Potentiation of aflatoxin B1 induced hepatotoxicity in male Wistar rats with ethanol pretreatment

The interaction of ethanol and aflatoxin B1 (AFB1)-induced hepatotoxicity was studied in male Wistar rats using the activity of plasma GOT and GPT, liver triglyceride and histopathologic changes of liver necrosis as indices. Pretreatment of four oral doses of ethanol (4.0 g/kg BW each) at 48, 45, 24 and 21 hrs prior to AFB1 (0.5 to 2.0 mg/kg BW) single i.p. administration caused a significant increase in the activity of PGOT (6 folds) and PGPT (5 folds), liver triglycerides (2 folds) and severity of liver necrosis at 48 hrs after AFB1 administration. Ethanol pretreatment potentiated AFB1-induced hepatotoxicity by increasing MFO enzymes, aniline hydroxylase and p-nitroanisole-O-demethylase activity and lipid peroxidation, and decreasing in cytochrome b5, epoxide hydrolase activity and hepatic glutathione content. However, it did not cause any significant change in the activity of NADPH-cytochrome c reductase and glutathione-S-transferase and cytochrome P-450. These results suggest that potentiation of ethanol pretreatment on AFB1-induced hepatotoxicity may be due to an increase in the metabolic formation of AFB1-2, 3-oxide and subsequent binding to DNA.     

双环醇对大鼠黄曲霉毒素B1代谢和肝毒性的影响

目的:研究抗肝炎新药双环醇对大鼠黄曲霉毒素B_1(AFB_1)代谢和肝毒性的影响.方法:大鼠灌胃双环醇300 mg·kg~(-1)·d~(-1),连服三日后腹腔注射黄曲霉毒素B_1 1.5 mg·kg~(-1).给黄曲霉毒素B_1 16小时后观察双环醇对黄曲霉毒素B_1引起肝损伤的防护作用以及对体外代谢的影响.结果:双环醇(300 mg·kg~(-1)·d~(-1),连服三日)可明显降低黄曲霉毒素B_1引起的大鼠血清转氨酶和肝脏MDA的升高,增加低毒代谢产物AFQ_1的生成.双环醇还可增加大鼠肝脏细胞色素P450总量和胞浆谷胱甘肽含量,诱导P450 CYP2B1介导的7-戊氧基香豆素脱烃酶和谷胱甘肽疏基转移酶的活性.此外,双环醇对P450 CYP3A介导的红霉素脱甲基酶和 P450 CYP1A介导的7-乙氧基香豆素脱烃酶也有诱导作用.结论:双环醇可通过增加大鼠肝脏对AFB_1代谢的解毒功能起到肝保护作用.     

Induced hepatotoxicity in female rats by aflatoxin B1 and ethynylestradiol interaction

Female Sprague-Dawley rats were treated with a single ip dose of aflatoxin B1 (AFB1) (3 or 6 mg/kg). Twenty-four hours later and weekly until killed, some of the rats treated with AFB1 were given ethynylestradiol (EE) by gavage at the dose of 13 mg/kg. One, three, six, and nine months following the beginning of the experiment, animals were killed. Light microscopy of liver and histochemical determinations of gamma-glutamyltransferase (GGT) as well as the measurement of hepatic drug-metabolizing enzyme activities were investigated. The results show that AFB1 induced only very weak changes in the levels of different constituents studied. Thus, the mycotoxin did not affect GGT activity and increased epoxide hydrolase activity by a maximum of 42%. In contrast, EE significantly and progressively decreased (20 to 50%) the activity of UDP-glucuronosyltransferase (UDPGT) as well as the concentration of cytochrome P-450 and microsomal proteins. However, the estrogen increased the activity of epoxide hydrolase up to 150% as well as the activity of the hepatic (400%) and plasma (175%) GGT. The results also indicate that AFB1 amplified the EE-induced increase in liver weight and enhanced the depressive effects of the estrogen on microsomal proteins, cytochrome P-450, and UDPGT. Foci of cellular alteration which consisted of clear, acidophilic and basophilic cell lesions were seen in the livers of treated rats examined by light microscopy. These lesions were more prominent in the livers of animals given combinations of AFB1 and EE; they were accompanied by a strong intensity of GGT staining in the periportal area and a marked increase of the enzyme activity in the plasma (324%). From the sixth month, the livers of some animals treated with the combinations of AFB1 and EE showed hyperplastic nodules. This study indicates that the interaction between chronic administration of EE and a single ip injection of AFB1 induces hepatic lesions considered as possible forerunners of liver cell carcinomas. It also shows that GGT is a potential marker of preneoplastic lesions and may be used, therefore, in epidemiologic surveys in humans exposed to liver carcinogens such as the aflatoxins.     

Enhanced hepatotoxicity of aflatoxin B1 in the rat by ethanol: ultrastructural changes.

The effect of ethanol pretreatment on aflatoxin B1 (AFB1)-induced ultrastructural alteration in hepatocytes was investigated in male Wistar rats. Pretreatment with 4 oral doses of ethanol (4.0 g/kg body wt. each) at 48, 45, 24 and 21 h prior to a single intraperitoneal administration of AFB1 (2.0 mg/kg body wt.) produced an alteration of fine structure more extensive than that observed after administration of AFB1 or ethanol alone. The organelles most affected were the mitochondria, endoplasmic reticulum and nucleus. The degree of nuclear edema, rough endoplasmic reticulum dilatation and mitochondrial swelling in hepatocytes from rats treated with ethanol and AFB1 was much greater than that of hepatocytes from rats treated with AFB1 or ethanol alone. Thus, ethanol may play a specific role in the potentiation of hepatic injury induced by AFB1 at the mitochondrial, endoplasmic reticular and nuclear level.     

Hepatocurative effect of picroliv and silymarin against aflatoxin B1 induced hepatotoxicity in rats

Single doses of aflatoxin B1 (2 mg/kg, i.p.) caused significant increases in the activities of tau-glutamyl transpeptidase, 5'-nucleotidase, acid phosphatase and acid ribonuclease, and decreases in the activities of succinate dehydrogenase and glucose-6-phosphatase in liver, after 8 weeks. The level of lipid peroxides, DNA, RNA, and cholesterol increased while glycogen decreased. It also increased the serum level of transaminases, sorbitol dehydrogenase, glutamate dehydrogenase, lactate dehydrogenase, acid phosphatase, alkaline phosphatase, and bilirubin. Oral administration of picroliv (25 mg/kg/day for 15 days), a standardised iridoid glycoside fraction of Picrorhiza kurroa, 6 weeks after aflatoxin B1 toxication, significantly prevented the biochemical changes induced in liver and serum of aflatoxin B1 treated rats. The hepatocurative effect of picroliv and silymarin, a plant based standard hepatoprotective are comparable.     

Effect of ethanol on the in vivo covalent binding and in vitro metabolism of aflatoxin B1 in rats

The binding of [3H]aflatoxin B1 (AFB1) to the DNA, RNA and protein of liver after i.p. administration to rats with and without ethanol pretreatment was studied. The quantities of AFB1 binding to DNA and RNA were significantly increased by ethanol pretreatment but the formation of protein adducts was not affected. AFB1 metabolism by hepatic microsomes from ethanol-treated rats to aflatoxins M1 (AFM1) and Q1 (AFQ1) was increased when compared to those of control microsomes. These results suggest that an increase in AFB1 binding to liver nucleic acids and AFB1 metabolism after pretreatment of ethanol resulted from an increase in hepatic mixed-function oxidases and a possible decrease in hepatic glutathione (GSH) content which subsequently lead to an increase in hepatotoxicity of AFB1.     

Protective activity of different hepatic cytosolic glutathione S-transferases against DNA-binding metabolites of aflatoxin B1

To evaluate the role of glutathione S-transferase (GST) isoenzymes in induced resistance of hepatocytes to aflatoxin B1 (AFB1), we compared DNA protective activities of different hepatic cytosol preparations and purified GSTs from normal rats, rats exposed to different polychlorinated biphenyls (PCBs), and rats with carcinogen-induced hepatocellular neoplasms, with cytosols or purified GSTs from mouse, rainbow trout, and human livers. These comparisons were performed in an in vitro assay for [3H]AFB1-DNA binding after activation by rat liver microsomes. Cytosol and S-hexylglutathione-affinity-purified GST preparations from livers of mice consistently had strong protective activity against AFB1-DNA binding. The majority of this activity was dependent on the presence of reduced glutathione (GSH) but some GSH-independent protection was observed in mouse hepatic cytosol, but not in purified GST preparations. We found that all of the GSH-dependent DNA-protective activity in mouse liver eluted as a single GST isoenzyme by hydroxyapatite chromatography. Preparations of cytosol and purified GSTs from normal rat liver, rainbow trout liver, and human liver had much less AFB1-specific DNA protective activity than GSTs found in mouse liver preparations. Cytosol from rats with carcinogen-generated liver neoplasms and livers induced with 3,3',4,4'-tetrachlorobiphenyl and 2,2',4,4',5,5'-hexachlorobiphenyl had more GST activity toward CDNB than cytosol from normal rat liver. When equivalent units of GST activity (CDNB) were compared, there was little difference observed between the DNA-protective activities of PCB-induced and normal rat liver cytosols, yet cytosol from rat liver neoplasms was more protective. Purified GST-P (7-7), the GST isoenzyme most induced in carcinogen-generated rat liver neoplasms, was not protective when added at protein concentrations found to be protective for total GSTs isolated from these neoplasms. These studies demonstrate that the resistance of mouse liver to AFB1 can be explained primarily by a single constitutive GST isoenzyme (YaYa or 4-4) with a relatively high activity toward DNA-binding metabolites of AFB1. GST isoenzymes with such high specific DNA protective activity against AFB1 metabolites were not evident in human, rat, or rainbow trout liver or in PCB-induced or neoplastic rat liver preparations.     

Effect of aflatoxin B1 on hepatic drug-metabolizing enzymes in female rats. Interaction with a contraceptive agent

1. The effect of a contraceptive on aflatoxin B1 toxicity has been studied in female rats treated for 15 consecutive days with repeated doses of aflatoxin (0.40 mg/kg/day), norethindrone (0.60 mg/kg/day) and ethynylestradiol (0.012 mg/kg/day). 2. Increases occurred in hepatic microsomal cytochrome P-450 content (31%), and in the activities of epoxide hydrase (77%), UDP-glucuronyltransferase (67%) and gamma-glutamyltransferase (78%). 3. Aflatoxin alone caused a 22% increase in GSH S-epoxide transferase activity, whereas the contraceptive given alone or combined with aflatoxin increased the hepatic reduced glutathione. 4. The effect of aflatoxin plus contraceptive was not additive. 5. The effects caused by aflatoxin and the contraceptive were similar, and the contraceptive (depending on its progestogen/estrogen ratio), may modify aflatoxin toxicity by increasing the drug-metabolizing enzyme activities and the concentration of hepatic glutathione.     

Effects of chronic exposure to aflatoxin B1 and aflatoxin M1 on the in vivo covalent binding of aflatoxin B1 to hepatic macromolecules

Induction of resistance to aflatoxin B1 (AFB1) binding to cellular macromolecules in the rat by chronic exposure to AFB1 and aflatoxin M1 (AFM1) was investigated. The binding of [14C]AFB1 to liver macromolecules was measured in F-344 rats fed 0.5 ppb or 50 ppb AFM1 or 50 ppb AFB1 for 41 wk. The animals then received an intragastric dose of [14C]AFB1 at 5 micrograms/kg and were sacrificed 6 h later. Hepatic DNA, RNA, and protein were isolated by chloroform-phenol extraction and hydroxylapatite chromatography. In animals preexposed to 50 ppb AFB1, labeled AFB1 binding to DNA, RNA, and protein was decreased by 72%, 74%, and 61%, respectively. Preexposure to AFM1 resulted in a small reduction in binding to nucleic acids. Glutathione transferase activity was increased by 133% in animals fed 50 ppb AFB1, by 48% in those preexposed to 50 ppb AFM1, and remained at control values in rats fed 0.5 ppb AFM1. These results suggest that the induction of detoxification enzymes following chronic exposure to aflatoxin might contribute to the reduction in covalent binding of AFB1 to macromolecules.     

Time-course effects of ethanol pretreatment on hepatic necrosis and fat accumulation induced by aflatoxin B1 in the rat

Effect of ethanol pretreatment on acute hepatotoxicity and hepatic fat accumulation induced by aflatoxin B1 (AFB1) was followed up to 120 h in male Wistar rats. Pretreatment with 4 oral doses of ethanol (4.0 g/kg body wt. each) at 48, 45, 24 and 21 h prior to AFB1 (2.0 mg/kg body wt.) single intraperitoneal administration caused a significant increase in the activity of plasma glutamic oxaloacetic transaminase (PGOT, 2.4-fold), plasma glutamic pyruvic transaminase (PGPT, 2.8-fold), liver triglycerides (2.3-fold) and the severity of liver necrosis at 72 h after AFB1 administration. The effect of ethanol pretreatment on an increase in the accumulation of liver cholesterol and cholesterol esters induced by AFB1 is additive in nature. In a time-course study, it was shown that liver necrosis and triglyceride, cholesterol and cholesterol ester accumulation occurred simultaneously in both groups of rats treated with AFB1 and ethanol-AFB1. These results suggest that fat accumulation per se is not a primary cause of liver necrosis induced by AFB1 and ethanol-AFB1.     

Effects of intermittent exposures of aflatoxin B(1) on hepatic and testicular glutathione S-transferase in rats

Glutathione S-transferase (GST) plays a major role in the detoxification of the potent hepatocarcinogen aflatoxin B(1) (AFB(1)). This study evaluated the effects of intermittent exposures to AFB(1) on hepatic and testicular GST in rats. Male Fischer 344 rats were fed diets containing AFB(1) (0, 0.01, 0.04, 0.4 and 1.6 ppm) intermittently at 4-week intervals up to 20 weeks. The control animals were fed an AFB(1)-free NIH-31 diet. Rats consuming diets with 0.01 ppm AFB(1) did not show the induction of hepatic or testicular GST activity. Intermittent exposures to AFB(1) at concentrations of 0.04-1.6 ppm significantly increased the GST activities. The increase of the enzyme activity was proportional to the dose and length of AFB(1) exposure.     

Prevention of aflatoxin B1-initiated hepatotoxicity in rat by marine algae extracts

Chemoprevention by extracts of Laurencia obtusa (E1) and Caulerpa prolifera (E2) collected from the Egyptian coast of the Red Sea against aflatoxin B(1) (AFB(1))-initiated hepatotoxicity in female Sprague-Dawley rats has been studied. Animals were fed aflatoxin-contaminated diet (3 mg kg(-1) diet) for 6 days then treated orally with pure aflatoxin B(1) (AFB(1)) (200 microg kg(-1) b.w.) for 4 days either in combination with or before E1 or E2 administration (50 mg kg(-1) b.w.). AFB(1) resulted in a signicant increase in serum alpha fetoprotein, carcinoembryonic antigen, tumor necrosis factor alpha, nitric oxide, interleukin-1alpha, procollagen III and lipid peroxidation level in the liver. It caused a signicant decrease in food intake, body weight, serum leptin, the activities of glutathione peroxidase, superoxide dismutase and DNA and RNA concentrations in the liver. Cotreatment with AFB(1) and E1 or E2 resulted in an obvious improvement in all tested parameters. Noteworthy, E2 was more effective than E1 in the protection against AFB(1)-induced hepatotoxicity.     

Protective effect of Aquilegia vulgaris L. on aflatoxin B(1)-induced hepatic damage in rats

The aim of the study was to investigate the effect of ethanol and ethyl acetate extract obtained from Aquilegia vulgaris L. on microsomal lipid peroxidation, reduced glutathione level and antioxidant enzymes activity in the liver of rats intoxicated with aflatoxin B(1) (AFB(1)). Animals were pretreated with 12 daily p.o. doses of the extracts tested (100mg/kg body weight). Then AFB(1) was administered intraperitoneally at a single dose of 1.5mg/kg b.w. to evoke the liver damage. α-Tocopherol was used as a positive control. Reduced glutathione (GSH) was depleted in aflatoxin-treated rats by 80% in comparison with that in the controls. The extracts restored the GSH concentration up to the basal level. Microsomal lipid peroxidation stimulated by Fe(2+)/ascorbate (assessed by measuring TBARS) was enhanced in AFB(1)-treated rats by 28% as compared to that in the control group. The extracts caused a decrease in TBARS level by 40% and 27%. Only two antioxidant enzymes were affected by AFB(1) administration. The activity of catalase was reduced by 24% and the activity of glutathione-S-transferase (GST) was increased by 33%. The pretreatment with ethyl acetate and ethanol extract reduced the GST activity by 76% and 30%, respectively. No significant changes in the activity of other antioxidant enzymes were observed in rats treated with the extracts and AFB(1). It can be concluded that multiple pretreatment with the extracts obtained from A. vulgaris attenuated aflatoxin B(1)-induced hepatic damage as evidenced by inhibition of lipid peroxidation and preventing reduced glutathione depletion.     

Residues of aflatoxin B1 in broiler meat: Effect of age and dietary aflatoxin B1 levels

This study describes the effect of dietary levels of aflatoxin B1 (AFB1) and age of the birds upon the residue level in liver and muscles of broiler chicks. In three different experiments broiler chicks of 7, 14 and 28 days of age were kept for 7 days on contaminated rations having 1600, 3200 and 6400 μg/kg AFB1. AFB1 residues were detected earlier in younger birds and those fed high AFB1 dietary levels. The highest residue levels in liver and muscles of young chicks fed 6400 μg/kg AFB1 was 6.97 ± 0.08 and 3.27 ± 0.05 ng/g, respectively. Maximum residue concentration was high in birds of young age and those kept on high AFB1 ration. After withdrawal of AF contaminated rations, residues clearance was slow and AFB1 was detectable in liver and muscles of birds for longer duration in younger birds and those fed high AFB1 dietary levels. AFB1 residues in poultry tissues may buildup to high levels in areas with no regulatory limits on AFB1 levels of poultry feed and may pose a risk to consumers health.     

Effect of aflatoxin B1 treatment in vivo on the in vitro activity of hepatic and extrahepatic glutathione S-transferase

The effect of aflatoxin B1 (AFB1) on the glutathione S-transferase activity (GST) and on non-protein thiol levels of different tissues was studied in adult male Wistar rats. Animals received a single dose of the toxin (100 or 500 micrograms/kg body wt., p.o.), and were studied 6 or 24 h after administration. GST was determined in liver, renal cortex, duodenum, jejunum-ileum and distal ileum, using 3 substrates: 1-chloro-2,4-dinitrobenzene (CDNB), trans-4-phenyl-3-buten-2-one (PBO) and 1,2-epoxyethylbenzene (STOX). The non-protein thiol content of all tissues tested increased with the lowest dose at 6 h, returning to normal values at 24 h, while the higher dose produced a significant decrease in reduced thiol levels at 6 h, returning to normal values at 24 h. AFB1 administration induced, independently of dose and tissue, total GST (CDNB) and epoxide-transferase activity (STOX) while A--C-type transferases (PBO) were inhibited. Almost all activities returned to normal values at 24 h. In cases of enzyme induction there was in general an increase in Vmax and a decrease in apparent Km. The opposite was seen in cases of inhibition. In conclusion, the results provide evidence that extrahepatic GST could be important in the overall process of detoxification of AFB1. The behavior seen in hepatic and extrahepatic tissues revealed the functions of catalysis (B-type transferases) and covalent bond formation, as well as inactivation by probable AFB1 metabolites (A--C-type transferases).     

The effects of dietary cabbage on xenobiotic-metabolizing enzymes and the binding of aflatoxin B1 to hepatic DNA in rats

The effect of dietary cabbage (Brassica oleracea) on the binding of aflatoxin B1 (AFB1) to hepatic DNA and on the activities of liver and intestinal microsomal and cytosolic enzymes was studied in weanling male Fischer 344 rats. Freeze-dried cabbage was fed to rats at a level of 25% in the diet for 21 days, while others received a basal diet. In the cabbage-fed group there was an 87% (P less than 0.01) reduction in the binding of AFB1 to hepatic DNA 2 hr after the ip injection of [3H]AFB1 (3 micrograms/kg). There was also a 41% (P less than 0.05) increase in liver weight expressed relative to body weight. Hepatic and intestinal glutathione S-transferase activities were significantly increased (2.1- and 2.3-fold, respectively) over those in rats fed the basal diet. Hepatic and intestinal microsomal epoxide hydrolase activities were significantly increased (2.6- and 1.4-fold, respectively) over the basal group. Intestinal aryl hydrocarbon hydroxylase (AHH) and ethoxycoumarin O-deethylase (ECD) activities were significantly increased (2.3- and 2.5-fold, respectively), over the basal group but dietary cabbage had no significant effect on hepatic AHH or ECD activities.     

Interactions between aflatoxin B1 and dietary iron overload in hepatic mutagenesis

BACKGROUND/AIM: Dietary aflatoxin B(1) (AFB(1)) exposure and iron overload are important causes of hepatocellular carcinoma in sub-Saharan Africa. The aim of this study was to investigate if the two risk factors have an interactive effect. METHODS: Four groups of Wistar albino rats were studied for 12 months. Group 1 (control) was fed the normal chow diet; group 2 (Fe) was supplemented with 0.75% ferrocene iron; group 3 (Fe+AFB(1)) was fed 0.75% ferrocene throughout and gavaged 25 microg AFB(1) for 10 days; group 4 (AFB(1)) was gavaged 25 microg AFB(1) for 10 days. Iron profile, lipid peroxidation (LPO), 8-hydroxydeoxyguanosine (8OHdG), oxidative lipid/DNA damage immunohistochemistry, superoxide/nitrite free radicals, cytokines IL6, IL-10, transaminases (ALT/AST) and Ames mutagenesis tests were performed. RESULTS: LPO and ALT showed a significant (p<0.05)/additive effect and 8OHdG a significant (p<0.05)/multiplicative effect in the Fe+AFB(1) group. IL-6 produced a negative synergy as against an additive antagonistic effect with IL-10. Massive deposits of 4-hydroxynonenal (4-HNE) and 8OHdG were observed in liver sections of the Fe+AFB(1) group, suggestive of multiplicative synergy. Significant levels of mutagenesis (p<0.001) were observed in the Fe+AFB(1) group. This multiplicative synergy was five-fold. CONCLUSION: Dietary iron overload and AFB(1) have a multiplicative effect on mutagenesis.