|Year : 2015 | Volume
| Issue : 1 | Page : 181-190
Influence of selenium on radiogenic collagen destruction and the degree of collagen tissue maturation in stage III oral squamous cell carcinoma patients undergoing therapeutic irradiation
Sonaa Elango1, Usha Subbiah2
1 Department of Radiological Science, College of Medical Science, Catholic University of Daegu, 13-13 Hayang-ro, Hayang-Eup, Gyeongsan-Si, Gyeongbuk 712-702, Korea
2 Department of Medical Microbiology, Faculty of Medicine, University of Malaya, Kuala Lumpur 50603, Malaysia
|Date of Web Publication||16-Apr-2015|
Department of Radiological Science, College of Medical Science, Catholic University of Daegu, 13-13 Hayang-ro, Hayang-Eup, Gyeongsan-Si, Gyeongbuk 712-702
Source of Support: None, Conflict of Interest: None
Introduction: We set out to assess whether selenium, an antioxidant mineral could influence radiogenic collagen maturation.
Materials and Methods: The study comprise of normal (Group I), untreated oral carcinoma cases (Group II) (n = 20), cases who underwent radiotherapy (Group IIa) n = 10 and cases supplemented with selenium along with radiotherapy (Group IIb) n = 10.
Results: Spectrophotometric estimation and luminescence spectral assignment of collagen showed improved collagen maturation status. Measurement of the mature collagen cross-links hydroxylysylpyridinoline and lysylpyridinoline by high-performance liquid chromatography on irradiated tissues showed a considerable decrease in the selenium Group IIb (P < 0.05) indicating a decrease in collagen fragments. Electron microscopic studies showed significant morphological alteration in the selenium group. The micro nucleus frequency, decreased in radiation group (P < 0.05) compared with untreated (P < 0.05). While much more decrease observed in the selenium group (P < 0.05).
Discussion: The results represent the effect of selenium treatment with a bearing on carcinogenic process to curtail it, thus enhancing the maturity of collagen.
Keywords: Antioxidant, collagen, oral cancer, radiotherapy, selenium
|How to cite this article:|
Elango S, Subbiah U. Influence of selenium on radiogenic collagen destruction and the degree of collagen tissue maturation in stage III oral squamous cell carcinoma patients undergoing therapeutic irradiation. J Can Res Ther 2015;11:181-90
|How to cite this URL:|
Elango S, Subbiah U. Influence of selenium on radiogenic collagen destruction and the degree of collagen tissue maturation in stage III oral squamous cell carcinoma patients undergoing therapeutic irradiation. J Can Res Ther [serial online] 2015 [cited 2017 Dec 11];11:181-90. Available from: http://www.cancerjournal.net/text.asp?2015/11/1/181/143328
| > Introduction|| |
Oral squamous cell carcinoma (OSCC) progresses from hyperplasic epithelial lesions through dysplasia to invasive carcinoma. Histologically, the disorders is characterized by a subepithelial chronic inflammatory reaction and an accumulation of dense collagen at the dermo epidermal function, hence the pathological changes in the overlying epithelium include increased keratinization, epithelial hyperplasia and/or atrophy and epithelial dysplasia.  Hence, the concept of "field cancerization" with molecular alterations can be applied to the oral cavity tumorigenesis. 
There remains much explained variability in the clinical course of patients with OSCC, like molecular changes at the wound margins, which predispose patients to malignancy and contribute to disease recurrence.  OSCC manage acute toxicities, including severe mucositis, pain, difficulties in swallowing and sticky saliva as well as tracheotomies and/or feeding tubes, and the potential long-term dysfunction, including limited diet, drymouth, compromised speech and disfigurement. 
Patients in addition prepare for and cope with the undesirable chronic sequelae of radiotherapy including, mucocutaneous changes (radiation-induced mucositis), xerostomia, osteoradionecrosis, radiation neuritis etc.  The detrimental consequences of ionizing radiation on wound healing is multifaceted: It has direct cytotoxic effects on various cellular/molecular components of wound repair. The initial effect of anticancer therapy, such as radiation and chemotherapy, is on the rapidly proliferating cells of the oral epithelium. As a consequence, the epithelium may show atrophy and ulceration. The sites of these alterations are related to the rate of epithelial proliferation. 
Apart from these complications the radical radiation on cancer cells stimulates free radicals production in normal cells exacerbating the free radical mediated damage in the system. This indirect effects through the production of a burst of free radicals, rearranges tissue components immediately, causing DNA damage and altering other complex molecules involved in tissue repair and regeneration.
Ionizing radiation provokes the decomposition reaction of water, producing a variety of reactive oxygen species (ROS) thereby hindering the maturation of collagen at the irradiated site. The major cytotoxic effect of free radical is supposed to be the peroxidation of lipid components of cellular and subcellular membranes resulting in the loss of cellular integrity which could lead to irreversible cell injury.  The radiation-induced damage through free radicals aggravates the cancer associated free radicals level.  Therefore, there is a need for a multifunction drug that conforms to all criteria of optimal radioprotector/antioxidants to overcome direct negative effects of ionizing radiation, which could be an important therapeutic strategy to improve healing of irradiated wounds.
Selenium is a natural antioxidant mineral with anticarcinogenic property. Several epidemiological studies have indicated inverse association between selenium and various neoplasm, including colorectal, skin, oral.  Selenium is a metalloid of considerable interest in the human from both a toxicological and a nutritional perspective, with a very narrow safe range of intake. The ability of certain Se species to exert toxic effects on cancer cell growth has been demonstrated in a number of laboratory studies,  leading to interest in the use of Se compounds as anticancer therapeutic agents, possibly as additions to standard chemotherapeutic regimens.  A growing body of evidence from human and laboratory studies indicates dramatically different biological effects of the various inorganic and organic chemical forms of selenium. NonHodgkin lymphoma patients reported that the addition of selenite to chemotherapy appeared to reduce recurrence risk.  In this study, inorganic form of selenium (sodium selenite) was selected owing to its toxic property over cancer cells.
However, there is no study over the effect of selenium on collagen maturation in radiation treated oral cancer cases. This study explores the collagen maturation status in radiation treated cases with and without selenium supplementation and prognosis of the patient.
| > Materials and methods|| |
Oral cancer patients were recruited for the study. This study consists of following groups. Group I consisted of control (normal healthy volunteers) (n = 10), Group II composed of oral cancer patients (untreated) (n = 20) (24 patients were recruited at the beginning, where there was four dropouts), Group IIa comprised of oral cancer patients who underwent radiotherapy alone (n = 10), and Group IIb constituted of oral cancer patients who underwent radiotherapy along with supplementation of selenium (n = 10). Both Group IIa and IIb were followed-up for 6 months. Oral cancer patients of stage (III) alone was chosen for the study, since they are with established disease condition and therefore the effect of any supplementation would be studied exactly. Stage IV cases had been avoided owing to the severity of the disease and the deceased condition of the patient. Participating cases and controls were interviewed in-person by trained personnel using a structured questionnaire. Different episodes of tobacco usage during patient's life time, with each episode representing a different frequency of use (elicited in terms of times per day, week, month or year) were recorded. The interview also elicited demographic characteristics and alcohol consumption. Anatomic sites and clinico pathological features of patients of patients with squamous cell carcinoma of the oral cavity selected for the study were given in the [Table 1]a and b. Patients excluded from enrollment of the study were the ones with history of systemic diseases or those who are taking medication that might interfere with nutritional status or mineral supplements. Exclusion was also based on intercurrent diseases (thoracic herpes zoster, salivary fistula requiring surgery, and other medical diseases) that led to an interruption of radiotherapy.
Radiotherapy was delivered with a tele-cobalt beam using anterior and lateral wedge pair or lateral parallel portals (theratron-780- 60 Co; phoenix- 60 Co; gammatron- 60 Co) at a dosage of 6,000 Gy (200 cGy/day) in five fractions per week for a period of 6 weeks. Selenium supply selenium capsules-per capsule contained 400 μg of selenium (sodium selenite). Patients in the selenium group received 400 μg selenium/d for a period of 6 months. Subjects were dispensed with selenium capsules at every month to ensure strict monitor over them of taking no medication or any other nutritional supplements and is there any discontinuity in routine intake of the selenium capsules.
Normal tissue specimens were collected from consented patients who were scheduled for oral surgery but who did not have a diagnosis of oral carcinoma. Normal samples were treated in the same fashion as the tumor samples. Mucosa dissected far from cancerous tissue was considered normal. All these normal control samples that were excised from surgical margins of patients with OSCC were histologically normal. (The data pertaining to these samples were not shown).
Fresh OSCC cancer tissue samples were collected from 20 patients. The cancer tissue specimens are solid chunks with random shape and size about 5 Χ 5 Χ 2 or smaller. Pathologic examination of hematoxyline and eosin-stained sections indicated that OSCC samples were comprised of at least 60-70% tumor.
A part of the collected tissue specimens were cut into small fragments, minced, homogenized in a warring blender and used to estimate the nuclei acid content, collagen fractions and hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP) content. The other part of the specimens were stored in the deep freezer at - 20C for luminescence spectral assignment, transmission electron microscopic (TEM) studies. The tissues were thawed to room temperature during experiment. Surface cells were scraped from, oral mucosa of the tumors from OSCC patients and from normal healthy volunteers. All scrapings of the oral mucosa were made with a clean wooden tongue depressor, which was moistened with water, prior to use. Scrapings from patients were obtained from areas covering the lesion proper and the immediate vicinity around the lesion and also scrapings from area far from lesion were taken (the data's pertaining to these samples were not shown). The scrape smears were done and proceeded for micronucleus count assay.
Under nucleic acid content analysis, DNA and RNA was estimated. , Collagen estimation was done.  Estimation of collagen fractions (neutral salt soluble, acid soluble, insoluble) was done.  The HP and LP content was quantified by reverse-phase high performance liquid chromatography.  Luminescence spectral assignment was analyzed.  TEM studies done.  Micronucleus frequency was studied employing the Fuelgen technique.  Detailed procedure of all the above mentioned methods were discussed elsewhere.
Statistical significance of differences between all the groups was determined by Students t-test and one-way analysis of variance using Statistical Package for the Social Sciences (version 7.5, SPSS Inc, Chicago, IL,USA). P <0.05 were considered to be significant and are expressed as mean standard deviation.
| > Results|| |
Nucleic acid content
[Figure 1] shows the tissue nucleic acid content of normal, untreated oral cancer, radiation treated and selenium supplemented groups. Elevated levels of DNA and RNA content in the tissues of untreated oral cancer patients in comparison to normal denoted more cell proliferation in the site (Group II). In comparison to untreated groups radiation group showed a decrease of both the contents P < 0.001 (Group IIa). Selenium supplementation resulted with much decreased tissue DNA and RNA content (P < 0.001) (Group IIb vs. Group IIa) after 6 months, indicating prognosis, through inhibition of cell proliferation.
|Figure 1: Nucleic acid content in carcinomateous tissues of normal and study groups. Group I: Normal; Group II: Untreated oral cancer patients Group IIa: Radiation treated-6 months Group IIb: Radiation treated + selenium supplemented-6 months values are expressed as mean ± standard deviation (aGroup II compared with Group I; bGroup IIa and IIb compared with Group II; cGroup IIb compared with Group IIa. Statistical significance are expressed as *P < 0.05; #P < 0.01; $P < 0.001)|
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[Figure 2] shows tissue total collagen content of normal, untreated oral cancer, radiation treated and selenium supplemented oral cancer patients. In [Figure 2] untreated oral cancer patients showed an increase in total collagen content (Group II) in comparison to normal (P < 0.05). A decrease in the total collagen content following radiation after 6 months was noticed (Group IIa). Selenium supplementation showed much decrease of collagen content (P < 0.05). [Table 2] comprehensively depicts the percentage of hydroxyproline of acid soluble, neutral and insoluble collagen fractions of all four groups. Compared to normal the untreated cancer patients showed diminishing level of insoluble fractions P < 0.05 except soluble and neutral which shoots up significantly P < 0.05.
|Figure 2: Total collagen content in tissues of normal and study groups. Group I: Normal; Group II: Untreated oral cancer patients Group IIa: Radiation treated-6 months Group IIb: Radiation treated + selenium supplemented-6 months values are expressed as mean ± SD. (aGroup II compared with Group I; bGroup IIa and IIb compared with Group II; cGroup IIb compared with Group IIa. Statistical significance are expressed as *P < 0.05; #P < 0.01; $P < 0.001)|
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|Table 2: Percentage of hydroxyproline in the collagen fractions in tissues of normal and study groups|
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In radiation group, with its side effects being subsided after 6 months a slight improvement in the % of hydroxyproline of insoluble collagen fractions was observed (P < 0.05) in relocation time (RT) group compared with untreated, while acid soluble and neutral fractions showed a slight decrease (P < 0.05). With selenium supplementation group appreciable increase of insoluble collagen fraction and a little more decrease of acid soluble and neutral fractions were observed in comparison to RT group (P < 0.05). All these data's indicate the collagen maturation and prognosis toward selenium therapy.
High performance liquid chromatography analysis
The concentrations of HP, LP in the collagen tissue [Table 3], of untreated showed increased level (2.61, 1.88), severe increase was observed in radiation group (24.6, 6.1). Selenium group showed a decline in the concentrations of HP, LP reverting back to near normal (4.22, 1.8). These data is found to be represented in the chromatogram. [Figure 3] is the chromatogram of the normal collagen tissue Group I (a), Untreated OSCC Group II (b), RT Group IIa (c), RT + selenium Group IIb (d).
|Figure 3: Chromatogram of the normal collagen tissue group I (a); untreated oral squamous cell carcinoma Group II (b); relocation time Group IIa (c); radiotherapy + selenium Group IIb (d)|
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|Table 3: The concentrations of HP, LP collagen tissue of normal and study groups|
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Luminescence spectral studies
[Figure 4] shows the excitation and emission luminescence spectra of normal and cancer tissues in the wavelength between 250 and 750 nm. [Figure 4]a shows the synchronous luminescence spectra of various endogenous fluorophores. [Figure 4]b shows the luminescence spectra of normal and OSCC tissues. The peaks at 299 nm and 350 nm with a small shoulder peak at 530 nm are assigned to trytophan [Figure 4]c, collagen and flavin adenine dinucleotide (FAD) [Figure 4]d of normal tissue. In the case of cancerous tissues the resolved peaks are at 303 nm [Figure 4]e, 527 nm and third peak at 350 nm [Figure 4]f with less intensity. This may be attributed to unfolding conditions of proteins with low peaks of tumor tissue endoplasmic reticulum (ER) and absence of keratinization and microfilaments (MF). The present study shows the characterization of cancer tissues of untreated, radiation treated, selenium supplemented group.
|Figure 4: (a) Synchronous luminescence spectra of various endogenous fluorophores (b) Synchronous luminescence spectra of normal and oral squamous cell carcinoma tissues (c) Excitation and emission spectra of normal oral tissues for tryptopzhan (d) Excitation and emission spectra of oral squamous cell carcinoma tissues for tryptophan (e) Excitation and emission spectra of normal tissues for collagen (f) Excitation and emission spectra of oral squamous cell carcinoma tissues for collagen and\flavin adenine dinucleotide (g) Scattered plot for tryptophan/collagen intensity ratio (h) Scattered plot for tryptophan/flavin adenine dinucleotide intensity ratio (i) Scattered plot for collagen/flavin adenine dinucleotide intensity ratio|
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Three ratio parameter I 301/I 350, I 301/I 527 and I 350/I 527 were used to classify the normal and tumor tissues of all groups. The ratio values of tryptophan/collagen intensity ratio, tryptophan/FAD intensity ratio, collagen/FAD intensity ratio were shown as a scattered plot in [Figure 4]g-i respectively. The critical values assigned to classify the normal from tumor in each group are shown in [Table 4] along with sensitivity and specificity obtained in each case. The luminescence spectral assignment data of collagen, tryptophan, FAD denotes the selenium supplemented group reverting to near normal.
|Table 4: The sensitivity, specificity characteristics of tryptophan, collagen and FAD of normal and study groups|
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Electron microscopic studies
The electron microscopic studies of the oral cancer tissues of untreated OSCC, radiotherapy and radiotherapy + selenium supplemented patients are represented in [Figure 5]. Well differentiated carcinoma showed elongated nuclei, scanty cytoplasm and wide intercellular spaces (ICS) with clumping of tono filaments [Figure 5]a, tubular mitochondria [Figure 5]b, keratinisation [Figure 5]c, tono filaments [Figure 5]d and desmosomal alterations with widened ICS [Figure 5]e. In radiotherapy patients, the tissue showed degenerative necrotic changes with irregular nuclear membrane (NM) [Figure 5]f, swollen mitochondria [Figure 5]g, dilated ER [Figure 5]h and less keratinisation [Figure 5]i. In selenium supplemented patients, the tissue showed pleomorphic nucleus with altered nucleus to cytoplasmic ratio, cytoplasmic granules, condensed chromatin, less dense intercellular junctions (ICJs) [Figure 5]j, globular ER, absence of keratinization [Figure 5]k and MF [Figure 5]l.
|Figure 5: Well-differentiated carcinoma (a) showing elongated nuclei, scanty cytoplasm, wide intercellular spaces (ICS) and clumping of tonofilaments ×3000, tubular mitochondria (b) ×45,000, Keratinization (k) (c) ×20,000, tonofilaments (d) ×3000 and desmosomal (d) alterations with widened ICS (e) ×4500 in oral squamous cell carcinoma patients. (f) showing degenerative, necrotic changes with irregular NM ×7000, swollen and irregular mitochondria (M) (g) ×45,000, dilated endoplasmic reticulum (ER) (h) ×10,000 and less keratinization (K) (i) ×10,000 in relocation time (RT) patients. (j) showing pleomorphic nucleus (n) with altered nucleus to cytoplasmic ratio, cytoplasmic granules, condensed chromatin, less dense intercellular junctions ×4500, globular ER, absence of keratinization (k) ×15,000 and microfilaments (l) ×15,000 in RT + selenium supplemented patients|
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Micronucleus frequency analysis in exfoliated cells of oral mucosa
Analysis of micronucleus frequency in exfoliated cells of the oral mucosa in normal, untreated oral cancer, radiation treated and selenium supplemented groups has been illustrated in [Figure 6] and [Figure 7]. Micronucleus occurrence in untreated oral cancer mucosal cells by several fold (Group II) was observed in comparison to normals (P < 0.05). Radiation induced micronucleus frequency declined after 6 months (P < 0.05). Interestingly, selenium supplementation for 6 months had more appreciable decrease in micronucleus count (P < 0.05).
|Figure 6: Micronucleus frequency in exfoliated oral squamous carcinoma cells of normal and study groups: (a) Normal (×20), (b) micronuclei (×20), (c) micronuclei (×40)|
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|Figure 7: Enumeration of micro nucleus count in the exfoliated cells of the oral mucosa of normal and study groups. Group I: Normal; Group II: Untreated oral cancer patients. Group IIa: Radiation treated-6 months. Group IIb: Radiation treated + selenium supplemented-6 months. Values are expressed as mean ± standard deviation (aGroup II compared with Group I; bGroup IIa and IIb compared with Group II; cGroup IIb compared with Group IIa. Statistical significance are expressed as *P < 0.05; #P < 0.01; $P < 0.001)|
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| > Discussion|| |
At various time points in recent history, selenium has been hypothesized to be a carcinogen, a cancer preventive agent, a cancer therapeutic agent, and to have no effect on human cancer. A recent study by,  conducted a double-blind, placebo-controlled trial to evaluate the incidence of second primary tumors in patients with resected nonsmall-cell lung cancer receiving selenium supplementation and reported it to have chemopreventive benefits in lung cancer. Inorganic form of Se, whose biological properties may be markedly different from those of other chemical forms of Se such as selenomethionine, an organic form commonly used in human trials. Toxicity of inorganic Se greatly exceeds that of organic Se.  The safe upper limit of intake of 400 μg/day in adults was suggested in 2000 by the Institute of Medicine. 
It is interesting to note that cancer cells are substantially more sensitive to selenite and more prone to apoptosis induction than normal cells, , and this gives an opportunity to use it as a therapeutic agent and moreover inorganic form of selenium sodium selenite is water soluble gets eliminated from the system. An ancillary study, involving patients added an arm assigning 400 μg/day Se supplementation in order to evaluate the effects of high Se exposure and possible dose-response trends.  The results showed a possible limited decrease of risk of nonmelanoma skin cancer in the 400 μg Se supplemented individuals.
Collagen produced by fibroblasts assists the wound in gaining tensile strength during repair. The subsequent changes in the oral mucosa reflect damage to connective tissue, including fibroblasts and blood vessels. This results in hyalinization of collagen, hypovascularity, and ischemia.  Previous records state that the increase in DNA content of wounds indicated hyperplasia (cellular proliferation) of cells.  In the present study, increase in levels of DNA and RNA was observed in the cancer tissues of untreated oral cancer patients. This may be due to abnormal cell proliferation and enhanced DNA- RNA polymerases. The hemi-body irradiation decreased hexosamine and DNA content.  with the cancer cell destruction in the cancer mass. The same is noticed in this study following radiation therapy after 6 months, with an appreciable decrease in cancer tissue nucleic acid level. Increase in the production of radiation mediated ROS led to a decrease in the nucleic acid contents in cancer site of radiation treated oral cancer group. Inactivation of enzymes like DNA, RNA polymerases leads to the impairment in the metabolism of nucleic acids due to single and double strand breaks.  It is also presumed that an enhanced damage to membrane as well as to DNA is associated with a decline in the production of adenosine triphosphate.
A significant decrease of the nucleic acid contents was observed in cancer tissue of the selenium supplemented groups (Group IIb). Selenium exerts its influence in reducing the nucleic acid content (Group IIb), through the formation of selenodiglutathione inside the cancer cells, where selenium undergoes reduction with the glutathione, which is normally present in excess inside the cancer tissue.  In its capacity as an antioxidant, selenium treatment could have elevated glutathione and certain antioxidant enzymes and reduce lipid peroxidation in irradiated wounds. This happens after the elimination of hypoxic tumor cells, where selenium acts as antioxidant in oxic atmosphere. Thus, selenium proves its dual role as an anticarcinogen and antioxidant and in addition as a radioprotective agent.
An increased fraction of soluble collagen represents newly synthesized collagen and increase in insoluble collagen signifies matured tissue. Oral malignancy has increased soluble collagen thereby decreased maturation.  In the present study the level of collagen in the OSCC patients is increased compared to normal tissue. And also there is an increase in the salt soluble collagen and acid soluble collagen but with a decrease in the insoluble collagen fraction in comparison with normal tissues. In the epidermis, the epithelial cell above the most basal cell layers has been shown to be stacked in columns of highly ordered structure. The filiform papillae seem to be formed through distortion of the columnar stacks of cells actually forming a chevron-like pattern.  Similar mode of formation of the chevron-like keratinization is seen in oral cancer. The normal dermis contains both type I and type II collagens with type one accounting for approximately 85%. It seems that newly formed collagen in dermal wounds consists primarily of type II collagen with also type I and that this ratio is not restored to normal till after several days. 
The healing of inflammation involves many complex mechanism, one of which is the enhanced deposition of collagen fibers with increased cross-links in granulated tissues.  The increased level of soluble collagen observed in oral cancer cases (Group II) was also found to be high in radiation treated group. The undesirable sequelae in consequence to radiotherapy includes xerostomia, granulocytopenia decreased saliva flow rate, mucositis so that the protective mucin coating of the epithelium is compromised.  These factors may be reasonable for the delay in maturation of the collagen at the treatment site, which overshadows the delay in wound healing.
Yet the meager improvement in collagen maturation shown by radiation group at 6 months, reflects the radiation effect getting subsided and also the decrease in the tumor mass. During the biosynthesis of collagen as chain synthesis proceeds and the polypeptide moves in to the cisternae of ER, various enzymes hydroxylate certain proline and lysine residues to hydroxyproline and hydroxylysine. Vitamin C, molecular oxygen and other factors are needed for this step. As antioxidant both enzymatic and nonenzymatic found to be deficient in oral cancer cases.  Vitamin C, iron deficiency and anoxia result in impaired collagen synthesis. This is evident that in oral malignancy the amount (percentage of hydroxyproline) of soluble collagen increases due to decreased maturation. It has been reported that there was an increase in the ratio of soluble collagen to insoluble collagen during periods of rapid growth. 
Studies have assessed that irradiation caused a dose-dependent delay in wound contraction and wound healing time. Ascorbic acid demonstrated that with increasing doses of gamma rays there was progressive destruction of the native collagen fibrils. Our data with the % of hydroxyproline goes in hand with these findings. Hexosamine, ground substratum for collagen synthesis, is known to increase during early stages of wound healing and decrease thereafter.  This could also be a considerable reason for the further increase of the hydroxyproline content in radiation group, which might have been showing a gradual decrease with elimination of tumor cells. A lengthy follow-up of patients would try to figure out the facts.
Healing phase is being characterized by the deposition of dense collagen bands. The degree of collagen maturation increased appreciably, corresponding to selenium supplementation (Group IIb), for 6 months. The radiation-induced cytotoxicity is mediated through the production of oxygen-derived free radicals. When ROS are overproduced, oxidative stress results with detrimental cytotoxic effects causing delayed wound healing. It can be concluded that in contrast to the increased levels of soluble collagen in the oral cancer tissue of the radiation group and the untreated oral cancer tissue, selenium group showed a decreased soluble collagen and increased insoluble collagen content indicating improvement in maturation of the collagen fibrils. This stands on to explain the scavenging property of selenium on oxidants and restoration of antioxidants.
This is supported by the luminescence spectral assignment which shows the altered biochemical, collagen structure and some biologically important molecules like FAD and tryptophan. Since the collagen tissues are highly heterogeneous, their architecture and biochemical composition may be altered as a function of metabolic and pathological conditions.  Selenium group showed reversion of collagen, FAD, tryptophan levels to near normal denoting maturation of the collagen fibrils.
The ultrastructural changes were prominent in the cancer condition. Elongated nuclei, scanty cytoplasm and wide ICS with clumping of tonofilaments and tubular mitochondria was observed in patients of the present study. Radiation treated groups showed degenerativenecrotic changes with irregular NM and swollen mitochondria. Disrupted ER with less keratinization, enlarged nuclei, mitochondrial hyperplasia with alterations of mitochondrial cristae was seen which may be due to the intense effect of radiation. Selenium supplemented groups showed pleomorphic nucleus, cytoplasmic granules condensed chromatin, globular ER less dense ICJs. These observations denote that selenium might have suppressed the keratinization patterns, reduced inter cellular spaces, maintained and preserved the intracellular organelles such as ER and mitochondria. These changes indicate the significance of selenium in mediating the morphological alteration at the cancer site following radiation, which in turn denotes the anticarcinogenic effect of selenium.
Micronuclei formation results from chromosomal damage by clastogenic agents present in tobacco and over production of ROS, carcinogens ascertaining genotoxic damage. There is a quantitative relationship between micronucleated cell (MNC) frequency and oral carcinoma. , In this study, a significant elevation in micronucleus was noticed in the exfoliated oral mucosal cells of untreated oral cancer patients and also in radiation group at initial month. This may be attributed to the chromosomal damage by carcinogens to dividing basal cells of the epithelium resulting in the production of micronuclei in the daughter cells, which migrates through the epithelium and are exfoliated. The frequencies of micro nucleated exfoliated cells in the radiation group showed a gradual decrease after 6 months of our study. Sensitive and resistant tumors to radiotherapy had different patterns of increase in MNC count. 
The drastic decrease in the micronucleus count observed in the selenium supplemented group after 6 months may probably due to binding of selenium to the carcinogenic metabolites and scavenging it or by converting them to stable compounds. Selenium was reported to have the capability of complexing with carcinogenic metabolites and eliminating them out of the system thus involving in detoxification mechanism and conferring protection to DNA and other cellular components from carcinogen attack. 
So owing to clastogenic effect of carcinogens and associated free radical induction with impaired antioxidant defense along with radiation mediated oxidative stress, the antioxidant therapy as an adjuvant to RT can be considered benevolent. Selenium as an antioxidant adjuvant could combat the oxidative stress considerably and improve the wound healing process, there by the degree of collagen maturation and thus have a beneficial effect on irradiated wounds and could be part of strategy to ameliorate radiation-induced delay in wound repair.
There are several possible explanations for alterations in wound healing after irradiation.
I Regarding synthesis of hexosamine and DNA contents, increased synthesis of hexosamine and DNA contents were speculated to be responsible for the accelerated healing of irradiated wounds. There are reports stating that ascorbic acid, aloe vera treatment significantly increased hexosamine and DNA contents at postirradiation, topical application and oral administration. In the present study the status with pretreatment of the antioxidant Selenium were not done and also the lengthy follow-up of selenium supplementation and its overall impact on the collagen maturation. So that the effect of selenium on synthesis of hexosamine and DNA contents on long term postirradiation period could have been estimated
II Wound repair results from a series of well-coordinated cellular and biochemical events, including increased synthesis of the bioregulatory molecule nitric oxide (NO). NO production promotes processes central to wound healing, such as inflammation, angiogenesis, fibroblast synthetic function, epithelial cell proliferation, regulation of collagen formation, and wound contraction in distinct ways. A direct negative impact of ionizing radiation on wound fibroblast proliferation, neovascularization, and collagen deposition has been reported. The pretreatment of mice with ascorbic acid improved collagen deposition, reduced the hyalinization, and increased the vascularity and fibroblast density in the ascorbic acid + irradiation group. Pretreatment of mice with ascorbic acid prior to irradiation enhanced NO levels significantly in the wound bed. This study did not overlook the above. With selenium treatment there could be a significant enhancement in the NO levels in the wound bed
III Ionizing radiation has been reported to cause severe damage to vital tissues, especially those with a high rate of cell division, such as the hematopoietic system.  Selenium might also provide strength to the regenerating wound by, improving the hematopoietic system causing early closure of the wound as revealed by wound contraction studies. This needs confirmation with selenium pre- post supplementations
IV DNA index is specific for malignancy. Patients with DNA diploid tumors have a better survival rate when compared with patients with aneuploid tumors.  Because "cancer" can override restriction points (R-Point) on cell cycles and consequently can avoid apoptosis or senescence.  The study on it may prove that DNA content in tissues may have a screening ability as diagnostic as well as prognostic significance. The drawback of this study is that we could not get cancer tissue of patients at the end of treatment (i.e.,) after 6 months of treatment. Hence changes in cell cycle parameters could not be evaluated
V Hypoxic cells present in many solid tumors can limit cure by ionizing radiation and perhaps by some drugs.  An indepth analysis in correlation to hypoxia would provide interesting as Selenium act as an anticarcinogenic agent undergoing thiol reduction forming anticarcinogenic metabolites in the hypoxic environment
VI The frequently overlooked differences in toxicity and biological activity of selenium compounds compels additional research efforts. Hence, an adequate assessment of various other selenium forms over its effect on collagen maturation, as an anticancer agent and to justify its administration in cancer therapy, remains relevant and merits further evaluation.
The above mentioned limitations, if done indepth could provide valuable informations pertaining to the putative impact of antioxidant mineral selenium on the cancer cells, in terms of wound healing and also in the retardation of the debilitating side-effects of radical radiotherapy.
| > Acknowledgement|| |
This research work was funded by University Research Funds of Catholic University of Daegu, Gyeongsan-Si, Gyeongbuk, Republic of Korea, in 2014.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
[Table 1], [Table 2], [Table 3], [Table 4]