Effect of portacaval anastomosis on the activities of hepatic enzymes related to cholesterol and bile acid metabolism in rats.

1. The effect of a portacaval anastomosis on the activities of hepatic enzymes related to cholesterol metabolism was investigated in rats. 2. Portacaval anastomosis led to a fall in body weight and liver weight/body weight ratio, and to a rise in the activities of hydroxymethylglutaryl-CoA reductase and cholesterol 7alpha-hydroxylase per g of liver. The net effect was to maintain a normal activity of both enzymes per 100 g of rat. Diurnal rhythm in the activities of both enzymes was maintained after portacaval anastomosis. 3. The rate of excretion of total bile acids, per 100 g of rat, in bile fistula rats was not significantly decreased by portacaval anastomosis.     

The influence of portacaval anastomosis on gonadal and anterior pituitary hormones in a rat model standardized for gender, food intake, and time after surgery

Portacaval anastomosis causes delayed growth, decreased testes and liver weights, and elevated estradiol serum levels in male rats compared with sham-operated controls. Female rats treated with portacaval anastomosis grow at a normal rate despite changes in liver weight and estradiol levels similar to those observed in the male rats. This study examined the pituitary gonadal axis in both genders in this animal model. The rats receiving portacaval anastomosis were compared with both pair-fed and sham-operated control groups. Portacaval anastomosis decreased serum testosterone and increased estradiol in the male animals, while both testosterone and estradiol were increased in the females compared with gender-matched pair-fed and sham controls. Because pair feeding lowers male testosterone to a lesser extent, impaired nutrition may partially account for the decrease in the males treated with portacaval anastomosis. The ratio of estradiol to testosterone increased following anastomosis in male rats, but it was decreased in similarly treated females. Portacaval and anastomosis decreased luteinizing hormone without changing follicle-stimulating hormone in both male and female rats compared with sham-operated controls. Growth hormone was significantly decreased in male portacaval-treated rats compared with sham- and pair-fed animals. Increased insulin levels were found in both male and female pair-fed and portacaval anastomosis-treated animals. These data suggest that following portacaval anastomosis in rats, growth, serum testosterone, estradiol to testosterone ratios, and growth hormone are altered in a gender-specific manner with gender-independent changes in insulin and luteinizing hormone levels. These gender-specific effects may protect the portacaval anastomosis-treated female rat from growth retardation.     

Effect of vitamin A dietary intake on in vitro and in vivo activation of aflatoxin B1.

The mechanism by which vitamin A prevents or delays in chemical carcinogenesis remains unclear. In the present study, we assess the suggestive role of vitamin A in the initiation phase of carcinogenesis. We have conducted a dose-effect relationship between vitamin A dietary intake and aflatoxin B1 (AFB1) genotoxicity measured both in vitro and in vivo. Thus AFB1-induced mutagenesis in Salmonella typhimurium TA98 was investigated and compared to AFB1-induced single-strand breaks (SSBs) in DNA of rat hepatocytes. Rats were fed ad libitum with diet containing 0, 5, 50 or 500 IU of retinyl palmitate for 8 weeks. The AFB1-treated rats were injected i.p. with 1 mg/kg body weight. In the Ames test conditions TA98 back-reversion was negatively correlated with the log of vitamin A concentration in liver S9 fractions from experimental groups. However, the activities of metabolizing enzymes which specifically activate or deactivate AFB1 were found to be significantly decreased in vitamin A-deficient animals and weakly modified in vitamin A-supplemented animals. For in vivo experiments, the DNA elution rate of both AFB1-treated and untreated rats was increased in vitamin A deficiency condition (+79% and +17% respectively) and was reduced with the higher vitamin A dietary level (-44% and -53% respectively). DNA damage measured in vivo showed a significant positive correlation with mutagenic activity measured in the Ames test. These results confirm that the vitamin A status of animals can influence AFB1 genotoxic activity in vitro and indicate that this phenomenon also occurs in vivo. Thus a similar mechanism may be considered for the protective action of vitamin A both in vitro and in vivo. However, this mechanism is unlikely to involve modulation of the microsomal enzyme system responsible for AFB1 metabolism. Therefore a protective mechanism at the cytosolic or nuclear levels may be suggested.     

Effect of portacaval surgical anastomosis on systemic and splanchnic hemodynamics in portal hypertensive, cirrhotic rats

The effect of surgical end-to-side portacaval anastomosis (PCSA) on systemic and splanchnic circulation has been studied in cirrhotic rats with portal hypertension (CCl4-phenobarbital method) and in control animals. Hemodynamics have been measured using the microsphere technique, with a reference sample for the systemic hemodynamic measurements, and intrasplenic injection for portal systemic shunting rate measurements. Compared with controls, sham-operated (SO) cirrhotic rats showed a hyperdynamic circulation with increased cardiac output (CO) and decreased mean arterial pressure and peripheral resistances. PCSA in control rats induced only a small change in systemic hemodynamics, with parallel decreases in arterial pressure and peripheral resistances, and a small, nonsignificant increase in CO. In cirrhotic rats, PCSA induced a decrease of CO to values similar to those of control rats, with an increase in total peripheral resistances. PCSA induced an increase in hepatic arterial blood flow in control and in cirrhotic rats, portal pressure becoming in this latter group not different from that of control rats. Blood flow to splanchnic organs was higher in SO cirrhotic than in SO control animals. Thus portal venous inflow was also increased in SO cirrhotic rats. PCSA induced an increase in portal venous inflow in control rats, which was only significant in cirrhotic rats when expressed as a percentage of CO. In SO control animals, a significant correlation was observed between total peripheral resistances and splanchnic arteriolar resistances and between CO and splanchnic blood flow. These correlations were not observed in cirrhotic rats. These results do not support the hypothesis that hyperdynamic circulation shown by cirrhotic rats is based on increases in splanchnic blood flow and (or) massive portal systemic shunting.     

Coexistence of Aflatoxicosis with Protein Malnutrition Worsens Hepatic Oxidative Damage in Rats

To investigate the effects of the coexistence of aflatoxin B1 (AFB1) and protein malnutrition in rat liver, weanling rats were fed either normal protein diet (20% protein), low‐protein (PEM) diet (5%), normal protein diet + 40 ppb AFB1, or low‐protein diet + 40 ppb AFB1. After 8 weeks, biomarkers of hepatic functions and oxidative stress, caspase‐3 activity, and tumor suppressor protein 53 (p53) were determined spectrophotometrically. Randomly amplified polymorphic DNA polymerase chain reaction (RAPD‐PCR) was employed to determine genomic alterations among the groups. Coexistence of aflatoxicosis and PEM significantly decreased glutathione, glutathione‐S‐transferase, glutathione peroxidase, and superoxide dismutase, while it increased peroxidase and catalase. RAPD‐PCR showed genomic alterations that were associated with significant increases in p53 level and caspase‐3 activity in rats fed PEM diet + AFB1. In conclusion, the coexistence of aflatoxicosis and protein malnutrition induced oxidative stress with concomitant genomic alterations in the liver of weanling rats.     

Aflatoxin B1-induced Hprt mutations in splenic lymphocytes of Fischer 344 rats. Results of an intermittent feeding trial

In a previous study, we found an increase in the mutant frequency at the Hypoxanthine phosphoribosyl transferase (Hprt) locus in the splenic lymphocytes of Fischer 344 rats acutely exposed to aflatoxin B1 (AFB1). Because an acute exposure may not reflect the exposure pattern of individuals whose diet may contain AFB1-contaminated foodstuffs, we sought to determine if the feeding regimen affected the induction of Hprt mutations in the rat splenic lymphocyte. Thus, Fischer 344 rats were fed either (A) a control diet, (B) various doses of AFB1 for three four-week periods interspersed with two four-week periods of the control diet, or (C) continuously fed 1.6 ppm of AFB1. Not only was a significant increase in the mutant frequency detected in the lymphocytes of rats fed a dose as low as 0. 01 ppm of AFB1, but the increase in the mutant frequency at the end of the 20-week experimental period was consistent with an accumulation of damage induced by AFB1. These results indicate that the rat lymphocyte/Hprt assay is useful for detecting chronic low level exposures. Further, these data suggest that an intermittent, low-level exposure to AFB1 may present a human health risk.     

Lack of effect of long-term glutathione administration on aflatoxin B1-induced hepatoma in male rats

The effect of long-term GSH administration on aflatoxin B1 (AFB1)-induced carcinogenesis in the livers of male Wistar II rats was evaluated. No significant effect of an 11 months period of reduced glutathione (GSH) administration was observed concerning both the survival curve and the incidence of liver tumors. Liver tissues of all animals were bearing tumors or nodular lesions 24 months after AFB1 treatment, regardless of GSH treatment. The capacity of the GSH conjugation system was elevated in the liver tissue of AFB1-treated animals both by an increase of GSH content and an increase of the specific activities of several GSH S-transferase isoenzymes. Likewise the specific activities of GSH related enzymes as GSSG reductase and gamma-glutamyltransferase (gamma-GT) and the activity of the GSH independent detoxication system NAD(P)H:quinone oxidoreductase were increased in the AFB1-treated livers, there was no significant effect of GSH treatment. These results demonstrate that long-term GSH treatment has no effect on the survival of AFB1-pretreated male rats on the incidence of liver tumors and on the activities of drug metabolizing systems. The hepatic detoxication capacity 24 months after AFB1 treatment is elevated.     

The effect of portacaval transposition of hepatic cholesterol-7alpha-hydroxylase activity in the rat.

Portacaval anastomosis in the rat results in an increase in the activity of the liver microsomal cholesterol-7alpha-hydroxylase enzyme system. The increase in the activity of this oxygenase occurs despite a decrease in the total amount of cytochrome p450 in the liver microsomes after portacaval anastomosis. It is possible to increase further the activity of the cholesterol-7alpha-hydroxylase enzyme in these portacaval shunted animals by feeding them on a diet containing a bile salt sequestering agent. This suggests that one of the factors influencing the activity of the liver microsomal cholesterol-7alpha-hydroxylase enzyme may be the concentration of bile salts reaching the liver from the blood plasma. Portacaval anastomosis in the rat tended to achieve a small decrease in the plasma cholesterol concentration.     

Non-invasive in vivo magnetic resonance imaging assessment of acute aflatoxin B1 hepatotoxicity in rats

Acute aflatoxin B1 (AFB1)-induced hepatotoxicity was assessed in vivo in male Sprague-Dawley rats (150-300 g) using magnetic resonance imaging (MRI). MRI results were compared to serum enzyme levels, histology and electron microscopy. Twenty-four hours following intraperitoneal delivery of AFB1 (3 mg/kg body weight in a saline/dimethyl sulfoxide (DMSO; 0.03 ml/kg body weight) solution), regions of damage, characterised by increased proton signal intensities in T2-weighted images, were observed in the vicinity of the hepatic portal vein (HPV) and in the right medial regions of the liver. Image analysis of regions of apparent damage around the HPV and right medial regions, following 24 h of AFB1 exposure, indicated statistically significant (P<0.05) increases in proton image signal intensities, when compared to saline/DMSO-treated rats. No significant difference in proton image signal intensities were observed 1-2 h following AFB1 exposure. Twenty-four hours following AFB1 exposure, histopathological assessment was characterised by portal/central vein/artery congestion, sinusoid congestion, nuclear pyknosis and karyolysis, and hepatocyte vacuolation; electron microscopy (EM) examination indicated nuclear debris, swollen cytoplasmic compartments, vacuolation, and the disappearance of the smooth endoplasmic reticulum, and elevated levels of serum aspartate aminotransferase and alanine aminotransferase were found to be significantly different (P<0.01) than controls.     

Caffeic acid phenethyl ester modulates aflatoxin B1‐induced hepatotoxicity in rats

Aflatoxin B1 (AFB1) is the most potent of the mycotoxins and is widely observed in nutrition abnormalities. There are some studies suggesting oxidative stress‐induced toxic changes on liver related to AFB1 toxicity. The aim of the present study was to evaluate whether antioxidant caffeic acid phenethyl ester (CAPE) relieves oxidative stress in AFB1‐induced liver injury in rat. Twenty‐four male rats were equally divided into three groups. The first group was used as a control. The second group received three doses of AFB1. The three doses of CAPE were given to constitute the third group with doses of AFB1. After 10 days of experiment, liver and serum samples were taken from all animals. Serum gamma glutamyl transferase (GGT), alkaline phosphatase (ALP), glutathione s‐transferase (GST), nitric oxide (NO) and sulfhydryl values were higher in the AFB1 group than in control, whereas serum GGT, ALP, GST and NO values were decreased by in the AFB1 + CAPE group than in AFB1 group. Liver GST, total oxidant capacity, sulfhydryl, apoptosis index and ischemia‐modified albumin values were higher in the AFB1 group than in control, whereas the GST activity and apoptosis index were lower in the AFB1 + CAPE group than in the AFB1 group. There were histopathological degeneration and apoptosis in hepatocytes of AFB1 group. The findings were totally recovered by CAPE administration. In conclusion, we observed that AFB1 caused oxidative and nitrosative hepatoxicity to hepatocytes in the rat. However, CAPE induced protective effects on the AFB1‐induced hepatoxicity by modulating free radical production, biochemical values and histopathological alterations. Copyright © 2013 John Wiley & Sons, Ltd.     

Differential effects of 4-hydroxyaminoquinoline-1-oxide on pancreatic and liver DNA synthesis in rats.

The effect of 4-hydroxyaminoquinoline-1-oxide (4-HAQO) on DNA synthesis in the pancreas and liver, target and non-target organs for 4-HAQO carcinogenesis, respectively, were compared. Pancreatic and liver DNA synthesis were simultaneously induced in rats fed a protein deficient diet containing 0.5% DL-ethionine for 18 days, and DNA synthesis in both tissues was inhibited by hydroxyurea. A single i.v. injection of 4-HAQO at a dose of 7 mg/kg body weight also inhibited DNA synthesis in both tissues within 4 h. In the pancreas the inhibition was maximum at a dose of 7 mg/kg, and DNA synthesis was less than in the pancreas of rats fed a control grain diet. This inhibition continued for the subsequent 5 days which were tested. In the liver, the degree of inhibition was less than in pancreas but the value remained higher than in rats fed control diet. The inhibition of liver DNA synthesis at a dose of 7 mg/kg completely recovered within 1 day. These results suggest that the lesions of DNA induced by 4-HAQO and its repair might be different between the pancreas and the liver. A pancreatic chemical carcinogen, 4-HAQO, might thus have the same cytotoxic effect that liver carcinogens have toward the liver resulting in failure to respond to mitotic stimuli. This might be causally related to the organotropism of 4-HAQO toward the pancreas.     

Effects of Dietary Amino Acids on Brain Amino Acids and Transmitter Amines in Rats with a Portacaval Shunt

The etiologic relationship between disturbances in metabolism of amino acids and amines and hepatic coma was investigated by examining the effects of diets containing various mixtures of amino acids on brain amine metabolism in rats with a portacaval shunt, using a method for simultaneous analysis of amino acids and amines. Rats with a portacaval shunt were fed on four different amino acid compositions with increased amounts of various amino acids suspected to be etiologically related to hepatic coma, such as methionine, phenylalanine, tyrosine, and tryptophan. The animals were killed 4 weeks after operation. During the experimental period, these animals did not become comatose, but exhibited various behavioral abnormalities. Marked increase in the plasma and brain levels of the augmented amino acids, especially methionine and tyrosine, were observed in rats with a portacaval shunt. Brain noradrenaline, dopamine, and serotonin levels were significantly decreased when the brain tyrosine level was increased. These results indicate that in rats with a portacaval shunt the dietary levels of amino acids greatly influence the brain levels of both amino acids and transmitter amines.     

Antimutagenic effect of eight natural foods on moldy foods in a high liver cancer incidence area.

Ames test procedures were used to test 8 natural food extracts for their antimutagenic activity against the mutagenic activity induced in S. typhimurium strains TA98 and TA100 by aflatoxin B1 (AFB1) or metabolic extracts from A. versicolor or A. ochraceus. The tested substances were extracted repeatedly with acetone. The revertants induced by AFB1, metabolic extracts of A. versicolor or A. ochraceus were significantly decreased when extracts of the 8 natural foods were added to the media. The results showed that these extracts had marked inhibitory effects on the mutagenic activity induced by AFB1 or metabolic extracts of the two molds and also suggested that antimutagenic substances were present in these natural foods. These experiments provide a scientific basis for the study of food substances for the prevention of carcinogenesis. It is considered that these 8 natural food extracts produce marked antimutagenic effects and are practically valuable in the field of chemoprophylaxis of liver cancer in humans.     

Euvolemic cirrhotic dogs in sodium balance maintain normal systemic hemodynamics

Dogs with chronic biliary cirrhosis and portal hypertension commonly develop plasma volume expansion, urinary sodium retention, ascites, and perturbed systemic hemodynamics, i.e., a rise in cardiac output and a fall in peripheral vascular resistance. Our laboratory has previously demonstrated that creating a side-side portacaval anastomosis in such animals, and so venting hepatoportal pressure, will prevent sodium retention and ascites formation and will maintain the animals euvolemic. In the present study, in four cirrhotic dogs with such an anastomosis, observations made at 12 weeks postbiliary duct ligation, and in the presence of grossly disturbed liver function and morphology, failed to demonstrate any change from control conditions in arterial blood pressure, cardiac output, or peripheral vascular resistance. We conclude that venting hepatoportal pressure in cirrhotic dogs with markedly disturbed liver function prevents the advent of a hyperdynamic circulation, possibly by preventing volume expansion.     

Systems responses of rats to aflatoxin B1 exposure revealed with metabonomic changes in multiple biological matrices

Exposure to aflatoxins causes liver fibrosis and hepatocellular carcinoma posing a significant health risk for human populations and livestock. To understand the mammalian systems responses to aflatoxin-B1 (AFB1) exposure, we analyzed the AFB1-induced metabonomic changes in multiple biological matrices (plasma, urine, and liver) of rats using (1)H NMR spectroscopy together with clinical biochemistry and histopathologic assessments. We found that AFB1 exposure caused significant elevation of glucose, amino acids, and choline metabolites (choline, phosphocholine, and glycerophosphocholine) in plasma but reduction of plasma lipids. AFB1 also induced elevation of liver lipids, amino acids (tyrosine, histidine, phenylalanine, leucine, isoleucine, and valine), choline, and nucleic acid metabolites (inosine, adenosine, and uridine) together with reduction of hepatic glycogen and glucose. AFB1 further caused decreases in urinary TCA cycle intermediates (2-oxoglutarate and citrate) and elevation of gut microbiota cometabolites (phenylacetylglycine and hippurate). These indicated that AFB1 exposure caused hepatic steatosis accompanied with widespread metabolic changes including lipid and cell membrane metabolisms, protein biosynthesis, glycolysis, TCA cycle, and gut microbiota functions. This implied that AFB1 exposure probably caused oxidative-stress-mediated impairments of mitochondria functions. These findings provide an overview of biochemical consequences of AFB1 exposure and comprehensive insights into the metabolic aspects of AFB1-induced hepatotoxicity in rats.     

Effect of probiotic fermented milk and chlorophyllin on gene expressions and genotoxicity during AFB₁-induced hepatocellular carcinoma

The aim of this study was to investigate the chemopreventive effect of probiotic fermented milk and chlorophyllin on aflatoxin B? (AFB?) induced hepatocellular carcinoma. In vivo trials were conducted on 200 Wistar rats allocated to eight groups. Rats in the positive control group were given intraperitoneal injection of aflatoxin B? at 450 μg/kg body weight twice a week for 6 weeks. The rats were sacrificed and dissected at 25th week of the experiment, and comet assay was carried out in hepatic cells to assess the genotoxicity or DNA damage. The tumour incidence was decreased by approximately one-third than AFB? control group. The expression of c-myc bax, bcl-2, cyclin D1, p53 and rasp-21 genes was also studied. A significant (P<0.05) reduction in DNA damage was observed in probiotic fermented milk with chlorophyllin group as compared to aflatoxin B? control group. The c-myc, bcl-2, cyclin D1 and rasp-21 level was found to be highest in AFB? control group as compared to the treatment group. The results advocate the enhanced protective potential of probiotic fermented milk and chlorophyllin against AFB?-induced molecular alterations in hepatic cells during carcinogenesis.     

Comparative genotoxicity of 3 procarcinogens in V79 cells as related to glutathione S-transferase activity of hepatocytes from untreated rats and those fed 2% butylated hydroxyanisole

When 7,12-dimethylbenz[a]anthracene (DMBA) and aflatoxin B1 (AFB1) were activated by hepatocytes from Fischer 344 rats fed a diet containing 2% butylated hydroxyanisole (BHA), frequencies of mutation to 6-thioguanine resistance (TGR) at the HGPRTase gene locus and to ouabain resistance (OuR) at the Na+,K(+)-ATPase gene locus in V79 cells were 30-70% less than those obtained with hepatocytes from untreated controls. A difference in the mutation frequency did not occur when dimethylnitrosamine (DMN) was activated by BHA induced- rather than control-hepatocytes. Analysis of hepatocytes from rats fed 2% BHA showed a small (1.5-fold), but significant, increase in glutathione levels over that in the controls but no change in activity of cytochrome P450. Cytosolic glutathione S-transferase (GST) activity was increased 2-3-fold in hepatocytes from rats fed the 2% BHA diet. These results suggest that mutagenic response to DMBA and AFB1 is reduced, at least in part, because of BHA-induction of hepatocyte GST activity; while activation of DMN can occur by pathway(s) unaffected by BHA-induction of these liver enzymes. In contrast to mutation frequencies, significant differences between BHA- and control-activation in the production of sister-chromatid exchange (SCE) and micronucleus formation (MN) were not detected with any of the genotoxins. It was concluded that the mechanism(s) by which SCE and MN occur are likely unrelated to the capacity of BHA to induced activity of hepatic enzymes, e.g. the GSH S-transferases, that directly or indirectly affect mutation end-points.     

Aflatoxin B1 induced hepatocarcinogenesis in neonatal rats.

Role of cell replication on aflatoxin B1 (AFB1) induced hepatocarcinogenesis was investigated in neonatal rats showing persistence of cell replication in the liver for 21 days of post natal life. Adult (8-10 weeks old) rats displaying no hepatocytic proliferation served as controls. Three doses of AFB1 were administered to both the groups at intervals of 48 hr with the doses starting on 10th day of age in the neonatal group. Appearance of phenotypically altered preneoplastic hepatocytes was quantitated in both the groups. A significantly higher incidence of preneoplastic foci was recorded in neonatal rats as compared to adult animals. The results suggest that presence of cell replication in neonatal rats at the time of AFB1 administration enhances the process of hepatocarcinogenesis.     

Protection of salvia miltiorrhiza against aflatoxin-B1-induced hepatocarcinogenesis in Fischer 344 rats dual mechanisms involved.

Extract of Salvia Miltiorrhiza (SM) has been widely used in traditional Chinese medicine for treating liver diseases. Recent experimental evidence indicates that it has anti-tumor potential. In this study, the effect of SM on alfatoxin B1 (AFB1)-induced hepatocarcinogenesis was investigated in male Fischer 344 rats. AFB1 (40 microg/100 g body wt, by gavage) was administered once a week for 24 weeks. In SM treatment group, rats were given SM (0.25g/100g body wt, 5 days/week by gavage) for a total of 28 weeks, including 4 weeks before and 24 weeks during AFB1 exposure. Results showed that the elevation of serum alanine aminotransferase and aspartate aminotransferase activities due to AFB1 dosing was almost completely abolished by the treatment of SM, indicating that SM could prevent AFB1-induced liver cell injury. It was further observed that SM substantially reduced glutathione S-transferase placenta form (GST-P) positive foci formation and GST-P mRNA expression caused by AFB1, which clearly suggests that SM is effective in preventing AFB1-induced hepatocarcinogenesis. Furthermore, the inhibition on AFB1 hepatocarcinigenesis was associated with a corresponding decrease in AFB1-DNA adducts formation as well as AFB1-induced oxidative DNA damage (8-hydroxydeoxyguanosine) in rat liver. Our results also indicate that the protective effect of SM might be mediated through dual mechanisms: (i) the enhancement of AFB1 detoxification pathway, especially the induction of GST-Yc2 mRNA expression, and (ii) the antioxidant property of SM.     

Cell and species differences in metabolic activation of chemical carcinogens

Metabolic activation and DNA binding of aflatoxin B1 (AFB1), N-nitrosodimethylamine (DMN) and benzo[a]pyrene (B[a]P) were compared in human, rat and mouse hepatocytes and human pulmonary alveolar macrophages (PAM). The degree of carcinogen activation by hepatocytes and PAM was measured by cell-mediated mutagenesis assays in which co-cultivated Chinese hamster V79 cells were used to monitor mutagenic metabolites. Hepatocytes from human, mouse and rat metabolized DMN and released the active metabolites to induce either ouabain- or 6-thioguanine-resistant mutation. The mutation frequencies mediated by hepatocytes of the 3 animal species were approximately 3-9 mutants/10(5) survivors at a concentration of 0.2 mM DMN. The variations of radioactivity bound to liver cell DNA were relatively small in cultured mouse, rat, and human hepatocytes exposed to 14C label DMN (0.5 mM) and the binding values were in a range of 6-12 X 10(3) pmoles/mg DNA. However, rat hepatocytes were at least 10-fold more effective than either human or mouse hepatocytes in generating mutagenic metabolites of AFB1 and also had a much higher AFB1 metabolite DNA-binding value. The AFB1 DNA-binding levels were 4.1, 12-27 (range), 120 pmoles/mg DNA respectively in mouse, human, and rat liver cells following AFB1 (3.3 microM) exposure for 20 h. Hepatocytes from the 3 animal species were unable to mediate mutation in the presence of 4 microM B[a]P; PAM activated B[a]P and effectively mediated mutation in the co-cultivated V79 cells. In contrast to results with hepatocytes, PAM failed to generate enough mutagenic metabolites of AFB1 (3.3 microM) and the mediation of mutations was seen only at very high concentration of DMN (80 mM). The genotoxic effects of the 3 carcinogens on hepatocytes from different species in vitro were in agreement with the in vivo animal experiments in that mice are relatively resistant to AFB1 carcinogenesis whereas rats are sensitive; B[a]P is not effective as a complete liver carcinogen in adult rat and mouse whereas DMN induces liver cancer.