EVALUTING THE ABILITY OF PLEUROTUS OSTREATUS AQUEOUS EXTRACT TO MODULATE GENOTOXICITY INDUCED BY CYCLOPHOSPHAMIDE IN MICE BONE MARROW CELLS

This study was amied to evaluate the ability of local oyster mushroom(Pleurotus ostreatus; ID:MF065715.1; cultivated until maturation in growth medium containing cumin extract) to modulate the genotoxicity induced by cyclophosphamid (CP) in bone marrow cells of mice. In vivo genotoxicity was assessed by quantifying the incidence of micronucleated polychromatic erythrocytes in the bone marrow cells of mice that were administered different doses of P.ostreatus extract (150, 200,250, and 300 mg/kg/day( respectively, individually, and in combination with CP (40 mg/kg body weight) according to the following three experimental protocols (pre 2h, post 2h, and concomitant treatment for 14 days , respectively). Analysis and microscopic examination of micronuclei (MN) revealed no mutagenic effect of P.ostreatus extract alone at all the doses evaluated. By contrast, CP administration significantly increased (P<0.05) incidence of MN.Importantly, co-administration of P.ostreatus extract with CP caused a significant and dose-dependent reduction in MN induced by CP in the murine bone marrow cells.These data suggest that P.ostreatus extract administration has a protective effect against genotoxic damage inflicted by CP. The dietary cumin may serve as a scavenger for free radicals generated by CP and may augment the antioxidant activity of P. ostreatus extract. These findings open up new avenues for the use of oyster mushroom in many applications, including pharmacological preparations and food


INTRODUCTION
Cancer is one of the leading causes of death worldwide. Commonly-used anti-neoplastic treatments either prevent the proliferation of or kill tumor cells. Cyclophosphamide (CP) , the most frequently used anti-cancer chemotherapeutic agent, is an alkylating agent used to treat a wide range of malignancies. CP's cytotoxicity results from its chemically reactive metabolites that alkylate DNA and proteins, and thereby generate cross-links (3). Damage to healthy somatic tissues and immunosuppression are the common sideeffects that limit the use of CP (17).The creation of free radicals and other reactive oxygen species, in addition to lipid peroxidation, have been identified as the main mechanisms of CP-induced toxicity (19).CP is generally used as a genotoxic agent since CP and its metabolites can bind to and damage DNA, leading to chromosome breaks, micronuclei (MN) formation, and finally, cell death (14).
Thus, co-administration of antioxidants and free radical scavengers may mitigate some of the adverse side-effects associated with CP treatment (22). Herbal antioxidants containing high concentrations of phenolic and flavonoid compounds, have been shown to have a strong chemo-protective influence against CP-induced DNA damage and oxidative stress in the bone marrow cells of mice (5,27). Numerous studies have demonstrated that high concentrations of antioxidants can alleviate oxidative stress that causes cellular damage, leading to the generation of malignant cells or accelerating aging (30). Oxidative stress stems from an imbalance between the production of free radicals in our bodies (due to a range of factors such as radiation exposure, food intake, and inhalation of certain substances from our environments), and the inability of our bodies to adequately counteract their damaging effects (6).One of the chemo-preventive strategies that can be readily incorporated into our daily lives to avert cancer is the utilization of natural compounds such as bioactive botanicals, food constituents,or functional foods (5).Mushrooms form an essential part of the normal human diet; they are a good source of a lot of minerals, digestible proteins, vitamins, and polysaccharides (12) .Mushrooms are a good source of several interesting biologically effective compounds that provide medical or health benefits such as disease prevention and treatment (12).Bioactive compounds of the mushroom have been found in cell wall polysaccharides (e.g.,b-glucans), among proteins, or the minor secondary metabolites including phenolic compounds, steroids, and terpenes. The efficiency and concentration of bioactive compounds differ among different mushroom types, depending upon conditions of fruiting (if cultivated), substrate, development phase/age, storage conditions ,cooking procedures, and age of the fresh mushroom (10).Oyster mushrooms comprise the most commonly consumed Pleurotus species, that are often described as oyster mushroom king. Oyster mushroom possess several biological functions due to the unique bioactive compounds they harbor, such as peptides, lipids, monoterpenes, polysaccharides, sterols, dietary fiber, and antioxidant components such as ascorbic acid, β-carotene, and α-tocopherol (20).These bioactive compounds have been reported to diminish the incidence of a variety of human diseases including cancers, inflammatory diseases, atherosclerosis, diabetes, and liver damage (2).Numerous studies have reported the medicinal properties of mushrooms, such as their anti-carcinogenic, anti-inflammatory, anti-viral, anti-bacterial, hypo-glycemic, anti-fungal, hepatoprotective, anti-neurodegenerative, anti-diabetic, antiangiogenic, and antioxidant properties (1,4).The objective of this study was to evaluate the ability of pleurotus ostreatus extract to modulate genotoxicity induced by cyclophosphamide in mice bone marrow cells.

MATERIALS AND METHODS Direct biochemical analysis of P.ostreatus mushroom
The nutritional components, including protein, over all fat,dietary fiber, and total phenolic content (TPC) , freeze-dried powder of P. ostreatus (ID:MF065715.1) were analyzed using the "American Oil Chemists' Society (AOCS)" standards (8). The TPC , statedas gallic acid equivalents (GAEs) was calculated according to Folin-Ciocalteu method (19). Laboratory animals: 108 mice were obtained from the National Center for Drug Control and Research, Ministry of Health, Baghdad. Their weight ranged from 25±2 g.They were assigned into six separate groups (as described in the "Experimental Design"section)under laboratory conditions. These mice were housed in polypropylene cages and were provided food in the form of commercial mouse food pellets, and water ad libitum throughout the experiment Cyclophosphamide treatment Cyclophosphamide (Endoxan; 500 mg) was intra-peritoneal injection into mice at a dose equivalent to 40 mg/kg defined as a genotoxic dose for mouse bone marrow cells (4).

Preparation of Cuminum cyminum extract
The cumin extract was prepared by mixing 1 kg of cumin seeds with 4 liters of distilled water in a glass beaker and then placed in ultrasound for 60 minutes at 40 ° C. the aqueous extract was filter then concentrated by using a rotary evaporator. A concentration of 20% was attended and was using in the treatment of mushroom medium Preparation of P. ostreatus extract The P. ostreatus mushroom (ID: MF065715.1) was cultivated in growth medium that contained cumin extract at (20%) concentration for 14 days, until mushroom maturation was complete (28). Whole mature mushrooms were dried and then powdered. The (5 g) powder was extracted by stirring with 90 mL of ethanol (96%) at 30°C for a time of 72 h. The final extracts were centrifuged at 20°C at 3,000 rpm for 15 min, the supernatant was filtered and concentrated in a rotary evaporator (BÜCHI R-114, Switzerland) at 40°C to dryness, and redissolved in water for antigenotoxic assay to starting concentration of 20.0 mg/mL (9).

Experimental design
The animals were randomly assigned to 6 groups (G): G1:Negative control(6 mice) were given phosphate-buffered saline(PBS). G2:Positive control (6 mice) were treated with 40 μg/kg CP for 24h. The animals were euthanized 2 h after the treatment. G3:Mice (6 mice per dose; 24 mice total) were treated with four doses of P. ostreatus extract (150, 200,250, and 300 mg/kg) delivered orally for 14 days. The animals were euthanized 2 h after the treatment.

Micronucleus (MN) test
The right and left femurs of the mice were dissected and fetal calf serum was used to flush out the bone marrow from the femoral cavity. Bone marrow cells were dispersed by gentle pipetting and pelleted through centrifugation at 1,000 rpm for 10 min. The cell pellet was re-suspended in a small volume of fetal calf serum that allowed cell smears to be prepared on microscope slides. The smears of cells were stained with Giemsa after complete air-drying, while, 2,000 polychromatic erythrocytes (PCEs) from every animal were inspected to quantitate micronucleated polychromatic erythrocytes (MN-PCEs) under 1,000× magnifications using the light microscope. Furthermore, toxicity to bone marrow was evaluated using the PCE/ normochromatic erythrocyte (NCE) ratio in 500 erythrocytes from each animal (11). The amount of antimutagens effects was measured according to (15) as follows: Antimutagens %

Statistical analysis
The Statistical Analysis System-SAS (2012) program was used to effect of difference factors in study parameters . Least significant difference -LSD test (ANOVA) was used to significant compare between means in this study.

RESULTS AND DISCUSSION
Cumin seeds is using as food for oyster mushrooms due the role it plays in improving the nutritional and treasury properties ,Cumin seeds are nutritionally rich; they provide high amounts of fat (especially monounsaturated fat), protein, and dietary fibre. Vitamins B and E and several dietary minerals, especially iron, are also considerable in cumin seeds Table1.The literature presents ample evidence for the biological and biomedical activities of cumin, which have generally been ascribed to its bioactive constituents such as terpenes, phenols, and flavonoids (16).  Table 3 shows the results of MN assay in the bone marrow cells of mice from our various experimental groups. the results showed significant differences (P>0.05) between groups that were treated with P. ostreatus extract alone and positive controls, on the other hand, We found no significant difference (P>0.05) between groups that were treated with P. ostreatus extract alone and our negative controls , suggesting that P. ostreatus extract does not cause significant genotoxicity on its own.     (25). Unfortunately, treatment with CP was found to encourage the incidence of secondary tumors that were treatment-related in human cancer survivors due to the drug's genotoxic effects on healthy somatic cells. Moreover, the toxic side-effects of CP were related to its metabolite acrolein (19).These adverse side-effects may have resulted from oxidative stress that may have diminished the antioxidant activities of key enzymes and caused a strong increase in free radicals and lipid peroxidation (30).The failure of cellular antioxidant defenses concomitant with a surge in oxidative stress due to ROS overproduction has been found to induce the formation of bone marrow MN and chromosomal breakage (5) ;therefore ,co-administration of antioxidants and free radical scavengers may help reduce treatment-associated genotoxicity. More recently, anti-cancer treatment regimens involving CP are increasingly incorporating other protective and detoxifying agents (5).In this study, a new local strain of the oyster mushroom P. ostreatus that was cultivated in growth medium containing cumin extract was used and the ability of P. ostreatus extract to modulate the genotoxic effects of CP on murine bone marrow cells was evaluated. The obtained data showing that even the highest dose of P. ostreatus extract did not cause any significant genotoxicity compared to the negative control group (Table 3) indicated that the P. ostreatus extract was neither mutagenic nor clastogenic; thus, the P. ostreatus extract can potentially be used as a protective agent for oxidative stress without any side-effects. Furthermore, administration of the P. ostreatus extract at doses of 150, 200, 250, and 300 mg/kg/day for 14 days prevented the formation of MN in the bone marrow cells of CP-treated mice in a dose-dependent manner, thereby revealing the anti-clastogenic effects of the mushroom extract as well as its ability to protect cells from the genotoxic effects of CP. Previous studies have noted that chemical composition of the same mushroom can differ between lineages (26).Hence, the conditions of growing, harvesting, preparing, and storing also have their influence on the composition and the mushroom's biological activity (17).Therefore, the cumin (Cuminum cyminum L.) extract was used to enhance the mushroom's antioxidant activity. Many researchers recorded that cumin which has a high phenolic content and an accepted antioxidant activity that can be used as a supplement for both nutritional purposes as well as for preservation of foods (9). Cumin's antioxidant effects may be associated with the presence of phenolic compounds, anthocyanin, and essential oils within cumin (2).Cumin seeds have anti-carcinogenic properties and can help prevent the development of stomach or liver tumors in laboratory animals (11).