|Year : 2018 | Volume
| Issue : 1 | Page : 60-70
Ameliorative role of ethanolic extract of Moringa oleifera leaf on aflatoxin B1-induced genotoxicity and biochemical alterations in rats
Ibrahim M Farag1, Hanaa M Roshdy1, Hasnaa A Radwan1, Inas S Ghaly1, Sherifa H Salah1, Emam A Abdel-Rahim2, Aboelfetoh M Abdalla3
1 Department of Cell Biology, National Research Centre, Giza, Egypt
2 Department of Biochemistry, Faculty of Agriculture, Cairo University, Giza, Egypt
3 Department of Horticultural Crops Technology, National Research Centre, Egypt
|Date of Submission||30-Nov-2017|
|Date of Acceptance||14-Mar-2018|
|Date of Web Publication||19-Jul-2018|
Hasnaa A Radwan
Department of Cell Biology, National Research Centre, Dokki, Giza 12311
Source of Support: None, Conflict of Interest: None
Background/aim The present study was conducted to assess the ameliorative role of Moringa oleifera leaf extract (MOLE) on genotoxicity and biochemical alteration of aflatoxin B1 (AFB1) in rats.
Materials and methods The rat groups involved negative control, control of DMSO, positive control that received AFB1 in DMSO (0.7 g/kg, body weight) four times weekly for 1 month, groups 4–6 that received the same dose of AFB1 in DMSO at the same period plus MOLE doses (3.3, 4.0 and 4.7 g/kg) daily for 1 month, and groups 7–9 that received MOLE alone at the same doses for 15 days after cessation of AFB1 treatment. Molecular genetic, cytogenetic, sperm, and biochemical studies were documented.
Results Genetic and sperm results revealed that AFB1 treatment induced significant elevation of genetic alterations and sperm abnormalities as compared with normal control. Biochemical studies showed that the treatment with AFB1 disturbed the parameters of liver functions, where aspartate-transaminase, alanine-transaminase, and alkaline phosphatase were activated and bilirubin contents as well as the rate of malondialdehyde were increased significantly, but the endogenous antioxidative system (catalase, superoxide-dismutase activities and glutathione as well as total antioxidant capacity) and protein profile were reduced significantly. Moreover, kidney functions (urea, uric acid, and creatinine contents) were elevated under AFB1 administration. The treatment with MOLE significantly minimizes the genetic alterations, sperm abnormalities, and biochemical destruction. These ameliorations were increased by increasing the dose level. Better findings were seen by using MOLE as a therapeutic agent than its using as a protective agent.
Conclusion This study revealed that MOLE contains therapeutic factors used in curing of genotoxicity induced by AFB1 in rats, and treatment of animals that were exposed to AFB1 with MOLE significantly ameliorated the genetic, sperm, and biochemical parameters as compared with animals treated with AFB1 alone.
Keywords: aflatoxin B1, biochemistry, genotoxicity, Moringa oleifera, rats
|How to cite this article:|
Farag IM, Roshdy HM, Radwan HA, Ghaly IS, Salah SH, Abdel-Rahim EA, Abdalla AM. Ameliorative role of ethanolic extract of Moringa oleifera leaf on aflatoxin B1-induced genotoxicity and biochemical alterations in rats. J Arab Soc Med Res 2018;13:60-70
|How to cite this URL:|
Farag IM, Roshdy HM, Radwan HA, Ghaly IS, Salah SH, Abdel-Rahim EA, Abdalla AM. Ameliorative role of ethanolic extract of Moringa oleifera leaf on aflatoxin B1-induced genotoxicity and biochemical alterations in rats. J Arab Soc Med Res [serial online] 2018 [cited 2019 Feb 23];13:60-70. Available from: http://www.new.asmr.eg.net/text.asp?2018/13/1/60/237216
| Introduction|| |
Aflatoxins (AFs) are considered to be a major class of mycotoxins. AFs are produced by filamentous fungi, especially the strains of Aspergillus flavus and Aspergillus parasiticus in feed stuffs . AFs include approximately five types: B1, B2, G1, G2, and M1. Aflatoxin B1 (AFB1) is a strong carcinogen and mutagen that induces hepatocellular carcinoma (HCC) and immunosuppression and causes also a lot of injuries in different organs and tissues. Testis and kidney as well as bone marrow cells were affected by exposure to AFB1 ,. The toxicity of AFB1 might be owing to that during its metabolism in liver, reaction oxygen species (ROSs) are generated and formed. ROSs were capable of attacking cell biomolecules including DNA, proteins, and lipids ,.
Darwish et al.  and Deabes et al.  detected high frequencies of chromosome aberrations in bone marrow and testis cells as well as spermatotoxicity in mice exposed to AFB1. Moreover, Abdel-Rahim et al.  observed high rates of DNA damage, micronuclei, chromosome aberrations, and sperm abnormalities in mice treated with AFB1 in comparison with untreated control. Eshak et al.  found in Japanese Quail that exposure to AFB1 leads to significant DNA and micronuclei damage in comparison with normal control. Moreover, Eshak et al.  reported high frequencies of DNA fragmentation in mice receiving AFB1 than those found in normal control. Induction of damage to the hepatic parenchyma might produce deleterious effects to the liver physiochemical functions ,. Deabes et al.  observed that the treatment with AFB1 significantly increased the liver tissue levels of malondialdehyde (MDA); however, the rate of glutathione (GSH) and activity of superoxide-dismutase (SOD) were significantly reduced in liver and kidney of mice. Moreover, Eshak et al.  reported a significant elevation of alanine-transaminase (ALT), aspartate-transaminase (AST), and MDA in liver tissues as well as significant increase of creatinine and serum uric acid concentrations in kidney tissues of mice exposed to AFB1 compared with those found in normal control. Thus, AFB1 induced DNA damage and mutation by oxidase systems which produce hydroxylated metabolites ,,.
Therefore, the adverse effect of carcinogen (AFB1) leads to increase of deterioration of metabolism of proteins, lipids, and carbohydrate ,, causing several health problems in humans and animals owing to the consumption of AF-contaminated diet . To detoxify or eliminate the toxic effect of AFB1 in the contaminated food, antioxidants were used without a reduction in the nutritional values. The nutritional additives were found to be important means for this purpose, where they contain a lot of natural antioxidants. These natural antioxidants were found to reduce the lesions induced by harmful toxicants . Moringa oleifera plant, especially its leaves, was found to be an excellent source of natural antioxidants, which included, vitamins, essential sulfur-containing amino acids (cysteine and methionine), flavonoids, minerals, glucosinolates, kaempferol, sterols, and other compounds that are considered to have health beneficial effects ,,. These Moringa oleifera leaf extract (MOLE) antioxidants were also revealed to have strong properties as antimutagenic, anticarcinogenic, anti-inflammatory, and antifungal activity . Rao et al.  observed that the percentages of micronuclei and chromosome aberrations were significantly minimized in the animals that are pretreated with M. oleifera leave extraction and then exposed to the radiation. Sathya et al.  reported that animals pretreated with MOLE and then injected with cyclophosphamide (CP) had significantly decreased rates of micronuclei and DNA damage compared with those treated with CP alone. Radwan et al.  showed protective effects of MOLE against toxicity of CCL4 to DNA, micronuclei, chromosome, and sperm shape compared with rats treated with CCL4 alone. So, this study was carried out to evaluate the modulatory ameliorating role of MOLE on genotoxicity and cytotoxicity of AFB1 in rats.
| Materials and methods|| |
AFB1 powder and DMSO (art no. 7029.1) were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Overall, 5 mg of AFB was dissolved in 250-ml DMSO .
Preparation of Moringa oleifera leaves extraction
Ethanolic extract of MOL was prepared according to Ugwu et al.  as following: the fresh leaves of M. oleifera plant were picked from trees grown on sand soil in El-Sharkia governorate, Egypt. The leaves were washed thoroughly with distilled water and dried at 29–35°C for 3 weeks, after which the leaves were pulverized into coarse form with acrestor high-speed milling machine. The coarse form (1000 g) was then macerated in absolute ethanol and was left to stand for 48 h. After that the extract was filtered through muslin cloth on a plug of glass wool in a glass column. The resulting ethanol extract was concentrated and evaporated to dryness using rotary evaporator at optimum temperature between 40 and 45°C to avoid denaturation of the active ingredients. The concentrated extract was diluted to 1000 ml using a polysaccharide as a carrier and stored in the refrigerator.
Male albino rats of Sprague-Dawley strain weighing 120–150 g were obtained from the Animal House, National Research Centre, Egypt. Animals were housed in an ambient temperature of 25±3.2°C on light/dark cycle of 12/12 h. All rats were kept in clean polypropylene cages and administered food and water ad libitum. All animals were cared for, and experiments were carried out in accordance with the European Community guidelines for the use of experimental animals and approved by the Ethics Committee of the National Research Centre, Egypt .
A total of 72 rats were used and divided into nine equal groups:
- Group 1 (control group) received saline intraperitoneal at dose of 0.7 g/kg four times (day by day) a week for 1 month.
- Group 2 (DMSO group) received (intraperitoneal) DMSO at dose of 0.7 g/kg four times a week (day by day) for 1 month.
- Group 3 (afla group) received (intraperitoneal) AFB1 in DMSO (5 mg of AFB1 in 250 ml DMSO) at dose of 0.7 g/kg four times (day by day) a week for 1 month.
- Group 4–6 (afla+M1, afla+M2, and afla+M3) received (intraperitoneal) AFB1 dissolved in DMSO at the same dose and way previously mentioned and for the same period. Starting on the first day of AFB1 administrations, rats in 4–6 groups were treated (orally) daily for 1 month with MOLE (3.3, 4.0, and 4.7 g/kg, respectively) of the crude material that are equivalent to 561, 680, and 799 mg, respectively, of the extract, as each gram of the crude material yield contains 170 mg of the extract. These groups 4–6 were used to evaluate the protective role of MOLE against AFB1.
- Group 7–9 (M1, M2, and M3 groups) received (intraperitoneal) AFB1 in DMSO in the same dose and way previously mentioned and for the same period, and then the rat groups were treated with MOLE (3.3, 4.0, and 4.7 g/kg, respectively) for 15 days. These groups 7–9 were used to evaluate the therapeutic effect of MOLE against AFB1. Rats had free access to food and drinking water during the study. At the end of the experiment, blood samples were collected for biochemical study and then rats were killed by cervical dislocation for studying of the molecular genetics, cytogenetics, and sperm examination.
Evaluation of DNA damage
Assaying of DNA fragmentation using spectrophotometer
Liver samples were collected immediately after killing the animals. The tissues were lysed in 0.5 ml lysis buffer containing 10 mmol/l tris-HCL (pH, 8), 1 mmol/l EDTA, and 0.2% triton X-100 centrifuged at 10 000 rpm (Eppendorf) for 20 min at 4°C. The pellets were resuspended in 0.5 ml of lysis buffer. To the pellets (P) and supernatant (S), 1.5 ml of 10% trichloroacetic acid was added, followed by incubation at 4°C for 10 min. The samples were centrifuged for 20 min at 10 000 rpm (Eppendorf) at 4°C, and the pellets were suspended in 750 μl of 5% trichloroacetic acid, followed by incubation at 100°C for 20 min. Subsequently to each sample, 2 ml of diphenylamine solution (200 mg diphenylamine in 10 ml glacial acetic acid, 150 μl of sulfuric acid and 60 μl acetaldehyde) was added, followed by incubation at room temperature for 24 h . The proportion of fragmented DNA was calculated from absorbance reading at 600 nm using the following formula:
Bone marrow slides were prepared according to the method described by Krishna and Hayashi . The bone marrow was washed with 1 ml of fetal calf serum and then smeared on clean slides. The slides were left to air dry and then fixed in methanol for 5 min, followed by staining in May–Grunwald and Giemsa for 5 min, and then washed in distilled water and mounted. For each animal, 2000 polychromatic erythrocytes were examined for the presence of micronuclei.
For chromosome analysis, both treated and control animals were killed by cervical dislocation at the end of experiment. An hour and a half or 2 h before killing, rats were injected intraperitoneally with 0.5 g colchicine/kg. Femurs were removed and the bone marrow cells were aspirated using saline solution. Metaphase spreads were prepared using the method of Preston et al. . Fifty metaphase spreads per animal were analyzed, for scoring the different types of chromosome aberrations.
For sperm-shape analysis, the epididymis was excised and minced in ∼8 ml of physiological saline, dispersed, and filtered to exclude large tissue fragments. Smears were prepared after staining the sperms with eosin Y (aqueous), according to the methods of Wyrobek and Bruce  and Farag et al. . At least 3000 sperms per group were assessed for morphological abnormalities. The sperm abnormalities were evaluated according to standard method of Narayana .
At the end of the first stage and then the end of the second stage (15 days) of the present experiment (30 days), blood samples were collected and centrifuged at 3000g to obtain serum for the determination of total cholesterol according to Watson , high-density lipoprotein cholesterol according to Burstein et al. , low-density lipoprotein cholesterol according to Schriewe et al. , and triglycerides according to Megraw et al. . The levels of creatinine , urea  and uric acid  were determined as indicators for kidneys function, whereas the activities of ALT, AST, and alkaline phosphatase (ALP) as well as the bilirubin content were determined according to Reitman and Frankel , Belfield and Goldberg , and Walter and Gerarde , respectively, as indicators of liver function. MDA content and the activities of catalase (CAT) and SOD were determined according to Ohkawa et al. , Beers and Sizer , and Nishikimi et al. , respectively. Total antioxidant capacity (TAC) had been determined by ELISA technique (kit no MBS733414; My BioSource Co.). The GSH content was determined according to Beutler et al. . Total protein and albumin were determined according to Gornnall et al.  and Doumas et al. , respectively, but globulin content was calculated by the following equation:
Statistical analysis was performed with SPSS software Inc., Chicago, IL, USA. Data were analyzed using one-way analysis of variance followed by Duncan’s post-hoc test for comparison between different treatments. Results were reported as mean±SE, and differences were considered as significant when P value of less than 0.05.
| Results|| |
Results of DNA fragmentation
The present data ([Table 1]) revealed that the percentages of DNA fragmentation significantly elevated in rats injected with AFB1 with respect to untreated control. In the rats that received MOLE as a protective or therapeutic agent, the values of DNA fragmentations significantly reduced as compared with those received AFB1 alone. This reduction of DNA fragmentation was increased by increasing the dose level. The highest dose caused amelioration of genetic material and gave the lowest percentages of DNA fragmentation in comparison with other doses of MOLE. Moreover, best results were occurred by using MOLE as a therapeutic agent rather than MOLE as a protective agent.
|Table 1 Shows the ameliorative role of Moringa oleifera leave extraction on aflatoxin B1-induced DNA fragmentation in rats|
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Results of micronucleated polychromatic erythrocytes
The present findings ([Table 2]) observed that the rate of induction of micronucleated polychromatic erythrocytes (MNPCE) significantly increased in the animals injected with AFB1 as compared with untreated control. In rat groups that were injected with AFB1 and received MOLE as a protective or therapeutic agent, the percentages of MNPCE significantly decreased in comparison with rat group injected with AFB1 alone. This decrease of induction of MNPCE was increased by increasing dose. The treatment with the highest dose (4.7 g/kg) gave the lowest percentage of MNPCE. Furthermore, the use of MOLE as therapeutic agent especially by using the highest dose enhanced more the genetic material recovery (by reducing the rate of MNPCE) and gave much better results than the use of MOLE as a protective agent.
|Table 2 The ameliorative role of Moringa oleifera leave extraction on aflatoxin B1-induced micronucleated polychromatic erythrocytes in rats|
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Results of chromosome anomalies
The data reported in [Table 3] showed that the treatment with AFB1 caused highly significant increases in the frequencies of chromosome aberrations as compared with untreated control group. The rat groups that were injected with AFB1 and received the MOLE as protective or therapeutic agent had significant decrease of chromosome aberrations in comparison with rat group injected with AFB1 alone. The decrease in chromosome aberrations was increased by increasing the dose level of MOLE. The highest dose of MOLE as protective or therapeutic agent resulted in the lowest percentages of chromosome aberrations. Moreover, the use of MOLE as a therapeutic agent gave more amelioration in genetic material by decreasing more the chromosome aberration than the use of MOLE as a protective agent.
|Table 3 Shows the ameliorative role of Moringa oleifera leave extraction on aflatoxin B1-induced chromosome aberrations in rats|
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Results of sperm abnormalities
The present investigation on sperm examination ([Table 4]) revealed anomalies in head and in tail of sperms. Head sperm abnormalities included amorphous and without hock, whereas tail sperm anomalies involved coiled tail, terminal droplet, and bent tail. The injection with AFB1 induced high significant of sperm abnormalities as compared with untreated control group. However, the treatment with MOLE as a protective or therapeutic agent caused significant minimization of sperm-shape abnormalities. This amelioration of sperm shape is increased by increasing of MOLE. The treatment with the highest dose (4.7 g/kg) led to more amelioration in sperm shape by decreasing the head and tail abnormalities than other dose levels. The use of MOLE as therapeutic agent was revealed to give the best results.
|Table 4 Shows the ameliorative role of Moringa oleifera leave extraction on aflatoxin B1-induced sperm abnormalities in rats|
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Biochemical results ([Table 5]) showed that AFB1 administration significantly changed liver function enzymes actively, such as ALP, AST and ALT. These paralleled with significant elevation in bilirubin content under the same conditions of the serum of AFB1-intoxicated rats relative to those of normal control. Moreover, induction significantly stimulated ALP, ALT, and AST activities. Bilirubin serum content was insignificantly increased compared with that of normal control. Rats subjected to AFB1 developed significant hepatocellular damage as evident from the serum bilirubin content and activities of ALP, ALT, and AST as compared with normal control, and they have been used as reliable markers of hepatotoxicity.
|Table 5 Liver and kidney functions of serum aflatoxin B1-intoxicated rats treated with Moringa oleifera leaf extract|
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The AFB1-intoxicated rats were treated with MOLE, which significantly alleviated the harmful effects of AFB1. This effect was dose dependent, but the values were still more than healthy controls. In case of groups 7–9, MOLE treatments were continuously ingested into intoxicated rats with AFB1. The aforementioned enzyme activity was more reduced companied with the plant extract ingestion. Their activities were around those of the normal health control. It means that the ingestion of MOLE with AFB1 was able to decrease AFB1 harmful effects. Regarding AFB1 effects on rat kidneys function, results in [Table 5] showed that urea, uric acid, and creatinine levels were unchanged for groups of DMSO relative to that of normal control, but in AFB1-intoxicated rats, creatinine, uric acid, and urea levels were highly significantly increased compared with normal control. All the treatments with MOLE ameliorated the undesirable effects of AFB1 on kidneys. The same trend of liver function improvement was observed because of the continuous ingestion of MOLE without AFB1. These effects were dose dependent. The best improvement was found for the ninth group indicate.
Concerning serum protein profile, [Table 6] illustrates the total protein, albumin, and globulin in serum of the experimental rat groups. Rats of the DMSO group showed that the three parameters of protein profile were not significantly changed relative to control, but these fractions were significantly decreased under the ingestion of AFB1 at normal control levels. The induction of AFB1 with different doses of MOLE reduced disturbance in serum protein profile. In addition, more treatments with MOLE without AFB1 removed the effects of AFB1 ingestion in which serum protein profile (total protein, albumin, and globulin) was around to the control value, especially in the last two groups (G8 and G9). All the aforementioned results were dose dependent.
|Table 6 Serum protein profile as well as endogenous peroxidation and antioxidant system of serum in aflatoxin B1-intoxicated rats treated with Moringa oleifera leaf extract|
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In case of endogenous peroxidation (MDA) and antioxidative system (SOD, CAT, and GSH) in the present experiment rats, results are shown in [Table 2]; DMSO (G2) induction insignificantly changed MDA and GSH contents as well as SOD and CAT activities in serum relative to normal content (G1). In contrast, AFB1 ingestion (G3) elevated significantly the level of MDA, but reduced the GSH content. Activities of SOD and CAT were decreased significantly under the induction of AFB1. The treatments by MOLE together with AFB1 slightly significantly ameliorated the adverse effects of AFB1-induced toxicity, such that MDA was decreased but GSH was increased. Moreover, SOD and CAT activities were stimulated relative to intoxicated control (G3). For the TAC ([Table 2]), the value had similar trend that TAC was unchanged by DMSO induction, but the other treatments significantly decreased by AFB1 ingestion. The intoxicated control group (G3) showed the lowest value of TAC, and this value was elevated and improved gradually by treatment with MOLE together with AFB1 ingestion. The data were dose dependent. The continuous treatments with MOLE without AFB1 (G7, G8 and G9) into intoxicated rats resulted in more amelioration and improved the endogenous oxidative and antioxidative effects, such as the MDA value which decreased significantly at intoxicated control (G3), but was still higher than that of normal control. However, GSH and TAC were improved and increased compared with G3, but their values were more than those of the normal control. The SOD and CAT activities, which were inhibited under the ingestion of AFB1, were stimulated again by MOLE treatments relative to intoxicated control (G3), either with or without AFB1 ingestion. The continuous treatments by MOLE without AFB1 induction resulted in more improvements in the both activities. The aforementioned results were dose dependent. The best treatment results were seen in G8 and G9 in which MOLE was administrated with the highest doses without AFB1.
| Discussion|| |
In the present study, the mutagenic effect of AFB1 in rat cells was observed by inducing high rates of DNA damage, micronuclei, chromosome aberrations, and sperm-shape abnormalities. These findings were similar with that observed in previous studies: concerning the inducing of DNA damage, the exposure to AFB1 had induced DNA fragmentation in different animal species ,. Moreover, high frequencies of micronuclei and chromosome aberrations were observed in animals treated with AFB1 as compared with untreated controls ,,,. Moreover, sperm-shape abnormalities were found to be significantly increased in animals receiving AFB1 in comparison with normal control groups ,,.
The genotoxic effect of AFB1 might be owing to that this carcinogen when metabolized in the liver and activated by the system of cytochrome P450 enzyme causes production of AFB1-8,9 epoxide. This epoxide binds with nucleophilic sites in DNA producing AFB1-DNA adduct and consequently leads to induction of DNA damage, micronuclei, chromosome aberrations, and sperm abnormalities ,. On the contrary, several studies concluded that during the metabolic processes for AFB1 in liver, the oxidative stress was induced by forming intracellular ROSs that include hydroxyl radical (−OH), hydrogen peroxide (H2O2) and superoxide anion (O−). These radicals attack cell components (DNA, proteins, and lipids) and cellular membrane causing DNA lesions, cytotoxicity, and impairment of cell functionality ,,. Moreover, Alvarez et al. , Saradha and Mathur , and Fsieh et al.  reported that ROSs can cause peroxidation of fatty acids in the sperms inducing lipid peroxidation, and this lipid peroxidation causes damage to phosphatides of cell membrane and consequently leads to sperm-shape abnormalities and decline of sperm count.
Fenske and Fink-Gremmels  reported that this toxin (AFB1) has the ability to accumulate in germinal cells leading to impaired spermatogenesis. Kumari and Sinha  emphasized that AFB1 is considered to be a reproductive toxicants. Furthermore, Bose and Sinha , Solti et al. , and Darwish et al.  concluded that mycotoxin (AFB1) has strong adverse effect on spermatogenesis or spermiogenesis.
From the present findings, it was found that the animals that were treated with AFB1 and received MOLE as a protective or therapeutic agent have significant reduction of rates of DNA fragmentation, micronuclei, chromosome aberrations, and sperm-shape abnormalities as compared with animals treated with AFB1 alone. These results prove the ameliorate effect of MOLE against the lesion effect of AFB1. These ameliorations of MOLE on genetic materials and sperms might be owing to its containing high percentages of natural antioxidants as polyphenols, which have important properties as antimutagens or antigenotoxins and anticarcinogens ,,. The action mode of antioxidants might be owing to its binding with the mutagen (AFB1) or its suppression to the activation of cytochrome enzyme system leading to reduction of DNA adduct and consequently causing minimization of abnormalities in genetic materials and sperms ,,. The decrease of DNA lesionsleads to reduction of micronuclei and chromosome aberrations , as a result of a decrease in suppression of DNA replication and consequently cause a decrease of sperm abnormalities ,. Moreover, the antioxidants of MOLE have been found to have potent strategies by scavenging the ROS or free radicals causing reduction of oxidative stress on cellular components including proteins, DNA, and lipids ,,, leading to decrease of genotoxicity and cytotoxicity.
In previous studies, the antimutagenic effects of MOLE phytoconstituents were revealed by minimization of genotoxicity and cytotoxicity that were induced in different animal species exposed to radiation , and treatment with each of CP ,. Moreover, Prasanna and Sreelatha  detected that the treatment with MOLE in cells of Saccharomyces cerevisiae reduced the oxidative stress (that induced by ROS H2O2) and enhanced the levels of antioxidant enzymes, SOD and CAT, and this led to decrease of cytotoxicity and formation of lipid peroxidation. Moreover, other studies ,, reported that the antioxidant properties of MOLE played an important role as anticarcinogens by reducing the risk of cancer in lung, prostate, and ovarian tissues in human and mice.
From the present study, it could be reported that liver and kidneys being their target organs respectively especially during the protective stage, but after the therapeutic stage of experimental periods, values were around those of the normal state in the highest treatment (ninth group). The intoxicated rats treated with the different doses of MOLE showed different improvements in dose-dependent manner than intoxicated control rats.
These results are in agreement with Corcuera et al.  who stated that there was statistically significant stimulation in ALP and ALT activities under the induction of AF relative to those of normal control. After AFB1 induction, histology and biochemistry of liver showed necrosis, focal inflammation, and an increase in liver function enzymes. The AFB1 induction produced the same trend in kidneys function like those of liver in which kidney biomarkers (urea, uric acid and creatinine levels of serum) had the same manner of liver parameters. Mansour et al.  reported that MOLE showed protective influences against AFB1 hepatotoxicity and nephrotoxicity (ALT, AST, urea and creatinine). Moreover, Sheikh et al.  found that M. oleifera leaves abrogated the xenobiotic induced harm in liver function (ALP, ALT and AST) and kidneys function (urea). It also improved the contents of glucose, triglycerides, cholesterol, high-density lipoprotein, and low-density lipoprotein as serum lipid profile.
Hepatic tissues of the liver absorb toxic substances from the blood stream and thus from circulation. AF, specifically AFB1, is eventually secreted in the liver where it has been shown to be toxic to cell. AF in the liver is degraded in two phases  biotransformation to a more toxic product  and detoxification to a less toxic and easily excretal product ,. Ingestion of AFB1 is a major risk factor for HCC and is an immune stimulant . In liver, AFB1 is biotransformed to various metabolites especially the active AFB1-exo-8.9-epoxide (AFBO).
DNA adduct formation by the AFBO can be diminished by formation of AFB1-glutation conjugates, mediated by glutathione-S-transferase ,. The accumulation of AFB1 and AFBO depletes the GSH owing to the formation of high amount of epoxides and other ROS. These could activate and deactivate the various epigenetic mechanisms leading to development of various cancers ,.
The MOLE treatments for AFB1-intixicated rats against the AFB1 harm may re-adjust the levels of protein profile by stimulation of protein biosynthesis. It may be possible that MOLE treatments led to enzymes induction necessary to detoxify the toxicant (AFB1 and its metabolites), which play an antagonistic effects against the present xenobiotic. AFB1 induction stimulates cellular metabolism to generate ROS and hydroxyl radical which can endogenously and exogenously attack lipid, protein, nucleic acids and other compounds simultaneously in the living cells . AFB1 and AFBO damaged DNA that occurred from forming DNA by glutathione-S-transferase adduct (oxidase systems) . Forth with, AFB1 and AFBO interact with DNA, RNA, and protein which cause breakages in these molecules, especially DNA, and disturb DNA-replication. These result in chromosomal aberrations and inhibit the protein biosynthesis system ,,,.
AFB1 induction produced a complex significant metabolic disorder and disturbance of organs function and endogenous oxidative and antioxidative system against xenobiotic harm. The accumulation of AFB1 and its metabolites depletes the GSH due to the formation of high amounts of epoxides and other ROS that could activate and deactivate the various epigenetic mechanism leading to development of various cancers ,. The main biological influences of AFB1 and its oxidative metabolites are carcinogenicity, immunosuppression, and teratogenicity and also cause injuries in body animal organs with production DNA mutation, and damage by oxidase systems results in hydroxylated metabolites ,. These hydroxylated metabolites with ROS radicals can attack the endogenous biological structure such as lipid, protein, and nucleic acids (DNA and RNA)  and also deteriorate the different metabolic pathways in the living cells , in which MDA content was increased but GSH was decreased, and also SOD and CAT activities were decreased.
M. oleifera attenuates the oxidative stress and enhances the levels of activity of antioxidative enzymes such as SOD and CAT. Moreover, it caused inhibition of the extent of lipid peroxidation (MDA) and stimulated the hydroxyl radical scavenging activity. M. oleifera leaves alcoholic extracts suppressed the effects against AFB1 and its metabolites induced HCC or apoptotic cellular changes . The plant as a herbal drug has been discussed in terms of redox imbalance and oxidative stress and may be useful in efficient killing of tumor cells leading to establishment of improved protocol in patients with cancer ,. M. oleifera leaves are renewable source of chlorophylls, tocoferol, phenolics, and β-carotein, saponin, tannins, and other compounds . Chlorophylls can act as an interceptor molecule through the formation of tight molecular complexes with AFB1 antioxidant to inhibit lipid peroxidation (MDA), and it is a potent inhibitor of cytochrome p450 enzymes .
Administration of MOLE as antioxidant agents ameliorated the hepatotoxicity and nephrotoxicity  induced by AFB1  and enhancement of the antioxidant defense mechanisms ,,.
| Conclusion|| |
This study revealed that MOLE contains therapeutic factors used in curing of genotoxicity induced by AFB1 in rats. The treatment of animals exposed to AFB1 with MOLE significantly ameliorated the genetic, sperm, and biochemical parameters as compared with animals treated with AFB1 alone.
Authors are grateful to STDF project (ID 5979), under title: ‘Recent the application in the utilization of Moringa oleifera and Moringa peregrina as a good nutritional, medicinal and industrial plant in Egypt’ for providing with the MOLE and contribution in preparation of Animal House.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]