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Year : 2016  |  Volume : 12  |  Issue : 1  |  Page : 386-394

Modulatory effect of Pleurotus ostreatus on oxidant/antioxidant status in 7, 12-dimethylbenz (a) anthracene induced mammary carcinoma in experimental rats - A dose-response study

Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai University, Chidambaram, Tamil Nadu, India

Date of Web Publication13-Apr-2016

Correspondence Address:
Mirunalini Sankaran
Department of Biochemistry and Biotechnology, Annamalai University, Annamalai Nagar, Chidambaram - 608 002, Tamil Nadu
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0973-1482.148691

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 > Abstract 

Background: Worldwide, breast cancer is the second most prevalent cancer among women and its incidence is increasing alarmingly.
Aim: To determine a dose-response effect of Pleurotus ostreatus on oxidant/antioxidant status in 7,12-dimethylbenz. (a) antheracene induced. (DMBA) mammary carcinoma in experimental rats.
Materials and Methods: Cancer bearing female Sprague Dawley rats were orally treated with Pleurotus ostreatus ethanolic extract (POEet) (150, 300 and 600 mg/kg body weight) for 16 weeks. By means of high performance liquid chromatography (HPLC) analysis, ergosterol (48.82%) were identified and quantified in POEet. Body weight of experimental rats in each groups and the biochemical parameters of plasma, liver and mammary tissues were carried out. Histopathological analyses were also determined.
Statistical Analysis Used: Results were analyzed using SPSS software package, version 16.0. The values were analyzed by one way analysis of variance (ANOVA) followed by Duncan's multiple range test (DMRT).
Result: The result showed that depleted activities of enzymatic and non-enzymatic antioxidant level and significant elevated TBARS level were observed in DMBA group of plasma, mammary and liver tissues of experimental rats. The effects were dose.dependent and the above noted parameters were renovated to near normal after supplementation with different dose of POEet (150 mg, 300 mg and 600 mg/kg bwt). The data obtained from the study indicate that POEet at a dose of 600 mg/kg bwt possesses optimum anticancer effects against DMBA induced mammary carcinogenesis.
Conclusion: Based on the scientific appraisal, we conclude that the POEet is having a potent antioxidant capacity; thereby it offers maximum protection against DMBA-induced mammary carcinogenesis

Keywords: 7, 12-dimethylbenz (a) antheracene, Antioxidants, high performance liquid chromatography, Lipid peroxidation, Pleurotus ostreatus

How to cite this article:
Krishnamoorthy D, Sankaran M. Modulatory effect of Pleurotus ostreatus on oxidant/antioxidant status in 7, 12-dimethylbenz (a) anthracene induced mammary carcinoma in experimental rats - A dose-response study. J Can Res Ther 2016;12:386-94

How to cite this URL:
Krishnamoorthy D, Sankaran M. Modulatory effect of Pleurotus ostreatus on oxidant/antioxidant status in 7, 12-dimethylbenz (a) anthracene induced mammary carcinoma in experimental rats - A dose-response study. J Can Res Ther [serial online] 2016 [cited 2021 Jan 19];12:386-94. Available from: https://www.cancerjournal.net/text.asp?2016/12/1/386/148691

 > Introduction Top

Cancer is one of the most fatal diseases, which involves morphological cellular transformation, uncontrolled cellular proliferation, deregulation of apoptosis, invasion, angiogenesis and metastasis.[1] Breast cancer is the most common malignancy and major cause of death in women, makes up about one-tenth of all new cancer diagnosis worldwide.[2] As estimated, in the US 2013 alone approximately 233,000 new cases of breast cancer and nearly 40,000 deaths occurred in women due to this disease.[3] In India, breast cancer is the second most common cancer next to cervical cancer with an estimated 1, 15, 251 new cases every year.[4] The etiology of breast cancer is multifactorial which include age, early age at menarche, late age of menopause, and late age at first pregnancy, obesity, oral contraception, hormone replacement therapy, diet, family history, lactation and prior history of benign breast diseases.[5]

In addition to the general risk factors, the major source of human exposure are cigarette smoke, charbroiled food, automobile exhaust and specially the environmental contaminant, which are produced during incomplete combustion of organic materials such as polycyclic aromatic hydrocarbons (PAHs) that are released from industries, domestic oil furnaces, gasoline and diesel engines are also known to cause breast cancer. Among PAHs, 7, 12-dimethylbenz [a] antheracene (DMBA) can be used to induce experimental mammary carcinogenesis in rats and this process involves disruption of tissue redox balance, in turn, this suggest that biochemical and pathophysiological disturbances may result from oxidative stress.[6] Moreover, oxidative stress may also result from increasing free radical generation and decreases the efficiency of antioxidant defence mechanism in the target cells and tissues that play an important role in chemical carcinogenesis.[7] Conversely, antioxidants can interact with oxidative process by reacting with free radicals, chelating free catalytic metals and acting as oxygen scavengers. Thus, antioxidant defensive systems have co-evolved with aerobic metabolism to counteract oxidative damage of reactive oxygen species (ROS) and product from cellular and molecular damages.[8]

Even though, a vast number of cancer research have been devoted in the development of antineoplastic drugs, the prognosis of the diseases is often challenging due to increased side effects. Therefore, there is an urgent need for alternate preventive approaches through dietary means and the natural agents with efficacy and acceptable toxicity are considered to be the winning strategy in reducing the morbidity and mortality of breast cancer.[9] In recent years, natural dietary agents especially fruits, vegetables, sprouts and mushrooms have drawn a great deal of attention both from researchers and from the public because of their potential antioxidant and anticancer activities.[10]

Pleurotus ostreatus (Jacq.ex.fr) P.kumm.is the second most important cultivated mushroom for food purposes throughout worldwide.[11] Nutritionally, it has unique flavor and aromatic properties and is considered to be rich in protein, fiber, carbohydrates, minerals and vitamins as well as low fat.[12],[13] Many researchers from different regions of the world confirmed that the Pleurotus mushroom having various bioactive compounds including polysaccharides, terpenoids, steroids, phenols, alkaloids, lectins and nucleotides, which have been isolated and identified from the fruit body, mycelium and culture broth of mushrooms. In addition, these active compounds have promising biological effects that protect the body against various disease aliments.[14]

In many cases, exploratory drug safety research is essential to evaluate the novel targets in pre-clinical investigation, if the biology of the targets is poorly understood, which suggest adverse and undesirable risk factors. The outcome of such assessments can be useful for the backup program where the goal is often an improved safety margin.[15] Based on this study, our recent toxicological study on P. ostreatus have been described the high margin of safety up to the dose of 5,000 mg/kg bwt on experimental animals.[16]

The polysaccharide fraction of P. ostreatus has potent antitumor activity in Ehlich tumor and sarcoma 180 and the antitumor substance was postulated to be the β-glucan fraction.[17] However, the anticancer effects of lipid fractions have not been well studied. To elucidate this, we aimed to investigate the anticancer effects of P. ostreatus against DMBA induced mammary cancer in Sprague Dawley rats; hence, we made an attempt to use this rapid and non-invasive administration procedure during the dose-dependent preclinical experiments and to highlight the issues that have to be considered before starting this type of study.

 > Materials and Methods Top


The 7, 12 – dimethylbenz (a) anthracene (DMBA), nitrobluetetrazolium (NBT), phenazine methosulfate (PMS), thiobarbituric acid (TBA) nicotinamide adenine dinucleotide phosphate (NADPH), 5, 5′-dithio-bis-nitrobenzoicacid (DTNB) were purchased from Sigma Chemical Pvt. Ltd., Bangalore, India. All other chemical reagents were of analytical grade.


Pleurotus ostreatus mushrooms were collected in and around areas of Udhagamandalam, Nilagiri district, Tamil Nadu. The mushroom was taxonomically identified and voucher specimen (No: 233) was deposited in the herbarium of Botany, Department of Botany, Annamalai University.

Preparation of mushroom ethanolic extract

The fresh fruiting bodies of P. ostreatus were dried in shade conditions and the dried materials were pulverized in a blender to get coarse powder. For P. ostreatus fruiting bodies ethanolic extraction, 5 g of the powder was extracted with 100 mL of 95% ethanol using a Soxhlet apparatus. The solvent was evaporated on a rotary evaporator (Buchi Rotarvapour, Switzerland) under reduced pressure and controlled temperature (40-50°C).[18] A dark semisolid material (6% yield) thus obtained was stored at 4°C until use. A known amount of the residual extracts were suspended in distilled water and was orally administrated to the animals by gastric intubation.

HPLC analysis

High performance liquid chromatography (HPLC) method was performed on Shimadzu HPLC class VP series with two LC-10AT VP pumps (Shimadzu). Variable wavelength programmable phot diode array detector SPD-M10A VP (Shimadzu) CTO-10 AS VP column oven (Shimadzu), SCL-10A VP system controller (Shimadzu) and reverse phase luna C-18 phenomonex column (250 mm × 4.6 mm) was used. The mobile phase components methanol: Water (80:20) were filtered through 0.2 µ membrane filter before use and were pumped from the solvent reservoir at a flow rate of 1mL/min, which yield column backup pressure of 260-270 Kgf/cm 2. The column temperature was maintained at 27°C. A total of 20µl of respective sample was injected by using Rheodyne syringe (Model 7202, Hamilton). The elution was carried out with gradient solvent system with a flow rate of 1 mL/min at ambient temperature (25-28°C). Total running time was 15 min, the sample injection volume was 20µl while the wavelength of ultraviolet–visible spectroscopy (UV-VIS) detector was set at 290 nm.

In vivo studies

The experiment was carried out with 6-week-old adult female Sprague Dawley rats, weighing approximately 130-150 g that were obtained from National Institute of Nutrition, Hyderabad and maintained in the Central Animal House, Rajah Muthiah Medical College and Hospital, Annamalai University. The rats were housed in polypropylene cages at room temperature (27 ± 2°C) with relative humidity 55 ± 5%, in an experimental room. In Annamalainagar, the LD (light: dark) cycle is almost 12: 12 h. The rats were maintained as per the principle and guidelines of the ethical committee for animal care of Annamalai University in accordance with the Indian National Law on animal care and use. The rats had free access to standard pellet diet (Amrut Laboratory Animal Feed, Mysore Feed Limited, Bangalore, India) and water ad libitum were available to the animals throughout the experimental period and replenished daily. The standard pellet diet comprised of 21% protein, 5% lipids, 4% crude fiber, 8% ash, 1% calcium, 0.6% phosphorous, 3.4% glucose, 2% vitamin and 55% nitrogen free extract (carbohydrate) and it provides metabolizable energy of 3600 kcal/kg.

The study was conducted in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH, 1978). Animal studies in the work have been performed as per the Institutional Animal Ethical Committee of Rajah Muthiah Medical College (Reg. No: 160/1999, Proposal number: 947 CPCSEA), Annamalai University, Annamalainagar.

Experimental design

Animals were assorted into six groups of six animals each according to the following experimental regimen. Animals in Group I were induced with DMBA 25 mg in 1mL of vehicle (0.5 mL of sunflower oil in 0.5 mL of saline).[19] The Group II, III and IV were subjected to DMBA induction as described in Group I. In addition, the animals received different doses of POEet (150, 300 and 600 mg/kg body weight), by gastric intubation. The Group V control animals received POEet (600 mg/kg bwt) alone throughout the experimental period. Group VI served as normal control animals.

The experiment was terminated at the end of the 16th week and all the rats were kept overnight fast and anesthetized using ketamine chloride (24 mg/kg body weight) by intramuscular injection and sacrificed by cervical decapitation between 8.00 am to 10.00 am. Blood was collected in clean dry test tubes with few drops of heparin and plasma obtained was used for various biochemical estimations. Tissues such as liver and mammary tissues were removed, cleared off blood and immediately transferred to ice-cold containers containing 0.9% Nacl. The tissues were homogenized in a appropriate buffer and used for the estimation of various biochemical parameters.

Histological assay

For histological assays, three rats from each group were perfused with physiological saline, followed by formalin (10% formaldehyde). The mammary tissues were excised immediately and fixed in 10% formalin. The mammary tissues were sliced and embedded in paraffin wax, 3-5μm thick sections were cut in a rotary microtome and were stained using hematoxylin and eosin stain. The specimens were evaluated with a light microscope. All histopathological changes were examined by the pathologist.

Biochemical assessments

The lipid peroxidation was estimated as evidenced by the formation of thiobarbituric acid reactive substances (TBARSs). The TBARSs in plasma, liver and mammary tissues were assayed by the method of Niehaus and Samuelson (1968).[20] The activities of enzymatic antioxidants such as superoxide dismutase (SOD) in plasma, mammary and liver tissues were done by the method described by Kakkar et al. (1984). The catalase (CAT) activity in plasma, mammary and liver tissue was assayed using the method analyzed by Sinha (1972), and the activities of GPx in plasma, mammary and liver tissues were determined by Rotruck et al. (1973).[21],[22],[23] The non-enzymatic antioxidant activities was estimated by reduced glutathione (GSH), vitamin C and vitamin E in plasma, mammary and liver tissues by the methods of Ellman's (1959) method, Roe and Kuether et al. (1943) and Baker et al. (1980), respectively.[24],[25],[26]

Statistical analysis

Statistical analysis was performed using Statistical Package for the Social Sciences SPSS software package, version 16.0. The values were analyzed by one way analysis of variance (ANOVA) followed by Duncan's multiple range test (DMRT). All these results were expressed as mean ± SD for six rats in each group. P < 0.05 were considered as significant.

 > Results Top

Identification and Quantification of ergosterol by HPLC fingerprint

HPLC associated with UV-Vis detector was employed to identified and quantify the active compound (ergosterol) present in POEet according to their retention time (RT) against the authentic standard. The extract was tested to determine its RT of ergosterol (RT: 6.742). The data presented in this study by HPLC fingerprint shows that P. ostreatus extract contain compound such as ergosterol (48.82%). The concentrations were determined by calculation the HPLC peak area, which are proportional to the amount of analytes in a peak. The ergosterol present in P. ostreatus was compared with the standard ergosterol (RT: 6.783 min). [Figure 1](a) and [Figure 1](b) show the HPLC chromatogram of P. ostreatus extract and standard ergosterol, respectively.
Figure 1: (a) HPLC chromatogram of POEet detection was at 290 nm. Peak: Ergosterol (b) HPLC chromatogram of standard Ergosterol (detection at 290 nm)

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Effect of POEet on bodyweight of DMBA-induced sprague Dawley rats

The body weight of Sprague Dawley rats in the control and experimental groups is represented in [Figure 2]. We observed that the body weight was decreased in DMBA group with respect to control group, whereas treatment with POEet (150, 300 and 600 mg/kg bwt) showed a significant improvement in the body weight of tumor- bearing groups. The maximum weight was observed at a dose of 600 mg/kg bwt. No significant changes were observed in control and POEet alone treated groups.
Figure 2: Effect of POEet on the changes of bodyweight of control and experimental rats. Group I: DMBA, Group II: DMBA + POEet (150 mg/kg bwt), Group III: DMBA + POEet (300 mg/kg bwt) Group IV: DMBA + POEet (600 mg/kg bwt), Group V: POEet alone (600 mg/kg bwt), Group VI: Control. Values are expressed as mean ± SD for six animals in each group. Values not sharing a common superscript differ significantly at P < 0.05 (DMRT)

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Gross appearance and histological alteration

The size of the tumor observed in DMBA + POEet (600 mg/kg bwt) treated rats was very small as compared to rats induced with DMBA alone. [Figure 3]a shows the gross appearance of mammary tumor of DMBA alone group and [Figure 3]b shows the gross appearance of mammary tumor of DMBA + POEet (600 mg/kg bwt) treated group. [Figure 4] represents the histopathological examination of mammary tissue section of control and experimental rats. The control and POEet alone treated group showed normal ductal architecture. Tumor-bearing animals induced with DMBA showed altered ductal architecture indicating invasive ductal carcinoma. Whereas, tumor-bearing animal treated with POEet showing improved ductal architecture, thereby proving the anticancer effect of POEet.
Figure 3: Gross appearance of DMBA and DMBA + POEet treated rats. (a) shows the mammary adenocarcinoma in DMBA induced female Sprague Dawley rat and (b) the gross appearance of the mammary carcinoma in DMBA + POEet (600 mg/kg bwt) treated female Sprague Dawley rats

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Figure 4: Photomicrographs of histopathological changes in the mammary tissues of control and experimental rats (H and E ×40). (a) (DMBA induced rats) showing invasive ductal carcinoma with abnormal cellular proliferation. (b) DMBA + POEet (150 mg/kg bwt) shows florid ductal hyperplasia (c) DMBA + POEet (300 mg/kg bwt) shows ductal hyperplasia (d) DMBA + POEet (600 mg/kg bwt) shows ductal dysplasia. (e) POEet alone (600 mg/kg bwt) showing normal ductal epithetlial architecture and (f) Control shows normal ductal epithelium cells

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Effect of POEet on the level of TBARS in control and experimental animals

The levels of TBARS in plasma, mammary and liver tissues are represented in [Figure 5]. The lipid peroxidation marker enzyme TBARS was significantly elevated in tumor-induced groups compared with control group. However, the oral administration of POEet at different doses of 150, 300 and 600 mg/kg bwt significantly reduced the level of TBARS when compared with DMBA groups. At the high dose of POEet (600 mg/kg bwt), the effect was being more noticeable. Furthermore, no significant difference on TBARS level in POEet alone treated group when compared to control animals.
Figure 5: Effect of POEet on lipid peroxidation in plasma, mammary and liver tissues of control and experimental rats. Plasma (mM/dL), mammary and liver tissues (mM/100 g tissue). Values are expressed as mean ± SD for six animals in each group. Values not sharing a common superscript differ significantly at P < 0.05 (DMRT)

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Influence of POEet on the activities of enzymatic antioxidants in plasma, mammary and liver tissues of control and experimental animals

The activities of enzymatic antioxidants like SOD, CAT, GPx in plasma, mammary and liver tissues of control and experimental animals are depicted in [Table 1]. In DMBA group, the enzymatic antioxidant levels were significantly decreased in plasma, mammary and liver tissues when compared with control group. However, oral supplementation of POEet at the different doses of (150, 300 and 600 mg/kg bwt) significantly reversed these changes to near normal, but the effect was more pronounced in DMBA treated POEet group at a dose of 600 mg/kg bwt. However, the administration of POEet alone animals did not show any significant changes on enzymatic antioxidant levels when compared with control group.
Table 1: Effect of POEet on the enzymatic antioxidants in control and experimental animals

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Effect of POEet on the level of non-enzymatic antioxidants in plasma, mammary and liver tissues of control and experimental animals

The data presented in [Table 2] shows the effect of POEet on the activities of non-enzymatic antioxidants in plasma, mammary and liver tissues of control and experimental animals. In DMBA group, the levels of GSH, Vitamin C and Vitamin E were significantly lower in plasma, mammary and liver tissues when compared with control groups. However, oral administration of POEet at different doses (150, 300 and 600 mg/kg bwt) significantly reversed these changes to near normal, but the effect was more pronounced in DMBA treated with POEet group at a dose of 600 mg/kg bwt, although POEet alone treated animals did not show any significant changes on non-enzymatic antioxidant levels when compared with control groups.
Table 2: Effect of POEet on the Non-enzymatic antioxidants in control and experimental animals

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 > Discussion Top

Phytoconstituents obtained from natural source have been gaining importance in day-to-day because of the vast chemical diversity. So, there is a need to ensure the quality, safety and efficacy of natural drugs. Phytochemcial standardization is one of the tools for the quality assessment, which include preliminary phytochemical screening and HPLC analysis.[27] Moreover, HPLC identification test are required to confirm the presence of the active constituents and potential adulterant in natural drugs. Therefore using newer analytical techniques as marker can be generated for the research to evaluate the quality of the phytoconstituents and also incorporate in pharmacopeias. The present study was intended, to evaluate the antioxidant potentials and anticancer effects of POEet on DMBA induced mammary carcinogenesis in Sprague Dawley rats. Furthermore, we have investigated the presence of ergosterol (48.82%) in POEet by HPLC analysis. In this view, Chiochhio et al., (2011) and Ana villares et al., (2014), have also reported that mushroom contain ergosterol, which is responsible for maximum antioxidant capacity.[28],[29]

Many clinical studies explore the potential of naturally occurring antioxidants act as radical scavenger and inhibiting other free-radical mediated process thereby protecting the human body from various diseases including cancer.[30] Redox disturbances are known to negatively affect body weight system through generation of reactive oxygen species (ROS).[31] Experimental data as well as clinical and epidermological findings also indicated that the elevated level of ROS have been implicated to damage many biomolecules and exert diverse cellular and molecular effects including mutagenicity, cytotoxicity and changes in gene expression that lead to initiation and promotion of carcinogenesis.[32]

In this study, the body weights of the animals were recorded from the day of tumor induction until the completion of the experimental period. In cancer bearing rats, there were substantial weight losses observed because of cancer cachexia, anorexia or malabsorption.[33] The weight loss is due to depletion of both adipose tissues and skeletal muscle mass; whereas the non-muscle protein compartment is relatively preserved, thus distinguishing cachexia from simple starvation.[34] In addition, muscle loss occurs due to increased amount of alanine originating from muscle degradation and cultivation for gluconeogenesis.[35] However, oral administration of POEet showed a gradual increase in body weight when compared with DMBA induced positive control. Results from the previous worker suggest that P. ostreatus, which mediate the favorable changes due to the presence of various bioactive compounds like fiber, carbohydrates, non-starch polysaccharides (β-glucan), steroids and triterpenoids as well as their antioxidant properties.[13],[36] In addition, earlier reports suggest that the P. ostreatus had a rich content of protein and the superior quality of this mushroom may be because of this genus contain complete proteins with the well distribution of essential amino acids, as well as non-essential amino acids, which might be the key factor for the improved body weight of the POEet-treated rats.[37]

Toxic manifestation of DMBA induced oxidative stress is associated with a wide range of macromolecular damages such as lipids, proteins and nucleic acids, thereby producing interrelated derangements of cellular metabolism including peroxidation of lipids. Thus numerous studies have demonstrated that peroxidation of membrane lipids are together associated with the initial stage of carcinogenesis altering the normal biochemical process.[38] Increased incidence of oxidative stress and lipid peroxidation are implicated as one of the basic mechanism for tissue damage and the extent of tissue damage can be estimated by TBARS.[39] We observed that elevated TBARS level of plasma, mammary and liver tissues in breast cancer rats can be related to overproduction and diffusion of free radicals from the damaged tumor tissues when compared with control rats. Conversely, the increased plasma, mammary and liver tissues TBARS were observed in experimental mammary carcinogenesis.[40],[41] Our results also corroborate with the above finding where DMBA induced tumor bearing animals showed increased TBARS level in plasma, mammary and liver tissues. Whereas, oral administration of POEet exhibited significant dose-dependent alteration in TBARS, which presumably is due to its ability to inhibit free-radical generation. Previous report also suggest that P. ostreatus exerts its potent scavenging action on hydroxy anion and superoxide anion in vitro antioxidant assays.[18],[42] Hence, from the above mentioned doses, it is speculated that POEet (600 mg/kg bwt) effectively scavenged free radicals thereby reducing the oxidative damage.

Antioxidant enzymes present in the biological system functions interactively and synergistically to neutralize free radicals and also involved in various action such as prevention, interception and repair.[43] Enzymatic antioxidants act as the primary line of defence against ROS, and their usefulness in estimating the risk of oxidative damage induced during carcinogenesis has been studied. The SOD is a major intracellular enzyme, which protects against free radicals by catalyzing the SOD free radicals and anion to hydrogen peroxide and oxygen, which is further removed by CAT and GPx. Similarly, the specific activity of CAT is a peroxisomal hemoprotein that catalyzes the decomposition of H2O2 to O2. GPx is an equally important antioxidant, which reacts with H2O2 thus preventing intracellular damages caused by the same.[44],[45],[46] During cancerous condition tumor cells have abnormal activities of antioxidant enzymes.[47] Several reports have showed decreased enzymatic antioxidant activity in various cancer studies.[48] Thus, decreased activity of these enzymes in cancerous condition may be due to super saturation of enzymes with a high concentration of ROS formation.[49] Our study also in line with the finding, as decreased enzymatic antioxidant activities in plasma and tissues in DMBA untreated groups. whereas, The administration of the POEet to breast cancer rats restored the antiperoxidative enzyme (SOD, CAT and GPx) activities to near normal levels, which may be due to its antioxidant potency of ergosterol against breast cancer-induced free radical generation.

The non-enzymatic antioxidants from the second line of defence mechanism to scavenge excessively generated ROS in the system. The antioxidant such as GSH, vitamin C and vitamin E play an excellent role in protecting the cell from oxidative damage. The GSH, an essential cofactor for GSH transferase/GSH-peroxidase/GSSG reductase system, protects cellular proteins against oxidation through the glutathione redox cycle and also directly detoxifies ROS and neutralizes reactive intermediates generation from exposure to xenobiotic including chemical carcinogens.[50] GSH serves as a marker for evaluation at both intracellular and extracellular levels.[51] Vitamin C (ascorbic acid) is an important H2O soluble antioxidant in biological fluid and an essential micronutrient required for normal metabolic functioning of the body. The availability of vitamin C is a determined factor in controlling and potentiating many aspects of host resistance against cancer. It is showed to directly react with superoxide, hydroxy radicals and singlet oxygen.[52] Decreased level of water soluble antioxidant in cancer bearing rats may be due to the utilization of antioxidant to scavenge free radicals.[53] Vitamin E the major lipid soluble antioxidant present in all cellular membranes acts as a powerful terminator of lipid peroxidation.[54] The decreased level of vitamin E was observed in plasma and tissue of DMBA rats, which may be due to reduced vitamin C and GSH levels that can result in reduced conversion of α- tocopheroxyl radical to α-tocophenol. Our present experimental investigation exhibited a significantly decreased in the level of GSH, vitamin C and vitamin E in plasma and tissues of DMBA induced untreated rats. Our present experimental investigation exhibited a significant decrease in the levels of GSH, vitamin C and vitamin E in plasma and tissues of cancer-induced groups. Our results were consistent with the earlier researches Padmavathi et al., (2006) and Pandi et al., (2010) have reported decrease in the levels of GSH, vitamin C and vitamin E in mammary gland carcinogenesis of Sprague Dawley rats.[55],[56] Administration of POEet significantly reversed the activities of abovementioned enzymes to near normal levels that indicate its antioxidant and anticancer properties. Thus, the anticarcinogenesis properties of P. ostreatus may be attributed due to its carbohydrates component mostly β-glucan content, terpenoids, proteins and steroids, which exerts its role through the enhancement of antioxidant system.[57] Existing reported state that ergosterol can act as anti-inflammatory and inhibiting cycloxygenase (COX) enzyme thus terminating the propagation of cancer reactions.[58] On the other hand, ergosterol besides acting as an antioxidant, enhances the antioxidant defence system thereby inhibiting oxidative stress and prevent tissue damage.[59] In addition, Subbiah et al., 2003 also reported that ergosterol provide significant antioxidant potential against the different cancer model.[60] Thus our results collaborate well with the above findings.

Taken together, DMBA induced mammary cancer increased oxidative stress by diminishing the cellular antioxidant which confirms precancerous stages in Sprague Dawley rats. Whereas, oral administration of POEet at dose of 600 mg/kg bwt was found to be optimal dose, which effectively restored the antioxidant levels to normal levels. This decreased oxidative damage which may probably suppress the progression of precancerous cells prior to malignant development.

 > Conclusion Top

Our finding is the first report on the antioxidant potential of P. ostreatus on mammary carcinogenesis in Sprague Dawley rasts, to our knowledge. We performed to hypothesize the in vivo oxidant/antioxidant capacity of P. ostreatus on DMBA-induced mammary cancer in a dose-dependent manner. In this connection, our results signify that the high dose of POEet was potent in inhibiting the mammary cancer than the low dose and the medium dose. Considering the antioxidant property of P. ostreatus, the bioactive compound derived from whole fruiting bodies of mushroom can be supplemented with anticancer medicines. On the basis of our data, P. ostreatus contain active constituent like ergosterol, which may be responsible for anticancer effects. It is worth emphazing that P. ostreatus have considerable anticancer potential for improving human health if used on a regular basis and also used in natural product pharmaceuticals. These preliminary data indicate needs for further elaborated research on the molecular mechanism of P. ostreatus, which remains to be studied in our laboratory.

 > Acknowledgments Top

The authors would like to acknowledge for the Finacial Support from University Grants Commission (UGC) - Major Research project (F. NO: G7/17342/2012 (SR)), New Delhi, India to carry out the work successfully.

 > References Top

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]

  [Table 1], [Table 2]


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