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PubMed1989

2(3)-tert-butyl-4-hydroxyanisole inhibits oxidative metabolism of aflatoxin B1 in isolated rat hepatocytes.

Previous studies indicate that dietary administration of phenolic antioxidants, 2(3)-tert-butyl-4-hydroxyanisole (BHA) and 3,5-di-tert-butyl-4-hydroxytoluene, inhibits the carcinogenic effect of a number of chemical carcinogens including aflatoxin B1 (AFB1). Induction of hepatic enzymes, such as glutathione S-transferase, UDP-glucuronyltransferase, and epoxide hydrolase, has been shown to be responsible for the reduction of AFB1 cytotoxic and carcinogenic effects. The effect of BHA on AFB1 activation was examined in vitro utilizing isolated rat hepatocytes and liver microsomes. In hepatocytes, the total AFB1 content and bound form of AFB1 were 3.4 and 1.4 pmol/10(6) cells, respectively. In the cell-free microsomal activating system, 2.2 pmol were activated per mg of microsomal protein during 60 min of incubation. BHA (0.1-0.5 mM) inhibited AFB1 activation and binding in both systems in a dose-dependent manner; in hepatocytes, 90% inhibition was observed at 0.5 mM. Analyzing various AFB1 adducts, BHA (0.25 mM)-treated hepatocytes contained a significantly reduced amount of AFB1 macromolecular adducts. The antioxidant neither stimulated nor inhibited the cytosolic glutathione S-transferase and microsomal UDP-glucuronyltransferase activities. Analysis of various hydroxylated (aflatoxins M1 and Q1 (AFM1 and AFQ1] and demethylated (aflatoxin P1 (AFP1] metabolites of AFB1 in both the conjugated and unconjugated form indicated that there was a 30-50% reduction of unconjugated AFP1, AFQ1, and AFM1, whereas AFB1 was increased 3-fold. There was no significant change of conjugated metabolites. The effect of BHA on AFB1 activation in hepatocytes was compared with that of other cytochrome P-450 inhibitors; the ED50 values of SKF 525A, BHA, and metyrapone were 9 microM, 40 microM, and 280 microM, respectively. In the cell-free microsomal system, biotransformation of AFB1 to AFP1, AFM1, and AFQ1 was also inhibited. Kinetic analysis of p-nitroanisole O-demethylase activity of rat liver microsomes demonstrated that BHA inhibited noncompetitively with an apparent Ki of 90 microM. In the absence of enzyme induction, the phenolic antioxidant, BHA, blocks the oxidative biotransformation of AFB1 in isolated hepatocytes.

Aflatoxin B1↗
PubMed1979

Extraction, cleanup, and quantitative determination of aflatoxins B1 ANd M1 in beef liver.

A method for the determination of aflatoxin B1 in eggs was applicable for aflatoxin B1 in liver, but ineffective for aflatoxin M1 in liver because of poor recovery of added aflatoxin and interferences in thin layer chromatography. The method was modified by the addition of citric acid to the extracting solvent and ammonium sulfate to the extract solution for removing protein. The elution system for silica gel column cleanup was also changed by substituting methanol for acetone, and adding a step for confirmation of aflatoxin M1 identity. The method has been used successfully for survey and research on aflatoxin residues in animal tissues.

Aflatoxins↗
PubMed1982

Aflatoxicol and aflatoxins B1 and M1 in the tissues of pigs receiving aflatoxin.

Aflatoxicol (AFL) and aflatoxins B1 and M1 were found in tissues (kidney, liver, and muscle) of feeder pigs given an estimated LD50 oral dose of B1 (1.0 mg/kg body weight) provided as a rice culture of Aspergillus flavus and of market-weight pigs fed a naturally contaminated feed, containing aflatoxin B1 at a level of 400 ng/g from corn, for 14 days. The residues in all tissues decreased with time after treatment in both groups, with no detectable residues (approximate detection limits, ng/g, B1 0.03, M1 0.05, AFL 0.01) in pig tissues from the feeding experiment 24 h after withdrawal of aflatoxin-contaminated feed. B1 and M1, when found in the feeding experiment, were at about the same levels in all tissues except the kidney, in which M1 was the dominant aflatoxin. The level of AFL, when detected, was about 10% of the B1 level.

Aflatoxin B1↗
PubMed1986

Feedlot performance and tissue residues of cattle consuming diets containing aflatoxins.

A comparative slaughter feeding trial was conducted using crossbred Hereford-Angus steers. Cottonseed meal known to be contaminated with aflatoxins was fortified with long-grain rice cultures of Aspergillus flavus to produce complete rations that contained 0, 60, 300 and 600 ppb aflatoxin B1 (AFB1). Forty steers, weighing approximately 250 kg were randomly assigned to four equal treatment groups. Animals were housed in individual pens and fed the assigned contaminated ration ad libitum for 155 d. On d 156 all animals were placed on the control ration for 14 d. Animals were weighed and blood samples collected every month for clinical chemistry analysis. Liver, muscle and fat biopsies were collected at 6-wk intervals. These samples were stored in liquid N2 prior to analysis for aflatoxin M1 and B1 content. Separate liver samples were taken for histopathological examination. Aflatoxin was withdrawn from the diet 2 wk prior to slaughter. After 1 wk of withdrawal liver, muscle and fat samples were collected and assayed for AFB1 and aflatoxin M1 (AFM1). Liver samples also were taken for histopathological examination. Growth rates and feed intakes in the 60 and 300 ppb aflatoxin treatments were not significantly different from controls. The 600-ppb treatment group had a decrease in feed intake and rate of gain. Rations containing 60 and 300 ppb of aflatoxin had no significant influence on blood components and enzyme patterns. The 600-ppb treatment caused a slight, but consistent, increase in serum glutamic oxaloacetic transaminase and alkaline phosphatase.(ABSTRACT TRUNCATED AT 250 WORDS)

Aflatoxins↗
PubMed1996

The kinetics of aflatoxin B1 oxidation by human cDNA-expressed and human liver microsomal cytochromes P450 1A2 and 3A4.

The combined presence of CYP1A2 and 3A4, both of which oxidize aflatoxin B1 (AFB1) to the reactive aflatoxin B1-8,9-epoxide (AFBO) and to hydroxylated inactivation products aflatoxin M1 (AFM1) and aflatoxin Q1 (AFQ1), substantially complicates the kinetic analysis of AFB1 oxidation in human liver microsomes. In the present study, we examine the reaction kinetics of AFB1 oxidation in human liver microsomes (HLMs, N = 3) and in human CYP3A4 and CYP1A2 cDNA-expressed lymphoblastoid microsomes for the purpose of identifying the CYP isoform(s) responsible for AFB1 oxidation at low substrate concentrations approaching those potentially encountered in the diet. AFBO formation by cDNA-expressed human CYP1A2 followed Michaelis-Menten kinetics (Km = 41 microM, Vmax = 2.63 nmol/min/nmol P450). Furthermore, the portion of AFBO formed in HLMs which was eliminated by furafylline, a specific mechanism-based inhibitor of CYP1A2, also followed Michaelis-Menten kinetics (Km = 32-47 microM, Vmax = 0.36-0.69 nmol/min/nmol P450). The formation of AFBO (activation product) and AFQ1 (detoxification product) in cDNA-expressed human CYP3A4 microsomes was sigmoidal and consistent with the kinetics of substrate activation. Accordingly, application of a sigmoid Vmax model equivalent to the Hill equation produced excellent fits to the cDNA-expressed CYP3A4 data and also to the data from HLMs pretreated with furafylline to remove CYP1A2. The Hill model predicted that two substrate binding sites are involved in CYP3A4-mediated AFB1 catalysis and that the average affinity of AFB1 for the two sites was 140-180 microM. Vmax values for AFQ1 formation were 10-fold greater than those for AFBO, and total substrate turnover to both was 67 nmol/min/nmol CYP3A4. Using the derived kinetic parameters for CYP1A2 and 3A4 to model the in vitro rates of AFB activation at low substrate concentrations, it was predicted that CYP1A2 contributes to over 95% of AFB activation in human liver microsomes at 0.1 microM AFB. The important role of CYP1A2 in the in vitro activation of AFB at low substrate concentrations was supported by DNA binding studies. AFB1-DNA binding in control HLMs (reflecting the contribution of CYP1A2 and CYP3A4) and furafylline-pretreated microsomes (reflecting the contribution of CYP3A4 only) catalyzed the binding of 1.71 and 0.085 pmol equivalents of AFB1 to DNA, respectively, indicating that CYP1A2 was responsible for 95% of AFB1-DNA adduct formation at 0.133 microM AFB. These results demonstrate that CYP1A2 dominates the activation of AFB in human liver microsomes in vitro at submicromolar concentrations and support the hypothesis that CYP1A2 is the predominant enzyme responsible for AFBO activation in human liver in vivo at the relatively low dietary concentrations encountered in the human diet, even in high AFB exposure regions of the world. However, because the actual concentrations of AFB in liver in vivo following dietary exposures are uncertain, additional studies in exposed human populations are needed. Quantitative data on the relative rates of AFM1 and AFQ1 excretion (potential biomarkers for CYP1A2 and 3A4 activity, respectively) in humans would be useful to validate the actual contributions of these two enzymes to AFB1 oxidation in vivo.

Adult↗
PubMed1991

Mouse strain differences in glutathione S-transferase activity and aflatoxin B1 biotransformation.

Previous studies have suggested that mice are resistant to the carcinogenic effects of aflatoxin B1 (AFB1) and that this resistance is largely the result of expression of an isoenzyme of glutathione S-transferase (GST) with high activity toward AFB1-8,9-epoxide. Significant interstrain differences in cytosolic GST activities toward a variety of substrates have been reported in mice. If such differences exist for the conjugation of AFB1-8,9-epoxide, then there may be significant mouse strain differences in susceptibility to AFB1-induced hepatocarcinogenicity. The hepatic microsomal and cytosolic biotransformation of AFB1 was studied in 8 different strains of mice fed a purified diet. GST-mediated conjugation of AFB1-8,9-epoxide with glutathione and GST activity toward 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), ethacrynic acid (ECA) and cumene hydroperoxide (CHP) were determined with cytosolic fractions from 8-10 pooled livers. Specific activities of cytochrome-P-450-mediated oxidation of AFB1 to aflatoxin Q1 (AFQ1), aflatoxin M1 (AFM1), and aflatoxin P1 (AFP1), as well as the reactive intermediate AFB1-8,9-epoxide, were determined with hepatic microsomal fractions from each mouse strain. No striking differences in specific activity between mouse strains were observed for any of the P-450- or GST-mediated enzymatic pathways measured, although some statistically significant differences were found. GST specific activities toward AFB1-8,9-epoxide, CDNB, DCNB, ECA and CHP ranged from 1.5-2.1, 2,830-5,370, 81-144, 38-69 and 32-73 nmol/mg protein/min, respectively. The rate of formation of AFB1-8,9-epoxide ranged from 208 to 465 pmol/mg protein/min. The specific activities of AFQ1,AFM1, and AFP1 formation by microsomes ranged from 36-70, 161-326, and 252-426 pmol/mg protein/min, respectively. Mice fed a standard rodent chow diet showed evidence of microsomal and cytosolic enzyme induction when compared to mice fed a purified diet. The lack of substantial differences in enzyme specific activities between mouse strains suggests that interstrain variations in the hepatocarcinogenic effects of AFB1 in mice should not be large.

Aflatoxin B1↗
PubMed1995

Effects of phyllosilicate clay on the metabolic profile of aflatoxin B1 in Fischer-344 rats.

The phyllosilicate clay, hydrated sodium calcium aluminosilicate (HSCAS), has been shown to prevent aflatoxicosis in farm animals by reducing the bioavailability of aflatoxin. The present study was designed to determine the effects of HSCAS on the metabolism of aflatoxin B1 (AFB1) in an aflatoxin-sensitive species. Male Fischer-344 rats were orally dosed with 1.0, 0.5, 0.25 and 0.125 mg AFB1/kg body weight alone and in combination with 0.5% HSCAS. Urine samples were collected after 6, 24, 36, and 48 h. Aflatoxin M1 (AFM1) and aflatoxin P1 (AFP1) were detected in most urine samples, with or without HSCAS. AFM1 was found to be the major metabolite. Metabolite concentrations were significantly decreased in the presence of HSCAS, and more importantly, no additional metabolites were detected. Our results suggest that the AFB1-HSCAS complex was not significantly dissociated in vivo, and support earlier findings that HSCAS tightly binds aflatoxin.

Aflatoxin B1↗
PubMedDate not supplied

Long-term administration of low doses of mycotoxins to poultry. 1. Residues of aflatoxin B1 and its metabolites in broilers and laying hens.

A study was performed to determine aflatoxin residues in tissues and organs of male broilers and hens that had been fed a diet contaminated with 50 micrograms/kg aflatoxin B1 (AFB1). Residue levels of AFB1, aflatoxicol (Ro), aflatoxin M1 (AFM1) and aflatoxin B2a (AFB2a) were determined by an HPLC method and, with the exception of AFB2a, were detected in the liver, kidney and thigh of both male broilers and hens. The highest levels found were for Ro in liver (1.10 and 0.60 micrograms/kg for male broilers and hens, respectively). On the other hand no detectable amounts of aflatoxins were found in any tissue after withdrawal periods of 14 and 33 days for male broilers and laying hens respectively.

Aflatoxin B1↗
PubMed1991

Evaluation of an indirect enzyme-linked immunosorbent assay for screening antibody against aflatoxins.

Enzyme-linked immunosorbent assay screening of antibody produced against aflatoxin was accomplished by a new and simple procedure. To demonstrate the new indirect ELISA technique used, antibody against aflatoxin M1 was produced in female BALB/CJ mice by immunization with an aflatoxin M1-bovine serum albumin conjugate. Instead of coating test-plate wells with purified antibody (direct ELISA) or synthesizing a second protein-aflatoxin conjugate (aflatoxin M1-poly-L-lysine) to coat test-plate wells, wells were coated with the readily available aflatoxin M1-bovine serum albumin and aflatoxin B1-bovine serum albumin. This method, applicable for any aflatoxin conjugated by the common cyclopentano-carboxymethoxyl-oxime technique, eliminates the more time-consuming and technically difficult portions of earlier direct and indirect ELISA. The new technique can be valuable in continued efforts toward development of new and improved immunoassays against aflatoxin metabolites.

Aflatoxins↗
PubMed1976

Metabolism of aflatoxin B1 to aflatoxins Q1, M1 and P1 by mouse and rat.

We have used a microtechnique to study (a) the metabolism of aflatoxin (AF)B1 by rat and mouse, and (b) the effect of phenobarbital treatment in vivo on the in vitro metabolism of AFB1 by hepatic microsomes from rat and mouse. The results indicate and AFP1, the O-demethylated product of AFB1, is a major metabolite produced by the mouse. Although it is detectable in rat, the amount produced is negligible and was calculated to be at least 10 times less than that produced by the mouse. Using several microincubations, AFP1 was prepared in sufficient quantities to verify its identity by UV spectroscopy and by thin layer chromatography against an authentic standard in six different solvent systems. Phenobarbital pretreatment resulted in an enhancement in the total metabolism of AFB1 as well as in the formation of AFM1, AFQ1 and AFP1.

Aflatoxins↗
PubMed1983

The metabolism of aflatoxin B1 by hepatocytes isolated from rats following the in vivo administration of some xenobiotics.

Isolated rat hepatocytes, an intact cellular system capable of performing phase I and phase II metabolism, have been used to investigate metabolism of aflatoxin B1. These cells were found to metabolise [14C]aflatoxin B1 to aflatoxins M1 and Q1, and to radiolabelled polar material, presumably conjugates, as analysed by h.p.l.c., t.l.c. and radioactive determination. In vivo administration of the mixed function oxidase inducers, phenobarbitone and 3-methylcholanthrene, resulted in enhanced hepatocyte phase I (microsomal) metabolism of aflatoxin B1. In contrast to metabolism of AFB1 by in vitro subcellular systems increased production of polar material (conjugated metabolites) derived from [14C]aflatoxin B1 was also detected in hepatocytes isolated from these pretreated animals. Formation of aflatoxin Q1 by isolated hepatocytes appeared to be mediated by cytochrome P450-linked enzymes whereas cytochrome P448-linked enzymes were apparently involved in aflatoxin M1 production. Chronic feeding of aflatoxin B1 to rats enhanced hepatocyte production of conjugated material only and did not elevate cellular cytochrome P450 levels, thus suggesting that aflatoxin B1 is not an inducer of its own primary metabolism.

Aflatoxin B1↗
PubMed1984

Aflatoxins in human breast milk.

Breast milk from 99 Sudanese mothers was analysed for aflatoxins. Aflatoxins M1 and/or M2 were detected in 37 of the milks. No other aflatoxin was detected. M1 occurred alone in 13 milks, (mean 19.0 pg/ml), M2 in 11 milks (mean 12.2 pg/ml), and in 13 samples both M1 and M2 were detected. There appeared to be a linear relationship between M1 and M2 where both were excreted. No aflatoxin was detected in subcutaneous abdominal wall fat removed during Caesarian section from 15 women, but was present in three out of 14 bloods taken during anaesthesia. The presence of aflatoxins in mothers' milk showed no correlation with duration of lactation, the infants' nutrition, presence of aflatoxin in mothers' blood, or the infant's blood and urine. It is concluded that some Sudanese women excrete aflatoxins in breast-milk at levels similar to or higher than those considered safe in animal milk, for human consumption.

Adipose Tissue↗
PubMed1999

Protective alterations in phase 1 and 2 metabolism of aflatoxin B1 by oltipraz in residents of Qidong, People's Republic of China.

BACKGROUND: Residents of Qidong, People's Republic of China, are at high risk for development of hepatocellular carcinoma, in part due to consumption of foods contaminated with aflatoxins, which require metabolic activation to become carcinogenic. In a randomized, placebo-controlled, double-blind phase IIa chemoprevention trial, we tested oltipraz, an antischistosomal drug that has been shown to be a potent and effective inhibitor of aflatoxin-induced hepatocarcinogenesis in animal models. METHODS: In 1995, 234 adults from Qidong were enrolled. Healthy eligible individuals were randomly assigned to receive by mouth 125 mg oltipraz daily, 500 mg oltipraz weekly, or a placebo. Sequential immunoaffinity chromatography and liquid chromatography coupled to mass spectrometry or to fluorescence detection were used to identify and quantify phase 1 and phase 2 metabolites of aflatoxin B1 in the urine of study participants. Reported P values are two-sided. RESULTS: One month of weekly administration of 500 mg oltipraz led to a 51% decrease in median levels of the phase 1 metabolite aflatoxin M1 excreted in urine compared with administration of a placebo (P = .030), but it had no effect on levels of a phase 2 metabolite, aflatoxin-mercapturic acid (P = .871). By contrast, daily intervention with 125 mg oltipraz led to a 2.6-fold increase in median aflatoxin-mercapturic acid excretion (P = .017) but had no effect on excreted aflatoxin M1 levels (P = .682). CONCLUSIONS: Intermittent, high-dose oltipraz inhibited phase 1 activation of aflatoxins, and sustained low-dose oltipraz increased phase 2 conjugation of aflatoxin, yielding higher levels of aflatoxin-mercapturic acid. While both mechanisms can contribute to protection, this study highlights the feasibility of inducing phase 2 enzymes as a chemopreventive strategy in humans.

Acetylcysteine↗
PubMed1989

Preparation of protein conjugatable aflatoxin oxime derivative from significantly reduced starting quantities.

A modification of an existing technique for the preparation of aflatoxin B1-(0-carboxymethyl) oxime is presented which allows for a significantly reduced starting amount of aflatoxin. About 80% of the aflatoxin was converted to its oxime. The new technique will be particularly valuable when protein conjugatable oximes of more expensive aflatoxin metabolites, such as aflatoxin M1, are required for immunoassay production.

Aflatoxin B1↗
PubMed1982

Metabolism and activation of aflatoxin B1 by reconstituted cytochrome P-450 system of rat liver.

Metabolism and activation of aflatoxin B1, a potent hepatocarcinogenic and mutagenic mycotoxin of Aspergillus flavus, were investigated in the reconstituted enzyme system composed of purified NADPH-cytochrome P-450 reductase and cytochrome P-450 or P-448 of rat liver. The aflatoxin M1 formation was strictly mediated by P-448 purified from the liver microsomes of polychlorobiphenyl- and 3-methylcholanthrene-treated rats, while the aflatoxin Q1 formation, as well as the binding of DNA, were catalyzed by both P-450 and P448. Differences between the kinetic data on metabolism and activation of aflatoxin B1 obtained with the reconstituted cytochrome systems and those obtained with the microsomal and nuclear systems were discussed, and the significance of these biochemical data in the in vivo carcinogenicity of aflatoxin B1 was evaluated.

Aflatoxin B1↗
PubMed1994

Determination of aflatoxin Q1 in urine by automated immunoaffinity column clean-up and liquid chromatography.

A liquid chromatographic system with an automated clean-up procedure for aflatoxin Q1 in human urine is described. The samples were cleaned up by using immunoaffinity columns originally designed for aflatoxin M1. The chromatographic system was a C18 column with an acidic mobile phase of acetonitrile-water containing potassium bromide. Fluorescence detection (365/440 nm) of aflatoxin Q1 was enhanced by addition of bromine, using post-column derivatization, which was studied by factorial designs. Average recovery of aflatoxin Q1 in spiked 10-ml urine samples was 88% (R.S.D. = 6.4%) at a level of 50 pg/ml. The determination limit was 49.5 pg/ml urine.

Aflatoxin M1↗
PubMedDate not supplied

Distribution and clearance of aflatoxins B1 and M1 in turkeys fed diets containing 50 or 150 ppb aflatoxin from naturally contaminated corn.

Turkeys were fed a diet containing 50 or 150 ppb aflatoxin for 11 or 13 weeks or fed these diets for 11 weeks and then the control diet for 1 or 2 weeks. Aflatoxins B1 and M1 were found in liver, kidney, gizzard, and feces of poults fed the diets for 11 or 13 weeks. However, in turkeys fed the control diet for 1 or 2 weeks after the 11-week feeding trial, no residues of aflatoxin were found in the feces or tissues, except for some aflatoxin B1 remaining in detectable amounts in the gizzard. No mortality was attributable to aflatoxin, and there were no notable differences among groups in weight gains, feed conversion, or histopathologic changes in selected tissues. The response to a second inoculation with sheep erythrocytes was significantly lower in poults given dietary aflatoxin than in controls. This reduced antibody response was not observed when a Pasteurella multocida vaccine was administered.

Aflatoxin B1↗
PubMed1975

Effect of aflatoxins on oxidative phosphorylation by rat liver mitochondria.

The in vitro effect of aflatoxins M1, B1 and G1 on oxidative phosphorylation by rat liver mitochondria with succinate as substrate has been studied. All these toxins inhibit the electron transport chain at a 1-10-4 M concentration and the site of inhibition is between cytochrome b and cytochrome c or c1. Aflatoxin M1 (AFM1) uncouples oxidative phosphorylation at a concentration of 1-10-6 M and reduces the ADP:O ratio, whereas aflatoxin B1 (AFB1) at 1-10-6 M concentration uncouples oxidative phosphorulation but does not affect the ADP:O ratio. At a concentration of 1-10-5 M, AFB1 also decreases the ADP:O ratio along with the uncoupling of oxidative phosphorylation. Aflatoxin G1 (AFG1) acts as an uncoupler at a relatively higher concentration of 1-10-4 M. Preincubation of mitochondria with these aflatoxins resulted in inhibition of respiration and uncoupling of rat liver mitochondria.

Adenosine Diphosphate↗