Bilirubin: its role in cytoprotection against oxidative stress

Bilirubin, the end product of heme catabolism in mammals, is generally regarded as a potentially cytotoxic, lipid-soluble waste product that needs to be excreted. However, in the last 10 years, in vitro and in vivo studies, have demonstrated that bilirubin exhibits potent anti-oxidant properties preventing the oxidative damage triggered by a wide range of oxidant-related stimuli. Therefore, the idea of a beneficial and physiological role for bilirubin in cytoprotection against short and long-lasting oxidant-mediated cell injury is highlighted here.     

Copper-mediated oxidative stress in rat liver

Copper is an essential trace element with various biological functions. Excess copper, however, is extremely toxic, leading to many pathological conditions that are consistent with oxidative damage to membranes and molecules. Exposure to high levels of copper results in various changes in the tissues. In liver, hypertrophy of hepatocytes, hepatitis, hepatocellular necrosis, and hepatocellular death are the results. Lipid peroxidation causes dysfunction in the cell membrane, decreased fluidity, inactivation of receptors and enzymes, and changes ion permeability. In this study, we aimed to determine the effect of copper on oxidative and antioxidative substances in plasma and liver tissue in a rat model. Sixteen male Sprague—Dawley rats were divided into two groups: Group 1 rats included control rats given tap water. Group 2 rats were given water containing copper in a dose of 100 μg/mL. All rats were sacrificed at 4 wk under ether anesthesia. Plasma and liver superoxide dismutase (SOD) activities, plasma and liver MDA (malondialdehyde) levels, and liver glutathione (GSH) levels were studied. Plasma and liver SOD activities were found to be higher in group 2 than those in group 1. Although plasma MDA levels were higher in group 2, MDA levels in liver tissues were comparable. Liver tissue glutathione levels were lower in group 2. It was concluded that although copper is needed in trace amounts, an excess amount is toxic for the organism. It increases lipid peroxidation and depletes GSH reserves, which makes the organism more vulnerable to other oxidative challenges.     

Melatonin protects against taurolithocholic‐induced oxidative stress in rat liver

Cholestasis, encountered in a variety of clinical disorders, is characterized by intracellular accumulation of toxic bile acids in the liver. Furthermore, oxidative stress plays an important role in the pathogenesis of bile acids. Taurolithocholic acid (TLC) was revealed in previous studies as the most pro‐oxidative bile acid. Melatonin, a well‐known antioxidant, is a safe and widely used therapeutic agent. Herein, we investigated the hepatoprotective role of melatonin on lipid and protein oxidation induced by TLC alone and in combination with FeCl₃ and ascorbic acid in rat liver homogenates and hepatic membranes. The lipid peroxidation products, malondialdehyde and 4‐hydroxyalkenals (MDA + 4‐HDA), and carbonyl levels were quantified as indices of oxidative damage to hepatic lipids and proteins, respectively. In the current study, the rise in MDA + 4‐HDA levels induced by TLC was inhibited by melatonin in a concentration‐dependent manner in both liver homogenates and in hepatic membranes. Melatonin also had protective effects against structural damage to proteins induced by TLC in membranes. These results suggest that the indoleamine melatonin may potentially act as a protective agent in the therapy of those diseases that involve bile acid toxicity. J. Cell. Biochem. 110: 1219–1225, 2010. Published 2010 Wiley‐Liss, Inc.     

Endogenous ferritin protects cells with iron-laden lysosomes against oxidative stress

Previous studies have shown that a variety of mammalian cell types, including macrophages, contain small amounts of redox-active iron in their lysosomes. Increases in the level of this iron pool predispose the cell to oxidative stress. Limiting the availability of intralysosomal redox-active iron could therefore represent potential cytoprotection for cells under oxidative stress.

In the present study we have shown that an initial 6 h exposure of J774 macrophages to 30 μM iron, added to the culture medium as FeCl₃, increased the lysosomal iron content and their sensitivity to H₂O₂-induced (0.25 mM for 30 min) oxidative stress. Over time (24-72 h), however, the cells were desensitized to the cytotoxic effects of H₂O₂; most likely as a consequence of both lysosomal iron exocytosis and of ferritin synthesis (demonstrated by atomic absorption spectrophotometry, autometallography, and immunohistochemistry). When the cells were exposed to a second dose of iron, their lysosomal content of iron increased again but the cells became no further sensitized to the cytotoxic effects of H₂O₂. Using the lysosomotropic weak base, acridine orange, we demonstrated that after the second exposure to iron and H₂O₂, lysosomes remained intact and were no different from control cells which were exposed to H₂O₂ but not iron.

These data suggest that the initial induction of ferritin synthesis leads to enrichment of lysosomes with ferritin via autophagocytosis. This limits the redox-availability of intralysosomal iron and, in turn, decreases the cells' sensitivity to oxidative stress. These in vitro observations could also explain why cells under pathological conditions, such as haemochromatosis, are apparently able to withstand high iron concentrations for some time in vivo.     

Inosine ameliorates the effects of hemin-induced oxidative stress in broilers

The objective of these studies was to determine whether inosine, a precursor of the antioxidant uric acid, can ameliorate hemin-induced oxidative stress. Dietary inclusion of inosine was begun either before or after hemin-induced oxidative stress. Broilers (4 weeks) were divided into four treatment groups (Control, Hemin, Inosine, Hemin/Inosine). Throughout the study control birds (n = 10) were injected daily with a buffer solution, while hemin birds (n = 10) were injected daily (i.p.) with a 20 mg/kg body weight hemin buffer solution. Leukocyte oxidative activity (LOA) and concentrations of plasma uric acid (PUA) were measured. Results from the first study showed that hemin birds had increased levels of LOA (P = 0.0333) and lower PUA (P = 0.1174). On day 10, control and hemin birds were subdivided into inosine birds (n = 5) and hemin/inosine birds (n = 5). These birds were given 0.6 M/kg of feed/day of dry inosine. Plasma concentrations of uric acid and LOA were then measured on day 15. Results showed that inosine raised concentrations of PUA (P = 0.0001) and lowered LOA (P = 0.0044) as induced by hemin. In the second study pretreatment of broilers with hemin prevented the increase in LOA induced by hemin (P = 0.0001). These results show that modulating the concentrations of uric acid can markedly affect oxidative stress.     

Protection against oxidative stress in liver of four different vertebrates.

The possible relation between respiratory capacity and antioxidant capacity and susceptibility to oxidative stress of the liver has been investigated in Rattus norvegicus, Gallus gallus domesticus, Lacerta s. sicula, and Rana esculenta. Accordingly, we measured oxygen consumption and cytochrome oxidase activity, glutathione peroxidase and glutathione reductase activity and overall antioxidant capacity, and lipid peroxidation and response to oxidative stress in vitro in liver. The order of liver oxygen consumption and cytochrome oxidase activity among the different species was rat > chick > lizard > frog. The antioxidant defenses supplied by the combined action of glutathione peroxidase and glutathione reductase were not adapted to the respiratory capacities. In particular, there was no correlation either between the activities of two enzymes or between their activities and oxygen consumption. In contrast, the overall antioxidant capacity of the liver appeared to be related to its oxidative capacity, and the malondialdehyde formation, an indirect measure of lipid peroxidation, was inversely related to antioxidant capacity. The response to oxidative stress in vitro indicated that the liver susceptibility to oxidative challenge is higher in ectothermic than in endothermic species. Such higher susceptibility appeared to depend on both lower antioxidant capacity and higher levels of free radical producing species. This finding is apparently in contrast with a higher content of cytochromes in endotherms, which are able to determine both respiratory characteristics and sensitivity to pro-oxidants. However, it could indicate the existence of species-related differences in the tissue content of either preventive antioxidants or hemoproteins able to trap the radicals produced at their active center. J. Exp. Zool. 284:610-616, 1999.     

Lutein and zeaxanthin as protectors of lipid membranes against oxidative damage: the structural aspects.

Two main xanthophyll pigments are present in the membranes of macula lutea of the vision apparatus of primates, including humans: lutein and zeaxanthin. Protection against oxidative damage of the lipid matrix and screening against excess radiation are the most likely physiological functions of these xanthophyll pigments in macular membranes. A protective effect of lutein and zeaxanthin against oxidative damage of egg yolk lecithin liposomal membranes induced by exposure to UV radiation and incubation with 2, 2'-azobis(2-methypropionamidine)dihydrochloride, a water-soluble peroxidation initiator, was studied. Both lutein and zeaxanthin were found to protect lipid membranes against free radical attack with almost the same efficacy. The UV-induced lipid oxidation was also slowed down by lutein and zeaxanthin to a very similar rate in the initial stage of the experiments (5-15 min illumination) but zeaxanthin appeared to be a better photoprotector during the prolonged UV exposure. The decrease in time of a protective efficacy of lutein was attributed to the photooxidation of the carotenoid itself. Both lutein and zeaxanthin were found to slightly modify mechanical properties of the liposomes in a very similar fashion as concluded on the basis of H(1) NMR and diffractometric measurements of pure egg yolk membranes and membranes pigmented with the xanthophylls. Linear dichroism analysis of the mean orientation of the dipole transition moment of the xanthophylls incorporated to the lipid multibilayers revealed essentially different orientation of zeaxanthin and lutein in the membranes. Zeaxanthin was found to adopt roughly vertical orientation with respect to the plane of the membrane. The relatively large orientation angle between the transition dipole and the axis normal to the plane of the membrane found in the case of lutein (67 degrees in the case of 2 mol% lutein in EYPC membranes) was interpreted as a representation of the existence of two orthogonally oriented pools of lutein, one following the orientation of zeaxanthin and the second parallel with respect to the plane of the membrane. The differences in the protective efficacy of lutein and zeaxanthin in lipid membranes were attributed to a different organization of zeaxanthin-lipid and lutein-lipid membranes.     

Bilirubin and oxidative stress in term and preterm infants

Hyperbilirubinemia is the most frequent clinical problem neonatologists must deal with during the newborn period. It has been suggested that bilirubin is involved in the balance between antioxidant and pro-oxidant agents due to its antioxidant properties. However, the relevance of these effects in vivo in term and preterm infants is still debated. We performed a literature review of studies that investigated the association between total serum bilirubin (TSB) and oxidative stress in newborn infants. We found that studies in term infants give contradictory results, while studies in preterm infants suggest that the TSB increase is associated with an oxidative stress increase due to concurrent factors other than bilirubin level, such as heme oxygenase (HO) activity. Moreover, it could be speculated that low physiologic TSB values are associated with antioxidant effects, while high pathologic TSB values are associated with pro-oxidant effects. Literature data do not allow the establishment of whether if the antioxidant properties of bilirubin are important from a clinical point of view and can affect the outcome in ill infants.     

Lindane-induced liver oxidative stress.

The development of an oxidative stress condition in the liver by lindane intoxication is discussed as a possible hepatotoxic mechanism of the insecticide. Lindane is metabolized by liver microsomal enzymes to a variety of metabolites, which are susceptible of conjugation for proper elimination. In addition, the interaction of lindane with the liver tissue results in the induction of the microsomal cytochrome P-450 system, together with enhanced rates of superoxide radical generation and a significant increase in indicators of lipid peroxidation. Concomitantly, lindane intoxication induces a derangement of some antioxidant mechanisms of the liver cell, including decreased superoxide dismutase and catalase activities and alterations in reduced glutathione content leading to depressed GSH/GSSG ratios. The time course study of the changes in hepatic lipid peroxidation and antioxidant parameters are closely interrelated and coincide with the onset and progression of morphological lesions.     

Naproxen-induced oxidative stress in the isolated perfused rat liver

We previously showed that naproxen induced the oxidative stress in the liver microsomes and the isolated hepatocytes of rats. In this study, the in situ effect of naproxen on the rat liver tissue was investigated, using the isolated perfused liver from the view-point of the naproxen-induced hepatotoxicity. The leakage of glutamic-oxaloacetic transaminase (GOT) from the perfused liver and appearance of thiobarbituric acid reactive substances (TBARS) in the perfusate increased with the progress of perfusion after a lag time of about 1h. The naproxen-perfusion of the liver decreased the biliary excretion of glutathione (GSH) and oxidized glutathione, glutathione disulfide (GSSG) prior to TBARS production and GOT leakage. GSSG content in the naproxen-perfused liver was significantly higher than in the control. TBARS appeared in the perfusate of the naproxen-perfused liver for 30 min, but not in the control. The biliary excretion clearance (CL(bile)) of indocyanine green (ICG), a reagent for testing the liver function, in the liver perfused with naproxen decreased to a half of that in the liver perfused without naproxen. Thus, the naproxen-induced oxidative stress in the liver was shown to affect the physiological function of liver through the impairment of biliary excretion, which is recognized as a detoxification system.     

Dapsone induces oxidative stress and impairs antioxidant defenses in rat liver

Dapsone (DDS) is currently used in the treatment of leprosy, malaria and in infections with Pneumocystis jirovecii and Toxoplasma gondii in AIDS patients. Adverse effects of DDS involve methemoglobinemia and hemolysis and, to a lower extent, liver damage, though the mechanism is poorly characterized. We evaluated the effect of DDS administration to male and female rats (30 mg/kg body wt, twice a day, for 4 days) on liver oxidative stress through assessment of biliary output and liver content of reduced (GSH) and oxidized (GSSG) glutathione, lipid peroxidation, and expression/activities of the main antioxidant enzymes glutathione peroxidase, superoxide dismutase, catalase and glutathione S-transferase. The influence of DDS treatment on expression/activity of the main DDS phase-II-metabolizing system, UDP-glucuronosyltransferase (UGT), was additionally evaluated. The involvement of dapsone hydroxylamine (DDS-NHOH) generation in these processes was estimated by comparing the data in male and female rats since N-hydroxylation of DDS mainly occurs in males. Our studies revealed an increase in the GSSG/GSH biliary output ratio, a sensitive indicator of oxidative stress, and in lipid peroxidation, in male but not in female rats treated with DDS. The activity of all antioxidant enzymes was significantly impaired by DDS treatment also in male rats, whereas UGT activity was not affected in any sex. Taken together, the evidence indicates that DDS induces oxidative stress in rat liver and that N-hydroxylation of DDS was the likely mediator. Impairment in the activity of enzymatic antioxidant systems, also associated with DDS-NHOH formation, constituted a key aggravating factor.     

Anticarcinogenic antioxidants as inhibitors against intracellular oxidative stress

Oxidative stress has been implicated in the pathogenesis of numerous diseases, including cancer. In the present study, the protective effect of natural antioxidants, such as quercetin and tea polyphenols, on intracellular oxidative stress was studied. Here we report a novel function of quercetin and tea polyphenols, as potential inhibitors of 4-hydroxy-2-nonenal (HNE)-induced intracellular oxidative stress and cytotoxicity. In rat liver epithelial RL34 cells, a potent electrophile HNE dramatically induced the productions of reactive oxygen species (ROS), which correlated well with the reduction in cell viability. We found that quercetin and tea polyphenols, such as epigallocatechin gallate and theaflavins and their gallate esters, significantly inhibited the HNE-induced ROS production and cytotoxicity. In addition, HNE induced a transient decrease in the mitochondrial membrane potential (Δψ), which was also retarded by the antioxidants. These data suggest that the antioxidants, such as quercetin and tea polyphenols, are inhibitors against mitochondrial ROS production.     

Anticarcinogenic antioxidants as inhibitors against intracellular oxidative stress

Oxidative stress has been implicated in the pathogenesis of numerous diseases, including cancer. In the present study, the protective effect of natural antioxidants, such as quercetin and tea polyphenols, on intracellular oxidative stress was studied. Here we report a novel function of quercetin and tea polyphenols, as potential inhibitors of 4-hydroxy-2-nonenal (HNE)-induced intracellular oxidative stress and cytotoxicity. In rat liver epithelial RL34 cells, a potent electrophile HNE dramatically induced the productions of reactive oxygen species (ROS), which correlated well with the reduction in cell viability. We found that quercetin and tea polyphenols, such as epigallocatechin gallate and theaflavins and their gallate esters, significantly inhibited the HNE-induced ROS production and cytotoxicity. In addition, HNE induced a transient decrease in the mitochondrial membrane potential (Δψ), which was also retarded by the antioxidants. These data suggest that the antioxidants, such as quercetin and tea polyphenols, are inhibitors against mitochondrial ROS production.     

New synthetic flavonoids as potent protectors against doxorubicin-induced cardiotoxicity.

A series of 3,7-disubstituted-2(3',4'-dihydroxyphenyl) flavones has been studied as potential cardioprotective agents in doxorubicin antitumor therapy. The influence of substituents on the 3 and 7 position of the flavone nucleus on antioxidant activity cytotoxicity and cardioprotective properties was explored to improve the activity of our lead compound 7-monohydroxyethylrutoside. In the protection against Fe(2+)/vitamin C-induced microsomal lipid peroxidation (LPO assay), IC(50) values ranged from 0.2 to 37 microM. In general, the 3-substituted flavones were the most potent compounds in this assay. The cytotoxicity of the new compounds was tested (up to 250 microM) in hepatocytes. LDH leakage ranged from 2.6-29.2%, whereas the GSH concentrations decreased to 87.3-41.3%. Only four compounds out of this series protected the isolated mouse left atrium against doxorubicin-induced toxicity. Because of the positive inotropic effect of 8d (N-(3-(3',4'-dihydroxyflavon-7-yl)oxypropyl)-N,N,N-trimethylammonium chloride) and 10c (3-hydroxyethoxy-7,3',4'-trihydroxyflavone) on the atrium, compounds 9i (3',4'-dihydroxy-3-glucosylflavone) and 10d (N-(3-(7,3',4'-trihydroxyflavon-3-yl)oxypropyl)-N,N,N-trimethylammonium chloride) were selected to be evaluated as cardioprotective agents in vivo.     

Effects of fasting on oxidative stress in rat liver mitochondria

While moderate caloric restriction has beneficial effects on animal health state, fasting may be harmful. The present investigation was designed to test how fasting affects oxidative stress, and to find out whether the effects are opposite to those previously found in caloric restriction studies. We have focused on one of the main determinants of aging rate: the rate of mitochondrial free radical generation. Different parameters related to lipid and protein oxidative damage were also analyzed. Liver mitochondria from rats subjected to 72 h of fasting leaked more electrons per unit of O₂ consumed at complex III, than mitochondria from ad libitum fed rats. This increased leak led to a higher free radical generation under state 3 respiration using succinate as substrate. Regarding lipids, fasting altered fatty acid composition of hepatic membranes, increasing the double bond and the peroxidizability indexes. In accordance with this, we observed that hepatic membranes from the fasted animals were more sensitive to lipid peroxidation. Hepatic protein oxidative damage was also increased in fasted rats. Thus, the levels of oxidative modifications, produced either indirectly by reactive carbonyl compounds (N^epsilon- malondialdehyde-lysine), or directly through amino acid oxidation (glutamic and aminoadipic semialdehydes) were elevated due to the fasting treatment in both liver tissue and liver mitochondria. The current study shows that severe food deprivation increases oxidative stress in rat liver, at least in part, by increasing mitochondrial free radical generation during state 3 respiration and by increasing the sensitivity of hepatic membranes to oxidative damage, suggesting that fasting and caloric restriction have different effects on liver mitochondrial oxidative stress.     

Catalase takes part in rat liver mitochondria oxidative stress defense

Highly purified rat liver mitochondria (RLM) when exposed to tert-butylhydroperoxide undergo matrix swelling, membrane potential collapse, and oxidation of glutathione and pyridine nucleotides, all events attributable to the induction of mitochondrial permeability transition. Instead, RLM, if treated with the same or higher amounts of H2O2 or tyramine, are insensitive or only partially sensitive, respectively, to mitochondrial permeability transition. In addition, the block of respiration by antimycin A added to RLM respiring in state 4 conditions, or the addition of H2O2, results in O2 generation, which is blocked by the catalase inhibitors aminotriazole or KCN. In this regard, H2O2 decomposition yields molecular oxygen in a 2:1 stoichiometry, consistent with a catalytic mechanism with a rate constant of 0.0346 s(-1). The rate of H2O2 consumption is not influenced by respiratory substrates, succinate or glutamate-malate, nor by N-ethylmaleimide, suggesting that cytochrome c oxidase and the glutathione-glutathione peroxidase system are not significantly involved in this process. Instead, H2O2 consumption is considerably inhibited by KCN or aminotriazole, indicating activity by a hemoprotein. All these observations are compatible with the presence of endogenous heme-containing catalase with an activity of 825 +/- 15 units, which contributes to mitochondrial protection against endogenous or exogenous H2O2. Mitochondrial catalase in liver most probably represents regulatory control of bioenergetic metabolism, but it may also be proposed for new therapeutic strategies against liver diseases. The constitutive presence of catalase inside mitochondria is demonstrated by several methodological approaches as follows: biochemical fractionating, proteinase K sensitivity, and immunogold electron microscopy on isolated RLM and whole rat liver tissue.     

Effects of fasting on oxidative stress in rat liver mitochondria

While moderate caloric restriction has beneficial effects on animal health state, fasting may be harmful. The present investigation was designed to test how fasting affects oxidative stress, and to find out whether the effects are opposite to those previously found in caloric restriction studies. We have focused on one of the main determinants of aging rate: the rate of mitochondrial free radical generation. Different parameters related to lipid and protein oxidative damage were also analyzed. Liver mitochondria from rats subjected to 72 h of fasting leaked more electrons per unit of O₂ consumed at complex III, than mitochondria from ad libitum fed rats. This increased leak led to a higher free radical generation under state 3 respiration using succinate as substrate. Regarding lipids, fasting altered fatty acid composition of hepatic membranes, increasing the double bond and the peroxidizability indexes. In accordance with this, we observed that hepatic membranes from the fasted animals were more sensitive to lipid peroxidation. Hepatic protein oxidative damage was also increased in fasted rats. Thus, the levels of oxidative modifications, produced either indirectly by reactive carbonyl compounds (N^epsilon- malondialdehyde-lysine), or directly through amino acid oxidation (glutamic and aminoadipic semialdehydes) were elevated due to the fasting treatment in both liver tissue and liver mitochondria. The current study shows that severe food deprivation increases oxidative stress in rat liver, at least in part, by increasing mitochondrial free radical generation during state 3 respiration and by increasing the sensitivity of hepatic membranes to oxidative damage, suggesting that fasting and caloric restriction have different effects on liver mitochondrial oxidative stress.     

Protective effect of ascorbic acid against oxidative stress induced by inorganic arsenic in liver and kidney of rat

Ascorbic acid treatment in arsenic trioxide treated rats increased arsenic excretion, inhibited lipid peroxidation, improved GSH status, regulated GSSG turnover and also restored glutathione-S-transferases activity in liver and kidney. Suitable mechanisms leading to ascorbic acid protection have been discussed. Upregulation of GSH dependent enzymes was found to be necessary for a protective effect. Protection is finally attributed to higher GSH levels observed in the liver and kidney of ascorbic acid and inorganic arsenic treated rats. It is also concluded that ascorbic acid protection is influenced by gender dependent factors. Arsenic poisoning is a global problem now. Gender differences need to be considered while applying therapeutic measures.     

Grape seed and skin extract mitigates garlic-induced oxidative stress in rat liver

Garlic is a commonly used spice in folk medicine that can exert adverse health effects when given at a high dose. Grape seed and skin extract (GSSE) exhibits a variety of beneficial effects even at a high dose. In the present study we evaluated the toxicity of high-dose garlic treatment on liver and the protective effect of GSSE. Rats were intraperitoneally administered either with garlic extract (5 g·(kg body weight)(-1)) or GSSE (500 mg·(kg body weight)(-1)) or a combination of garlic and GSSE at the same doses daily for 1 month. Plasma and hepatic levels of cholesterol, triacylglycerol, and transaminases and liver antioxidant status were evaluated. Data showed that a high garlic dose induced liver toxicity and a pro-oxidative status characterized by increased malondialdehyde and decreased antioxidant enzyme activities as catalase, peroxidase, and superoxide dismutase. Garlic increased intracellular H(2)O(2) but decreased free iron and Ca(2+). GSSE alone or in co-treatment with garlic had the reverse effect and counteracted almost all garlic-induced deleterious impacts to near control levels. In conclusion, a high garlic dose induced a pro-oxidative state characterized by the Fenton reaction between H(2)O(2) and free iron, inducing Ca(2+) depletion, while GSSE exerted antioxidant properties and Ca(2+) repletion.     

Ameliorative effects of thyme and calendula extracts alone or in combination against aflatoxins-induced oxidative stress and genotoxicity in rat liver

The aims of the current work were to evaluate the hepatoprotective effect of calendula flowers and/or thyme leave extracts on aflatoxins (AFs)-induced oxidative stress, genotoxicity and alteration of p53 bax and bcl2 gene expressions. Eighty male Sprague–Dawley rats were divided into eight equal groups including: the control group, the group fed AFs-contaminated diet (2.5 mg/kg diet) for 5 weeks, the groups treated orally with thyme and/or calendula extract (0.5 g/kg b.w) for 6 weeks and the groups pretreated orally with thyme and/or calendula extract 1 week before and during AFs treatment for further 5 weeks. Blood, liver and bone marrow samples were collected for biochemical analysis, gene expression, DNA fragmentation and micronucleus assay. The results showed that AFs induced significant alterations in oxidative stress markers, increased serum AFP and inflammatory cytokine, percentage of DNA fragmentation, the expression of pro-apoptotic gene p53 and bax accompanied with a decrease in the expression of bcl2. Animals treated with the extracts 1 week before AFs treatment showed a significant decrease in oxidative damage markers, micronucleated cells, DNA fragmentation and modulation of the expression of pro-apoptotic genes. These results suggested that both calendula and thyme extracts had anti-genotoxic effects due to their higher content of total phenolic compounds.