Effect of aflatoxin metabolism and DNA adduct formation on hepatocellular carcinoma among chronic hepatitis B carriers in Taiwan

BACKGROUND/AIMS: Aflatoxins (AFs) are established hepatic carcinogens in several animal species. This study was performed to establish whether aflatoxin exposure may affect the risk of developing hepatocellular carcinoma in chronic hepatitis B virus carriers. METHODS: Urinary AF metabolites were measured for 43 HCC cases and 86 matched controls nested in a cohort of 7342 men in Taiwan. Thirty hepatocellular carcinoma cases and 63 controls were also tested for AFB1-albumin adducts. RESULTS: There was a dose-response relationship between urinary AFM1 levels and risk of hepatocellular carcinoma in chronic hepatitis B virus carriers. Comparing the highest with the lowest tertile of urinary AFM1 levels, the multivariate-adjusted odds ratio (OR) was 6.0 (95% confidence interval (CI) = 1.2-29.0). The hepatocellular carcinoma risk associated with AFB1 exposure was more striking among the hepatitis B virus carriers with detectable AFB1-N7-guanine adducts in urine. Compared with chronic hepatitis B virus carriers who were negative for AFB1-albumin adducts and urinary AFB1-N7-guanine, no elevated risk was observed for those who were positive for either marker. But an extremely high risk of hepatocellular carcinoma among those having both markers was found (OR = 10.0, 95% CI = 1.6-60.9). The proportion of AFB1 converted to AFM1 decreased with the progress of liver disease, whereas the formation of AFP1 increased. The difference in patterns of AFB1 metabolite formation was an independent risk factor for hepatocellular carcinoma after adjustment for total AFB1 excretion. There was a synergistic interaction between glutathione S-transferase M1 genotype and AFB1 exposure in hepatocellular carcinoma risk. CONCLUSIONS: AFB1 intake and expression of enzymes involved in AFB1 activation/detoxification may play an important role in hepatitis B virus-related hepatocarcinogenesis.     

The value of synthetic linear epitope analogues of La/SSB for the detection of autoantibodies to La/SSB; specificity, sensitivity and comparison of methods

Aflatoxin M1 (AFM1) is the principal hydroxylated aflatoxin metabolite present in the milk of dairy cows fed a diet contaminated with aflatoxin B1, (AFB1) and the metabolite is also present in the milk of human nursing mothers consuming foodstuffs containing the toxin. AFM1 is usually considered to be a detoxification product of AFB1 and this appears warranted if the biological endpoints involved are carcinogenicity and mutagenicity. However, it may not be a valid conclusion in the case of cytotoxicity. The metabolism of AFM1 and AFB1 have been studied in vitro using human liver microsomes. Formation of primary metabolites associated with metabolic activation to the respective epoxides reflected the differences between the carcinogenic potentials of the two toxins and, similar to AFB1, the conjugation of AFM1 epoxide with reduced GSH was catalyzed by mouse, but not human liver cytosol. Although the majority of the binding of [3H]AFB1 to microsomal protein was dependent on metabolic activation, a high level of retention of [3H]AFM1 by microsomes, nonextractable in methanol and unrelated to metabolic activation, was observed. It appears possible that this property is related to the high cytotoxicity of AFM1. Experiments using human cell line cells either expressing or not expressing human cytochrome P450 enzymes in assays of acute toxicity (MTT assays) have demonstrated a directly toxic potential of AFM1 in the absence of metabolic activation, in contrast to AFB1. Caution therefore needs to be exercised in designating the formation of AFM1 as essentially detoxification when considering a biological response in which cytotoxicity may play a significant role, e.g., immunotoxicity.     

Absence of mutations in the p53 tumor suppressor gene in woodchuck hepatocellular carcinomas associated with hepadnavirus infection and intake of aflatoxin B1

Infection with hepadnaviruses and exposure to aflatoxin B1 (AFB1) are considered major risk factors in the development of hepatocellular carcinoma (HCC) in humans and in animals. A high rate of mutations in the p53 tumor suppressor gene in hepatocellular carcinomas of predominantly hepatitis B virus (HBV) carrier patients has been recently related to dietary aflatoxin. Another member of the hepadnavirus family, the woodchuck hepatitis virus (WHV), infects woodchucks in a manner similar to that of HBV in humans. Therefore, it was of particular interest to determine whether the p53 gene in woodchuck HCCs associated with hepadnavirus infection and with exposure to AFB1 is affected in the same manner as in human HCCs. By direct PCR-sequencing, we analyzed exons 4-9 of the p53 gene in 13 HCCs from 12 woodchucks (two uninfected, ten WHV carriers). Six WHV carrier and two uninfected woodchucks were treated with AFB1. None of the analyzed HCC samples exhibited mutations, either in p53 gene exons 4-9, or in splicing donor-acceptor sites. The present data are consistent with our previous study that indicated a low rate of p53 mutations in HCCs of AFB1-treated ground squirrels, either infected or not infected with ground squirrel hepatitis virus, and in WHV carrier woodchucks not exposed to AFB1. Overall, our findings indicate that in woodchucks and in ground squirrels exposure to aflatoxin may affect the development of p53 mutations less than in humans.     

Death from heroin overdose: findings from hair analysis

BACKGROUND: Morphine analysis of hair is used in forensic toxicology to study the addiction history of heroin addicts. To clarify the features underlying fatal heroin intake, we measured hair morphine content in a group of deceased heroin addicts, to verify a possible correlation between fatal heroin overdoses and the addiction behaviour of these individuals before death. METHODS: 91 deaths were attributed to heroin overdose in Verona, Italy, in 1993-96. We analysed the hair of 37 of these individuals, and of 37 active heroin addicts, 37 former heroin users abstinent from the drug for several months, and 20 individuals with no evidence of exposure to opioids. From each individual, a hair sample of about 150 mg was analysed by RIA and high-performance liquid chromatography, to measure the morphine content. FINDINGS: The mean morphine content in the hair of the addicts who had died was 1.15 ng/mg (SD 2.35 ng/mg; range 0-12.25 ng/mg) compared with 6.07 ng/mg (4.29; 1.15-17.0) in the active heroin addicts, 0.74 ng/mg (0.93; 0.10-3.32) in the abstinent former addicts, and values below the detection limit in the non-exposed group. Hair morphine content among those who had died was significantly lower than that in active heroin consumers (p<.00001), but not significantly different from that in the former addicts (p=0.978). INTERPRETATION: Although our findings may be subject to selection bias, since suitable hair samples were available for only 37 of the 91 addicts who had died, these findings support the theory of high susceptibility to opioid overdose after periods of intentional or unintentional abstinence, due to loss of tolerance. Medical staff running detoxification programmes should be aware of the risk inherent in relapse to heroin after a period of abstinence. Moreover, occasional heroin use without a build-up of tolerance could also give a high risk of overdose.     

Occurrence of aflatoxins, fumonisin B1, and zearalenone in foods and feeds in Botswana

Sorghum and maize form the main dietary staple foods in Botswana. Other products such as peanuts, peanut butter, phane (an edible larval stage of an emperor moth Imbrasia belina Westwood), and pulses (cowpeas and beans) are also widely used as food and for the manufacture of feeds. These important food and feed commodities were analyzed for the presence of aflatoxins, fumonisin B1, and zearalenone. Aflatoxins were detected in 40% of the samples analyzed. The concentration of total aflatoxins ranged from 0.1 to 64 microg/kg. The mean concentration ranged from 0.3 microg/kg in sorghum to 23 microg/kg in peanut butter. Peanut butter samples were the most contaminated (71%). No aflatoxins were detected in maize. Fumonisin B1 was detected in 36% of the samples. Maize samples were the most contaminated (85% of the samples) with the concentration ranging from 20 to 1,270 microg/kg. No fumonisin B1 was detected in peanuts, phane, and beans. Zearalenone was only found in 2.6% of the samples analyzed at 40 microg/kg. Aflatoxins were the most common toxins detected in foods and feeds in Botswana. However, fumonisin B1 was more prevalent in maize than aflatoxins or zearalenone.     

Aflatoxin B1-adduct formation in rat and human small bowel enterocytes

BACKGROUND/AIMS: Hepatic CYP3A enzymes have been implicated in the bioactivation of aflatoxin B1 (AFB1) to DNA binding metabolites. CYP3A enzymes are also abundant in the small bowel, and we therefore examined the ability of this tissue to form intracellular AFB1 adducts. METHODS: Immunohistochemistry using a antibody to the stable AFB1-DNA adduct was performed on small bowel sections obtained from rats orally gavaged with AFB1 and on human small bowel biopsy specimens maintained in explant culture. 3H-AFB1 was instilled into a loop of small bowel of untreated rats and rats pretreated with the CYP3A inducer dexamethasone during vivisection. DNA was isolated from the loop 2 hours later and assayed for specific activity. RESULTS: In both rats and humans, AFB1-adducts were detected exclusively in mature enterocytes in a pattern similar to the distribution of CYP3A enzymes. Induction of enterocyte CYP3A in rats resulted in an increase in enterocyte immunoreactive AFB1 adducts and in a 1.8-fold increase in 3H-AFB1-nucleic acid adducts (P = 0.01). CONCLUSIONS: Intracellular AFB1 adducts are formed in the small intestine, and this reflects, at least in part, the catalytic activity of CYP3A enzymes. Because these AFB1 adducts should ultimately pass in stool, enterocyte CYP3A may represent a regulatable barrier to dietary aflatoxins.     

Role of ultrasound and harness treatment in the management of developmental dysplasia of the hip

INTRODUCTION: Pustular candidiasis in heroin addicts is a rare entity in dermatology. We report a case. CASE REPORT: A 29-year-old female heroin addict developed a painful pustular growth on the scalp. There was no fever. Multiple follicular pustulae measuring 2 to 3 mm were associated with hyperesthesia of the scalp and painful cervical nodes. Biopsy showed acute ostiofolliculitis with a few blastospores and mycelial filaments. Candida albicans was isolated from the pustulae and the buccal cavity. Candida serology was positive (indirect immunofluorescence 1/100, coelectrosyneresis: 4 archs). Search for other localizations and HIV serology were negative. The last injection of brown heroin had been taken 15 days earlier; lemon had been added. Treatment with flucanazole (400 mg/d) led to improvement within 48 hours. DISCUSSION: Sudden development of pustulae or nodules in pilous zones in a heroin addict should suggest the diagnosis. Outcome depends on early treatment after diagnosis and search for other localizations. Our case presented two particular aspects: ostiofollicular localization of the pustulae and a long delay (15 days) between the (presumably) last injection and the development of the lesion. Folliculitis develops almost exclusively in addicts who use brown heroin. Contamination by Candida albicans results from the lemon used to improve solubility at injection.     

MMPI differences of male Hispanic-American, Black, and White heroin addicts.

Tested 2 hypotheses: (a) that Hispanic-American heroin addicts are better adjusted than White (majority group) addicts and (b) that Hispanics characteristically are less open about expressing psychological symptoms and are more masculine in their interests. All Ss were given the MMPI. Both hypotheses were confirmed in MANOVAs and analyses of covariance among 41 Hispanic-American, 161 White, and 268 Black heroin addicts, with age (mean age of Ss 30.13 yrs), education, and socioeconomic status serving as covariates. (8 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Alterations of immune functions in heroin addicts and heroin withdrawal subjects

Conflicting results, both decreased and increased, have been reported concerning the function of T-lymphocytes in heroin addicts. We investigated the alterations of T-lymphocyte proliferative responses and immunophenotypic markers on lymphoid cells in heroin addicts and during different periods of heroin withdrawal in addicted subjects. This study has demonstrated a decrease in the response of T-lymphocytes to 1.2, 2.5, 5 and 10 microg/ml of phytohemagglutinin stimuli in heroin addicts and 1- to 5-day heroin withdrawal subjects compared with controls. Similarly, in an in vitro study, 10(-4), 10(-6) and 10(-8) M concentrations of morphine were shown to suppress 0.6 and 2.5 microg/ml of PHA-stimulated T-lymphocyte obtained from naive subjects. This inhibitory effect of morphine on PHA stimulation was completely abolished by 100 microM naloxone. The immunological parameters of total T-lymphocytes (CD3), T-helper cells (CD4), cytotoxic T-cells (CD8), B-cells and natural killer cells that are the immunophenotypic markers studied by flow cytometric analysis were altered in heroin addicts, 15- to 21-day and 6- to 24-month heroin withdrawal subjects, when compared with controls. These results suggest that heroin addicts and short period (15 to 21 days and 6 to 24 months) of heroin withdrawal have decreases in their immune system functioning and that the heroin withdrawal subjects seem to gradually reverse their immunological parameters to normal levels when withdrawal was sustained >/=2 years. This is the first report examining immune function in heroin withdrawal subjects using the "cold turkey" method. The results are beneficial for further study of the mechanism responsible for the opioid-induced changes in immune function.     

Direct synthesis of aflatoxin B1-N7 guanine adduct: a reference standard for biological monitoring of dietary aflatoxin exposure in molecular epidemiological studies

Aflatoxin B1-N7-guanine and aflatoxin B1-human serum albumin adducts have been established as biomarkers of dietary aflatoxin exposure in epidemiological studies. Earlier chemical oxidants were used to synthesize aflatoxin B1-8,9-epoxide in vitro and its subsequent interaction with DNA or synthetic oligodeoxynucleotide was used as a source of authentic aflatoxin B1-N7-guanine adduct. In the present communication we report a simple single step procedure for the synthesis of aflatoxin B1-N7-guanine adduct using free guanine and m-chloroperbenzoic acid as the chemical oxidant for the production of AFB1-8,9-epoxide. At a molar ratio of 1:1 of AFB1-8,9-epoxide and guanine the recovery of the AFB1-N7-guanine adduct was found to be 60% while at higher molar ratios (1:2 and 1:4) of guanine the recovery of the AFB1-N7-guanine adduct was found to be low (30-40%). HPLC analysis of the AFB1-N7 guanine adduct showed a retention time identical with the retention time of the AFB1-N7-guanine adduct synthesized using calf thymus DNA. TLC-fluorodensitometric analysis indicated that the Rf of the AFB1-N7-guanine adduct was zero. Spectral analysis of the adduct synthesized showed an excitation wavelength of 360 nm and emission wavelength at 440 nm in phosphate buffer (100 mM, pH 7.4). Further, the formation of the AFB1-N7-guanine adduct was confirmed by perchloric acid treatment resulting in the destruction of the adduct. The AFB1-N7-guanine adduct thus synthesized was stable in both acidic as well as lyophilized conditions over a period of 2 weeks. The antibody capture assay showed that the antibodies produced against the antigen BSA-guanine-N7-AFB1 also cross-reacted with calf thymus DNA-AFB1 adduct, indicating specificity to the guanine-N7-AFB1 moiety. The method developed may find immediate application as a source of authentic reference standard in molecular epidemiological studies.     

Prehospital treatment of heroin intoxication in Oslo in 1996

The number of heroin overdoses among drug addicts in Oslo is increasing. In 1996 overdoses counted for 1,248 (12%) of all emergency call-outs by the ambulance service. Heroin can cause fatal respiratory insufficiency, and in 1996 a total of 104 deaths related to heroin overdoses were reported in Oslo. Heroin overdoses are treated on site by ambulance personnel. Advanced cardiopulmonary resuscitation was started on 18 of the 79 addicts who were found unconscious, and 11 persons were treated successfully. A total of 846 drug addicts had to be given the antidote naloxone, and among these 678 (80%) persons were found in a coma. Only 29 persons had to be transported to hospital. Early treatment probably prevented both morbidity and mortality, no time being wasted transporting the patients to hospital. Ambulance personnel treat all drug addicts with the same respect as they do other patients. They have no police escort; they are familiar with the addicts and their environment and they have gained their confidence. Prehospital treatment saves on health services resources, and should, in our experience, be carried out in collaboration with a hospital or other health institutions for mutual and optimal benefit.     

Withdrawal in opiate addicts in an internal medicine hospital unit

Withdrawal of opiates drug addicts in Internal Medicine is unusual in France. Four main preliminary conditions are requested: 1--Drug addict preparation and self motivation, 2--Inter and intra institution team collaboration, 3--Opening the hospital towards community agencies, 4--Hospital staff recruited on volunteer basis. Within two years (1992-1993), 210 opiates drug addicts were hospitalized for withdrawal. Two third were males, median age was 27, median years of addiction was 7. Thirty percent were seropositive for HIV, 70% for HCV. Hospitalisation lasted 7 days for heroin addicts and 10 days for morphin, codein or buprenorphin addicts. Successful withdrawn was observed for 70% patients but six months after withdrawal, only 15% remained abstinent.     

Comparison of three methods for determining aflatoxins in melon seeds

The suitability of 3 methods for determining aflatoxins in melon seeds was examined. The first 2 are the Contaminants Branch (CB) method and the Best Foods (BF) method, both official methods for determining aflatoxins in peanuts and peanut products. The third method, the modified CB method-Rapid Modification of the Cottonseed (CB-RCS-Mod) method, devised in this work, was derived by combining steps from the CB method and the Rapid Modification of the Cottonseed method. The CB method was superior to the other 2 methods for quantitation of aflatoxins. It gave better recoveries and cleaner extracts that exhibit less fluorescent interference for thin-layer chromatography (TLC) than the BF method. Also, its solvent efficiency was better than that of the CB-RCS-Mod method. With the CB method, recoveries from spiked samples were 85.0% for aflatoxin B1 and 90.0% for aflatoxin B2. Recoveries of G aflatoxins were more variable, averaging 90.0% for aflatoxin G1 and 72.5% for aflatoxin G2. Total aflatoxin recovery was 86.5% for the CB method. At a low aflatoxin contamination level (8 micrograms B1/kg sample), aflatoxin B1 was detectable by the CB method but not by the BF method. Detection of aflatoxins in BF method sample extracts by TLC was not improved by the use of chloroform-acetone-water (88 + 12 + 1), benzene-ethanol-water, or ether-methanol-water (96 + 3 + 1) in place of the standard chloroform-acetone (88 + 12) developer. Use of ether-methanol-water (96 + 3 + 1) for detecting aflatoxins by TLC in the CB method extracts increased interference compared with the standard chloroform-acetone (88 + 12) developer.     

Elevated serum-calcium and parathormone-levels in HIV afflicted female heroin addicts

Alterations of calcium and bone metabolisms have been observed in numerous studies of small groups of male HIV-infected patients. However, our knowledge regarding the manifestation of AIDS-associated hypoparathyroidism in female subjects is limited. In order to investigate the influence of heroin on the calciotropic hormones we performed a cross-sectional study on 45 female patients with proven HIV infection. The following criteria were used for exclusion from the study: age less than 20/ more than 50 years; confinement to bed; wasting symptoms; treatment with agents containing ketoconazole, renal or hepatic insufficiency; clinical or echographic signs of liver cirrhosis; endocrine diseases, or treatment with drugs known to influence calcium metabolism. A reduced parathormone (PTH) level was found among the female HIV-infected patients. Additional long-term use of heroin resulted in a significant increase of PTH compared to sex- and age matched controls and a second group of non-HIV-afflicted heroin dependent females. Significantly lowered serum magnesium concentrations were found in all three groups. Both serum calcium and urinary excretion of calcium were elevated in the group of HIV-infected heroin addicts and were independent from low vitamin D3 levels (1,25-dihydroxycholecalciferol) and alterations of protein metabolism. Therefore, it is concluded that the changes of PTH secretion are mainly due to mechanisms both of the impaired immune defense of HIV-infected females and the additional effect of opiates.     

Robotic automated analysis of foods for aflatoxin

Immunoaffinity column-based sample preparation procedures for determination of aflatoxins B1, B2, G1, and G2 in several food matrixes and aflatoxin M1 in milk have been automated by using flexible automation, or robotics. Components used to assemble the system were purchased commercially or developed and built in-house. A liquid-level sensor developed in-house to assist elution of the immunoaffinity column is described. After immunoaffinity column cleanup, aflatoxins are separated by reversed-phase liquid chromatography and determined by fluorescence without derivatization. Mean recoveries of aflatoxins B1, B2, and G1 added to corn and nuts at 9-36 ng/g total aflatoxins were > 85% (coefficient of variation [CV] = 16%). Recoveries of aflatoxin G2 averaged 50% (CV = 28%). Recoveries of aflatoxin M1 added to milk at 0.12-0.50 ng/mL averaged 78% (CV = 19%). The ability of the automated system to reproduce its results is demonstrated by the fact that the CV of replicate assays is generally better than 10%. Comparability between the automated procedure and the AOAC official method is demonstrated.     

The apomorphine test in heroin addicts

Chronic administration of opiates to laboratory animals induces supersensitivity of the dopamine receptors in the cerebral areas innervated by the mesotelencephalic dopamine pathways. In humans, the in vivo study of the sensitivity of the dopamine neurotransmitter system in Parkinson's patients can be done by means of the apomorphine test, which consists of measuring the number of yawns induced by the subcutaneous administration of low doses of apomorphine (0.005 mg/kg). If chronic opiate use in humans, as in experimental animals, results in supersensitivity of the dopamine systems, the apomorphine test could differentiate between heroin addicts and healthy volunteers, with the former showing greater number of yawns. In order to test this hypothesis we carried out the apomorphine test in two groups of subjects: a group of male heroin addicts attending our Addiction Treatment Centre for detoxification and the other group consisting of healthy volunteer male university students. Results showed that subcutaneous apomorphine administration induced a greater number of yawns (p < 0.05) in the group of heroin addicts as compared with the group of healthy volunteers, suggesting that heroin addicts present an enhanced sensitivity of the dopamine nuerotransmitter system.     

What effects does methadone maintenance treatment have on the rehabilitation of heroin addicts?]

The purpose of the study was to determine the effect of methadone maintenance treatment on heroin addicts. By searching in Medline and Cochrane Library two randomized controlled trials were found where methadone was the main intervention in the rehabilitation of heroin addicts. The trials were found among more than 2,350 articles on various aspects of methadone and were the only ones that met our criteria for inclusion in the study. The two studies comprised 347 participants. Both trials showed that methadone maintenance treatment had a positive effect on continuing participation in the treatment programme. One of the trials also showed that the treatment lowered the rates of opioid and cocaine use. It is alarming that only two randomized controlled trials could be found evaluating the effect of methadone maintenance treatment on the rehabilitation of heroin addicts. No trials demonstrating the effect of the treatment on mortality, crime, prostitution or risk behaviour related to communicable diseases were found.     

Personality differences and sociopathy in heroin addicts and nonaddict prisoners.

Examined measurable personality differences between carefully selected samples of 40 heroin addicts and 40 nonaddict prisoners. Composite mmpi profiles were compared for statistical differences between groups, and individual mmpi profiles were classified using a system of differential diagnosis reported by P. E. Meehl. Results suggest that there are measurable personality differences between heroin addicts and nonaddict prisoners, especially with respect to the incidence and extent of sociopathy. The 2 samples did not differ from each other in terms of age, educational level, intellectual level, or chronicity of antisocial behavior as indicated by time served in prison. (16 ref.) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Measurement of antibacterial activities of T-2 toxin, deoxynivalenol, ochratoxin A, aflatoxin B1 and fumonisin B1 using microtitration tray-based turbidimetric techniques

Various mycotoxins were tested for their antibacterial activity by evaluating growth delays using a fully automated microturbidmetric method. Ten different strains of the genera Escherichia, Streptococcus, Staphylococcus, Yersinia, Salmonella, Erysipelothrix and Lactobacillus were used as test micro-organisms. T-2 toxin, deoxynivalenol (DON), ochratoxin A (OTA), aflatoxin B1 (AFB1) and fumonisin B1 (FB1) were used as representative mycotoxins. The inhibitory effect in vitro was defined as the difference between the growth rate without mycotoxins and the growth rate in the presence of a mycotoxin. Among the tested strains, Streptococcus agalactiae was found to be sensitive to all the toxins, with the exception of OTA. T-2 toxin and FB1 were the most effective in slowing down the growth of Staphylococcus aureus. AFB1 affected the growth of Yersinia enterocolitica. The growth rate of Escherichia coli and Salmonella infantis was decreased by FB1. Among the bacterial strains used in this study, only the growth of Erysipelothrix rhusiopathiae was inhibited by OTA. Thus, using appropriate tester strains it should be possible to set up a broad-range microtubidimetry assay for individual mycotoxin screening in vitro. We concluded that the microtitration technique provides a rapid, convenient and high-throughput capacity system to analyse bacteria-mycotoxin interactions.     

Chemoprevention of aflatoxin B1-induced carcinogenesis by indole-3-carbinol in rat liver--predicting the outcome using early biomarkers

Indole-3-carbinol (I3C) was examined for its ability to inhibit aflatoxin B1 (AFB1)-induced hepatocarcinogenesis in male Fischer rats when administered either before or after the carcinogen. After 13 weeks, animals pretreated with I3C (0.5% in the diet) for 2 weeks prior to administration of AFB1 and with continuing treatment during exposure to the carcinogen were protected from development of preneoplastic lesions, as determined by the classical markers gamma-glutamyltranspeptidase (GGT) and glutathione S-transferase (GST) P. In animals receiving AFB1 for 6 weeks before treatment with I3C, there was no obvious protective effect at 13 weeks compared with animals receiving only AFB1. Using cytokeratin 18 expression as a marker, animals fed AFB1 alone had a small number of positive foci at 13 weeks. However, no cytokeratin-positive foci were visible in the majority of livers from either group receiving I3C in combination with AFB1 and after 43 weeks all animals in these groups were protected from liver tumour formation. These results suggest that expression of cytokeratin 18, a later phenotypic change in foci than induction of GST-P and GGT, correlates more closely with tumour outcome in this model. I3C appeared to retard progression of AFB1-induced carcinogenesis at both the initiation and promotion stages. Continuous treatment with I3C for 13 weeks caused significant induction of CYP1A1, 1A2, 3A and 2B1/2, GST Yc2, aflatoxin B1 aldehyde reductase and quinone reductase. Such alteration of the drug metabolizing capacity of the liver by I3C contributes to blocking of initiation, while the observed inhibition of ornithine decarboxylase, a rate limiting enzyme in polyamine biosynthesis, and of tyrosine kinase activity may contribute to the suppressive effect of I3C.