Combined toxic effects of aflatoxin B2 and the protective role of resveratrol in Swiss albino mice

In this study, potential toxic effects of AFB₂ on albino mice were investigated by using body weight and organ weight, serum parameters, oxidant/antioxidant dynamics and cytogenetic parameters in albino mice and the protective effects of resveratrol against AFB₂ toxicity were studied. In addition, radical scavenging activity of resveratrol were investigated and associated with its protective effect. Resveratrol exhibits antioxidant activity by neutralizing the free radicals. Free radical scavenging is one of the important mechanism of antioxidant activity. In this study, free radical scavenging activity of resveratrol was tested against superoxide, DPPH and H₂O₂, which have important radical properties.

Radical scavenging activity of resveratrol

There is a close relationship between the protective role of resveratrol and its antioxidant activity. Radical scavenging activity plays an important role in antioxidant activity mechanism. The radical scavenging property of resveratrol tested against superoxide, DPPH and H₂O₂ and the results are presented in Fig. 1. The scavenging effect of resveratrol also increased with the increasing dose and the highest scavenging effect was observed against DPPH (%79). DPPH, a stable free radical, accepts an electron or hydrogen to become a stable diamagnetic molecule, and 1 mg/mL resveratrol and BHT exhibited 79% and 88% DPPH scavenging activity, respectively. Among free radicals, superoxide is a strong oxidizing agent, and resveratrol exhibited a stronger superoxide scavenging activity than the standard antioxidant compound. Especially at high doses (0.5–1 mg/mL), resveratrol exhibited higher superoxide scavenging activity compared to the BHT. H₂O₂ itself is not very reactive, however, it can sometimes be toxic to cells as it mediates the formation of the hydroxyl radical²². Resveratrol showed a moderate hydrogen peroxide scavenging activity and the peroxide scavenging activity of 1 mg/mL resveratrol was 49%. Resveratrol has electron donor properties to neutralize free radicals and provides the formation of stable products with its reducing properties. Its reducing property prevents the initiation of radical chain reactions and protects cells against oxidation²².

Literature studies show that resveratrol is a good radical scavenging agent^20,21. Gülçin²² reported that resveratrol has a higher superoxide scavenging activity compared to standard antioxidants such as α-tocopherol and trolox. Murcia and Matinez-Tome³⁶ indicated that resveratrol tested at different concentrations did not exhibit H₂O₂ removal activity, whereas Gülçin²² reported that 30 μg/mL resveratrol had 19.5% H₂O₂ scavenging activity. These results indicate that resveratrol is a powerful antioxidant and will have protective properties against AFB₂ toxicity.

Physiological parameters

The effects of AFB₂ and resveratrol applications on body weight and organ weights are given in Table 2. In control group, the initial body weight average was 33.50 ± 1.80 g, and a weight increase of approximately 12.40 g was observed at the end of the application period. Similar increase was observed in Groups II and III where 10 µg/kg and 20 µg/kg b.w resveratrol were administered, respectively. This result shows that resveratrol application alone does not cause any toxic effect on weight gain in organisms. The most significant reduction in body weight was observed in the group treated with 20 µg/kg b.w AFB₂, and a 23% reduction observed compared to the control group. Abnormalities caused by AFB₂ application on body weight were also observed in liver and kidney weights. While no statistically significant difference observed in the organ weights of groups I, II and III (p > 0.05), statistically significant reductions were found in 20 µg/kg b.w AFB₂ treated group (p < 0.05). Liver and kidney weights decreased by 46.9% and 46.3% times, respectively, in group IV compared to the control group. This result indicates that liver and kidney weights were similarly affected by AFB₂ toxicity. The negative effects of AFB₂ treatment on body weight and organ weights in albino mice may be associated with anorexia, fatigue, impaired protein/lipid metabolism, protein synthesis and inhibition of lipogenesis. Lipolysis-lipogenesis balance plays an important role in increasing body weight and weight gain is increased by lipogenesis and fat accumulation^37,38. Lipid metabolism disorder, which may arise as a result of the hepatotoxic effects of aflatoxins and their negative effects on other tissues, may significantly affect the weight gain and organ weights in organisms. Although there is no study in the literature about the effect of AFB₂ application on weight gain, there are important data on the effects of other aflatoxin species. Similarly to our results, Arvind and Churchil³⁹ found that there was a 33.94% reduction in weight gain of chickens fed with 1 ppm AFB₁ for 6 weeks. In another study, Dimitri et al.⁴⁰ reported that exposure to AFB₁ and AFG₂ caused an average of 539 g (16%) body weight loss in rabbits, and this was related to impaired protein synthesis and DNA synthesis. Cardoso et al.⁴¹ stated that the mixture containing 62.9% of AFB₁ and 37.1% of AFG₁ significantly decreased the body weight gain in broiler chicks, did not cause a change in liver anatomy in terms of shape, texture and color, however, it caused an increase in relative liver weight.

Another important result observed in the body weight and organ weight analysis is that resveratrol application together with AFB₂ caused weight gain and an increase in organ weights. An increase observed in the body weights of Groups V and VI, in which resveratrol and AFB₂ were applied together, compared to the group treated with 20 µg/kg b.w AFB₂. This improvement was more pronounced in Group VI, which received 20 mg/kg resveratrol. While an increase of 1.78 g in body weight observed only in the group receiving AFB₂, this increase determined as 7.5 g in the group in which AFB₂ and 20 mg/kg resveratrol were administered together. The increasing effect of resveratrol on body weight was also observed in kidney and liver organ weights. In Group VI, where 20 mg/kg b.w resveratrol was applied, liver and kidney weights increased by 45.9% and 55.8%, respectively, compared to the group treated with AFB₂. This healing property of resveratrol can be explained by the suppression of toxic effects induced by AFB₂. Resveratrol exhibits an important protective role in lipid metabolism by preventing lipid peroxidation and LDL oxidation, decreasing total cholesterol and low-density lipoprotein cholesterol levels in plasma, and increasing plasma high-density lipoprotein (HDL) levels^17,42. This protective role reduced the negative effects of AFB₂ on lipid metabolism and caused a re-increase in body weight. Resveratrol exhibits an important protective role in tissues such as kidney and liver, particularly by reducing oxidative damage and inflammation⁴³. The fact that the decrease observed in the weight of kidney and liver tissues as a result of AFB₂ administration increased again with resveratrol can be explained by this protective feature.

Liver and kidney functions analysis

The effects of AFB₂ and resveratrol application on selected serum parameters are given in Fig. 2 and Table 3. Among the serum parameters, the liver marker enzymes AST and ALT, and the kidney damage indicators BUN and creatinine levels were examined. No abnormality was found in AST and ALT levels in groups I, II and III, and the differences between the groups were statistically insignificant (p > 0.05). Significant increase was found in AST and ALT levels in the 20 µg/kg b.w AFB₂ treated group. AST and ALT activities increased by 52.8% and 69.4%, respectively, in the AFB₂-treated group compared to the control. These statistically significant increases in AST and ALT levels indicate that AFB₂ administration causes serious liver damage. Besides carcinogenic and mutagenic effects of aflatoxins, hepatotoxic and nephrotoxic effects are also known. Aflatoxins inhibit polymerase activity in hepatocytes, cause biochemical and histopathological changes in liver and cause hepatocellular necrosis⁴⁴. All these effects induced by aflatoxin cause damage to liver, resulting in cell destruction and organ failure. These abnormalities observed in hepatocytes cause the enzymes in these cells to enter the blood and increase their levels in the serum. AST is found in a variety of tissues, including liver, brain, pancreas, heart, kidney, lung and skeletal muscle. After damage to these tissues, AST passes from the tissue cells to the bloodstream and the blood level increases. Increased AST levels are indicative of tissue damage, but do not indicate liver damage alone. ALT is predominantly found in the liver, and any increase in the blood level of ALT is accepted as a direct indicator of liver damage⁴⁵. The increase in both enzymes in the AFB₂ applied group is evidence of liver damage. Similarly, Eraslan et al.⁴⁶ reported that a mixture of 500 μg/kg aflatoxin (80% AFB₁, 10% AFB₂, 6% AFG₁, 4% AFG₂) exposure significantly increased serum ALT, AST, ALP and LDH enzyme activities and these increases were associated with liver damage. Han et al.⁴⁷ reported that 20 μg/kg and 40 μg/kg AFB₁ administration caused a significant increase in AST and ALT enzymes, and this increase caused by the transition of enzymes from hepatocytes to the blood as a result of liver damage.

AFB₂ administration also caused an increase in BUN and creatinine levels, which are indicators of kidney damage. BUN and creatinine levels increased by 33.9% and 134.4%, respectively, in 20 µg/kg b.w AFB₂ treated group compared to the control group. Aflatoxins cause different damages in kidney such as glomerular capillary damage, obstruction in cortical blood vessels, coagulation necrosis, inflammation, focal bleeding⁴⁸. BUN and creatinine levels are used to predict potential kidney damages. Creatinine is the breakdown product of creatinine phosphate in muscle and is usually produced by the body at a constant rate depending on muscle mass and excreted through the kidneys. Since creatinine cannot be filtered sufficiently in kidney damage or failure, its level increases in the blood. Similarly, BUN levels increase in kidney disease or failure, fever and shock, obstruction of the urinary tract due to kidney stones⁴⁹. In this study, administration of 20 µg/kg AFB₂ caused significant increases in BUN and creatinine levels, indicating kidney damage. Supporting these findings, Eraslan et al.⁴⁶ reported that 500 μg/kg aflatoxin exposure resulted in significant increases in uric acid, BUN and creatinine levels in albino rats, indicating kidney damage. Verma and Kolhe⁵⁰ reported that 15 mg/kg aflatoxin isolated from Aspergillus parasiticus significantly increased the serum creatinine level in rabbits and caused nephrotoxicity.

While AFB₂ administration alone caused a significant increase in liver and kidney indicator parameters, resveratrol application together with AFB₂ improved alterations in these parameters. ALT and AST levels decreased by 27.1% and 18.7%, respectively, in Group VI, compared to the group that only applied AFB₂. Despite this decrease, it was determined that AST and ALT levels were still higher than the control group. When the kidney marker parameters were examined, BUN and creatinine levels were decreased by 17.9% and 33.5%, respectively, compared to the AFB₂ applied group. The decline in increased ALT, AST, BUN and creatinine levels after resveratrol application can be explained by the protective properties of resveratrol. Resveratrol exhibits an important protective role in kidney and liver, especially by reducing macromolecular oxidation, lipid peroxidation and inflammation⁴³. Although there is no study on the protective effect of resveratrol against AFB₂-induced damages in the literature, it has been reported that resveratrol reduces liver and kidney damages induced by many chemicals. Şener et al.⁵¹ reported that the administration of 100 mg/kg of resveratrol treatment exhibited an antioxidant protection and prevented liver damage by reducing induced oxidative damage in rats. Palsamy and Subramanian⁵² found that 5 mg/kg resveratrol resulted in an improvement in creatinine clearance and had protective properties on the kidney by reducing oxidative stress and inflammation.

Antioxidant/oxidant dynamic

Similar to the negative effects of AFB₂ administration on physiological and serum parameters, a toxic effect was also observed on antioxidant/oxidant dynamic. The effects of exogenous and endogenous oxidant molecules in the body neutralized by antioxidants. The increase in exogenous oxidant molecules and an insufficiency of endogenous antioxidants cause deterioration in antioxidant/oxidant balance. In order to examine the effects of AFB₂ on this balance, MDA and GSH levels in liver and kidney were examined and the results are given in Figs. 3 and 4. It was observed that MDA levels were close to each other in the control group and only resveratrol treated groups. 20 µg/kg b.w AFB₂ administration caused a significant increase in MDA levels of kidney and liver. When compared with the control group, AFB₂ exposure significantly increased the MDA levels in liver and kidney compared to control. Many studies have demonstrated that aflatoxin species cause oxidative stress^9,53,54. Oxidative stress causes damage and lipid peroxidation in cells. The cellular structure most affected by lipid peroxidation is the cell membrane, which contains saturated and unsaturated lipids. MDA is a mutagenic product formed as a result of peroxidation of membrane lipids^55,56. At the end of the normal biochemical process, low levels of MDA can occur in cells and can be removed with the antioxidant system.

Increased MDA level in AFB₂ applied group indicates that AFB₂ cause oxidative damage and lipid peroxidation in liver and kidney. Similarly, Shen et al.⁵⁷ reported that cell damage and lipid peroxidation occurred in the liver of rats exposed to AFB₁. In a similar study, it was reported that the serum MDA level increased in albino rats treated with aflatoxin mixture, and this increase was associated with the formation of free radicals and the weakening of cellular antioxidant defense mechanisms⁴⁶. In this study, liver MDA levels decreased by 11.2% and 21.9% in the groups V and VI treated with 10 mg/kg b.w and 20 mg/kg b.w resveratrol, respectively, compared to the group that received only AFB₂. In Group V and VI, the decrease in kidney MDA levels was 20.9% and 33.7%, respectively. The most significant improvement in liver and kidney MDA levels observed in the 20 mg/kg b.w resveratrol group (Group VI), but MDA levels in this group were still higher than in the control group. The decreasing effect observed in MDA level can be explained by the antioxidant role of resveratrol. Resveratrol exhibits a protective effect by directly suppressing oxidant molecules and radicals, increasing the expression and activity of antioxidant enzymes such as glutathione peroxidase, superoxide dismutase and catalase^15,16. Resveratrol reduces lipid peroxidation and can also prevent deterioration in cell membrane integrity¹⁷. The decrease in MDA levels is closely related to the radical scavenging activity of resveratrol. The ability of resveratrol to scavenge free radicals was determined by previous analyzes in this study. Resveratrol exhibited a significant scavenging effect against superoxide and H₂O₂ that cause oxidation in the cell and especially in cell membrane lipids. Supporting our results, Kasdallah-Grissa et al.⁵⁸ reported that increased MDA levels as a result of induced lipid peroxidation in liver, heart, brain and testicular tissues decreased by 72.7–40.5% with resveratrol administration.

The effects of resveratrol and AFB₂ application on GSH levels are given in Fig. 4. It was observed that GSH levels were close to each other in the control group and only resveratrol treated groups. Resveratrol application was increased the GSH level at a low level in Group II and III, but this increase was statistically insignificant (p > 0.05). 20 µg/kg b.w AFB₂ administration caused a decrease in GSH levels in liver and kidney. Compared with the control group, AFB₂ treatment reduced GSH levels in liver and kidney tissues by 24.6% and 57.1%, respectively. Considering the increasing effect of AFB₂ on MDA levels of liver and kidney and its decreasing effect on GSH levels, it can be said that the oxidant/antioxidant dynamic was disrupted. The increase in MDA level is evidence of oxidative damage, and antioxidant molecules play a role to neutralize this damage. GSH is a non-enzymatic tri-peptide antioxidant and is an essential part of the antioxidant defense in free radical removal. The reduced-GSH protects the cell against oxidative damage by neutralizing free radicals. As a result of this reaction, GSH molecules turn into the oxidized GS-SG state and the level of reduced-GSH decreases⁵⁹. In short, the increase in the MDA ratio and the decrease in reduced-GSH prove the oxidative damage caused by AFB₂. GSH conjugation also plays an important role in aflatoxin metabolism, and conjugation contributes to the decrease in the level of reduced-GSH⁸. In the literature, it is reported that aflatoxin exposure causes changes in the levels of antioxidant molecules. Kheir Eldin et al.⁶⁰ reported that 250 μg/kg AFB₁ treatment in rats decreased GSH level and increased lipid peroxidation. Eraslan et al.⁴⁶ found that aflatoxin exposure in rats caused a significant decrease in antioxidant enzyme levels in the liver, thereby disrupting the antioxidant balance. In this study, it was determined that resveratrol application provided improvement in impaired reduced-GSH levels and also with increasing the resveratrol dose, recovery is more predominant. 20 mg/kg b.w resveratrol application provided 11.9% and 28.8% improvement in liver and kidney GSH levels, respectively, compared to the AFB₂-treated group. Resveratrol scavenges the oxidative stress and free radicals induced by AFB₂ and prevents the decrease in GSH level. Similarly, Şener et al.⁵¹ reported that 30 mg/kg of resveratrol administration in mice regulates the impaired antioxidant balance and leads to improvement in decreased GSH levels.

Genotoxic effects

The genotoxic effects of AFB₂ and the protective effect of resveratrol against these effects were evaluated by examining MN formation in buccal mucosa epithelium, erythrocyte and leukocyte cells and CAs frequency in bone marrow cells.

MN assay

Frequencies and the appearance of MN are presented in Fig. 5. A statistically significant level of MN formation was not observed in the control group and groups II and III, where only resveratrol was applied. These results show that resveratrol application alone does not induce MN formation. High levels of statistically significant MN frequencies were obtained in the group treated with 20 µg/kg b.w AFB₂ (p < 0.05). Among the three cells tested, the highest MN formation observed in leukocyte cells. MN frequencies ​​observed in buccal mucosa epithelium, erythrocyte and leukocyte cells in the group treated with 20 µg/kg b.w AFB₂ were 31.46 ± 3.28, 57.92 ± 5.85 and 80.35 ± 7.70, respectively. MN formation caused by chromosomal fragments, double-stranded DNA breaks, single-strand breaks, or lagging chromosomes. In the telophase stage of mitosis, a nuclear envelope formed around the delayed chromosomes and fragments, resulting in smaller MN structures resembling the main nucleus. The increase in MN formation and frequency indicates the presence of toxic effects on the cell. Therefore, MN test is used to evaluate the toxicity of chemicals to which cells are exposed^61,62. The size of MN formed in a cell provides information about the mechanism of toxicity and also enables the determination of the aneugenic or clastogenic effect of the chemicals. MNs resulting from centromere division errors and spindle defects as a result of the aneugenic effect are larger in size. MN’s formed as a result of clastogenic effect have smaller dimensions since they originate from chromosome fragments or breaks^61,63. MN formation in buccal mucosa, erythrocyte and leukocyte cells belonging to groups treated with AFB₂ indicates that AFB₂ has a genotoxic effect. The observation of MN formations in both large and small sizes indicates that both aneugenic and clastogenic effects of AFB₂. This result shows that AFB₂ can induce MN formation by causing centromere division errors, spindle defects or chromosome breaks. Similarly, Ezekiel et al.⁵³ reported that the frequency of MN in erythrocytes increased in mice treated with AFB₁ and that AFB₁ caused mutagenic and genotoxic effects. Corcuera et al.⁶⁴ observed a toxic effect occurred on bone marrow cells and f MN frequency increased in rats treated with AFB₁.

In order to determine the genotoxic effects of AFB₂, CAs frequency in the bone marrow were examined and the results were given in Table 4. In Group I, II and III, no CAs were found except for low level of gap formation (p > 0.05). Similar MN levels of the control group, Groups II and III show that resveratrol did not cause genotoxic effects at doses of 10 mg/kg body weight and 20 mg/kg body weight. High level and statistically significant CAs frequency were detected in the group treated with 20 µg/kg b.w AFB₂ (p < 0.05). Among these abnormalities, fragment occurred at the highest rate as a level of 53.18 ± 5.50. When all abnormalities are evaluated together, it is possible to sort the CAs according to the frequency of occurrence as break > fragment > acentric chromosome > dicentric chromosome > gap > ring. Observation of these abnormalities proves the genotoxic effect of AFB₂. The genotoxic effects of aflatoxins are closely related to oxidative stress and damage in living systems. Aflatoxins are metabolized to aflatoxin-8,9-epoxide, which is a reactive form that binds to DNA and forms aflatoxin-N7-guanine structures by binding with high affinity to guanine bases in DNA. Aflatoxin-N7-guanine generates guanine-thymine transversion mutations in DNA and negatively effects the p53 repressor gene in the cell cycle. Bone marrow cells are susceptible to oxidative damage and clastogenic chemicals, so they are widely used for mutagenicity and/or antimutagenicity screening^65,66. High level of breaks and fragments observed in bone marrow cells indicates the clastogenic effect of AFB₂. Chromosome and chromatid breaks resulting from clastogenic effect also cause the formation of fragments^67,68. The high MN level observed in the AFB₂ applied group was also closely related to the formation of breaks and fragments. In this study, the high level of MN frequency and fragments observed as a result of AFB₂ application also support each other. Ring chromosomes arise when a chromosome is broken twice and the broken ends are re-united. In mitosis, if the ring chromosomes of some cells cannot be attracted to the poles, chromosome loss/excess may occur in the cells and this can cause genome instability. Gap formations are defined as achromatic lesions that do not exceed the width of a chromatid. Dicentric and acentric chromosomes are also formed as a result of breakage, and the dicentric chromosome mostly occurs as a result of the rearrangement of chromosome breaks. Increased frequency of gaps and dicentric chromosomes are considered important indicators of toxicity^68,69. When all these effects are evaluated together, it can be said that most of the CAs originate from fragments or breaks. In this study, AFB₂ caused the most breaks and other abnormalities were observed as a result of the rearrangement of these breaks. There are many studies in the literature reporting the genotoxic effect of other aflatoxin species, especially AFB₁. Fetaih et al.⁶⁸ reported that AFB₁ induced the macro-DNA damages such as gap, fracture, deletion, dicentric chromosome, stickiness, hypopolyploidy, centromeric rearrangements in the bone marrow cell of rats. Fadl-Allah et al.⁷⁰ observed that AFB₁ (5–25 μg/mL) exposure increased the frequency of CAs such as stickiness, bridges, laggards, unequal division, fragments.

Another important result found in genotoxicity analyzes is that resveratrol application exhibits protective effect against AFB₂-induced CAs and MN formation. The frequency of CAs and MN formation decreased with the increase of resveratrol dose. Application of resveratrol together with AFB₂ reduced MN frequency in buccal mucosa epithelium, erythrocyte and leukocyte cells. Similarly, there was a decline in CAs frequency in bone marrow after resveratrol application. The most prominent protective effect of resveratrol was observed against gap formation. 20 mg/kg b.w resveratrol exhibited a reducing effect in the range of 49.34–80.5% in CAs frequency induced by AFB₂. This healing effect observed against MN and CAs formations can be explained by the multi-biological properties of resveratrol.

Recovery effects of resveratrol

RE values of 20 mg/kg b.w resveratrol in all tested parameters are given in Fig. 6. Resveratrol, which exhibits a RE value in the range of 40.9–80.55%, exhibited the highest protective role against ring formation. Although RE values against GSH decrease in kidney, MN formation in leukocyte and fragment formation in bone marrow were below 50%, resveratrol exhibited a RE over 50% in all other toxicity parameters. Resveratrol ensured the normalization of antioxidant/oxidant balance and resulted significant improvements in kidney and liver marker parameters. This protection can be explained by the suppression of oxidative damage induced by AFB₂. The protective role of resveratrol on the kidney and liver was proven by causing an improvement in organ weights, a decrease in organ MDA and an increase in GSH. In the literature, resveratrol has been reported to have a protective role in the liver by alleviating oxidative stress, induced apoptosis, necrosis and inflammation⁷¹. It is also reported that resveratrol administration reduces oxidative damage in kidney by enhancing the antioxidant defense⁷². Resveratrol also exhibited a significant RE against the formation of MN and CAs and the highest RE values were observed against gap and ring formations. The protective role of resveratrol against cytogenetic toxicity can be explained by its strengthening effect on antioxidant system and its regulatory role on the cell cycle. Resveratrol has a powerful antioxidant activity that scavenges free radicals and induces antioxidants such as GSH and CAT^20,21. The strong radical scavenging properties of resveratrol have been demonstrated by different analyzes in this study. Its protective role against CAs and MN damages is also due to this scavenging effect. Resveratrol also has a regulatory role in the cell cycle. It enables the cell to remain in the G₀/G₁ phase or S phase by suppressing the cell cycle in damaged cells. It may also regulate the cell cycle by inhibiting the expression of cyclin D₁, D₂, E and decreasing the expression of cyclin-dependent kinases^73,74. In this way, it can reduce the frequency of DNA damage and CAs by preventing damaged cells from dividing. Similar studies in the literature also support our results. Macar et al.⁷⁵ reported the attenuating effect of resveratrol administration against CAs such as fragment, sticky chromosome, unequal chromatin distribution, bridges and abnormal polarization.