study concentrated on the effect of different doses of gamma radiation on seedling injury, morphology, photosynthetic pigments like chlorophyll and carotenoid and alterations in free polyamine content in Vigna radiata (L.) Wilkzek. Gamma radiation induced stress resulted in seedling injury, decrease in germination percentage and alteration in level of photosynthetic pigments. Statistically significant altered levels of Polyamines have been correlated with stress response of Vigna radiata.

Abstract No. 191

Deinococcus radiodurans: World's most radioresistant bacterium offers novel opportunities for basic research and applications

Shree Kumar Apte

Molecular Biology Division, Bhabha Atomic Research Centre, Mumbai-400085, India. Email: aptesk@barc.gov.in

The Gram positive, deep orange-red pigmented bacterium Deinococcus radiodurans finds mention in the Guiness Book of Records as the world's most radioresistant bacterium. Strains of this microbe easily survive severe DNA damage caused by ionizing radiations, UV and prolonged desiccation. Genome sequence of this superbug in 1999 revealed a great deal of multiplicity and redundancy with 4-10 copies each of one large and one small chromosome, one megaplasmid and a smaller plasmid per cell. The extreme radioresistance of this microbe is consequent to its unbelievable ability to stitch back its completely fragmented chromosomes and plasmids following intense DNA damage. How this remarkable feat is achieved is not known very well. Deinococcal genome harbors over 3000 genes of which almost one-third are unique to this organism. The known genome sequence does not reveal much about its phenomenal DNA repair ability. In fact, the deinococcal genome lacks many classical DNA repair genes and pathways routinely present in other bacteria. The attention has therefore turned to the unknown and hypothetical genes in its genome in the hope that they probably hold the secret of its radioresistance. We have conducted a detailed study to elucidate the kinetics and dynamics of proteomic changes accompanying the post-irradiation recovery to learn some new facts. The organism also offers a tremendous potential for bioremediation of nuclear waste. We have genetically engineered deinococcal strains for bioprecipitation and recovery of traces of uranium from acidic, neutral and alkaline radioactive waste. Novelties underlying its extreme radioresistance and possibilities for its biotechnological exploitation in high radiation environments will be discussed.

Abstract No. 197

Biological consequences and health concerns from nuclear radiation exposures in mice and humans

Taisei Nomura

Osaka University and National Institute of Biomedical Innovation, Osaka, Japan. Email: n5nomura@nibio.go.jp

This paper reviews our published and unpublished data; (1) the direct effect of nuclear radiations in mice and human tissues maintained in Super-SCID (severe combined immunodeficient) mice, and (2) transgenerational (hereditary) effects via germ cells in mice and human populations after nuclear explosion and accidents.

Briefly, nuclear radiations induced cancer, malformation and mutations dose dependently in mice and also morphological and functional changes and changes in gene expression in human thyroid tissues. Fission neutron and tritium β-rays showed higher RBE. Dose rate effects of ionizing radiation were apparent in mice and human tissues, but not in double-strand break repair deficient SCID mice. Thus, human thyroid tissues exhibited strong repairing ability to radiation damage and we found 14 genes which specifically respond to radiations.

Parental exposure to nuclear radiations induces mutations (ordinary and microsatellite), cancer and malformations dose dependently in the mouse progeny. Fission neutron showed higher RBE. Spermatogonial stages are less sensitive than post-meiotic stages and apparent dose rate effects were observed at spermatogonial stages. Cancer incidence in the progeny of mice exposed to radiation was strikingly enhanced by postnatal treatment with environmental cancer promoting agents. In humans, there are several reports on the increase of cancer and/or malformations in the children of parents who had been exposed to nuclear and diagnostic radiations before conception. However, these findings have not been proven in the children of atomic bomb survivors. Minisatellite mutations were detected in the progeny of the inhabitants near the nuclear bomb test sites and nuclear accidents, but not in those of liquidators and atomic bomb survivors. Similar results were observed in our microsatellite studies in Chernobyl and Hiroshima/Nagasaki.

These technologies are useful for studying the effects of cosmic radiation on the human tissue carried into the space by SCID mice and on the next generations; a few male mice launched into the space produce large numbers of offspring after returning to the earth for rapid detection of genetic effects. Human cancer tissue and its normal tissue maintained in SCID mice are good targets to examine the efficacy of radiotherapy and side effects of radiations in that organ.

(Supported by JSPS, Japan Space Forum, Takeda Science Promotion Fund and Yasuda Medical Award)

RADIATION INDUCED BYSTANDER EFFECTS AND GENOMIC INSTABILITY

Abstract No. 35

Focused vertical microbeam system for proton irradiation of living cells for radiobiological research at NIRS

Teruaki Konishi 1 , Masakazu Oikawa 1 , Noriyoshi Suya 1 , Takahiro Ishikawa 1 , Takeshi Maeda 1 , Alisa Kobayashi 1 , Naoko Shiomi 1 , Kumiko Kodama 1 , Tsuyoshi Hamano 1 , Shino Homma-Takeda 1 , Mayu Isono 1,2 , Kotaro Hieda 3 , Yukio Uchihori 1 , Yoshiyuki Shirakawa 1

1 National Institute of Radiological Science, Chiba, Japan, 263-8555. 2 Metropolitan University of Tokyo, Tokyo, Japan, 116-8551. 3 Rikkyo (St. Paul's) University, Tokyo, Japan, 171-8501. Email: tkonishi@nirs.go.jp

Microbeam irradiation system is a powerful tool for radiation biological studies in the field of low dose effects studies, such as bystander effects using mammalian cells and also has the potentials to contribute in variety of ways to other biological studies. The development of Single-Particle Irradiation system to CEll (SPICE), a microbeam irradiation system, has been completed at the National Institute of Radiological Sciences (NIRS). SPICE can generate 3.4 MeV proton microbeams with a beam size of approximately 2 micrometer in diameter. Cell targeting system consists of fluorescent microscope, voice coil motor (VCM) stage, particle detector and a beam shutter. The functions of these components are synchronized to irradiate all or only desired fraction of cells in the targeted area with a programmable number of protons. Standard procedures for irradiation experiments, such as cell image capturing, position recognition (calculation of X-Y coordinates) and irradiation is mostly automated. Thus, cells with fluorescent dyed nuclei or with other fluorescence, such as green fluorescent protein, prepared in specially designed Mylar film base dish can be easily targeted for microbeam irradiation. Moreover, SPICE can complete the routine procedure within 10 ~ 15 minutes to irradiate approximately 3000 cells, at irradiation speed of 400 cells per minute. Specifications of SPICE, and ongoing radiobiological studies will be presented.

Abstract No. 54

Cytokine profile of conditioned medium from human tumor cell lines after acute and fractionated doses of gamma radiation and its effect on survival of bystander tumor cells

Sejal Desai, Amit Kumar, S. Laskar1 and B. N. Pandey

Radiation Biology and Health Sciences Division, Bhabha Atomic Research Centre, Mumbai - 400085. 1 Department of Radiation Oncology, Tata Memorial Hospital, Mumbai - 400012, India. Email: sejaldesai8@gmail.com

Cytokines are known to influence various steps of cancer progression. Evidences suggest differences in cytokine profiles of cancers with diverse origin: however, a comprehensive study of the cytokine profile after acute and fractionated doses of gamma-radiation is missing in literature. In this study, we have evaluated the cytokine profiles of human tumor cell lines (HT1080, U373MG, HT-29, A549 and MCF-7) either before (basal) or after acute (2, 6Gy) and fractionated doses (3x 2Gy) of gamma-irradiation of these cells by multiplex bead assay/ELISA. Based on the screening of 28 analytes, our results showed that the basal profiles of these cell lines varied considerably in terms of the number and magnitude of secreted factors. Moreover, CM caused increase in clonogenic survival of respective tumor cells (HT1080 > U373MG > HT29 > A549 > MCF-7), which was correlated with the levels of IL-1β, IL-6, IL-8, GMCSF and VEGF in their CM. After irradiation, the level of most of the cytokines increased markedly in a dose dependent manner, which was lower in magnitude for fractionated than respective acute dose, except in MCF-7. Amongst these cell lines, the radiation-induced fold increase in cytokine levels was highest in ICM of A549 cells. Furthermore, bystander A549 cells treated with respective ICM showed dose dependent decrease in clonogenic survival. In conclusion, our study revealed the similarities and subtle differences in basal and radiation-induced cytokine profile of different tumor cell lines, and its effect on growth of respective bystander cells.

Abstract No. 86

Role of ATM in bystander signaling between human monocytes and lung adenocarcinoma cells


Dr. Somnath Ghosh

Radiation Biology & Health Sciences Division, BARC, Mumbai. India. Email: somnath@barc.gov.in

The response of a cell or tissue to ionizing radiation has been shown to be mediated by direct damage to cellular components like DNA, lipids, proteins and small molecules as well as indirect damage mediated by radiolysis of water. It is now apparent that the target for ionizing radiation is not solely the irradiated cells but also includes the surrounding cells and tissues. Radiation-induced bystander effect is defined by the observance of biological effects of radiation in cells that were not themselves in the field of irradiation.

To establish the contribution of the bystander effect in the survival of the neighboring cells, the lung carcinoma A549 cells were exposed to 2Gy gamma-irradiation. The medium from irradiated cells was then transferred to unirradiated cells. Irradiated A549 cells as well as unirradiated A549 cells cultured in the presence of medium from irradiated cells showed decrease in survival and increase in γ-H2AX & pATM foci, indicating presence of bystander effect. Bystander signaling was also found to exist between different cell types. Phorbol-12-myristate-13-acetate (PMA) stimulated and gamma-irradiated U937 (human monocyte) cells induced bystander response in unirradiated A549 (lung carcinoma) cells as was evident by decrease in survival and increase in γ-H2AX & pATM foci. The unstimulated and/or irradiated U937 cells did not induce such effect in unirradiated A549 cells.

Since ATM protein was activated in irradiated cells as well as bystander cells, it was of interest to understand its role in bystander effect. Suppression of ATM with siRNA in A549 cells completely inhibited bystander effect in bystander A549 cells. On the other hand suppression of ATM with siRNA in U937 cells caused only a partial inhibition of bystander effect in bystander A549 cells. These results indicate that apart from ATM, some additional factor may be involved in bystander effect between different cell types.

Abstract No. 100

Role of soluble factors from tumor cells on survival and radiosensitivity of bystander cells


Sejal Desai, S. Laskar 1 and B. N. Pandey

Radiation Biology and Health Sciences Division, Bhabha Atomic Research Centre, Mumbai 400 085, 1 Department of Radiation Oncology, Tata Memorial Hospital, Parel, Mumbai. Email: bnp@barc.gov.in

To understand the bystander interaction in tumor cells through secretable factors, we have studied secretion profile of human lung carcinoma (A549) cells. Our results suggest that conditioned medium (CM) obtained from A549 is rich in pro-inflammatory cytokines, chemokines and growth factors. In this study, the self-CM is shown to affect survival and proliferation of tumor cells as studied by clonogenic assay and flow-cytometry based cell cycle analysis. The CM-induced changes in growth pattern of A549 were found to be regulated by MAP kinases (p38 and JNK). Upstream to MAPKs activation, CM treated cells showed increase in the levels of cAMP. Downstream to MAPKs, CM also induced phosphorylation and nuclear translocation of activating transcription factor 2 (ATF-2). However, sub-cellular localization of STAT-3 and c-Jun was not affected, suggesting major involvement of ATF-2 signaling in the observed conditioning effect. Furthermore, we examined the effect of CM on the expression of proliferative and anti-apoptotic proteins which are the major targets of ATF-2 like cyclin A, cyclin D1 and bcl-2 and bcl-xl. Interestingly, their levels were found to be increased in CM-treated A549 cells. The observed conditioning effect was abrogated when tumor cells were grown in conditioned medium treated with neutralizing antibodies against putative mediators like IL-8 and VEGF. The enhanced basal survival is also reflected in radio-resistance, as cells irradiated in the presence of CM showed higher survival than that of in PM. These can be attributed to the activation of aforementioned signalling pathway and induction of pro-survival factors.

Abstract No. 144

The physics and radiobiology of space-time fractionated radiation therapy


Xiaodong Wu

Biophysics Research Institute of America. Email: drxiaodongwu@yahoo.com

Purpose: Traditional fractionation in radiotherapy has been mostly configured in time domain. Clinical data from spatially fractionated schema, albeit less abundant, have suggested important advantages in terms of normal tissue sparing and tumor bystander effect. The Space-Time Fractionation (STF) proposed herein is a novel concept that combines the strengths of fractionations in both the temporal and spatial domains, aiming at maximum tumor control with substantially reduced normal tissue toxicity. Any external beam radiotherapy (EBRT) modalities such as 3DCRT or IMRT can be delivered in STF through an STF collimator. Materials and Methods: In the STF approach, treatment volume is divided into segments or slices, consecutively numbered 1,2,3,4,5,6,7,8…, with typical slice thickness of 1-2 cm. Alternated time-fractions are delivered to their corresponding spatial segments, i.e. the time-fractions 1,3,5,7,9,... are delivered to spatial slices 1,3,5,7.., and the time-fractions 2,4,6,8,.. are delivered to spatial slices 2,4,6,8… To demonstrate the normal tissue sparing effect, three groups of C57/BL6 mice were used: untreated; STF, 500cGy/fx x 10 delivered through a block of multiple equally spaced open/closed slits of 2 mm width; and Conventional 310cGy x 10, open field. The two irradiated groups have the same BED delivered to the whole abdomen. Two mice were sacrificed immediately, 24 hr, 10 days and 30 days after the last fraction of radiation and intestinal, kidney and liver tissues were collected for pathological analysis. Results: Of all the treated groups, pathological analysis of the tissues clearly showed more damage with open field irradiation compared to STF. With the open-field, intestinal epithelial cells showed severe damage with cell loss in intestinal crypts and thus villi were shortened and blunted. One month after irradiation, amphophilic cytoplasm, variation in the size of the nuclei, clumping of chromatin and prominent nucleoli were observed in the intestinal tissues. The number of mitotic figures increased with time indicating regeneration but recovery was only partial. With STF, negligible damage was observed at all the time points studied. A similar level of differentiation in the response of kidney and liver tissues were observed. Conclusions : In STF, only partial volumes of normal/critical structures are exposed at each dose fraction. This could facilitate the repair of radiation injury in normal tissues and thus reduces toxicity. Our results are concordant with this hypothesis. Additional non-conventional biological effects associated with STF are currently under active investigation and have potentially radical impact in tumor control.

Abstract No. 170

Studies on bleomycin induced bystander response and their significance in cancer chemotherapy

P. Venkatachalam, M. Chinnadurai and Solomon F.D. Paul

Department of Human Genetics, Sri Ramachandra University, Porur, Chennai - 600 116. Email: venkip@yahoo.com

Radiation induced second malignancies occurring after oncological treatments have become a concern; it was found that the cumulative incidence of second malignancy was as high as 20% of patients treated by radiotherapy and 7% by chemotherapy. This increased risk of second cancer, may in part reflect the induction of genome destabilizing mutations during the treatment although the potential mechanism remains speculative. Expression of DNA damages in the cells which are not directly exposed to ionizing radiation is termed as radiation induced bystander response; a well-established phenomenon in radiation biology has been suggested as a possible mechanism for therapy induced second malignancy. Similar to ionizing radiation, chemotherapeutic drugs like mitomycin, phleomycin, actinomycin and bleomycin have also been shown to induce non targeted effects; the observation in response to chemotherapeutic agents underpins its considerable importance in cancer therapy, in which not all tumor cells are targeted and indirect killing of non-targeted cells is required to ensure maximal tumor cell kill. In terms of consequences to the exposed tissue or organism, non-targeted bystander responses may be considered both detrimental and beneficial. Hence, we have investigated the bleomycin (used to treat many types of cancer) induced bystander response in normal skin fibroblasts (WI-38), human bone marrow stem cells (BMSC), human blood lymphocytes, lung adenocarcinoma cells (A-549, NCI-H23), leukemic cells (CCRF-CEM, HL-60) and glioma (BMG-1) using co-culture methodology. The DNA damages were quantified using MN assay and gene expression by flow cytometry. The obtained results shows that the bleomycin induced bystander response are cell line dependent. Over all, the paradigm shift due to non-targeted effects of therapeutic agents have increased concerns of primary therapy with chemicals and radiation, as they may increase the risk for the occurrence of secondary malignancies.

Abstract No. 177

Glycolytic inhibitor 2-Deoxy-D-Glucose (2-DG) inhibits tumor associated angiogenesis

Saurabh Singh 1 , Amanpreet Singh Chawla 2 , Sanjay Pandey 1* , Daman Saluja 2 , B S Dwarakanath 1

1 Institute of Nuclear Medicine and Allied Sciences, Delhi 110054; 2 Ambedkar Centre for Biomedical Research, University of Delhi, India. Email: saurabh_11may@yahoo.co.in

Background: Tumorogenesis disturbs the precise balance of growth promoting and inhibitory factors of angiogenesis also activates pro-angiogenic pathway to overcome nutrient and size limitations thus promoting metastasis. Stimulated angiogenesis involves proliferation, migration, and capillary formation by endothelial cells (ECs); requiring increased energy demand. Moreover, angiogenic switch significantly upregulates EC expression of glucose transporter-1 (Glut-1) and glucose uptake where almost all the energy (ATP) is derived from the catabolism of glucose generated by glycolysis. Glucose metabolism of the tumor as well as the EC can be modulated by dietary regimen or by use of Energy Restriction Mimetic Agents (ERMA) such as 2-Deoxy-D-Glucose (2-DG) without causing reduction in calorie intake. Our earlier studies have shown that 2-DG as a dietary component reduces the incidence as well as the growth of implanted tumors such as Lewis Lung Carcinoma (LLC), Ehrlich Ascites Tumor (EAT). Present studies were undertaken to investigate the anti-angiogenic effect of 2-DG that may partly contribute to the inhibition of tumor growth. Objective: To investigate the effects of chronic (dietary) administration of 2-DG on tumor associated angiogenesis. Materials and Methods: In-vitro studies : Migration (scratch) assay and matrigel tube formation assay was performed to assess whether or not 2-DG affected the tube formation property of human umbilical vein endothelial cells (HUVECs). Cell invasion, cell viability and cytotoxicity assays are being performed presently. In vivo matrigel plug assay : Lewis lung carcinoma cells (0.5million) were injected (s.c) along with matrigel at right leg in C57BL6/J mice to evaluate the in vivo effects of 2-DG on tumor angiogenesis. 2-DG administration (0.2% and 0.4% w/v) was started 24 hrs after tumor implant in drinking water followed till the termination of the study (7 days). Matrigel plugs were excised at different time points and photographed, quantified for angiogenesis by FITC-Dextran method. Results and Conclusion: 2-DG inhibited tube formation and cell migration in both the conditions of normoxia and hypoxia clearly indicating that it alters the functioning of the endothelial cells (HUVECs). In vivo matrigel assay showed reduced levels of micro vasculature around the tumor in the 2-DG fed mice. 2-DG not only inhibited the tumor growth but also the neovascularisation in the tumor containing matrigel plugs. Quantification of tumour angiogenesis in matrigel plugs by FITC-Dextran injection (tail vein) revealed decreased angiogenesis in mice fed with 2-DG. Thus 2-DG appears to function as a potent anti-angiogenic agent that may partly contribute to its antitumor effects.

RADIATION INDUCED OXIDATIVE DAMAGE, DNA DAMAGE AND APOPTOSIS

Abstract No. 16

Antioxidant capacity of melatonin on the oxidative damages caused by a potent oxidant Uvc: An in Vivo and iIn Vitro study

S. Goswami, S. Sharma, and C. Haldar

Pineal Research Lab., Department of Zoology, Banaras Hindu University, Varanasi, India. Email: soumik.goswami911@gmail.com

UV radiation a known oxidant causes inflammation due to the release of inflammatory cytokines which in turn generates reactive oxygen species (ROS) that damages lipids, proteins and DNA. Melatonin a potent antioxidant from the pineal gland acts as a free radical scavenger following UVC radiation. Our data suggests that melatonin significantly protected against UVC induced suppression of contact type hypersensitivity (CTH) response in tropical rodent F.pennanti. It also protected the rodents from UVC increased TBARS level in splenocytes accompanied with a significant decrease in SOD activity indicating the superoxide anion mediated damages following UVC exposure. Melatonin reduced the radiation induced oxidative stress in the splenic tissue as analysed by reduced lipid damages and higher SOD activity. Under in vivo conditions, melatonin supplementation (100μg/100gm body wt.) was more potent in protecting lymphatic tissues from radiation induced cell death when compared with the in vitro condition.

In conclusion we suggest that melatonin may be one of the potent antioxidant as defence mechanism that reduces UV induced toxicity to the cells due to higher oxidative load.

Abstract No. 32

Gamma radiation induced reactive oxygen species and DNA damages in HaCat cells under euoxic and hypoxic condition

Praveen Joseph 1 , N N Bhat 2 and Y Narayana 1

1 Department of Studies in Physics, Mangalore University, Mangalagangotri-574 199. 2 RPAD, Bhabha Atomic Research Centre, Mumbai - 400 085. India. Email: narayanay@yahoo.com

The gamma radiation induced Reactive Oxygen Species (ROS) generation in human keraninocyte (HaCat) cells has been assessed using the fluorescent probes under euoxic and hypoxic condition. The DNA damage induced under the both irradiation condition also has been quantified using alkaline comet assay. ROS produced in the cells were determined by measuring fluorescence after loading with 2', 7' dichlorofluorescin diacetate (DCHF-DA), a dye that is oxidized into a highly fluorescent form in the presence of peroxides. Studies reveal that in HaCat cells radical generation occurs when exposed to ionizing radiation and it increases linearly with dose under euoxic and hypoxic irradiation. However, significant reduction in the rate of ROS generation as well as DNA damage induction was observed under hypoxic irradiation compared to that under euoxic condition. A linear relation between ROS generation and DNA damage induction was observed at lower doses. However at higher doses, even though the ROS generation is linear with dose a reduction in DNA damage induction was observed, which can be attributed to the occurrence of radical- radical interactions due to high radical concentration at higher doses thereby minimizing the damage to the DNA. The study clearly shows the importance of ROS in DNA damage induction and the results allow establishment of a relationship between specific DNA damage levels induced by ROS. Abstract No. 43

Prediction of radiotherapy induced normal tissue acute toxicity: Molecular significance of DNA double strand breaks and its repair

BS Satish Rao

Division of Radiobiology & Toxicology, Manipal Life Sciences Centre, Manipal University, Manipal, 576104, India. Email: satishraomlsc@gmail.com

Improved chemo-radiotherapy treatment outcome has come at the expense of increased normal tissue toxicity. Despite the progress in the treatment techniques, normal tissue toxicity still persists and varies widely from patient to patient. As the number of long-term cancer survivors increases, preventing or reducing side effects of chemotherapy and/or radiotherapy has become a major concern. Early and late normal tissue toxicities are considered as the major limiting factors in radiotherapy. About 20% of patients who undergo radiotherapy will develop adverse reactions to the therapy. Therefore, identification of these radiosensitive and radioresistant individuals by cellular and molecular based techniques before radiotherapy will play a pivotal role in controlling normal tissue adverse effects in radiosensitive group and also may permit the dose to be increased in nonsensitive patients.

Response to ionizing radiation is non-homogeneous in population as well as at individual level. There exist interindividual difference in double strand break (DSB) induction,rejoining capacity and the need to determine DSBs using methods capable of differentiating the minor interindividual differences in damage induction and repair has a significant translational relevance. Also, at the molecular level, single nucleotide polymorphisms (SNP) in DNA damage and repair genes, pro-fibrotic and inflammatory cytokines, detoxification and endogenous antioxidant enzymes contribute to variations inradiation response, resulting in differential radiosensitivity.

In this regard, DNA double strand break induction and its repair kinetics in lymphocytes of healthy individuals, breast as well as head and neck cancer patients undergoing chemo-radiotherapy was analysed by γ-H2AX foci counting, neutral comet assay and a modified version of neutral filter elution assay. Further, the genotyping of SNPs in selected candidate genes were done by Polymerase Chain Reaction- Restriction Fragment Length Polymorphism (PCR-RFLP).

In healthy individuals, the variation in double strand break induction and repair kinetics was observed with coefficient of variation of 13.65%, 24.40% and 24.27% for 0.25, 3 and 6h post irradiation respectively. The NBN rs1805794 and RAD51 rs1801321 polymorphisms the variant allele was found to associate with the increased radiosensitivity. Further, our results also suggest that γ-H2AX analysis may have predictive implications for identifying the acute reactions in breast and head and neck cancer patients prior to the initiation of radiotherapy. The molecular studies to associate the role of SNPs to acute reactions among cancer patients are being carried out.

Our experimental findings indicate a range of DNA repair capacity in healthy humans and demonstrate that polymorphisms in double strand break repair pathway genes significantly contribute to radiation sensitivity individually. Further, the combination of such SNPs contributes further to the compromised repair capacity and thereby increased radiosensitive phenotype. Also, our study suggests that the radiosensitivity of lymphocytes, quantified by γ-H2AX assay may have prognostic implications in assessing the severity of mucositis and skin reactions at least for stratifying overreactors in HNC patients prior to the initiation of radiotherapy.

Acknowledgements: The financial support from Department of Biotechnology, Government of India (BT/01/COE/06/02/07) & Manipal University is gratefully acknowledged.

Abstract No. 49

Silymarin can be used as a safe radiation countermeasure in the event of radiological emergencies: Evidence from in vitro and in vivo studies

Manish Adhikari 1none , Veselin Ivanov 2 , J.S. Adhikari 1 , Y. Karamalakova 2 , T. Zheleva 2 , R.Kumar 1 , R.K. Sharma 1 , V. Gadjeva 2 and Rajesh Arora 1

1 Radiation Biotechnology Group, INMAS, Brig. S.K. Mazumdar Marg, DRDO, Delhi-110054, India. 2 Department of Chemistry and Biochemistry, Medical Faculty, Trakia University, Armeiska 11, Stara Zagora 6000, Bulgaria. Email: manishadhikari85@gmail.com

In view of the non-availability of safe radiation countermeasure (RCM) agents for the management of radiation emergencies, the search for efficient such agents is on-going. Silymarin, derived from Silybum marianum (L.) Gaertner (Milk thistle), comprises mainly of flavonolignans (flavonoids and other polyphenolic compounds). The aim of the present investigation was to assess the ability of silymarin against radiation-induced stress. Viability studies by MTT assay and Annexin V-PI studies showed that pre-incubation of HEK cells with silymarin protected them from gamma-irradiation mediated killing and reduction in apoptosis (76.3%). It also decreased the percentage of radiation-induced micro nucleated cells and frequency of micronuclei (> 69%; p < 0.05). Measurement of intracellular reactive oxygen species (ROS) by H2DCFDA revealed reduction in ROS (21%) generated by gamma- irradiation (0.5h). Cell cycle analysis data revealed G1 block in unirradiated control, which declined in pre-treated silymarin irradiated group (0.5h). Plasmid relaxation assay revealed protection of DNA by maintaining its integrity in a dose-dependent manner against gamma-irradiation. Survival studies using Swiss albino Strain A mice confirmed that silymarin exhibits maximum protection at 70mg/kg b/w against 8Gy gamma-irradiation. Silymarin exhibited an increased CD4: CD8 ratio in pre-treated irradiated group as compared to radiation group; while it exhibited a decrease in Tregs (CD4+ CD25high) population as compared to radiation-treated group in a significant manner (p < 0.05). From results it concluded that silymarin used as radiation countermeasure agent.

Abstract No. 53

In vitro raman spectroscopy of gamma irradiated HeLa cells

Priyanka Sathe
1 , Donil Domnic 2 , Jayant Sastri Goda#, Supriya Chopra#, C.M. Krishna*

1 Chilkapati Lab, 2 Clinical biology Lab, Department Of Radiation Oncology, ACTREC, Tata Memorial Centre, Kharghar, Navi Mumbai - 410 210, India. Email: priyankasat@gmail.com

Raman spectroscopy (RS) is a vibrational spectroscopic method known to be sensitive to molecular composition of a given substance. Several studies have demonstrated its efficacy to identify normal and malignant conditions, HPV +ve & HPV -ve cell lines and different cell lines expressing MDR phenotypes. In the present study we have explored potentials of Raman spectroscopy in classifying HeLa cells irradiated at different doses and its correlation with biological assays (Clonogenic assay, γ-H2AX assay). HeLa cells were grown under standard conditions to achieve 70% confluency. Cells were irradiated with single fraction (0 Gy, 2Gy, 4Gy, 6Gy, 8Gy and 10Gy) by Co-60 therapy source. Cells were trypsinized to obtain pellet for Raman spectroscopic analysis at 24 hours post-irradiation. Raman spectra were acquired with 40x objective coupled HE-785 commercial Raman instrument. Spectral acquisition parameters were: λex-785 nm, laser power-40 mW, integration time: 15 seconds, number of accumulations-3. Approximately 10 spectra were recorded from single pellet. A total of about 180 spectra were acquired over 3 independent experiments. Pre-processed spectra (in region 800-1800 cm-1) were subjected to Principal Component Analysis (PCA). Parallely clonogenic and γ-H2AX assay were performed. PCA gave exclusive clusters for HeLa cells irradiated at low doses i.e. 2Gy, 4Gy and control. But overlapping clusters were observed for HeLa cells irradiated at higher doses i.e. 6Gy, 8Gy and 10Gy. In-vitro clonogenic assay correlated very well with Raman spectroscopic analysis in the dose range of 0- 4 Gy. The surviving fraction of HeLa cells decreased in a dose dependent manner till 6 Gy, after 6 Gy plateauing of survival curves was observed with increasing radiation dose. Similar results were observed with γ-H2AX analysis, the mean γ-H2AX foci intensity increased linearly with dose administered till 6Gy, Beyond 6 Gy, there was saturation in the intensity and the mean number of γ-H2AX foci. Preliminary work supports the feasibility of RS to classify HeLa cells that were subjected to different doses of radiation and results showed good correlation with biochemical assays.

Abstract No. 55

Identification of a novel inhibitor of nonhomologous DNA end joining: Implications in DNA double-strand break repair and cancer

Sathees C. Raghavan

Department of Biochemistry, Indian Institute of Science, Bangalore - 560 012, India.

DNA Ligase IV is responsible for sealing of double-strand breaks (DSBs) during nonhomologous end joining (NHEJ). Abrogation of this step could result in amassing of DSBs. In the present study, we identify a novel molecule, SCR7 that inhibits joining of various DSBs in a cell-free repair system. SCR7 blocks joining by purified Ligase IV by interfering with its binding to DNA, but not of T4 DNA Ligase or Ligase I. Importantly, inhibition is restored by addition of purified Ligase IV/XRCC4 complex. Inhibition of NHEJ by SCR7 within cells leads to accumulation of unrepaired DSBs, thereby activating intrinsic pathway of apoptosis. Interestingly, SCR7 impedes tumor progression in different mice models and when coadministered with existing DSB inducing therapeutic modalities, enhances their sensitivity significantly. Thus, for the first time, we identifiedt an inhibitor, which targets NHEJ by disrupting joining of DSBs by Ligase IV, and can potentially be developed as a strategy for the treatment of cancer.

Abstract No. 64

Mechanism of DNA double-strand break repair: Biochemical characterization of classical NHEJ and microhomology mediated alternative DNA end joining

Sheetal Sharma and Sathees C. Raghavan

Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India. Email: sheetal@biochem.iisc.ernet.in

Maintenance of genomic integrity is very important for the survival of an organism. Double-strand breaks (DSBs) are the most deleterious type of DNA damages, as they can result in chromosomal rearrangements, genomic instability and cancer. Nonhomologous DNA end joining (NHEJ) is one of the major DSB repair pathways in higher eukaryotes. Though the protein machinery required for NHEJ is well understood, little is known about the joining mechanisms of different DSBs in normal tissues. In the present study, we have established a novel NHEJ assay to evaluate the efficiency and mechanism of DSB repair in normal tissues. Different DNA substrates containing appropriate DSBs were incubated with cell-free extracts from various rat tissues and the joining products were evaluated. Results showed that substrates containing 5'-compatible ends joined more efficiently than blunt, 5'-5' or 5'-3' noncompatible ends. Testis, followed by lungs, possessed maximum NHEJ efficiency and followed a conservative mode of repair. In contrast, heart, liver and kidney exhibited least NHEJ which followed extensive modifications at the junctions.

Recently, another pathway known as alternative NHEJ has been discovered and suggested to utilize microhomology for DNA end joining. However, the exact mechanism and enzymatic requirements for this pathway are not well understood. In the current study, we show that DNA substrates possessing various lengths of microhomology regions predominantly get repaired using microhomology, irrespective of the type of tissue. Importantly, the joining was dependent on MRE11, NBS1, LIGASE I, LIGASE III, XRCC1 and PARP1 proteins, both in vitro and inside cells. Besides, robust MMEJ mediated DSB repair was observed in heart tissues which was also consistent with the elevated expression of several proteins involved in this pathway. Further, we find that irradiation of rats with increasing doses of gamma-rays (5, 10 and 20 Gy) resulted in upregulation of MMEJ in a dose-dependent manner. Hence, our study provides new insights into both classical and nonclassical NHEJ in somatic tissues and their implications in genomic instability.

Abstract No. 79

Regulation of cellular redox systems under conditions of oxidative stress induced by ionizing radiation

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R. S. Patwardhan, Rahul Checker, Deepak Sharma and S. Santosh Kumar

Radiation Biology & Health Sciences Division, BARC, Trombay, Mumbai, India. Email: rs.patwardhan27@gmail.com

Reactive oxygen species are products of normal metabolism, xenobiotic exposure or exposure to ionizing radiation. Depending upon their concentration, "ROS" can be beneficial or harmful to living systems. To respond to cellular redox changes various redox systems such as glutathione/glutathione reductase, thioredoxin/thioredoxin reductase exist that transduce signals to molecules which regulate cellular redox. There are a number of mammalian transcription factors that are regulated by changes in cellular redox e.g. APE-1/AP-1/ NF-kB/Nrf-2/p53 etc. Nrf-2 is a redox-sensitive transcription factor and a master regulator of battery of antioxidant enzymes. Recent findings have reported that ionizing radiation leads to Nrf-2 activation. Although, studies have been carried out explaining the role of individual redox regulatory systems in different contexts. To our knowledge no study has been carried out explaining behavior of all redox regulatory systems under irradiated conditions. In the present study we have investigated the effect of exposure of human T cell lymphoma cells (Jurkat) to ionizing radiation on cellular redox systems in terms of their spatio-temporal gene & protein expression and enzymatic activity. We have determined GSH/GSSG ratio, total cellular thiol and phosphorylation status of Nrf-2 in irradiated cells which is indicative of oxidative stress in a time dependent manner. mRNA and protein levels of thioredoxin and thioredoxin reductase along with GCLC, hemoxygenase-I were estimated. Enzymatic activity of thioredoxin, thioredoxin reductase and superoxide dismutase was estimated. Our present study demonstrates that, there are time dependent changes in redox regulatory systems of cell after exposure to ionizing radiation, which may be responsible for redox restoration. Our results pave the way for better understanding of biology of redox couple and their relative behaviour.

Abstract No. 84

Role of PARP-1 in high LET radiation induced DNA damage in cultured HeLa cells

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Atanu Ghorai 1none , Asitikantha Sarma 2 , Nitai P. Bhattacharyya 3 , Utpal Ghosh 1

1 Department of Biochemistry & Biophysics, University of Kalyani, Kalyani - 741 235, 2 Inter-University Accelerator Center, Aruna Asaf Ali Marg, New Delhi - 110 067, 3 Saha Institute of Nuclear Physics, 1/AF Bidhannagar, Kolkata - 700 064, India. Email: atanu_ghr@yahoo.com

To investigate the role of PARP-1 in response to high LET 12C6+ ion beam (62 MeV, average LET 287 kev/μm), we measured the colony forming ability, cell viability, DNA breaks by comet assay, cell cycle alteration, induction of apoptosis by DNA ladder and Caspase-3 activation in both parental and PARP-1 knocked down HeLa cells (HsiI). The survival fraction of HsiI cells was significantly less than that of parental HeLa within the range of doses used (0- 4 Gy) as detected by colony forming ability. The DNA breaks produced was significantly higher in HsiI cells compared with HeLa counterpart as detected by comet assay, indicating that PARP-1 is involved in DNA repair. Furthermore, our cell cycle data showed that HsiI cells were arrested at G2/M phase for 0 - 2 Gy of carbon ion beam while nomal HeLa counterpart showed no arrest within the said range of dose. Nucleosomal ladder was found in both cell lines after twenty four hrs of treatment but HsiI cells showed intense ladder compared with HeLa. Caspase-3 activation was also observed significantly higher in HsiI than parental HeLa cells. Importantly, activation of Caspase-3 in untreated HsiI was significantly higher than untreated HeLa. These data implicating that HsiI cells were more sensitive to high LET radiation and PARP-1 might be involved in DNA damage response pathway.th

Abstract No. 109

Apigenin, a dietary flavone, modulates gamma radiation-induced cellular and molecular changes in human peripheral blood lymphocytes and swiss albino mice


Rajendra Prasad

Annamalai University, Annamalainagar, Tamilnadu, India. Email: drprasadnr@gmail.comp

The present study was aimed to explore the antioxidant potential of apigenin and its preventive effect on radiation-induced oxidative damage in human peripheral blood lymphocytes (HPBL) and whole body irradiated (WBI) Swiss albino mice. Apigenin (4, 5, 7-trihydroxy flavone) effectively scavenges hydroxyl (OH•), superoxide anion (O2•- ), 1, 1-diphenyl- 2-picrylhydrazyl (DPPH•) and 2, 2-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid radical cation (ABTS•+ ) in a concentration-dependent manner (37.2-186 μM). Gamma-radiation (3 Gy) exposure increased the levels of reactive oxygen species (ROS), % apoptotic cells and decreased the mitochondrial membrane potential in HPBL. On the other hand, apigenin treatment (37.2 μM) 60 min prior to irradiation significantly decreased ROS generation in HPBL. It has also been observed that apigenin treatment significantly reduced gamma-radiation-induced apoptotic incidence and modulates loss of mitochondrial membrane potential in HPBL. Furthermore, apigenin treatment significantly maintains cellular antioxidant status (superoxide dismutase, catalase, glutathione peroxidase activities and reduced glutathione level) and protects cellular milieu from gamma-radiation-induced lipid peroxidation, dicentric and micronuclei formation in irradiated HPBL. Radiation exposure increased p53, p21, Bax and NF-kB expressions and decreased Bcl-2 expression in HPBL. Conversely, an increase in Bcl-2 along with a decrease in p53, p21, Bax and NF-kB expression was observed in apigenin plus irradiated HPBL. It has also been observed that radiation induced XRCC-1 expressions were effectively modulated by apigenin pretreatment in HPBL. In Swiss albino mice, apigenin (15 mg/kg. wt.) increased radiation LD50 from 8.2 to 10 Gy indicating the dose modifying factor of 1.21. It was also observed that Hb content, WBC and RBC was decreased in irradiated Swiss albino mice. Apigenin pretreatment prevented radiation induced histological and inflammatory signaling elements in WBI mice GI tissue. Taken together, the present study indicates that apigenin showed strong antioxidant property in free radical scavenging systems and protects HPBL and Swiss albino mice from radiation-induced oxidative damages at cellular and molecular level.

Abstract No. 141

Radiation Response Mechanisms of Highly Radioresistant Insect Cells: Learning Nature's Lessons for Radioprotection


Sudhir chandna

NRRM Group, Division of Radiation Biosciences, Institute of Nuclear Medicine and Allied Sciences, Delhi, India. Email: sudhirchandna@yahoo.com

Lepidopteran insect cells are excellent model system for understanding biological radiation responses since these displays 50-100 times higher radioresistance than mammalian cells despite numerous homologies among the two systems. Extensive studies are being conducted in our laboratory for understanding the cellular responses & molecular pathways induced by radiation/ stress in these cells. Cellular radiation/stress responses of Sf9 ovarian cell line derived from Spodoptera frugiperda, the Fall armyworm (order Lepidoptera; class Insecta) have been studied which include DNA & -radiation doses up to 500Gy-1000Gy, with the iso-effect doses inducing DNA damage varying by up to 10 times. However, cell death was induced only at 1000Gy or higher, about 100 times higher than radiation doses lethal for human cells. Mitochondrial/ calcium disturbances were detected only at death-inducing doses, leading to typical apoptosis. Mitochondrial pathway of apoptosis was also induced by actinomycin-D with a excessive sensitivity in Sf9 cells, and cytosolic cytochrome-c release was Bax-mediated/ mPTP-independent in nature. Sf9 cells have significantly stronger antioxidant mechanisms accompanied by a prominent absence of NOS-induced NO-mediated response. Peculiar differences in chromatin organization may also prevent DNA damage or enhance DNA repair, which is also strongly indicated from our study. These cells also showed dependence on certain micro-RNAs known to regulate cell death in Drosophila, indicating presence of alternate/additional mechanisms regulating radiation responses. Lepidopteran radioresistance seems to have developed at important crossroads during the evolution of stress response mechanisms, and is revealing novel features of cellular radiation response. These studies may greatly help the Radiobiologists in developing more effective modalities for biological radioprotection or modulation.γ-cytogenetic damage, DNA repair, oxidative stress, nitrosative stress, NOS pathway, micro-RNA regulation, chromatin regulation as well as modes and mechanisms of cell death. Sf9 cells display excessive resistance.

Abstract No. 155

Identification of microRNA-31 in the radioresistant Sf9 insect cells and its bim-bax mediated / p53-independent role in radiation-induced apoptosis

Ashish Kumar , Mauli Chandna, Sudhir Chandna

Natural Radiation Response Mechanisms Group, Division of Radiation Biosciences, Institute of Nuclear Medicine & Allied Sciences,Delhi, India. Email: ashish_222006@yahoo.co.in

Introduction: A variety of cellular factors are known to contribute in the excessive radioresistance of Sf9 cells derived from the Lepidopteran insect Spodoptera frugiperda. However, the role of micro-RNAs is poorly understood. In this study, we aimed at identifying the role of miR-31 in radiation induced apoptosis in Sf9 cells. Materials and Methods: Micro-RNAs in Sf9 cells were detected using biotinylated probes (in-vitro transcribed) in RNase protection assay followed by chemiluminescence blot using HRP-streptavidin labeling. Alterations in micro-RNA expression were detected by real-time PCR. The level of Bax and Bcl-2 was observed by western blot, the translocation of Bax to mitochondria was studied by immuno-fluorescence microscopy. Interaction of Bax and Bim was observed by co-immunoprecipitation experiments. Results: We chose Bombyx mori as a reference organism for in-silico screening of miRNA having role in apoptosis regulation as no miRNA database was available for Spodoptera. Using ClustalW, we studied sequence conservation of these mi-RNAs in different insect orders and found very high sequence conservation in mature miRNA sequence between different orders. These mature miRNA sequences were used for designing biotinylated probes that facilitated the identification of five micro-RNAs (Let-7, Bantam, miR-14, miR-31, miR-34) yet unreported in Sf9 cells. Two probable isoforms of miR-31 were identified which were different from the Bombyx mori miR-31. The expression of miR-31 increased in a dose-dependent manner, which was corresponded well with the increased caspase activity and induction of cell death at radiation doses 1000Gy and onwards. Suppression of miR-31 expression by using antisense (AS) RNA could rescue the cells from radiation-induced apoptosis. In order to further find the possible target of miR-31, we performed in-silico analysis and identified p53 as one of the putative targets. However, p53 was not able to regulate its transcriptional targets and may not be responsible for apoptosis regulation via miR-31. Further we checked for the mechanism of apoptosis suppression mediated by miR-31 AS and found that miR-31 regulates translocation of Bax to mitochondria by manipulating the expression of Bim, despite the expression of Bax and Bcl-2 being unaffected by radiation. Conclusion: Over-expression of miR-31 regulates radiation-induced death in Sf9 cells primarily through activating Bim/Bax pathway in p53-independent manner at very high radiation doses that induce apoptosis in these highly radioresistant cells.

Abstract No. 157

Hypo-acetylated state of core histones regulates radioresistance in Sf9 insect cells

Kanupriya

Institute of Nuclear Medicine and Allied Sciences, Delhi, India. Email: kanupriya22j@yahoo.com

Chromatin architecture can directly affect induction as well as repair of DNA damage which is critical for cell survival and death. In the present study, we investigated the role of chromatin architecture as a regulator of 'radiosensitivity' in highly radioresistant Lepidopteran Sf9 insect cells derived from the ovaries of Spodoptera frugiperda, the Fall-armyworm. Despite similarities in the cellular organisation and apparent homologies in certain molecular stress response pathways, these cells display 50-100 times higher radioresistance as compared to human cells. We studied the role of core histones, their specific post translational modifications and histone-DNA interactions in Sf9 cells and compared it with mammalian cells. We first developed a specialized protocol utilizing modified single cell gel electrophoresis to evaluate the effect of ionizing radiation on specific histone-depleted Sf9 cells (which was achieved by employing empirically determined salt concentrations in vitro). Using this strategy, we observed a distinct DNA damage pattern in AA8 cells and Sf9 cells. Only 2M salt concentration which depletes all the histones in Sf9 cells could yield detectable DNA damage which potentially implies stronger DNA-protein interactions and/or increased chromatin compaction in Sf9 cells. This could be achieved by comparing frequency histograms of the tail length measurements from the neutral comets of gamma- irradiated AA-8 cells and Sf9 cells. We have also performed comparative profiling of histones extracted from Sf9 and mammalian cells and found a more compact basic chromatin structure in Sf9 cells. The histones were separated on the basis of their native charge by AUT gel electrophoresis and probed with modification specific antibodies like total lysine acetylation, H2A acetylation, H2B acetylation etc. Quite significantly, these experiments revealed a distinctive hypo-acetylated state of core histones in Sf9 cells.

Abstract No. 160

Differential expression pattern of organelles proteome in irradiated lepidopteran Sf9 cells

Shravan Kumar Singh

Institute of Nuclear Medicine and Allied Sciences (INMAS), Delhi-54, India. Email: shravanrb@gmail.com

Background: Lepidopteron Sf9 cells are known to show 10-100 times more radioresistance than human cells despite numerous homologies between two cell types. Ionizing radiation (IR) is known to induce macromolecular damage, which further altered the expression pattern of cellular and organelles proteome. The mitochondria and nucleus play multifaceted role in cell survival and death. Objective: Understanding the mechanisms responsible for hyper-radioresistance for improving biological radioprotection. Materials and Methods: Comparative study of irradiated Sf9 cellular proteome was done using 2D-PAGE & DIGE techniques. The differential expression analysis was carried out by DeCyder software and identification of proteins was attempted using MALDI-TOF & western blotting. Results: Significant differential expression pattern of cellular and organelles proteome was observed in irradiated Sf9 cells at various high radiation doses (10, 50, 200 Gy) and time points (4, 8, 12, 24 h). A total of 858 proteins (50Gy) and 1065 proteins (100Gy) were differentially expressed in cell lysate proteome. Comparing mitochondrial proteome of 10Gy-irradiated HEK and Sf9, a total of 486 and 435 proteins were differentially expressed at the same doses, respectively. Nuclear study also revealed the nature of differential expression pattern of proteome. Few of the significant differential spots/proteins of Sf9 mitochondrial and nuclear proteome were identified through MALDI-TOF and western bloting, e.g., PCNA, chaperones like HSP70, cytoskeletal protein Dynin and death regulatory protein AIF, besides other proteins. Conclusion: Identifying differentially expressed proteins will help in delineating novel molecular targets and pathways contributing in Lepidopteran resistance against radiation.

Abstract No. 161

Screening of some Indian medicinal plants as anticancer agents against colorectal adenocarcinomas


Radhika Raheja


C. U. Shah College of Pharmacy, India. Email: bhagwati72.r@gmail.com

Herbal medicines, because of their safety and efficacy, are being investigated for treatment of diseases such as cancer where the current modality of treatment including radiation and chemotherapy is known to cause severe side effects.

The current work includes screening of ten plants (about 30 extracts) belonging to families Acanthaceae, Orchidaceae, Geraniaceae, Compositae, Zingiberaceae and Asclepiadaceae for cytotoxicity against colorectal adenocarcinomas. It also involves understanding the mechanism of cytotoxicity exerted by the active extract/s leading to apoptotic death in cancer cells, whilst causing minimal cytotoxic effects on normal cells of similar type.

The prepared extracts were screened for cytotoxicity in vitro by the MTT assay on the colorectal adenocarcinoma cell lines Colo205 and HT-29. The active plant extracts were further tested on INT407, an intestinal cell line of embryonic origin so as to test their toxicity potential on normal intestinal cells.

Efficacy of the active extract was compared with 5-Fluoro uracil.

To understand the cytotoxic mechanisms, effect of the extract on

Reactive Oxygen Species (ROS) generation

Changes in Mitochondrial Membrane Potential (MMP)

Ability to induce apoptosis and Cell cycle was also investigated.

The active extract was further fractionated using HPTLC technique. Two steroidal moieties have been isolated from this plant which could be responsible for the activity shown by the extract.

It was found that of the 30 extracts, 4 extracts were active. The dichloromethane extract of the aerial parts of the plant from family Orchidaceae was cytotoxic against the colorectal adenocarcinoma cell lines, whilst exerting minimal toxicity on the normal embryonic cell line INT 407 at the IC50 values. Also, as compared to 5- Fluorouracil, this plant exerts similar toxicities on the cancer cells at much lower concentrations.

This extract was found to:

Have varied responses to reactive oxygen species (ROS) generation.

Decrease mitochondrial membrane potential (MMP).

Cause cell death via the apoptotic mechanism.

Arrest the cells in G0/G1 stage of cell cycle.

Two steroidal moieties have been isolated from the active extract. It is seen that one of these is responsible for causing cell cycle arrest and the other is capable of inducing apoptosis. Hence, this plant exhibits a tremendous potential for further investigations in the treatment of colorectal adenocarcinomas.

Abstract No. 165

Non-canonical DNA damage and radiation signaling responses indicated in highly radioresistant insect cells

Ayushi Guleria, Shashank Misra, Shubhankar Suman 1 , Vijaypal Singh and Sudhir Chandna

Natural Radiation Response Mechanisms Group, Division of Radiation Biosciences, Institute of Nuclear Medicine and Allied Sciences, Brig. S.K. Mazumdar Marg, Delhi - 110 054, India; 1 Present Address: Georgetown University, Washington DC, USA. Email: ayushiguleria@gmail.com

Lepidopteran insect cells, Sf9 ovarian cell line derived from Spodoptera frugiperda, the Fall armyworm (order Lepidoptera; class Insecta) are an excellent model system displaying up to 100 times higher radioresistance than mammalian cells. Factors regulating DNA damage induction and DNA repair mechanisms, oxidative stress response, as well as modes and mechanisms of cell death seem to be amongst the important factors that regulate radioresistance of these cells. Sf9 cells display excessive resistance to γ-radiation doses up to 1000Gy which is about 100 times higher than the death-inducing doses in mammalian cells. However, the iso-effect doses inducing DNA damage vary only by up to 10 times, which clearly implies that there are additional factors besides DNA damage that contribute to the excessive radioresistance of these cells, and may include certain non-canonical responses downstream to damage induction. Mitochondrial/ calcium disturbances were detected only at death-inducing doses, and nitrosative stress could not be induced in these cells even at very high death-inducing doses. The modes of cell death also showed unusual variations, with a certain sub-population undergoing a different manner of death than the conventionally known modes/mechanisms. The DNA damage response proteins, including gamma-H2AX, did not however show a good homology with the human system. Investigations on ATM/ATR pathway are in progress to understand the radiation response mechanisms, while the cell cycle checkpoints induced in these cells are homologous to human cells. Investigations on the calcium regulating mechanisms further revealed a higher level of resistance in these cells against the inhibitors of intracellular calcium channels such as SERCA inhibitors. Therefore, the Lepidopteran radioresistance seems to be effected at various levels of molecular responses and mechanisms. These studies are revealing intriguing differences existing in these model cells that might be imparting excessive stress/radiation resistance despite these cells possessing many of the canonical molecular cascades, which ultimately seem to be responding with very subtle alterations.

Abstract No.

Dynamics of G1-phase specific loss of H3-Serine10 phosphorylation in response to IR induced DNA damage in human cells

Ajit K Sharma, Tejkiran Sagwekar, Shafqat A Khan, Bharat Khade and Sanjay Gupta

Cancer Research Institute, Advanced Centre for Treatment Research and Education in Cancer (ACTREC), Tata Memorial Centre, Kharghar, Navi Mumbai 410210, India.

Studies in different models, from yeast to human cells imply that chromatin does not only act as a barrier to DNA repair processes but also dynamically help in DNA damage response. Chromatin organization facilitates efficient repair and participate in checkpoint signalling through histone modifications and even nucleosome remodelling. In this study we have unravel the site-specific histone modification(s) in response to IR-induced DNA damage and repair in G1 enriched human liver cells. Our data shows decrease in phosphorylation of H3ser10 and acetylation(s) of H3 and H4 in response to DNA damage. We show that IR induced DNA damage favors condensation of chromatin organization in repair phase with restoration of nucleosomal structure in restoration phase. The loss of H3Ser10P during DNA damage shows an inverse correlation with γH2AX in a dose-dependent manner. Significantly, we demonstrate loss of H3ser10P as a universal phenomenon because it is independent of origin of cell lines and nature of genotoxic agents. Interestingly, we demonstrate dephosphorylation of H3Ser10P is cell cycle and variant specific. Irradiated cells enriched in S and G2/M-phase does not show loss of H3Ser10P. The G1-enriched liver cell line shows loss and regain of phosphorylation in damage and recovery phase, respectively at serine10 residues from H3.2 and H3.3 variants. The dual phosphorylation of H3Ser10 and H2AX may destabilize chromatin structure and favour cell death of G2/M phase cells, whereas cells in G1 maintains single phosphorylation thereby survive in response to DNA damage. Finally, we show that the dephosphorylation of H3S10P is mediated through MKP1 and its restoration in phase of recovery is mediated through MSK1 in response to DNA damage. These results suggest that phosphorylation of H3ser10 is an essential component for efficient repair of DNA breaks, particularly during G1 phase of cell cycle.

RADIATION PROTECTION AND RADIOSENSITIZERS

Abstract No. 23

Development of organoselnium compounds as new class of radioprotectors

K. Indira Priyadarsini and Amit Kunwar

Radiation & Photochemistry Division, Bhabha Atomic Research Centre, Trombay, Mumbai-400085, India. Email: kindira@barc.gov.in

Selenium is an essential trace element. It is present in the active site of the redox active antioxidant enzymes like glutathione peroxidase (GPx) and thioredoxin reductase (TRx). Researchers are exploring new organoselenium compounds as GPx mimics and antioxidants. Due to the similarity in the reactivity of sulfur and selenium, we proposed to develop organoselenium compounds that show antioxidant activity, as new generation radioprotectors. A number of water soluble aliphatic organoselenium compounds, viz., selenoethers, diselnides and selenocystine derivatives have been synthesized and examined for antioxidant activity, GPx like enzyme activity, in vitro radioprotection studies.

After screening a number of selenium compounds substituted with different functional groups like, amine (-NH2), carboxylic acid (-COOH) and alocohol (-OH), diselenodipropionic acid (DSePA), was selected for in vivo toxicity and radioprotection. [1] At non-toxic dose of 2mg/kg body weight, DSePA was administered to mice five days prior to whole body irradiation to lethal doses of γ-radiation. DSePA significantly improved the thirty day survival of the mice and showed very efficient protection to radiosensitive organs like gastrointestinal tract (GI), and hematopoietic system. It inhibited radiation induced lipid peroxidation and protein carbonylation in hepatic tissue, restored villi height in small intestine and reversed the radiation-induced alterations in expression of pro-apoptotic BAX and anti-apoptotic Bcl-2 genes in spleenocytes, favoring towards survival. [2] Additionally the compound ameliorated the radiation-induced inflammation and restored the immune balance in irradiated mice. [3] These findings clearly confirmed that DSePA exhibits protective effects against whole-body γ-radiation and the underlying mechanisms responsible for this have been attributed to inhibition of oxidative stress, apoptosis, and inflammation. Currently we are pursuing the pharmacokinetic behavior and radiomitigative effects of DSePA in animals. Some of these results will be presented in the conference.

References

Prabhu P, Bag PP, Singh BG, Hodage A, Jain VK, Iwaoka M, et al Effect of functional groups on antioxidant properties of substituted selenoethers. Free Radic Res 2011;45:461-8.

Kunwar A, Bansal P, Kumar SJ, Bag PP, Paul P, Reddy ND, et al. In vivo radioprotection studies of 3,3'-diselenodipropionic acid, a selenocystine derivative. Free Radic Biol Med 2010;48:399-410.

Kunwar A, Bag PP, Chattopadhyay S, Jain VK, Priyadarsini KI. Anti-apoptotic, anti-inflammatory and immunomodulatory activities of 3,3'diselenodipropionic acid in mice exposed to whole body radiation. Arch Toxicol 2011;85:1395-405.

Abstract No. 31

Protection by 4-amino-4H-1, 2, 4-triazole-3-thiole against electron beam radiation induced ssDNA breakage in human lymphocyte

Madhu LN 1 , Suchetha Kumari N 2 , Vijay R 3

1 Department of Allied Health Sciences, Nitte University, Deralakatte, Mangalore. 2 Department of Biochemistry, K. S. Hegde Medical Academy, Nitte University, Deralakatte, Mangalore. 3 Central Research Laboratory, Nitte University, Deralakatte, Mangalore, India. Email: lnmadhu802@yahoo.com

Protection of biological systems against radiation damage is of paramount importance during accidental and unavoidable exposure to radiation. Chemicals capable of scavenging free radicals, relieving oxidative stress, promoting antioxidant activity and modulating immune response have been some of the radioprotectors extensively investigated with limited success. It has long been known that some of the most effective radioprotective agents are those which contain sulphydryl groups. The present study reports an electron beam radiation induced DNA damage in human lymphocytes and protective effect of 4 -amino-4H-1, 2; 4-triazole-3-thiole (100μg/ml) against the Electron beam radiation (EBR) induced damage. The compound was assessed for its radical scavenging property by DPPH radical scavenging assay and Ferric ion reducing power assay. DNA damage was assessed by comet assay parameters. The lymphocytes were incubated with 100μg/ml triazole for one hour before exposure to 2Gy and 4Gy electron beam radiation (EBR). The comet assay was performed for 2, 4 and 6 hour post irradiation time. The compound was showed very good scavenging property when compared with standard glutathione. The pre treatment of the cells with triazole showed a reduction in the Comet length, Olive tail moment, % of DNA in tail when compare to the radiation control group. These results show the protective effect of 4 -amino-4H-1, 2; 4-triazole-3-thiole against EBR induced damage.

Abstract No. 33

Treatment with EUK 207 mitigates pneumonitis after single dose of total body irradiation and fibrosis after thoracic irradiation in rats

Lakhan Kma , Feng Gao, Brian L. Fish, Aniko Szabo, Susan R. Doctrow, Robert C. Molthen, John E. Moulder, Elizabeth R. Jacobs and Meetha Medhora

Department of Biochemistry, North-Eastern Hill University, Umshing, Shillong 793 022, India. Email: lakhonkma@gmail.com

In the event of a radiological accident or terrorist attack, whole- or partial-body exposure can injure the lungs. To simulate such an incident, we used a single fraction of total-body irradiation (TBI) or whole-thoracic irradiation to induce pneumonitis or pulmonary fibrosis, respectively, in a rat model. The superoxide dismutase and catalase mimetic EUK-207 was given by subcutaneous injection (20 mg/kg/day, 5 days per week, once daily) starting at 7 days after irradiation and stopping before pneumonitis developed. After TBI, morbidity and the increase in breathing rates associated with pneumonitis were significantly improved in the rats treated with EUK-207 as compared to rats receiving irradiation alone. At 7 months after whole-thoracic irradiation, EUK-207 reduced synthesis of collagen as assessed by the Sircol collagen assay and Masson's trichrome staining. Our results demonstrate promise for EUK-207 as a mitigator of radiation pneumonitis and fibrosis.

Abstract No. 34

Nardostachys jatamansi ameliorates the severity of radiation induced liver and renal dysfunction

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Damodara Gowda K.M 1none , Latika Shetty 2 , Krishna A.P 1 , Suchetha Kumari N 3 , Naveen P 4 , Madhu L.N 4 , Ganesh Sanjeev 5 .

1 Department of Physiology, K.S.Hegde Medical Academy, Nitte University, Deralakatte, Mangalore, India. 2 Department of Radiology, K.S.Hegde Medical Academy, Nitte University, Deralakatte, Mangalore, India. 3 Department of Biochemistry, K.S.Hegde Medical Academy, Nitte University, Deralakatte, Mangalore, India. 4 Central research laboratory, Nitte University, Deralakatte, Mangalore, India. 5 Microtron centre, Mangalore University, Mangalagangothri, Mangalore, India. Presenting author Email: damodar001@gmail.com

Background: Radiation is increasingly used for medical purposes and is an established weapon in the diagnosis and the therapy of cancer. Radiation therapy injures or destroys cells in the area being treated by damaging their genetic material. Objective: The aim of the present study was to investigate the amelioration of the severity of radiation induced liver and renal dysfunction by Nardostachys jatamansi Extract (NJE). Materials and Methods : Albino wistar rats were administered with different dosages of NJE for 15 days before and after exposure to whole body electron beam irradiation (3Gy). Six hours after the last administration of NJE, the blood was collected by cardiac puncture, serum was obtained and used for the estimation of Total Protein, Albumin, A:G ratio, Total Bilirubin, Direct Bilirubin, SGOT,SGPT, Urea, Uric acid and Creatinine level. Data were analyzed statistical significance using SPSS, Version 16.0 and p less than 0.05 was considered the level of significance. Results : NJE has attenuated the severity of radiation induced liver and renal dysfunction by maintaining the level of all the parameters studied. Increase in the total protein and albumin levels suggesting the stabilization of Endoplasmic Reticulum leading to protein synthesis in rats treated with NJE. Regulation of the level of Urea, Uric acid and Creatinine level shows the protection of kidney. This indicates that NJE has the potential component to regulate the metabolic machinery. Conclusion: The results suggest that NJE may be a candidate offering protection in radiation induced liver and renal dysfunction.

Abstract No. 36

Radioprotective properties of Mesua ferrea on lymphocytes against electron beam radiation

th Shama Rao 1none , Prajna Palangana 2 , Rama Bhat 2 , Suchetha Kumari N 3

1 Department of Allied Health Sciences, Nitte University, Deralakatte, Mangalore, 2 Department of Biotechnology, Alva's College Moodbidri, 3 Department of Biochemistry, K. S. Hegde Medical Academy, Nitte University, Deralakatte, Mangalore, India. Email: shabtech14@yahoo.in


Radiation therapy remains an important component of cancer treatment with approximately 50% of all cancer patients receiving radiation therapy during their course of illness; it contributes towards 40% of curative treatment for cancer. The main goal of radiation therapy is to deprive cancer cells of their multiplication (cell division) potential. On the same time there is also a risk of damaging normal surrounding tissues and haematopoitic stem cells.

In this study we selected a Indian medicinal plant Mesua ferrea ( in sanscrit Nagakesari) Methanol soxhlet extraction of flower was examined for its antioxidant scavenging activity it gave very good result that 99% of radical scavenging activity in 100μl of 0.25 mg/ml sample followed by ABTS method Methanolic flower extracts of Mesua ferrea showed good antioxidant activity. 25μg of methanolic extract showed 99% radical scavenging activity in ABTS method. Based on this antioxidant study methanolic flower extracts were examined for radioprotective activity study using human lymphocytes against electron beam radiation. The lymphocytes were incubated with 50μg/ml Mesua ferrea extract for one hour before exposure to 4Gy electron beam radiation (EBR). The comet assay was performed for 2, 4 and 6 hour post irradiation time. The post irradiation study with Mesua ferrea showed a reduction in the Comet length, Olive tail moment, percentage of DNA in tail when compare to the radiation control group. These results show the protective effect of plant extract against EBR induced damage.

Abstract No. 37

4 amino-5-methyl-4H-1,2,4-triazole-3-thiol accelerates the red blood cell turn over by protecting the erythropoietic progenitor cells against electron beam radiation

th Suchetha Kumari N 1 , Madhu LN 2

1 Department of Biochemistry, K. S. Hegde Medical academy, Nitte University, Mangalore, 2 Department of Allied Health Sciences, Nitte University, Deralakatte, Mangalore, India. Email: suchethakumarin@gmail.com

The development of radioprotective agents has been the subject of intense research in view of their potential for use within a radiation environment, such as space exploration, radiotherapy and even nuclear war. With this background the present study was carried out to evaluate the effect of 100mg of 4 amino-5-methyl-4H-1,2,4-triazole-3-thiol (TR1) on Swiss albino mice exposed to 6Gy whole body electron beam radiation (EBR). The red blood cell turn over and hemoglobin level directly influenced by the status of erythropoietic progenitor cells such as polychromatic erythrocytes (PCE) and Non chromatic erythrocytes (NCE). The compound was assessed for its radical scavenging property by DPPH radical scavenging assay. The level of RBC, Hemoglobin and PCE/PCE+NCE ratio was assessed in Triazole treated animals on 16th post irradiation day and the obtained results were compared with control group animals. The compound was showed very good scavenging property (79.14% for 1mg/ml) when compared with standard glutathione (92.09% for 1mg/ml). The RBC and Hb count was found to be decreased in 6Gy irradiated animal, but it was found to be recovered in treated animals. Treatment of mice with 100mg of TR1 for 15days before irradiation significantly (p < 0.05) reduced the frequency of micronucleus formation in bone marrow cells. The results conclude that, the protection of progenitor cells against EBR by TR1 helps to accelerate the RBC turn over. The protective effect of TR1 is mainly due to the presence of sulphydryl functional group in it, which acts as a radical scavenger.

Abstract No. 39

2-Deoxy-D-Glucose induced modulation of genetic damage in irradiated cell lines in cultures

Jigni Sayeda

School of Biological Sciences, BITS Pilani, K K Birla Goa Campus, Zuarinagar, Goa, India. Email: sayedajigni@gmail.com

2-deoxy-D-glucose (2-DG) an antimetabolite of glucose was investigated for its radiosensitizing effect (pre and post 2-DG treatment) in human hepatoma (HepG2) and cervix carcinoma (SiHa) cells. Cytotoxic and antiproliferative assays (MTT and clonogenic) showed significantly decreased radiomodulation effect by 2-DG in HepG2 and SiHa cells with increasing doses of radiation in post treatment group when compared with the pre or IR alone with the dose modifying factor (DMF) of 1.19 and 1.26. Further, 2-DG treatment caused a significant increase in the genotoxicity as evidenced by the concentration-dependent elevation in the Olive Tail Moment (OTM) values. Post treatment of 2-DG showed the maximum DNA damage when compared with pre treatment.

A significant concentration-dependent decrease in the various intracellular antioxidant (GSH, GST, SOD and catalase) and mitochondrial membrane potential (MMP) levels with an increase in dichlorofluorescein (DCF) fluorescence after 2DG treatment confirm the ability of 2-DG to generate intracellular reactive oxygen species. Further, to elucidate the mechanism involved in the 2-DG induced radio modulation, cell cycle kinetics was performed by flow cytometry. 2-DG in combination caused G2M arrest significantly in post treatment of 2DG when compared with pre treatment group. 2-DG/Radiation-induced apoptotic and necrotic mode of cell death was demonstrated by oligonucleosomal ladder formation, microscopic analysis and increase in the hypodiploid fraction (sub Go peak in DNA histogram) analyzed by flow cytometry. The observation from the present study suggests that the combination of 2-DG after radiation could facilitate significant increase in percentage of apoptosis.

In conclusion, this study presents evidence that the radio modulating effects of 2-DG in HepG2 and SiHa cells. The maximum radio sensitization was observed when 2-DG was present after irradiation at clinically relevant radiation doses.

Abstract No. 60

Identification and evaluation of radiation countermeasures from protozoa fractions using High Throughput Screening (HTS) Technology

th Damodar Gupta, Vijay Singh, Rajesh Arora, RP Tripathi

Department of Radiation Biosciences, Institute of Nuclear Medicine & Allied Sciences, Brig SK Mazumdar Marg, Timarpur, Delhi, India. Email: damodar.gupta@gmail.com

Introduction: Screening of bioactive fractions prepared from protozoa by cell based High Throughput Screening (HTS) assays for modification of radiation effects is a thrust area in both clinical and nonclinical scenario. Identification of cellular and molecular targets for radio-modification and unravelling the mechanism of action will help in the development of radiation-countermeasures for human use. During present investigation protozoa fractions were screened and the identified 'Hits' was studied further for their radio-modification potential. Objectives: HTS for the identification of bioactive fractions with potential NFkB activation and evaluation of their efficacy as survival (colony formation) in vitro conditions. Materials and Methods: Cells (293T-NFkB lacZ) were treated with bioactive fractions prepared from protozoa for 6 hrs and thereafter NFkB activation was measured at 405nm using ONPG as substrate. Immortalized normal cells (TLR expressing; Kidney and urinary bladder origin) and transformed cells (HCT 115, Daudi) were treated with lead fractions (as identified by HTS), followed by irradiation (LD90) and thereafter media was replace from plates with fresh media and incubated till colony formation. Results and Discussion: Over 3-4 fold increases in NFkB activation was considered as 'Hit' and was tested further for radiation protection studies. Cells treated with 'Hit' (two fraction from protozoa viz. 9 and 10) showed over 60% increase in cell survival in both normal cells. However no protection was observed in case of transformed cells. The obtained results needs to be conformed in vivo, however in vitro observations suggests that the identified 'hits' protect normal cells which have potential benefits in development of protectors for human use.

Abstract No. 66

Bioinformatics database on radiomodifiers

Amruta Joshi and P M Dongre

Department of Biophysics, University of Mumbai, Vidyanagari, Santacruz (E), Mumbai 400098 India. Email: amrutajoshi_2007@rediffmail.com

Radiomodification has come up as a strategy to add to the cell killing effect of radiation and to overcome the shortcomings of radiotherapy. Radiomodifiers can either sensitize abnormal cells to radiation (radiosensitizer) or protect normal cells from hazardous effect of radiotherapy (radioprotectors). Better control on local tumor growth has been achieved with the help of this strategy and cytotoxic effect of radiotherapy is enhanced even in hypoxic core regions of solid tumours, both of which are considered as some of the major limitations of radiotherapy.

Several radiomodifiers (radioprotectors and radiosensitizers) have been investigated in vivo & vitro system and there is no bioinformatics database resource available. Hence our laboratory has decided to develop bioinformatics database resource for radiomodifiers. The database has been designed/ established in such way that one can retrieve biological information such as physico-chemical properties, Dose Modifying Factor (DMF), clinical study, target site/ targets, side effect, pharmacokinetics study, interaction with DNA/protein/membrane, LD50 of radiomodifiers by a single click on computer. Further database has been connected to internet hyperlink as result of which an investigator can quickly retrieve full text/ abstract. This database would prove useful to researchers, clinicians and others who work related with radiomodifiers.

Abstract No. 70

Apigenin modulates radiation-induced apoptotic signaling and accelerates recovery of intestinal mucosa in whole body irradiated Swiss albino mice

Naziya Begum, Rajendra Prasad N

Department of Biochemistry and Biotechnology, Annamalai University, Annamalainagar-608 002, Tamilnadu, India. Email: nazbiochemist@gmail.com

The development of effective pharmacological countermeasure is crucial to reduce the intestinal toxicity associated with possible exposure to ionizing radiation. We previously demonstrated that administration of apigenin at a non-toxic dose provided protection against acute radiation toxicity in human blood lymphocytes and Swiss albino mice. Radiation induced gastrointestinal (GI) damage occurs due to the destruction of the clonogenic crypt cells and eventual depopulation and denudation of the villi. The present study was designed to determine the possible protective effect of apigenin against GI damage induced by gamma-radiation in Swiss albino mice. Mice were treated with apigenin, at an intraperitoneal dose of 15 mg/kg body wt., once daily for seven consecutive days and on the seventh day 1 hour before to 7 Gy whole body irradiation (WBI). The histological observations indicated a decline in the villus height, villus and crypt cell numbers with an increase in goblet and dead cell population in the irradiated group, which was normalized by apigenin pretreatment. Radiation exposure (7 Gy) increased p53, p21, Bax and NF-kB expressions and decreased Bcl-2 expression in the intestine of experimental animals. Conversely, apigenin pretreatment prevented radiation induced apoptotic and inflammatory signaling elements in WBI mice GI tissue. This study demonstrates that apigenin protects the radiation-induced damage in the small intestine of mice and may be a useful adjunct to protect GI of cancer patients during cancer radiotherapy.

Abstract No. 85

Ameliorative effects of Podophyllum hexandrum on radiation inflicted injuries to hematopoietic system of mice

Savita Verma , Rashmi Saini and Manju Lata Gupta*

Department of Rradioprotective Drug Development Research, Institute of Nuclear Medicine and Allied Sciences, DRDO, Delhi, India. Email: vermasavi@rediffmail.com

*Corresponding Author: Dr Manju Lata Gupta, Sc F & Head of the department, Email: drmanjugupta2003@yahoo.com

Background: Increased threat of nuclear or radiological exposure by the multifaceted use of radiation has emerged the need of safe and effective radioprotector. The main cause of mortality in radiation induced hematopoietic syndrome is sepsis, haemorrhage and impaired immune system. The formulation prepared by combination of two bioactive principles from Podophyllum hexandrum (G-003M) was found rendering > 90% survival in lethally irradiated mice. Objectives: The study was aimed to evaluate the effects of Podophyllum hexandrum (G-003M) formulation on radiation induced injuries to hematopoietic system of mice. Materials and Methods: C57 female mice were administered G-003M intramuscularly, 1h prior to irradiation. Animals were anesthetized and dissected at different time intervals (4h, 24h, 72h, 5 th day, 10 th day & 30 th day). Spleens, thymus and femur were excised out and fixed in 10% buffered formalin for histological observations. Tissues were cut into 5μm sections and stained with H & E. ROS was also estimated in all the three organs at 1h after radiation exposure by DCF-DA staining and analyzed by flow cytometer. Results: Pretreatment of Podophyllum hexandrum (G-003M) formulation significantly recovered the radiation induced depletion in cellularity of the studied hematopoietic organs (bone marrow, spleen and thymus). Damage in lymphatic area of spleen and cortex of thymus in radiation treated mice, clearly demonstrated dysfunctionality of lymphoid organs. In G-003M pretreatment group, proliferation of the hematopoietic cells started within ten days of radiation exposure. Fibrous tissues were relatively less evident in studied hematopoietic organs of pretreated groups. ROS formation was found significantly declined in all the three hematopoietic organs of G-003M pretreated irradiated mice. Conclusion : The present study demonstrates that Podophyllum hexandrum (G-003M) formulation significantly protects hematopoietic stem cells from radiation cytotoxicity and enhances their proliferation and differentiation.

Abstract No. 87

Restoration of radiation inflicted damage to pulmonary system of mice by podophyllum hexandrum mediated Reactive Nitrogen Species (RNS) reduction

Rashmi Saini, Savita Verma and Manju Lata Gupta#

Department of Radioprotective Drug Development Research, INMAS, DRDO, Delhi, India.

#Corresponding Author: Dr Manju Lata Gupta,Sc F & Head of the department, Email: dr.rashmisaini@gmail.com / drmanjugupta2003@yahoo.com

Background: Radiation induced reactive nitrogen species (RNS) is reported to cause lung injuries such as pneumonitis and fibrosis which may be fatal at times. Cellular profiles and measurement of inflammatory cell infiltrates of irradiated BALF have shown correlations with lung pathology.The purified preparations of P.hexandrum rhizomes have reported delivering > 90% whole body protection in mice against radiation lethality. Objectives: To explore the modulatory effect of P.hexandrum formulation (G-003M) on nitric oxide mediated lung injury after exposure to moderate dose (7 Gy) of γ- radiation. Materials and Methods: Cell counts were determined in BALF of mice exposed to 7 Gy radiation and pretreated with G-003M. ROS generation was measured by DCF-DA in flow cytometer. Immunostaining of iNOS in BALF and lung sections was observed under microscope. Protein, LDH, phospholipid content in BALF & GSH, GR, catalase and lipid peroxidation were estimated in lung tissues. Results: G-003M significantly decreased neutrophil counts in BALF which was found 35.4% increased by radiation. Radiation mediated enhanced ROS generation, iNOS immunostaining in BALF, protein, LDH and phospholipid were restored in G-003M pretreated animals. Parenchymal fibrosis, an architectural distortion of the lung tissue with edema, infilteration of inflammatory blood cells with increased immunolabelling of iNOS was observed in irradiated mice. In G-003M pretreated group, iNOS immunostaining was decreased along with decline in NOS activity. Formulation pretreatment also significantly countered radiation mediated increase in lipid peroxidation and decrease in GR, catalase and GSH in mice. Conclusions: Current study demonstrates possible role of P. hexandrum in restoring NO induced lung pathogenesis by decreasing radiation induced RNS generation.

Abstract No. 90

Evaluation of the radioprotective effect of punica granatum (L). in cultured human peripheral blood lymphocytes exposed to electron beam radiation by single cell gel electrophoresis

Satheesh Kumar Bhandary

Department of ENT, K.S. Hegde Medical Academy, India. Email: satheeshbhandary@gmail.com

Ionizing radiations interact with mammalian cells by inducing a wide range of detrimental effects, with the most important damage occurring to the cellular DNA. Radiation is a well-known inducer of free radicals and compounds that can scavenge free radicals may reduce radiation-induced DNA damage. Pomegranate (Punica granatum L.) fruit is widely consumed as fresh fruit and juice. The potent antioxidant capacity of pomegranate and its components has been reported by numerous scientists using multiple invitro assay systems.

The aim of the present study was to investigate the radioprotective effect of ethanolic extract of pomegranate whole fruit (EPWF) in cultured human peripheral blood lymphocytes (HPBLs) by the comet assay. The optimum protective dose of the extract was selected by treating HPBLs with 50 and 100microg/ml EPWF after exposure to 2Gy electron beam radiation and then evaluating the frequency of DNA damage in HPBLs using Single cell gel electrophoresis (Comet Assay). To understand the mechanism of action of EPWF separate experiments were conducted to evaluate the free radical scavenging of OH, DPPH, and Fe3+ in vitro. EPWF was found to inhibit free radicals in a dose-dependent manner up to a dose of 400microg/ml for the majority of radicals as observed by the in vitro free radical scavenging assays. The irradiation of HPBLs with 2Gy dose of electron beam radiation caused an increase in the frequency of DNA damage while treatment of HPBLs with different concentrations of EPWF reduced the frequency of DNA damage significantly with the greatest reduction being observed for 100microg/ml when compared with the irradiated control. Our study demonstrates the potential of EPWF as an effective agent against radiation induced DNA damage.

Abstract No. 93

G2M block and radioprotective efficacy of active principles of podophyllum hexandrum in human blood lymphocytes: A cytogenetic study

Sangeeta Dutta, Dr. Manju Lata Gupta*

Radioprotective Drug Development Research Group, Institute of Nuclear Medicine and Allied Science, SK Mazumdar Marg, Delhi-54, India. Email id: sangeetadutta92@gmal.com

*Corresponding author Email: drmanjugupta2003@yahoo.com

Background: Chromosomes, most critical part of the cell containing genetic information are highly radiosensitive. Structural aberrations involving breakage and rejoining of chromosomes leads to unequal division of genetic material during cell division producing nonviable and abnormal cells. Formulations from rhizomes of Podophyllum hexandrum have been found rendering > 90% protection in lethally irradiated mice and assisting in fast recovery of many potential organs. Objective: The aim of the present study is to evaluate the radioprotective properties of Podophyllum hexandrum formulation (G-003M) against radiation induced cytogenetic damage in human blood lymphocytes. Materials and Methods: Blood was collected from healthy donors. Cultures were processed by following the standard protocol for dicentric assay with or without pre-incubation with G-003M before irradiation. Metaphases were assessed for dicentrics, rings, acentric fragments and chromosomal breaks. For CBMN assay standard procedure of 72hr culture was followed with addition of cytochalasin B at 44hrs. In case of CBMN assay binucleated cells were observed for micronuclei, nucleoplasmic bridges (NB) and nuclear buds (NuB). Most effective concentration was studied for its toxic effects. G-003M was studied separately for its cell cycle block characteristic at G2M phase. Result: Dicentrics in metaphases and micronuclei in binucleated cells shows linear quadratic increase with radiation dose (0.5-5Gy). All the other type of aberrations (NB, NuB) showed dose dependency. G-003M pretreatment was found to reduce radiation induced chromosomal damage significantly at all the radiation doses studied. Effective dose in terms chromosomal aberrations and micronuclei did not express any toxic effect and results were comparable to control samples. Metaphase arrest induced by G-003M was comparable to the colchicine at half of its concentration. With increase in G-003M dose (0.25, 0.5, 1.25 & 2.5μg/culture) metaphase frequency also increased. However at higher concentration % of premature centromeric division (PCD) also increased showing its toxic effect. The concentration indicating toxicity is however more than 10X of effective dose. Conclusion: The results clearly indicate effectiveness of Podophyllum hexandrum formulation (G-003M) in protecting chromosomes from radiation induced damages in human blood cells. The effective dose was significantly less than the toxic dose. Metaphase arrest property of G-003M was also revealed when the drug was replaced by colchicine. DMF of G-003M studied in human blood lymphocytes (exposed up to 3Gy) by analyzing micronuclei and dicentric was observed as 1.56.

Abstract No. 103

Radiation mediated DNA double strand breaks signaling modulation by active principles of podophyllum hexandrum: An in vitro study in human blood leukocytes

Nitya Nand Srivastava, Sandeep Kumar Shukla, Manju Lata Gupta*

Radioprotective Drug Development Research Group, Institute of Nuclear Medicine and Allied Scienc, SK Mazumdar Marg, Delhi-54, India. Email: drmanjugupta2003@yahoo.com

*Corresponding author

Background: Prophylactic administration of various formulations prepared from isolated active principles of Podophyllum hexandrum (G-002M) potentially protects most of the vital organs of lethally irradiated animals. Most of the studies conducted to explain its radio protective nature are focused on mouse model system. Since the formulation is intended to use for protection of defence and civil populations, it is therefore pertinent to perform some studies in human samples. This will also support in understanding the underlying mechanism. Objective: Current study is focused on evaluation of modulatory effect of G-002M on radiation mediated DNA double strand breaks signalling in human blood leukocytes. Materials and Methods: Blood collected from healthy human male volunteers, pre-incubated with G-002M, exposed to various doses of radiation was measured for double strand breaks biomarkers γ-H2AX and P53BP1 through flow cytometry. Irradiated blood was also processed at different time intervals to study time and radiation dose kinetics. Semi quntitative PCR was carried out at varied time points to measure the expression of DNAPK, Ku80, ATM and P53BP1 genes involved in DNA damage and repair signalling. Further flow cytometry analysis of ATM, DNA-PKcs, Ku80 and SMC 1 proteins were also performed. Results: Pretreatment of G-002M to irradiated blood leukocytes resulted in significant reduction in phosphrylated forms of γ-H2AX and P53BP1 indicating decrease in radiation mediated double strand breaks. PCR study has shown the significant up-regulation of DNAPK and Ku80 and down regulation of ATM and P53BP1 genes reflecting role of G-002M in DNA repair. Flow analysis data indicated decrease phosphorylated form of ATM, DNAPKcs, Ku80 and SMC1 in G-002M pretreated samples when compared with radiation alone groups. Conclusion: Current study has conveyed significant protection rendered by G-002M to DNA of human blood leukocytes. The DNA protective ability of G-002M could be due to its chemical constituent's lignans and flavonoids, known to possess properties like free radical scavenging, and cell cycle arrest at G2M leading to DNA repair.

Abstract No. 107

Role of rosemary leaves extract against hepatic injury induced by gamma radiation in mice

Dhanraj Soyal, Swafiya Jahan and P. K. Goyal*

Radiation and Cancer Biology Laboratory, Department of Zoology, University of Rajasthan, Jaipur - 302 004, India. *(E-mail: pkgoyal2002@gmail.com, dhanraj_soyal@rediffmail.com)

Over the past few decades, research in the development of radioprotectors world wide has focused on screening a plethora of chemical and biological compounds. Several synthetic compounds have been tested for their protective action against radiation. Owing to their inherent toxicity, these have not been successful in the field of clinical radiotherapy. Thus, it is considered important to explore alternatives to the synthetic compounds that would be radioprotective at non-toxic doses.

Search for non- toxic protectors from natural sources have indicated that some of the commonly used medicinal plants could prove to be valuable sources of clinically useful radio protectors as their ratio of effective dose to toxic dose is very high. Dietary ingredients may be useful if they are found to protect against the deleterious effects of gamma radiation, as they are widely acceptable, do not add an extra foreign substance into the body, and can be safely manipulated without toxic manifestations.

Rosemarinus officinalis plant has shown to be safe in toxicity studies in animal models when added as an antioxidant to food. It has a history of medicinal use as a tonic and stimulant, analgesic, antireumatic, carminative, diuretic, expectorant, anti-epileptic, anti-spasmodic in renal colic, dysmenorrhoea, relieving respiratory disorders effects and for effects on human fertility.

The present study has been focused to evaluate radioprotective potential of leaves extract of Rosemarinus officinalis against radiation-induced biochemical variations in liver of mammals.For this purpose, adult Swiss albino mice were selected from an inbred colony and were divided into two groups. Rosemarinus officinalis extract (ROE) was orally administered at the dose of 1000 mg/kg. by wt. once daily for 5 consecutive days in the animals of experimental group, whereas the irradiated control animals received an equal volume of DDW in similar manner. Thirty minutes after the last administration of DDW or ROE, animals of both the groups were exposed to 6 Gy gamma radiation and necropsied at 12 hrs, 24 hrs, 3rd, 5th, 10th, 20th and 30th day post-irradiation. Homogenate of liver was used to determine the glycogen, proteins and cholesterol contents.

In irradiated control animals, an elevation in glycogen and proteins contents was found till day 5th but thereafter decreased at successive intervals. Cholesterol level was found to be lower than the normal till 3rd day; then after it increased at further intervals, however the normal level could not be restored. A similar trend of variations in these biochemical parameters was observed in ROE pretreated irradiated animals also but to a lower extent and values were very close to near normal level by the end of experimentation. Furthermore, the rosemary extract treatment enhanced glutathione contents but inhibited depletion in lipid peroxidation in the liver and blood both against radiation-induced variations. Results from the present study demonstrate that the administration of ROE prior to gamma radiation is potential to provide protection against radiation-induced hepatic biochemical lesions in mammals.

Abstract No. 108

Protective Activity of Alstonia scholaris Against Radiation-induced Clastogenic Instability

Swafiya Jahan, Dhanraj Soyal and P.K. Goyal*

Radiation & Cancer Biology Laboratory, Department of Zoology, University of Rajasthan, Jaipur-302 004 India. Email: *pkgoyal2002@gmail.com, swafiajahan@gmail.com

The development of effective radioprotectors and radiorecovery drugs is of great importance in view of their potential application during both planned and unplanned radiation exposure. The systemic toxicity of the synthetic molecules in most cases that force researchers to relook at nature in the search for new chemical entities. The radioprotective drugs are no exception to this. In the search for novel, non-toxic, inexpensive and easily available radioprotectants from plants, were evaluated for their radioprotective potential. The herbal drugs offer an alternative to the synthetic compounds that have been considered either non-toxic or less toxic than their synthetic counterparts. This has given impetus to screen herbs for their radioprotective ability.

Alstonia scholaris, a non toxic herbal preparation, has been reported to be clinically effective in treating syphilis, insanity and epilepsy. It encouraged us to conduct further experiments to prove its radioprotective potential. The present study has been performed to verify the radioprotective capacity of A. scholaris bark extract (ASE) on radiation-induced clastogenic alterations in terms of chromosomal aberrations and micronuclei induction.

For this purpose, one group of male Swiss albino mice was exposed to 2.5 Gy gamma radiation to serve as the irradiated control while the other group received Alstonia scholaris bark extract/ ASE (100 mg/kg b. wt.) orally for 5 consecutive days before irradiation to serve as experimental. Such animals were autopsied between 12 hrs to 30 days and slides were made from their bone marrow to score chromosomal aberrations and micronuclei.

Results indicated that the dicentrics and chromosomal exchanges had increased appearance at 12 hrs postexposure in irradiated control as well as in the experimental group but the percentage of such aberrations was less marked in the ASE pretreated animals. The occurrence of chromatid breaks and acentric fragments was the maximum at 12 hrs. The incidence of chromosome breaks and centric rings increased till twenty four hrs of exposure after which it declined gradually and reached at zero level from day 7 th onwards. These aberrations were significantly lowered in ASE treated group at the early intervals.

The number of cells bearing micronuclei increased from 12 hrs and attained peak value on twenty four hrs, later declined continuously throughout the post-irradiation period. The normal value of micronuclei was regained on day 15 in ASE treated experimental group, however, it could not restored to normal even till the end of experimentation. Furthermore, a significant elevation in the catalase and superoxide dismutase level in liver and blood was recorded in experimental animals as compared to irradiated control. From the results, it is concluded that such plant extract has the potentiality to reduce radiation-induced cytogenetic lesions in mammals.

Abstract No. 116

Camptothecin sensitization studies in 177Lu-EDTMP treated bone osteosarcoma cells

Chandan Kumar , Aruna Korde, Kusum Kumari, Tapas Das, Grace Samuel, MRA Pillai

BARC, Mumbai, India. Email: ckumar@barc.gov.in

Introduction: Phosphonates labeled with beta emitting therapeutic radionuclides such as 153Sm, 177Lu are used as bone pain palliating agents in metastasized cancers. These radiopharmaceuticals intercalate with bone matrix and irradiate neighboring metastatic tumor cells. Camptothecin is an alkaloid which exhibits its anti-tumor activity via DNA damage by reversibly binding to enzyme DNA topoisomerase I. Biochemical pathways in response to the non repairable DNA damage results in proteases which are able to cleave apoptosis marker protein PARP (poly ADP ribose polymerase). The aim of the present study isto assess the sensitizing effect of camptothecin on bone osteosarcoma MG63 cell lines treated with 177Lu -EDTMP. We report here the in-vitro cellular uptake, toxicity studies and detection of PARP protein as a apoptosis marker. Materials and Methods: Bone osteosarcoma (MG63) cells were grown in MEM supplemented with 10% serum and antibacterial/antimycotic solution. Nearly 3x10 4 cells/well were seeded in 6-well plate and cultured upto 21 days in the presence of dexamethasone, ascorbic acid and beta glycerophosphate to induce bone mineralization. Cell binding with 177Lu-EDTMP was carried out after confirming bone mineralization with alizarin dye. Cell proliferation study was carried out for 48 h in MG63 cell line with camptothecin to determine IC50 value. MG63 cells (1x10 6 ) were exposed to 10 nM of camptothecin which was 50% C. Cells were harvested and cell toxicity was estimated by LDH and MTT assay. Protein was harvested by lysis of whole cells in cell lysis buffer and equal amount of protein was loaded on 8% SDS PAGE. The separated protein was transferred to nitrocellulose membrane by electroblotting. Western blotting was carried out to detect actin and PARP protein. Of the IC50 concentration along with 3.7 and 37MBq of 177Lu -EDTMP activity, and incubated for 48 h at 37΀C. Results: Alizarin dye staining confirmed sufficient mineralization in MG 63 cells after twenty one days. About 20% of activity was found to be associated with mineralized matrix after 2 h incubation with 177Lu -EDTMP. IC50 value of camptothecin was 20 nM as determined by MTT assay. Toxicity as well as PARP cleavage estimated after 48h exposure showed that cells treated with camptothecin + 177Lu -EDTMP were higher in comparison to those treated with either 177Lu-EDTMP or camptothecin alone. Conclusion: Anti cancer drug camptothecin can act as a potential sensitizer for 177Lu -EDTMP in bone osteosarcoma cells as studied in vitro.

Abstract No. 118

Whole body electron beam irradiation alters hematopoietic system in albino wistar rats

Sharmila K. P.

Email: sherlypoonacha@gmail.com

Background and Objective: The widespread use of radiation in diagnosis, industry, energy sector and inadvertent exposure during air and space travel, nuclear accidents & nuclear terror attacks found to cause enormous physiological alterations. Aim of the present study was to investigate the effects of whole body electron beam irradiation on Hematopoietic System. Materials and Methods: Male albino wistar rats were used in the present study and the Whole body Electron beam irradiation was done at MICROTRON CENTRE, Mangalore University at the dosage of 1Gy, 2Gy and 3Gy respectively with n = 6 in each group. Later blood was collected and used for determining the Peripheral blood counts (RBC, WBC), hemoglobin, thrombocyte count and Hematocrit percentage at 6 hr, 12 hr, 24 hr, 48 hr and 5, 10, and 15 days of post- Irradiation using automated hematology analyzer (Sysmex Co., F-820,Japan). Statistical analysis used: Statistical Analysis was done by one way ANOVA followed by Tukey's HSD Post Hoc Tests for Multiple Comparisons within the groups. P value less than 0.05 was considered the level of significance. Results: All the haematological parameters have shown an initial increase followed by a significant decline (p = 0.000) at the end of the experiment when compared to nonirradiated state. Conclusions: Whole body electron beam irradiation stimulates bone marrow during early hours of post-irradiation, followed by a significant suppression of hematopoiesis.

Abstract No. 121

Modulation of radiation affected cd34 expression in mouse bone marrow by isolated active principles of podophyllum hexandrum mediated hematopoietic protection

Rajiv Ranjan, Sandeep Kumar Shukla, Abhinav Singh, Manju Lata Gupta*

Radioprotective Drug Development Research Group, Institute of Nuclear Medicine and Allied Science, SK Mazumdar Marg, Delhi-54, India.

*Corresponding author Email: drmanjugupta2003@yahoo.com

Background: CD34 antigens are expressed on hematopoietic stem cells, progenitors, endothelial and fibroblasts cells. Studies on Expression of the stem cell antigen CD34 have attracted intense focus due to its capability to reconstitute the hematopoietic lineages in the lethally irradiated recipients. CD34 positive population has been observed to play a leading role in regeneration and maintenance of lympho-myelopoiesis after clinical transplantation. Objective: Present study was undertaken to decipher the mechanism of hematopoietic radioprotection conferred by a formulation prepared from isolated compounds from rhizome of Podophyllum hexandrum (G-003M).CD34 positive cells were measured as a hematopoietic marker in mouse bone marrow for evaluation of G-003M mediated radioprotection. Materials and Methods: 6-8 weeks C57BL/6 male mice were divided in to various groups. Mice intramuscularly pre-administered with G-003M against lethal radiation were sacrificed at various time intervals (24hr, 7, 13, 21 and 33 days). Bone marrow cells were flushed out from both femurs for CD34 measurements by Flow cytometry. Results: CD34 expression in bone marrow cell population of lethally irradiated and drug treated + irradiated mice indicated significant changes depending on the treatment type and time intervals. In drug pre-treated groups the expression was found comparable to the untreated mouse bone marrow on 33 day of experimentation.

Abstract No. 122

Nuclear Factor-ĸb (NF- ĸb) regulation by active principles of p. hexandrum in radiation protection: A study in irradiated mice jejunum

Bhargab Kalita, Ajaswrata Dutta and Manju Lata Gupta*

Radioprotective Drug Development Research Department, Institute of Nuclear Medicine and Allied Sciences, Delhi - 110 054, India.

*Corresponding author: drmanjugupta2003@yahoo.com

Background: Exposure of normal cells to radiation results in morbidity, mortality and various adverse side effects. Efforts into the development of a safe radioprotector have been going over many decades; however, associated toxicity with therapeutic concentrations has precluded their use from bench to bed. Current study is focused to assess the involvement of a key transcription factor, Nuclear Factor-ĸB (NF- ĸB) in modulation of radiation induced cellular damages by a potent radioprotective formulation prepared by a combination of two active principles (G-003M) isolated from P. hexandrum. Materials and Methods: 6-8 weeks old male C57BL6/J mice were exposed to ionizing radiation (9Gy γ-rays)/ pretreated with single dose of P. hexandrum formulation 1 hr prior to radiation exposure. Expression of transcription factor NF- ĸB and its target proteins (COX-2 and iNOS) were studied by western blotting in mice gastrointestinal tissue (jejunum). Nuclear translocation of NF- ĸB was also assessed during these studies. The involvement of NF- ĸB was further ascertained by immunohistochemistry. The role of NF-κB in radiation induced cell death and inflammation was studied by flow cytometry and western blotting. Results: A significant upregulation of NF-κB was observed within 1hr of exposure to 9 Gy radiation and maximum expression was evident at 6hr in mice GI. Pre-treatment with G-003M prior to radiation markedly decreased the radiation induced NF-ĸB expression and also restricted the translocation of NF-κB to the nucleus from cytoplasm. COX-2 and iNOS expression was also modulated by G-003M formulation. G-003M pretreatment further suppressed radiation induced apoptosis and also regulated expression of inflammatory cytokines.

Conclusion: Based on the results obtained, it can be hypothesized that decrease in ionizing radiation-induced NF-ĸB activation in intestinal epithelium by G-003M pretreatment may result in restraining prolong inflammatory response, leading to significant reduction in radiation mediated gastrointestinal tissue damage.

Abstract No. 127

Protective efficacy of emblica officinalis linn. against radiation and lead induced changes in the jejunum of swiss albino mice

R.K. Purohit

Radiation Biology Laboratory, Department of Zoology, Govt. Dungar College, Bikaner, Rajasthan, India. PIN -334003. Email: dr_rajendra_purohit@yahoo.co.in

Emblica officinalis
(EO) enjoys a hallowed position in Ayurveda-an Indian indigenous system of medicine. According to believe in ancient Indian mythology, it is the first tree to be created in the universe. It belongs to the family Euphorbiaceae. It is also named as Amla, Phyllanthus Emblica or Indian gooseberry. The fruits of EO are widely used in the Ayurveda and are believed to increase defense against diseases. It has its beneficial role in cancer, diabetes, liver treatment, heart trouble, ulcer, anemia and various other diseases. Similarly it has application as antioxidant, immunomodulatory, antipyretic, cytoprotective, antitussive and gastroprotective.

In light of the above, the present study was planned to evaluate the protective effect of Emblica against radiation and lead induced histopathological and biochemical alterations in the jejunum of Swiss albino mice. The animals were exposed to 3.0 Gy and 6.0Gy of gamma rays with or without lead acetate treatment. The Emblica was administered seven days prior to irradiation or lead acetate treatment.

The animals from all the experimental groups were sacrificed by cervical dislocation at each post-treatment intervals of 1, 2, 4, 7, 14 and 28 days. Immediately after the autopsy the small intestine (jejunum) was taken out. Contents of it were flushed out by the physiological saline. It was blotted and kept at -20 0 C for various biochemical estimations. Some of the pieces were kept in the Bouin's fluid for histopathological studies

The value of total proteins, glycogen, cholesterol, DNA and RNA decreased up to day-14, thereafter it increased on day-28, whereas the value of acid phosphatase activity and alkaline phosphatase activity increased up to day-14, thereafter it decreased on day-28 in the non drug treated groups. Whereas in the Emblica officinalis treated groups these values decreased up to day-7, then it increased on day-14 and continued so up to day-28. Similarly, the value of acid phosphatase activity and alkaline phosphatase activity increased up to day-7 then decreased on day-14, which continued up to day-28.

The histopathological changes included looseness of musculature, hydropic degeneration in submucosa and lamina propria, hyperaemia and haemorrhage in submucosa, pyknotic cells, cytoplasmic degranulation and vacuolation, abnormal mitotic figures. Karyolysis, karyorrhexis and chromatolysis were also observed in crypt cells at higher dose.

After combined treatment of radiation and lead synergistic effects were observed. The changes were found dose dependent. The jejunum of Emblica treated animals exhibited less severe damage as compared to non-drug treated animals at all the corresponding intervals. An early and fast recovery was also noticed in Emblica pretreated animals. Thus, it appears that Emblica is potent enough to check lead and radiation induced lesions in the jejunum of Swiss albino mice.

Abstract No. 132

Antioxidant potentiates radiation injury while pro-oxidant protect against toxic effects of radiation

Nazir M Khan

Bhbha Atomic Research Centre, Mumbai, India. Email: nazirkhan29@gmail.com

Ionizing radiation is known to alter the various biomolecules of cells and lead to cellular damage. Reactive oxygen species (ROS) generated by exposure of ionizing radiation play a major role in initiating the radiation injury. Of the total injury caused by radiation exposure to the cells, about 75% damage is mediated through ROS production. Since ROS are major denominator of radiation injury, antioxidant molecules having ROS scavenging ability are expected to protect against radiation injury while pro-oxidants worsened the radiation effects. Although this conventional wisdom for the modulation of radiation injury stands true in some instances, but this may not be the unified concept. Our results showed that unconjugated bilirubin (UCB), an endogenous antioxidant potentiated the radiation injury by augmenting radiation induced apoptosis, immunosuppression and infection susceptibility in the experimental mice. Further, our results demonstrated that well known pro-oxidants like hydrogen peroxide (H2O2), diethyl-maleate, t-butyl hydroquinone, and 1, 4 naphthoquinone (NQ) protected against radiation injury by activating multiple pro-survival pathways including redox sensitive transcription factor NF-E2-related factor 2 (Nrf2).

UCB, an endogenous degradation product of heme is considered as one of best known antioxidant of body, possessing 10,000 time better antioxidant property than that of GSH which is considered as major antioxidant and cytoprotector of body. In spite of having such strong antioxidant property, UCB at clinically relevant concentration and molar ratios showed strong immunotoxic effects in murine model via activation of GSH-p38MAPK-CD95/Bax-Caspase pathway. Further our results showed that treatment of mice with UCB potentiated whole body irradiation (WBI) induced splenic atrophy, bone marrow aplasia, lymphopenia, thrombocytopenia, neutropenia, decreased counts of peritoneal exudate cells and different spelnocyte subsets like CD3+T, CD4+T, CD8+T, CD19+B and CD14+macrophages. In an acute bacterial peritonitis model, UCB pretreatment of mice significantly increased radiation induced proinflammatory cytokines, nitric oxide and peritoneal bacterial load resulting in increased infection and death. Studies using pharmacological inhibitor of p38MAPK, demonstrated the involvement of p38MAPK activation in the inflammatory cascade of peritonitis.

Multiple cell viability assays used in our experiments demonstrated the strong radio-protective effects of various pro-oxidants like H2O2, diethyl-maleate and NQ in murine model. All Trans retinoic acid which is inhibitor of Nrf2 pathway, completely abrogated the radioprotective effect of NQ, suggesting that radioprotective activity of NQ may be due to activation of Nrf2 signaling pathways. Further, confocal microscopy, quantitative real time PCR and electrophoretic mobility shift assay showed the NQ mediated Nrf2 activation in lymphocytes. On knockdown of Nrf2, lymphocytes showed sensitivity to radiation. NQ also increased the expression of Nrf2 dependent cytoprotective genes like hemeoxygenase1, MnSOD, catalse. Expression of the Nrf2 dependent proteins is critical to maintain cellular redox homeostasis through elimination of radiation induced toxicants. NQ increased ERK phosphorylation which is upstream to Nrf2 and this ERK activation was through increased intracellular calcium levels. Further, administration of NQ to mice offered protection against WBI induced apoptosis and restored the changes in hematological parameters and functional responses of lymphocytes. Importantly, NQ rescued mice against WBI induced mortality.

Therefore, our results opened a new arena for the discovery of novel radiomodifiers. Even an antioxidant may worsened the radiation injury whereas pro-oxidant can protect radiation insult via activation of redox sensitive survival signaling pathways. [1-3]

References

Khan NM, Sandur SK, Checker R, Sharma D, Poduval TB, Sainis KB. Pro-oxidants ameliorate radiation-induced apoptosis through activation of the calcium-ERK1/2-Nrf2 pathway. Free Radic Biol Med 2011;51:115-28.

Khan NM, Poduval TB. Bilirubin augmentes radiation injury and leads to increased infection and mortality in mice: Molecular mechanisms. Free Radic Biol Med 2012;53:1152-69.

Khan NM, Poduval TB. Immunomodulatory and immunotoxic effects of bilirubin: Molecular mechanisms. J Leukoc Biol 2011;90:997-1015.

Abstract No. 139

Mitigating deleterious effects of ionizing radiation by phytoceuticals and nutraceuticals

C.K.K. Nair

Pushpagiri Institute of Medical Sciences & Research Centre, Tiruvalla 689101, Kerala, India. Email: ckknair@yahoo.com

Living systems are constantly exposed to a variety of physical and chemical agents which cause cellular damages including genetic alterations leading to mutations and cell death. Among the physical agents, ionizing and non-ionizing radiations are the major ones which cause damage to living organisms. Exposures of living systems to ionizing radiation cause a variety of damages to DNA and membranes due to generation of free radicals and reactive oxygen species. The radioprotectors elicit their action by various mechanisms such as: i) by suppressing the formation of reactive species, ii) detoxification of radiation induced species, iii) target stabilization and iv) enhancing the repair and recovery processes. The radioprotective compounds are of importance in medical, industrial, environmental, military and space science applications. Radiation protection might offer a tactical advantage on the battlefield in the event of a nuclear warfare. Radioprotectors might reduce the cancer risk to populations exposed to radiations directly or indirectly through industrial and military applications. They are required to reduce the normal tissue injury during radiotherapy of cancer. The recent incidences of terrorism have increased the fear of nuclear terrorism and the risk associated with the possible radioactive contamination with dirty bombs. This has created a global awareness for emergency preparedness and need for finding radioprotectors for human application and recent literature reveals enormous interest in this field. Many natural and synthetic chemicals have been investigated for their efficacy to protect radiation induced damages in biological systems and found to protect cells, membranes and biomolecules such as DNA and proteins in vitro. However many of these though show promising results in laboratory studies are of limited human application due to several factors such as toxicity, difficulties in administering required amounts, lack of preference for normal tissues, limited availability etc. To avoid the problems of toxicity and side effects of synthetic radioprotectors attention has been focused on phytochemicals, herbal medicines, diet etc Extracts of several medicinal plants such as Glyzyrrhizia glabra, Terminalia chebla, Hemidesmus indicus, Rubia cordifolia, Centilla asiatica, Aconitum heterophyllum, Holarrhena atidysentrica etc have been found to possess good antioxidant, free radical scavenging and radioprotection property. Also, a variety of compounds- both natural and synthetic - with different molecular structures, therapeutic activities, and metabolic functions are shown to have radioprotective action. These include a number of phytoceuticals such as curcumin, glyzyrrhizic acid, ferrulic acid, flavanoids, polyphenols, gallic acid, elagic acid; and nutraceuticals such as vitamin A, E, C and their derivatives etc. The talk will focus on the application of some of the phytoceuticals for protecting mammalian systems from the deleterious effects of lethal and sublethal ionizing radiation exposures.

Abstract No. 187

Regulation of endogenous antioxidant system by bacopa monnieri results in mitigation of ionizing radiation-induced Conditioned Taste Aversion (CTA) in sprague dawley rats

Jyoti Sharma, M. Adhikari, Raj Kumar, Rakesh Kumar Sharma, Rajesh Arora.

Email: jyotisharma86@gmail.com

Ionizing radiation exposure produced Conditioned Taste Aversion which have been proposes as behavioural endpoints mediated by harmful effect of radiation on peripheral systems. CTA in rodents is considered to be the equivalent of emesis in humans. Behavioural radioprotectors development is of imperative since radiation-mediated behavioral alterations result in emesis, performance decrement and anxiety in humans. CTA in rodents is considered to be the equivalent of emesis in humans. To investigate neurobehavioral protective efficacy of a Bacopa monnieri dietary natural product used in diet (BMA) in mitigating gamma radiation-induced CTA in Sprague-Dawley rats and its correlation with up-regulation of endogenous antioxidant defence system. Bacopa monnieri was analyzed for its neurobehavioral protective efficacy using the CTA assay. Adult male Sprague Dawley rats (12-15 weeks old, 300-350g), kept under standard laboratory condition with photoperiod of 12h/day at temperature of 25±2 o C, were trained for 23.5h water deprivation schedule for 10 days. On 10 th day rats were given a choice between 0.1% solution and tap water for 30mins. Animals having intake of more than 50% of saccharin of total fluid were further used. The different groups of animal were injected with BMA 1h before irradiation (2Gy), data was recorded upto 5 post-irradiation days. On fifth post-irradiation day, rats were sacrificed and levels of endogenous antioxidants (Cat, SOD, GST, reduced glutathione, Ascorbate, MDA) estimated. Gamma radiation was effective in blocking saccharin consumption response upto 5 post treatment observational days. The results established the efficacy of BMA in modulating radiation-induced CTA. Depleted level endogenous antioxidants activity and increased level of MDA was restored to baseline when treated with BMA. In vivo and ex vivo studies indicate that BMA holds immense potential as behavioural radiprotector for clinical application, radiologicial/nuclear rescue missions/emergencies.

Abstract No.

A semiquinone rich fraction isolated from bacillus sp. INM-1 protects bio-macromolecules from radiation induced oxidative stress

Saurabh Mishraa 1 , Deen D Bansala 1 , Poonam Malhotraa 1 , Ashutosh K Guptaa 1 , Praveen K Singha 1 , Neha Chhachiyaa 1 , Anil K Mishra 2 , Saleem Javedc 1 , Raj Kumara 1

Radiation Biotechnology Group, Institute of Nuclear Medicine & Allied Sciences, Brig S K Mazumdar Road,Delhi - 110 054, 2 Department of Cyclotron and Radiopharmaceuticals, Institute of Nuclear Medicine & Allied Sciences, Brig S K Mazumdar Road, Delhi - 11, India.

Protein and enzymes are the basic units in living systems. Radiation-induced damages in protein and enzyme molecules can have an impinging effect on cellular systems. Due to loss of their biocatalytic properties it undergoes radiation induced dysfunctioning. Radiation induced damage and modulation by SQGD pretreatment of Bovine Serum Albumin (BSA) was evaluated by UV-Vis, Circular Dichroism (CD) spectroscopic and SDS-PAGE analysis. Protection to functional activity of restriction enzymes (EcoRI, HindIII, BamH1) by SQGD was analysed through Agarose gel electrophoresis. While as protection to fluorescein, an organic dye and radiation induced decay of its fluorescence was analysed by fluorescence spectrophotometer. Significant increase in absorption maxima with irradiated (600-1200 Gy) BSA was observed which returned to normal level upon SQGD pretreatment. CD spectroscopic analysis showed shift towards higher wavelength upon irradiation. While after interaction with SQGD, shift towards the lower wavelength was observed. NATIVE-PAGE analysis showed a clear smearing in irradiated BSA which was found to reverse upon SQGD pre-treatment. Loss of digestion activity of restriction endonuclease was found at radiation exposure while SQGD helps to restore its digestion activity. Current study suggests that SQGD carried excellent protection to functional activities of enzymes and proteins against ionizing radiation and could be an ideal drug candidate for biological radioprotection in future

Abstract No.

Evaluation of antioxidant and radioprotective properties of hydroxyectoine

Saurabh Mishraa, Deen D Bansala, Poonam Malhotraa, Ashutosh K Guptaa, Praveen K Singha, Neha Chhachiyaa, Saleem Javedb, Raj Kumara

1 Radiation Biotechnology Group, Institute of Nuclear Medicine & Allied Sciences, Brig S K Mazumdar Road,Delhi-110054 India. 2 Department of Biochemistry, Jamia Hamdard, New Delhi - 110 062. India.

The prime purpose of the present study was to evaluate the antioxidant and radioprotective efficacy of a novel osmoregulant hydroxyectoine produced by extremophilic bacteria. To evaluate the antioxidant and radioprotective activities of hydroxyectoine, nitric oxide radicals neutralizing, hydroxyl radicals scavenging, superoxide radicals scavenging, DPPH reducing assay, and protein radioprotection assays with native PAGE analysis was carried out. Results of the study were demonstrated that hydroxyectoine exhibited nitric oxide radicals scavenging activity (12.23±3.215488%). OH - radicals scavenging efficacy of hydroxyectoine was estimated in terms of % inhibition in deoxy D-ribose degradation by non-site-specific assay. The maximum (16.2± 2.07869855%) inhibition of deoxy D-ribose degradation was observed in non-site-specific manner, at 5.0μg/ml concentration of hydroxyectoine. DPPH assay was carried out to evaluate the electron donation potential of hydroxyectoine. Maximum ~ 31.17% reduction in DPPH was observed at 500 μg/ml concentration of hydroxyectoine. Similarly, maximum (14.98%) hydroxyectoine mediated inhibition in superoxide radicals concentration was observed at 400μg/ml concentration. Significantly high degree of hydroxyectoine mediated protection to BSA was observed against ionizing radiation (1000-2000Gy).

In view of the observations, it can be concluded that hydroxyectoine possesses strong antioxidative and radioprotective activities and may be a potential candidate for radioprotection to cellular proteins in future.

Abstract No. XX

Evaluation of radioprotective efficacy of a novel semiquinone derivative rich bacterial fraction isolated from radioresistant bacterium Bacillus sp. INM-1

Raj Kumar 1 , DD Patel1, DD Bansal 1 , Saurabh Mishra 1 , Pravin Kumar Singh 1 , Ashutosh Kumar Gupta 1 , Rajesh Arora 2 , Ashok Sharma 1 , Kasyap Dubey 3 , Saleem Javed 4 , Swatantra Kumar Jain 5 R.P. Tripathi 1

1 Institute of Nuclear Medicine and Allied Sciences (INMAS), Brig. S.K. Mazumdar Road, Delhi. 2 Scientist-' E', SO to CCLS & IC, DRDO Head Quarter, DRDO Bhawan, Ddcelhi. 3 Associate Professor, Maharishi Dayadand University, Rohtak, Haryana. 4 Associate Professor, Department of Biochemistry, Jamia Hamdard, New Delhi. 5 Professor, Department of Biotechnology, Jamia Hamdard, New Delhi, India. Email: rajkumar790@yahoo.com

Extreme radioresistant in certain bacteria suggests an extraordinary capability of them to synthesis novel radioprotective biomolecules which may protect against lethal irradiation. In view of that, a radioresistant bacterium Bacillus sp.INM-1 was isolated and characterized. A potent radioprotective fraction rich in semiquinone derivatives was purified and evaluated for its radioprotective efficacy in vitro and in vivo models. Radioprotective activities of bacterial fraction was evaluated in terms of its in vitro and in vivo antioxidant activity, protein, enzymes and DNA radioprotecting efficacy, immunostimulatory properties, male reproductive, gastrointestinal radioprotective, hematopoietic system radioprotective capabilities and whole body survival assay. As the results of the experimental studies, radioprotective bacterial fraction demonstrated excellent antioxidant activities in terms of SOD, Catalase, Glutathione-S-transferase and Glutathione reductase activities in vitro as well as in vivo model. Similarly, bacterial fraction administration to the mice before irradiation significantly enhanced G-CSF, GM-CSG and M-CSF level in serum, spleen and bone marrow of the mice. Further, administration of bacterial fraction to the irradiated mice significantly increased the gene expression of cytokines as IL-12p40, IL23p19, Rel A etc. and thus stimulates TH1 type cellular immunity in irradiated mice. Pre-administration of bacterial fraction to the irradiated mice protects their gastro-intestinal and male reproductive system. Protein expression analysis indicated significant increase in the expression of p21, PARP, NF-kB, Bcl2, along with significant decrease in p53 and Bax protein expression in testicular and gastrointestinal system of the mice administered by bacterial fraction prior to irradiation compared to only irradiated mice. Finally, to estimate the overall survival of the irradiated mice pretreated by bacterial fraction whole body survival assay was carried out. More than ~ 80% protection against lethal dose (10Gy) of gamma radiation was observed in Swiss Albino strain A, C57BL/6, (male /female) mice compared to 0% survival with irradiated mice not administered by bacterial fraction.

Bacterial fraction under reference has excellent radioprotective activities. Further studies on chemical characterization, mode of action, route of administration and formulation development is underway.

Abstract No. 243

Protection by chlorophyllin against radiation induced hemapoietic injury: Cellular and molecular mechanisms

K. B. Sainis

Bio-Medical Group, Bhabha Atomic Research Centre, Trombay, Mumbai-400085, India. Email: kbsainis@barc.gov.in

The hematopoietic system comprising of immature stem cells, precursor cells, mature lymphocytes (B cells and T cells), granulocytes (neutrophils, eosinophils, basophils) monocytes, macrophages, NK cells etc is the most radiosensitive tissue. The suppression of haematopoiesis and immune function by radiation is considered to be one with the most life-threatening consequences. The recent progress in identifying markers expressed on hematopoietic stem cells, multi-lineage progenitors and lineage restricted progenitors provides an excellent opportunity to experimentally ascertain the radioprotective ability of specific formulations against hematopoietic injury. We used chlorophyllin, a water-soluble derivative of green plant pigment chlorophyll, to mitigate radiation induced hematopoietic injury. Chlorophyllin was found to enter the mature lymphocytes and it scavenged radiation derived reactive oxygen species. It also inhibited radiation induced apoptosis in splenic lymphocytes. The radiation mediated suppression of immune functions was significantly prevented by chlorophyllin. In control unirradiated animals, chlorophyllin enhanced the innate as well as adaptive immune responses mediated by phagocytes and lymphocytes respectively. These protective effects were attributed to its antioxidant action and increased expression of anti-apoptotic genes in lymphocytes after chlorophyllin treatment. Chlorophyllin treatment also significantly attenuated total body irradiation induced suppression of functional responses like proliferation of splenic lymphocytes and colony-formation by bone marrow cells ex vivo. Further, chlorophyllin treatment also prevented the TBI induced suppression of endogenous colony-forming ability of bone marrow cells in vivo. Most importantly, treatment with chlorophyllin resulted in 100% survival after a lethal dose of radiation. Mechanistic studies revealed cell cycle arrest in Lin-CD150+CD48-CD244- hematopoietic stem cells of chlorophyllin treated mice allowing more time for repair of radiation induced damage. Chlorophyllin significantly prevented radiation-induced suppression of hematopoietic stem cell proliferation. Chlorophyllin may thus qualify as "dual utility" drug that can be effective as a radiation countermeasure or during radiotherapy and also act as an immunostimulatory agent in immunodeficient individuals or patients undergoing myeloablative therapy.

RADIATION SIGNALLING

Abstract No. 51

Transmission of signals between irradiated and non-irradiated organisms: Mechanisms and significance

Carmel Mothersill

McMaster University, 1280 Main Street West, Hamilton, Ontario, Canada. Email: mothers@mcmaster.ca

Inter-animal signaling from irradiated to non-irradiated organisms has been demonstrated for whole body irradiated mice and also for fish. Endpoints studied include clastogenic damage, and reproductive cell death. For fish, proteomic data has also been produced showing the induction of a protective proteome in both medaka and rainbow trout. The aim of the current study was to determine the effect of medical microbeam irradiation using the ESRF synchrotron at Grenoble to the right brain hemisphere of the rat to determine whether abscopal effects could be produced in the rest of the animal and also in cage mates housed with it. The results show strong signal production in the contra-lateral brain hemisphere with less strong effects in the distant bladder of the irradiated rats. Signal strength was the same or stronger in the cage mates housed for 48 hrs with the irradiated rats. The proteomic study showed that the abscopal-associated proteins included A. an increase in the expression of NADH dehydrogenase, aconitase, ubiquinone and Glial fibrillary acid proteins, which are known to be present in gliomas, B. a reduction of the Prohibitin protein, which is thought to be a tumor suppressor in humans C. an increase on the expression of Heat shock cognate 71kDa protein, which is known to be involved in the disassembly of clarithin-coated vesicles and D. a decrease in the expression of Tubulin alpha-1A chain protein, which is known to be one of the major components of microtubules. All of these proteins would suggest a pro-carcinogenesis proteome. However in the cage mates, tubulin and aconitase were present but in different isoforms, HSP 71 was also induced. Significantly, pyruvate kinase, fructose biphosphate and aldolase were also present, suggesting mobilization of energy reserves. This also happened in the bystander fish. One brain specific protein which was suppressed in the cage mate brain is dihydropyrimidinase. This protein is prone to oxidation and the oxidised molecule is associated with Alzheimer's disease. A reduction could be seen as beneficial in terms of reducing the overall extent of oxidased dihydropyrimidinase. On the other hand dihydropyrimidinase is a vital neurotransmitter so a reduction could also be seen as detrimental to cognitive brain function. Whatever way the induced changes in the cage mates influence the health of the animals, it is very clear that proximity to an irradiated animal induces proteomic changes in an unirradiated partner. The transmitting agent in fish and rats is thought to be urine. If similar signaling occurs between humans, the results could have implications for caregivers and hospital staff treating radiotherapy patients.

Abstract No. 146

MicroRNA modulation in single versus fractionated doses: Hunting for novel targets for radiation sensitization

Norman Coleman

Radiation Research Program, National Cancer Institute, National Institutes of Health, 6130 Executive Blvd, Rockville, USA. Email: ccoleman@mail.nih.gov

We have previously demonstrated that prostate carcinoma cells exposed to fractionated radiation (MF) differentially expressed more genes compared to single-dose radiation (SD). To understand the role of miRNA in regulation of radiation-induced gene expression, we analyzed miRNA and mRNA expression in LNCaP, PC3 and DU145 prostate cancer cells and also in an in vivo mouse breast cancer tumor model treated with SD and MF radiation by microarray. Selected miRNAs were studied in RWPE-1 normal prostate epithelial cells by RT-PCR. MF radiation significantly altered more miRNAs compared to SD. Downregulation of oncomiR-17-92 cluster was observed only in the p53 positive LNCaP and RWPE-1 cells treated with SD and MF. Comparison of miRNA and mRNA data by IPA target filter analysis revealed an inverse correlation between miR-17-92 cluster and several targets including TP53INP1 in p53 signaling pathway. The base level expressions of these miRNAs were significantly different among the cell lines and did not predict the radiation outcome. Tumor suppressor miR-34a and let-7 miRNAs were upregulated by MF in radiosensitive LNCaP (p53 positive) and PC3 (p53-null) cells indicating that radiation-induced miRNA expression may not be regulated by p53 alone. Our data support the potential for using MF radiation to induce molecular targets and radiation-induced miRNAs may have a significant role in molecular targeted therapy.

Abstract No. 182

Molecular determinants of radiation response in human oral cancers

Tanuja Teni 1 , Sanchita Mallick 1 , Vinayak Palve 1 , Mohd. Yasser 1 , Sagar Pawar 1 , Sadhana Kannan 1 , Jai Prakash Agarwal 2 and Shubhada Kane 2

>1 Advanced Centre for Treatment, Research and Education in Cancer (ACTREC) Kharghar, Navi Mumbai-410210, India. 2 Tata Memorial Hospital, Tata Memorial Centre, Parel, Mumbai 400012, India. Email: tteni@actrec.gov.in

Apoptosis is a critical factor in radiation-induced cell death and defects in the apoptosis pathways provide opportunity for selection of genetically unstable cells, raising the threshold for cell death and conferring radioresistance. The ability of the tumor cells to respond to radiotherapy depends upon their intrinsic radiosensitivity, which may be partly governed by molecules of the intrinsic cell death pathway. To identify the defects in this pathway in oral cancers, transcript expression analysis of the pathway members was done using the Ribonuclease protection assay in oral cell lines and tumors. The intrinsic apoptosis pathway was found to be deregulated in oral cell lines and majority of oral tumors with altered expression of Mcl-1, bclxl, survivin, p53 and p16 mRNA. To identify factors associated with radiosensitivity, p53 mutation analysis and differential gene expression profiles of radiation-treated versus untreated oral cell lines of differing radiosensitivities was carried out. To assess the predictive value of above altered molecules in radiotherapy outcome in oral cancer patients, pretreated biopsies from thirty nine oral cancer patients were examined for the expression of the apoptotic markers using immunohistochemistry and their expression was correlated with the clinicopathological parameters. High expression of Mcl-1 (p = 0.05) and PCNA (p = 0.007) was seen to be associated with poor disease free survival. High expression of Bcl-xL was associated with poor response to radiotherapy treatment. PCNA (p = 0.04) and Mcl-1 (p = 0.05) emerged as independent prognostic markers for predicting disease free survival in oral cancers treated with primary radiotherapy. A predominant overexpression of anti-apoptotic Mcl-1L over pro-apoptotic Mcl-1S isoform was observed in the oral cancer cell lines and oral tumors. An inverse correlation was observed between Mcl-1L expression and apoptosis induction in AW8507 cell line post-radiation treatment supporting its pro-survival role. A rapid and short induction of Mcl-1L versus a sustained induction of Mcl-1L was observed in the relatively more radiosensitive FBM versus AW8507 respectively. SIRNA treatment in combination with IR demonstrated significant induction of apoptosis. The colony formation assays demonstrated a synergistic effect of Mcl-1 down regulation with IR. Our studies indicate that Mcl-1 expression influences survival and radiosensitivity of oral cancer cells. High expression of Mcl-1 was also observed in the radioresistant sublines generated by fractionated ionizing radiation The sustained expression of Mcl-1L protein in the more radioresistant cell line indicates a possible role for Mcl-1L in radioresistance and as a potential therapeutic target in oral cancers.

Radiobiology of Cancer Stem Cells

Abstract No. 173a

Cytotoxic and radio-sensitizing effects of polyphenolic acetates in human tumor cell lines


Amit Verma 1 , th K. Venkateswaran 1none , A. Farooque 1 , A.N. Bhatt 1 , A. Prasad 2 , V.S.Parmar 3 , H.G.Raj 2 , M.B. Arya 4 , B.S.Dwarakanath 1

1 Institute of Nuclear Medicine & Allied Sciences Brig. SK Majumdar Marg Timarpur Delhi, 2 VP Chest Institute Delhi, 3 Department of Chemistry, University of Delhi, India.

Introduction : The plant polyphenols have been widely used as chemo-preventive agents and also investigated for their chemo/radio-sensitization potential of tumor cells by altering the damage response pathways, regulating dynamic interplay between various processes like induction & repair of DNA damage, activation of cell cycle arrest & apoptosis. However, the biological effects of polyphenols are restricted due to problems of limited specificity, efficacy & bioavailability. Therefore, many semi-synthetic polyphenol derivatives have been designed & to achieve improved therapeutic gain. Acetylated polyphenols are reported to have better pro-oxidant property than the parental compound & are also demonstrated to modify the activities of a number of proteins (enzymes) like cyto-P-450, NADPH- cyto C reductase, glutathione S transferase & nitric oxide synthase by a novel acetoxy drug: calreticulin transacetylase acetylation system. Objectives : To investigate the cytotoxic & radio-sensitizing effects of polyphenolic acetates in a human glioma cell line (BMG-1). Materials & Methods : We investigated the cytotoxic & radio-sensitizing effects of two polyphenolic acetates {7, 8 diacetoxy-4-methyl coumarin (DAMC) & 7- monocetoxy-4-methyl coumarin (7-AMC)} in BMG-1 cells with clonogenic survival, growth inhibition, cell cycle perturbation & immuno-fluorescence as parameters of response. Results : DAMC & 7-AMC showed a dose dependent decrease in the metabolic viability after 24h drug exposure, with DAMC being more potent (IC50 ~ 70μM) than 7-AMC (IC50 ~ 100μM). The clonogenic survival & cell proliferation data corroborated the effects on viability & 7-AMC induced a higher level of anti-apoptotic (NFkB) regulator than DAMC suggesting that it perhaps has a better anticancer potential. DAMC significantly enhanced the radiation-induced cell death as compared to 7-AMC, which was accompanied by a higher degree of cell cycle perturbations and an increase in hypodiploid population with suggesting enhanced apoptosis Further, DAMC significantly altered the removal of radiation-induced DNA double stranded breaks (measured by γH2AX immuno-fluorescence) & enhanced the cytogenetic damage in the form of micronuclei, which appears to be a significant contributing factor for the enhanced cell death. Further, studies to elucidate the molecular mechanisms underlying the radio-sensitizing effects of DAMC are in progress. Conclusion: The results show the anticancer potential of DAMC & its ability to modulate the cellular responses to ionizing radiation implicating its role as an adjuvant during radio-therapy.

Abstract No. 173b

Mitochondrial uncoupling delays ageing in batch culture of cells by inducing glycolysis

th Kavya Venkateswaran,
B. S. Dwarakanath, Namita Kalra and Anant Narayan Bhatt

Division of Radiation Biosciences, INMAS, Delhi - 110054, India. Email: anbhatt@yahoo.com

Ageing is a complex process that involves every cell and organ of the body that leads to the deterioration of many body functions over the lifespan of an individual. Mild mitochondrial uncoupling using 2, 4-dinitrophenol (DNP) enhances longevity in animals, however the underlying molecular mechanisms anti ageing effects at the cellular level is not clear. In our endeavour to unravel the underlying mechanisms of DNP induced radio-resistance, we established oxidative phosphorylation modified cells (OPMBMG) from a human glioma cell line (BMG-1) as a cellular model with enhanced endogenous glycolysis by growing BMG-1 cells in DNP for several passages. In vitro studies suggest that transient elevation of glycolysis by DNP in the parental cells and OPMBMG cells are more resistant to radiation as compared to the parental cells (BMG-1). OPMBMG cells showed approximately 80% live cells on 8 th day as compared to 15% in parental cells, in a batch culture, which correlated with clonogenic survival of these cells plated on different days of batch culture. OPMBMG cells showed a relatively higher level of NADH and reduced DCF fluorescence, suggestive of improved redox balance under these conditions. OPMBMG cells also showed strong activation of Nrf2 signalling and increased expression of MnSOD, which appeared to be partly responsible for improved redox balance and cellular longevity. Our results demonstrate that mild mitochondrial uncoupling induced glycolysis is a highly effective antioxidant strategy to minimize oxidative stress induced ageing.

Abstract No. 175

The effects of amifostine analogues on etoposide induced toxicity in human tumor cell line


Aastha Arora 1 , Ravi Soni 1 , Uma Pathak 2 , R Vijay Raghavan 2 , M P Kaushik 2 , Shweta Singh 1 , Deeksha Sharma 1 , Namita Kalra 1 , Richa Bhardwaj 1 , Anant Narayan Bhatt 1 and BS Dwarakanath 1+

1 Institute of Nuclear Medicine and Allied Sciences, DRDO, Timarpur, Delhi-110054. 2 Defence Research and Development Establishment, DRDO, Jhansi Road, Gwalior - 474 002, India.

+ Corresponding author Email: bsd@inmas.drdo.in

Background : Amifostine (S-2(3-aminopropylamino) ethyl phosphorothioate), a well known radio-protector as well as cytoprotecor against many chemotherapeutics has also been demonstrated as an oral prophylactic agent against Sulfur mustard (SM) but the associated toxicity limits its utility for human application. Therefore, a series of analogues of DRDE-07 have been synthesized for developing more effective and less toxic compounds against SM. Since, the mechanisms of damage induction and the associated cellular responses underlying by SM shares a great deal of similarity with various anti-cancer drugs, we investigated effects of these analogues DRDE-07 & 35 on etoposide induced toxicity in a human tumor cell line. Objective : To investigate the effects of DRDE 07 & 35 on etoposide induced toxicity in human a glioma cell line (BMG-1). Materials and Methods : Metabolic viability, Clonogenicity, Cell proliferation & Cell cycle distribution, DNA damage & Repair and cell death (Annexin-V/PI and TUNEL assay) were investigated as parameters of response following treatment of exponentially growing BMG-1 cells with DRDE analogues and etoposide. Results and Conclusion : Metabolic viability, clonogenicity and cell proliferation studies showed that both the analogues (DRDE-07 & 35) did not significantly alter etoposide toxicity at low and non-toxic concentration, while they enhanced the toxicity at higher concentrations (i.e at IC50). Analysis of cell cycle perturbations suggested that enhancement in the etoposide-induced growth inhibition by the analogues was mainly due to the arrest of cells in G2/M phase. Further, these studies also indicated that apoptosis and necrosis both contributed to the overall cell death under the present experimental conditions. Investigations on DNA damage and repair using γ-H2AX assay indicated that both the analogues marginally reduced the etoposode-induced DNA damage at lower (non toxic) concentrations, which resulted in a decrease in the cytogenetic damage (micronuclei fraction). Taken together, current results suggest that DRDE analogues do not compromise the effects of etoposide in tumor cells (like BMG-1) at lower doses, while they enhance the etoposide toxicity at higher doses. Further studies are required for a better understanding of their chemo-modifying and in vivo evaluation in animal models before contemplating clinical applications.

Abstract No. 179

Immuno-modulation linked to the depletion of T regulatory cells contributes to the radio-sensitization of tumors by the glycolytic inhibitor 2-Deoxy-D-Glucose


Abdullah Farooque 1,2 , Niharika Singh 1 , Amit Verma 1 , Shishir Agrahari 1 , Sanjay Pandey 1 , Aashish Kumar 1 , Farhat Afrin 2 , J S Adhikari 1 , Sudhir Chandna 1 , BS Dwarakanath 1

1 Division of Radiation Biosciences, Institute of Nuclear Medicine and Allied Sciences, Delhi 110054, 2 Department of Biotechnology, Jamia Hamdard University, New Delhi 110062, India. Email: niharikasingh128@gmail.com; bsd@inmas.drdo.in; abdullah.farooque@gmail.com

Increased aerobic glycolysis facilitating rapid proliferation and defence against death is a prominent phenotype generally observed in cancer cells. Targeting this phenotype using glycolytic inhibitor, 2-deoxy-D glucose (2-DG), has been found to enhance the effects of radiation and chemotherapeutic drugs in vitro. However, a heterogeneous response in the form of complete response (CR) and partial response (PR) has been found for the local tumor control in vivo. Immune system plays an important role in the therapeutic response and is known to depend on glucose metabolism for its activation. Since induced T regulatory cells (T regs) profoundly influence the immune system in tumor bearing mice, it has been suggested that alterations induced by 2-DG on the T regs status is an important contributing factor for the differential radio-sensitizing effects by-2DG. CR observed in 50% of the animals was associated with extensive apoptotic death of tumor induced T regs in peripheral blood and decreases in the spleen, lymph node as well as tumor with concomitant increase in the antitumor immunity. Further, T regs related cytokine (TGF-β) and antibody (IgA) were also found to be down regulated with increase in antitumor Th1 related cytokines (IFN-γ, TNF-α) and antibody (IgG2a). Moreover, depletion of tumor infiltrated T regs following the combined treatment resulted in a massive infiltration of NK and CD8+ cells suggesting an involvement of cell mediated immunity in CR, which was absent in PR. While depletion of Tregs using cyclophosphamide after the combined treatment (2-DG+Radiation) enhanced the local tumor control from 50 % to 80%, stimulation using rapamycin resulted in 0% CR, lending support to the proposition of Tregs involvement in the heterogeneous response. These findings clearly demonstrate that immune-modulation by way of Tregs depletion is partly responsible for the in vivo radiosensitization by 2-DG.

Abstract No. 181

How do you increase radio-sensitivity in radio-resistant tumor cells?


Srikanta Basu, Amol S. Hosing, Elphine Telles, Samrat T. Kundu, Prajakta Gosavi, Prasanna Venkatraman and Sorab N. Dalal

KS215, ACTREC, Tata memorial Centre, Kharghar, Navi Mumbai, India. Email: sdalal@actrec.gov.in

Our laboratory has focussed on understanding the processes by which a mammalian cell prevents progression through cell cycle upon DNA damage. Our work has shown that two 14-3-3 proteins, 14-3-3ε and 14-3-3γ, are required to prevent cell cycle progression upon the induction of double strand breaks (DSBs). These two proteins perform this function by inhibiting the activity of the dual specificity phosphatase, cdc25C, which is required for entry into mitosis. Loss of either 14-3-3ε or 14-3-3γ, leads to an override of DNA damage induced G2 arrest and leads to cell death. These results suggest that inhibiting complex formation between cdc25C and 14-3-3 proteins could lead to an increase in radio-sensitivity.

Most metastatic tumors acquire resistance to commonly used therapeutic strategies such as radiation leading to a decrease in patient survival. Our laboratory has demonstrated that loss of the desmosomal plaque protein plakophilin3 (PKP3) leads to an increase in tumor growth and metastasis. Cells with a loss of PKP3 also show an increase in resistance to γ-radiation. We have attempted to identify the mechanisms downstream of PKP3 that are required for the increase in radio-resistance. Experiments directed at understanding these pathways will be presented at the meeting.

RADIOBIOLOGY OF HIGH LET RADIATION

Abstract No. 72

Dosimetry and initial biological evaluation of automated alpha particle irradiator developed for radiobiological studies


S. G. Shinde, R. Vasumathy, Manjoor Ali, Amit Kumar, R. V. Kolekar 1 and B. N. Pandey Radiation Biology and Health Sciences Division, 1 Radiation Safety Systems Division, Bhabha Atomic Research Centre, Mumbai 400 085, India. Email: manjoor1982ali@yahoo.co.in

Studies pertaining to effects of alpha radiation to biological systems are highly relevant in radiation risk assessment. Due to short path length traversal of alpha radiation in air, in-vitro experiments could not be conducted with accuracy, which requires special irradiator assembly. Hence, an automated alpha particle irradiator (named as 'BARC BioAlpha') was developed at BARC for in vitro radiobiological studies. 241Am source (0.13 microCi; 5 mm diameter) developed was loaded in the irradiator. The dosimetry and uniformity of the source was evaluated using CR-39 track dosimeter. The dose rate was found to be 1.5 microGy/sec in 1 cm diameter central region of CR-39 and was decreased in bigger diameter areas. In 3 cm diameter central area, ~ 75 % alpha flux was vertical, suggesting collimated nature of alpha flux in the irradiator. The theoretical calculation of energy at the target (cell) was 4.27 MeV. Special irradiation dishes were designed and fabricated to culture and irradiate cells under sterile conditions. Human lung cancer cells (A549) were cultured on these dishes followed by irradiation with alpha or gamma (2 Gy) to compare the pattern of DNA damage induced by these radiation types. Our results showed that in gamma-irradiated cells, the pattern of gamma H2AX foci was uniformly distributed throughout the nucleus compared to alpha radiation, which showed localized foci depending on number of alpha particles traversed. Moreover, it was interesting to observe that the size of foci was larger in case of alpha- than that of gamma-irradiated cells. These results suggest suitability of alpha irradiator for radiobiological experiments.

Abstract No. 174

Poisoning the sweetness of tumors with 2-Deoxy-D-Glucose: Excitement and challenges

BS Dwarakanath

Institute of Nuclear Medicine and Allied Sciences, Delhi 110054, India. Email: dwarakanathdrbs@gmail.com

Higher rates of glucose usage often found in several types of malignant tumors correlate with poor prognosis as well as resistance to therapy. The glucose analogue, 2-deoxy-D-glucose (2-DG) an inhibitor of glucose transport and glycolytic ATP production widely used for targeting this phenotype depletes energy, disturbs redox balance and alter N-linked glycosylation leading to unfolded protein responses, collectively resulting in changes in the gene expression and phosphorylation status of proteins involved in signaling, cell cycle control, DNA repair, calcium influx and apoptosis. Inhibition of cell proliferation and induction of apoptosis has been observed as cytotoxic effects in a wide variety of tumor cells in vitro, while sensitization of tumor cells to ionizing radiation and certain chemotherapeutic drugs is associated with enhanced mitotic as well as apoptotic cell death induced by the primary therapeutic agent. Studies with animal tumors have shown enhanced local tumor control without significant damage to the normal cells (and tissues) thereby suggesting that 2-DG is an ideal adjuvant to improve the efficacy of radio- and chemotherapy. Therefore, there has been a considerable amount of interest in developing 2-DG as a therapeutic agent and adjuvant in the radiotherapy and chemotherapy therapy of tumors. Following encouraging results on the tolerance and toxicity (acute as well as late effects) of the combination (2-DG and hypofractionated radiotherapy) in Phase I and II clinical trials, its efficacy has been evaluated in Phase III clinical trials of glioma patients, while protocols for employing 2-DG as primary therapeutic agent as well as adjuvant in radiosurgery, chemotherapy and other therapies have been designed that requires evaluation.

Alterations in cell signaling linked to tumor physiology and host-tumor interactions contribute significantly to the radio- and chemosensitization of tumors by 2-DG and heterogeneity in these aspects has been found to result in variations in local tumor control induced by the combined treatment (2-DG+Radiaton). Immune-modulation in the form of restoration of CD4+ / CD 8+ ratio, decrease in i-T-regs, shift from TH2 to TH1 immune functioning and stimulation of macrophages has been found to significantly contribute to the radiosensitization by 2-DG in vivo. Implications of these findings and associated challenges in designing protocols using 2-DG as adjuvant in radio-and chemotherapies will be discussed.

SPACE RADIATION BIOLOGY

Abstract No. 45

Space radiation and what we learn from small model organisms


Sharmila Bhattacharya 1 , Anuran Chatterjee 1 , Oana Marcu 1,2 , Ruth K. Globus 1

1 Space Biosciences Research Branch, NASA Ames Research Center, Moffett Field, CA USA, 2 Carl Sagan Center, SETI Institute, Mountain View, CA. USA.

Astronauts undertaking spaceflight missions are at risk of being irradiated by protons from solar particle events, as well as with high-energy charged particles (HZE) from galactic cosmic rays. The effects of such radiation could be severe depending on the duration of the mission, the orbit of the spacecraft and whether any countermeasures can be implemented within the restrictive environment of the space flight. Several studies have been conducted on the ground, with Earth-based radiation sources, to predict the effects of some of these highly ionizing particles on biological systems. Just a hand-full of experiments have been undertaken to determine the biological effects of space radiation during space flight. They have utilized an array of organisms including insects, worms, yeast, plants, mice, mammalian cells and tissues and have yielded varying results.

We have studied the proteomic changes in the serum of mice that were irradiated with 56 Fe six months prior to analyses. We will discuss the short and long term effects of radiation in mice and also discuss the effects of combining simulated microgravity with radiation treatment.

In addition, we have studied the effects of proton irradiation in the fruit fly, Drosophila melanogaster, with the long-term aim of developing it as an experimental system for quantifying biological damage from space radiation and to understand underlying molecular mechanisms to screen for potential countermeasures. Specifically we have studied (a) tumor formation and (b) alterations in behavior, following proton irradiation, and this data will also be discussed in the context of the effects of space radiation.

Abstract No. 136

Model calculations of GCR environment in space: Radiation biological assessment


Prof. Premkumar Saganti

Prairie View A&M University and NASA-JSC, PO Box 519, Prairie View, Texas, USA. Email: pbsaganti@pvamu.edu

Variations in the space radiation environment due to GCR (Galactic Cosmic Ray) from the past solar cycle to the current one has been intriguing in many ways, with an unprecedented recent long duration of the solar minimum condition. Model calculated radiation data and assessment of variations in the particle flux - protons, alpha particles, and heavy ions of the GCR environment is essential for all current and future intended human exploration missions as well as ground based radiation biology research. Over the past few years, we have been developing radiation dose estimates based on NASA's HZETRN (High Z and Energy Transport) code along with the newly expanded quantum multiple scattering fragmentation (QMSFRG) model. Particle flux calculations and comparisons with other observed measurement trends for the current and historical solar minimum (solar cycle # 23) showed significantly higher particle flux and the contributed dose. Also, for the current solar cycle (#24), it is expected that the solar maximum peak will be much lower (around June 2013) and hence higher radiation dose contribution is anticipated. We present our model calculated variations in the GCR particle flux and dose contribution for 2012 and beyond in the context of recent radiation biological studies.

TRANSLATIONAL RESEARCH AND CANCER BIOLOGY

Abstract No. 20

Delineation of breast cancer cell hierarchy identifies the subset responsible for dormancy

Pranela Rameshwar

UMDNJ-New Jersey Medical School, USA. Email: rameshwa@umdnj.edu

The bone marrow (BM) is a major organ of breast cancer (BC) dormancy and is a common source of resurgence after years of disease-free survival. Dormancy of BC in BM has been associated with gap junctional intercellular communication (GJIC) between BC cells (BCCs) and stroma and, change in growth-promoting cytokines. This study sought the identity of the responsible BCCs with preference for dormancy. Here we describe a hierarchy of BCCs with the most immature subset showing chemo resistance and a dormant phenotype. This subset showed properties consistent with stem cells: self-renewal, serial passage, cycling quiescence, long doubling time, asymmetric division, highly metastatic and invasive. More importantly, both in vitro and in vivo studies showed the subset with stem cell properties showed preference for GJIC with BM stroma. The stem cell subset was heterogeneous with respect to CD44 and CD24 and were Oct4hi/CD44hi/med/CD24-/+. The relevance of these studies was shown by the presence of a similar cell type in the blood of patients, despite aggressive treatment and in a patient with a relatively large tumor but no lymph node involvement. In brief, our studies identified a novel BCC subset with stem cell properties, with preference for dormancy and in the circulation of patients. The findings have prognostic and diagnostic implications.

Abstract No. 38

Alterations in gene expression profile during chemoradiation of cervix and comparison with recurrent tumors in locally advanced carcinoma cervix

Santam Chakraborty 1 , Geo Franscis 1 , Prashant Sood 2 , Sujith KM 1 , Geetha M 1 , Milind Kumar 1 , Satheeshan B 1

1 Malabar Cancer Centre, Thalassery, Kerala. 2 All India Institute of Medical Sciences, New Delhi. Email: drsantam@gmail.com

Background and Objectives: Major alterations in gene expressions spanning multiple pathways is expected after a major genotoxic stress like chemo-radiation. The present study aims to elucidate changes in gene expression patterns across twenty candidate genes in cervical cancer to assess their predictive utility in reliably identifying patients likely to have recurrence after treatment. In addition we also aim to assess if the gene expression in these candidate genes undergoes further alterations in residual and recurrent tumours as compared to their expression during the chemo-radiotherapy phase. Materials and Methods : Fifty patients of carcinoma cervix (stages IIB-IVA) will be selected for this study over a period of one year, after study specific informed consent. Twenty candidate genes (IMUP, IGF-2, ARHD, DLG1, AF085868, C16orf5, GADD45A, PXN, CA9, Icthyin, LGALS7, CCL3, IGFBP3, GAPD, GUSB, B2microglobulin) known to have significantly altered significant differences in expression between responders and non responders (Knoll et al.) will be studied. One sample will be retrieved before the start of chemo-radiotherapy and the second after 48 hours of starting chemo-radiotherapy. After completion of treatment, all patients with evidence of residual or recurrent disease will undergo a third tissue biopsy. Cervical tissue samples patients who do not demonstrate any residual or relapsed tumor will also be sought at completion of one year follow up. Real time reverse transcriptase polymerase chain reaction will be employed prospectively in these samples to study gene expression patterns of the 20 candidate genes in all the samples. Data Analysis : Student's t test, Wilcoxon rank test, ANOVA, Fischer's test, regression analysis and log-rank test will be utilized to analyze, compare and model appropriate genetic and clinical variables. Poisson distribution and hypergeometric distributions will be mapped out between appropriate sub-cohorts to compare results and draw final conclusions. Expected Outcome : Mutations arising as a consequence of therapeutic selection pressure are likely to determine resistance to treatment and recurrence. Our study aims to comprehensively analyse expression patterns in 20 cervical cancer candidate genes and assess their utility for predicting cancer resistance and relapse. We believe that such pattern recognition studies can help us elucidate and develop novel genetic biomarkers, predictive models and protocols for better management of cervical cancer patients. Timeline : Malabar Cancer Centre sees 80 - 100 new patients with carcinoma cervix annually. Thus it is expected that accrual can be completed within a span of 1 year and 1.5 years will be required for completing the mandatory one year followup for all patients. The total duration of the study is thus expected to be around 2.5 years.

Abstract No. 73

Ubiquitin conjugating enzyme E2C is potential therapeutic target for cervical cancer


Mayil Vahanan Bose 1 , G. Gopal 1 , G. Selvaluxmy 2 , T. Rajkumar 1

1 Department of Molecular Oncology, Cancer Institute (WIA), 2 Dept. of Radiation Oncology, Cancer Institute (WIA), Chennai, India. Email: go2mayil@gmail.com

Aim : To find the expression levels of Ubiquitin conjugating enzyme E2C (UBE2C) in cervical cancer cell lines and to correlate their radiation response. To investigate the effect of dominant negative Ubiquitin conjugating enzyme E2C (DN-UBE2C) in cell proliferation and radiation response. Materials and Methods: Taqman Real time PCR was done for UBE2C levels in cervical cancer cell lines. Immunohistochemistry was used to study the protein expression of UBE2C in normal, dysplasia and cancer of the cervix. Radiation sensitivities of cervical cell lines were analyzed by assessing their cell survival after irradiation with different doses by MTS assay. Growth curve and radiation response of DN-UBE2C transfected SiHa and HeLa cell lines were also assessed by MTS and clonogenic assay. Results: Quantitation of UBE2C levels revealed at least a four-fold over expression in SiHa cells and more than 2 fold in HeLa, C33A and ME180 relative to the HEK293 cells. Using IHC, we found UBE2C to be overexpressed in 87.5% (28/32) cancers and 18.1% (2/11) in CIN3/CIS. Radiation response studies reveal, SiHa as most resistant cell line showing 30 % survival at 10 Gy, whereas C33A cells were most sensitive showing 30 % survival even at 4 Gy. The growth rate of SiHa and HeLa transfected with DN-UBE2C was significantly reduced compared to vector control. Similarly, DN-UBE2C mediated radiosensitivity was correlated with a significant decrease in resistance to radiation by SiHa (2, 4, 8Gy) and HeLa cells (2, 4Gy) after transfection with the DN-UBE2C when compared to control cultures. Overall significance level of (p value = 0.05) was observed between all groups. Conclusion: These results suggest that UBE2C may be a potential therapeutic target for cervical cancer.

Abstract No. 147

Novel signal transduction targets for radiation sensitization


Dr. Stephen Yoo

Radiation Research Program, National Cancer Institute, National Institutes of Health, USA. Email: yoosu@mail.nih.gov

The potential for radiation modifiers in cancer therapy not only relates to the key goals of improved local tumor control, reduced normal tissue injury and improved survival, but it also provides increased use and return-on-investment for molecular-targeted therapy. Radiation modifiers utilize the extraordinary technological capabilities of radiation delivery and the biological properties of radiation can be applied in the "focused biology" concept whereby imaging is merged with treatment for accuracy and efficacy. The Molecular Radiation Therapeutics Branch (MRTB) is an integral component of the Frederick National Laboratory for Cancer Research (FNLCR). By networking to other programs within NCI, worldwide radiation oncology and biology investigators, pharmaceutical companies and the National Clinical Trials Network, MRTB serves as a resource for accelerating the translation of novel molecular, cellular and immunological therapeutics into the clinical arena to achieve improved cancer treatment outcomes. Thus, the MRTB program is to facilitate clinical trials with radiation modifiers by generating strong preclinical evidence for combining molecularly-targeted agents with radiation and also by providing unbiased parallel evaluation of certain candidate drugs in a given class. The MRTB effort will be presented by a recent few examples of classes of molecularly targeted agents as potential radiation modifiers.

Abstract No. 148

Requirement of pre-clinical evidence for radiation oncology clinical trials


Dr. Bhadrasain Vikram

Radiation Research Program, National Cancer Institute, National Institutes of Health, USA. Email: vikramb@mail.nih.gov

Radiation therapy with or without surgery/chemotherapy is employed in the initial definitive treatment of many kinds of cancers. For many locally advanced cancers, however, local/regional failures remain too common and often cause the death of the patient. Some examples include glioblastoma, HPV negative cancers of the head & neck, cancers of the lung, esophagus, liver and pancreas. For some cancers, such as rectal cancers and soft tissue sarcomas, the pathological complete response rates after radiotherapy or radiochemotherapy are low, requiring morbid surgery such as abdominoperineal resection or amputation.

Adding novel targeted agents that improve tumor control without excessive toxicity will prolong patients' survival and improve the quality of life. As a minimum the novel agent should be shown to enhance the effect of radiation in a relevant cancer cell line in vitro. The drug concentration employed in those studies should be what is clinically achievable. If the putative mechanism of action involves the tumor microenvironment then in-vivo demonstration of radiation enhancement is necessary.

More sophisticated models such as orthotopic xenografts, patient-derived tumor grafts, genetically engineered mouse models, etc are currently being investigated in efforts aimed at increasing our ability to predict success in the clinic.

Abstract No. 152

Cellular radiosensitivivty and its assosiation with clinical radiosensitivity in breast cancer subjects


Kamalesh D Mumbrekar

Division of Radiobiology, Manipal Life Sciences Centre, Manipal, India. Email: kamaleshdm@gmail.com

In breast cancer with a high patient survival rates, the quality of life becomes a major topic of concern. To improve the quality of life one need to reduce the suffering from severe normal tissue adverse reactions caused by radiotherapy. Ability to identify radiosensitive patients before treatment would allow personalized treatment that will decrease side effects. Assays measuring the DNA damage are more useful as the DNA damage and repair is the major contributing factor for radiosensitivity. The aim of this study was to evaluate the ability of γH2AX assay (for assessing DNA damage and repair) to stratify patients undergoing radiotherapy as overreactors and non-overreactors based on their DNA damage and repair profile.

Breast cancer patients (n=76) planned for radiotherapy were prospectively recruited. Lymphocytes isolated from blood were used to measure the cellular radiosensitivity after a challenge dose of 2Gy X-rays. Various repair parameters like baseline damage, initial damage (0.25h), and residual damage (3h & 6h) were measured using γH2AX assay. Adverse skin reactions were assessed and classified following the criteria set by RTOG.

A significant interindividual variation was observed at 6h repair time points with a % CV of 18, compared to 8 and 13 for 0.25 and 3h respectively. The repair kinetics between the non-overreactors and over reactors differed significantly (p = 0.02) at 3h and 6h repair post irradiation. DNA repair capacity decreased with increase in acute reaction grading (p = 0.001). Stratified analysis showed that the upper quartile of residual damage consisting of 40% overreactors compared to 10% non-overreactors. In conclusion we could able to associate the residual damage with clinical radiosensitivity using γH2AX assay. Stratification of patients with greater or less than average risk of complications before radiotherapy will allow prescribed radiation dose to be adjusted for increase local control and cure. Therefore, our studies indicated the clinical implication of γH2AX assay for predicting the normal tissue toxicity.

Acknowledgements: The financial support from Department of Biotechnology, Government of India (BT/01/COE/06/02/07) to carry out research work and Manipal University for providing the research facilities is gratefully acknowledged.

Abstract No. 156

Mapping the fate of umbilical cord derived MSCs after transplantation using 51Cr labeled cells

Archana Mukherjee 1none , Shabari Tipnis 3 , Haladhar Dev Sarma 2 , Chandra Viswanathan 3 , Grace Samuel 1

1 Radiopharmaceuticals Division, 2 Radiation Biology and Hea, 3 Radiopharmaceuticals Division, BARC,Mumbai, India. Email: archanamkhrj@gmail.com

Introduction: Stem cell therapy is a promising modality for treatment of several clinical conditions including cancer to replenish cells after therapy. However, there are many challenges for translation of stem cell therapy to a viable clinical therapy. Hence, a method to monitor homing as well as the fate of the delivered stem cells is desired. Although, stem cells are injected loco regionally to the diseased site for therapy, it is reported that when injected via intravenous (i.v) route, stem cells preferentially localize to the site of injury. To provide proof of principle, biodistribution and imaging studies after i.v injection are desired in animal models of injury. Umbilical cord (Human) mesenchymal stem cells (UCMSCs) are promising candidates for allogenic cell therapy. Radiolabelling of cells with 51Cr (Eγ= 320 KeV, T΍ = 27.7d) was considered for tracking cells for a longer duration. Herein, we report radiolabeling of UCMSCs with 51Cr and studies to map the fate of stem cells after i.v injection in normal animals and in animal models of injury. Experimental: Radiolabelling of stem cells UCMSCs were isolated from the cords using an in-house established protocol. Post activity optimization studies, 9.25 MBq of 51Cr was added to 107 UCMSCs and incubated at 37ΊC for 1h for animal studies. After centrifugation and washing of the cells, the radioactivity associated with cells was measured. Viability of cells was also studied at 3h post labelling. Development of animal models: Animal model of kidney injury was developed by using 10μg/Kg Cisplatin dose in Swiss mice. After 24h, kidney injury was confirmed by various methods. Inflammation model was developed by injecting 150 μl of turpentine in the right thigh of Wistar rats. After 24h, redness, swelling and difficultly in mobility was observed indicating inflammation at the injection site. Biodistribution & imaging studies: To map the fate of 51Cr labeled UCMSC cells, ~ 150μl (370KBq/1.5x106 cells) of radiolabelled cells were injected in Swiss mice via tail vein for biodistribution studies. Animals were sacrificed at 1h, 3h, 24 h & 48h post injection and % ID/organ was estimated. Set of animals were also injected with 51Cr for comparing distribution of the radionuclide. Similarly, biodistribution and imaging studies were carried out in animals with kidney injury and inflammation at various time intervals p.i. Results: Studies with 51Cr labeled UCMSC's when injected via i.v route revealed initial homing of cells in the lungs which cleared with time. In animal model of kidney injury, kidney activity at 24h was 1.17±0.24 % compared to 0.72±0.62% in normal animals. Uptake in lungs and liver was also high compared to normal animals. In case of animals with inflammation, activity was observed in inflamed muscle of the animal with T/NT ratio of 1.5 at 24h p.i. Slow clearance of activity from lungs and uptake in liver and GI tract was observed. Conclusion: These studies indicate that 51Cr labeled stem cells could be used for in vivo tracking. Significant uptake in the inflamed tissue was not observed due to reasons such as stem cells may not be actively involved in repair during inflammation compared to other conditions like in infarction or injury caused by chemicals. Further studies are desired to conclusively prove preferential localization of stem cells to the site of injury.

Abstract No. 168

High-dose hypofractionated radiation therapy: Radiobiological concepts


Seema Gupta

Biophysics Research Institute of America, Miami, FL, USA. Email: sgjija@gmail.com

It is widely believed that radiation dose-escalation is critical in improving loco-regional control and survival of the patients with advanced cancer. However, the dose-escalation is limited by the tolerance of surrounding normal tissues. Therefore, a strategy such as high-dose-per-fraction radiation is an effective alternative means of dose-escalation. Recent clinical trials have been using high-dose hypofractionated radiation therapy and important and encouraging data are emerging from these studies. However, the mechanisms responsible for the observed clinical outcomes are still lacking. Targeting the tumor microenvironment with these novel strategies such as LATTICE radiotherapy will induce different biological events such as abscopal/bystander effects, activation of immune system, endothelial cell death, re-organization of hypoxic and oxygenated regions and minimal normal tissue toxicity. Together, these events may make high-dose hypofractionated radiation therapy more effective than conventional radiation therapy for treatment of advanced cancers. In this talk, the results obtained from in vitro and pre-clinical studies will be discussed.

Abstract No. 172

The first cut is the deepest

Colin Seymour

McMaster University, 1280 Main Street West, Hamilton, Ontario, Canada. Email ID: seymouc@mcmaster.ca

This was originally a song title by Cat Stevens in 1967. I will argue in this paper that it should be regarded as the guiding principle for radiation and chemo therapy. I shall demonstrate that the accepted principles of fractionation (equal effects per dose) are wrong, and that possibly the most important treatment is the initial treatment. I shall use "in vitro" experiments performed in our laboratory to demonstrate the treatment rationale we propose.

Abstract No. 180

A study of clinical and cellular radiosensitivity and DNA repair pathway genes in women undergoing radiotherapy for breast cancer


Rima Pathak

Tata Memorial Hospital, Mumbai, India. Email: drrimapathak@gmail.com

Purpose: To prospectively study the factors influencing the acute radiation toxicity in women undergoing adjuvant radiotherapy for breast cancer and to examine the association of Single Nucleotide Polymorphisms (SNP) in XPD (rs13181), XRCC1 (rs25487), XRCC4 (rs1478485) and XRCC4 (rs 13180316) with individual radiosensitivity and breast cancer risk. Materials and Methods: All patients in the study were accrued prospectively and treated at TMC. Patient characteristics and treatment details were recorded. Computed Tomography (CT) based radiotherapy treatment planning was performed for most of our patients. Individual genetic polymorphisms were determined by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR- RFLP) and by Real-time PCR (RT-PCR) for all patients (n=195) and their age matched healthy controls (n=200). Cellular radiosensitivity was determined using γ-H2AX assay for a few patients (n=23). The development of moderate to severe acute skin reaction (RTOG Grade II/ III) associated with genetic polymorphisms was modelled using Cox proportional hazards, accounting for cumulative Biologically Equivalent radiation Dose (BED). All other factors were analyzed for their association with acute radiation toxicity using Chi square test and Spearman's correlations. For factors which were significant or approaching significance on univariate analysis were analyzed using Binary Logistic Regression for their association with development of grade II/III acute radiation toxicity. Key Results: Patient and radiotherapy treatment related factors that were significant with acute radiation toxicity of skin were, the type of surgery (p = 0.008) where Breast Conserving Surgery (BCS) was associated with higher incidence of grade II/ III toxicity and BMI (p = 0.045) where higher BMI was associated with higher incidence of acute radiation toxicity. After stratification on the basis of type of surgery, for patients in the MRM group, factors that were associated with higher incidence of grade II/ III acute radiation toxicity of skin were, presence of polymorphic alleles for XPD and XRCC4 (rs13180316) (p = 0.002) and presence of maximum dose >107% of the prescribed dose, inside or outside PTV (p = 0.022). For patients with conserved breast factor significantly correlating with acute grade II/ III toxicity of skin were BSA > 1.6 (p = 0.03) and BMI > 25 (p = 0.06). XRCC1 heterozygous genotype correlated significantly (p = 0.04) with development of radiation mucositis. The odds of development of breast cancer was 1.535 (0.966- 2.444) with heterozygous and homozygous genotype of XRCC4 (rs13180316). Conclusion: Multiple factors correlated significantly with development of moderate to severe acute radiation toxicity of skin like the irradiation of conserved breast compared to chest wall, BMI > 25 kg/m2, heterozygous and homozygous genotype of XPD and XRCC4 (rs13180316) and BSA > 1.6 m2 all which have been described in the literature previously. Heterozygous genotype of XRCC1 (rs 25487) significantly increases the incidence of radiation mucositis. Presence of polymorphic allele of XRCC4 (rs13180316) increased the odds by 1.5 times, of development of breast cancer.

Abstract No.199

Radiation modifiers (2DG & Ocimum): Taking pre-clinical evidence to clinical trials


Rajiv Sarin

ACTREC, Tata Memorial Centre, Navi Mumbai, India. Email: rsarin@actrec.gov.in

Conventional approaches to improve therapeutic ratio (TR) of cancer radiotherapy using improvisation in physical dose distribution; altered fractionation and concurrent chemotherapy have reached a plateau for several tumour types. Many tumours fail locally despite using maximum tolerated doses and many normal tissues exhibit clinically significant acute or late toxicity with standard doses used in cancer radiotherapy. There is a long history of a large number of natural and synthetic radiation modifiers which have been found to be very effective in pre-clinical models but their clinical evaluation was either not possible or negative. This has prompted search for novel radiation modifiers - both sensitizers for enhancing tumour killing and protectors of normal tissue. We have taken forward two pre-clinical leads through the process of phase II and phase III randomized clinical trials.

The novel radiation modifier 2-Deoxy-Glucose (2DG) exploits the differences in glucose metabolism in tumour. With unequivocal pre-clinical data produced by Jain, Dwarakanath and others it was an ideal molecule for clinical evaluation. Similarly the strong radioprotective effect of the flavanoids orientin and flavin in Tulasi - the Holy Basil (Ocimum Sanctum) had shown in pre-clinical models by Uma Devi and others required clinical validation. The issues pertaining to the development and conduct of clinical trials for 2-De-Oxy-Glucose in glioblastoma and the radioprotector - Ocimum Sanctum in patients undergoing Hemi Body Irradiation (HBI) for bone metastasis will be presented. Issues in developing and choosing clinical model system, endpoints for efficacy and toxicity and the trial results will be discussed. The effect of the HBI model on various clinical and laboratory endpoints and the time course of recovery from radiation injury will be presented to highlight the utility of this clinical model for testing novel radioprotectors and for developing biodosimetry.




 

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