Some mass-spectral and n.m.r. analytical studies of a glutathione conjugate of aflatoxin B1.

A system for the formation of an aflatoxin B1-reduced glutathione conjugate in vitro was developed, capable of yielding 80% conversion of aflatoxin B1 into the conjugate. A reverse-phase high-pressure-liquid-chromatography system was also devised that not only facilitates improved resolution of the compound but that, by manipulation of the pH, is also capable of an extensive purification of the compound from other aflatoxin B1 metabolites in a single step. Material produced by these techniques, after further purification, has been used in 1H-n.m.r. and mass-spectroscopic studies. Results were obtained that support the proposed linkage of the aflatoxin B1 to reduced glutathione in a 1:1 molar ratio via a thioether linkage. Amino acid analyses were also consistent with this structure. The absence of a Schiff-base linkage of aflatoxin B1 8,9-dihydrodiol to glutamate was further demonstrated by the presence of a gamma-glutamyltransferase-catalysed-transferable glutamate moiety. These data are consistent with the structure 8,9-dihydro-8-(S-glutathionyl)-9-hydroxy-aflatoxin B1.     

Reactivity of aflatoxin B2a antibody with aflatoxin B1-modified DNA and related metabolites.

Aflatoxin B2a (AFB2a) antiserum has been previously used in an enzyme-linked immunosorbent assay (ELISA) for the quantitation of AFB1 and AFB2a. The present investigation examined the reactivity of the antiserum toward those adducts and metabolites of AFB1 believed to play a major role in aflatoxicosis and carcinogenesis. 2,3-Dihydro-2-(N7-guanyl)-3-hydroxyaflatoxin B1 (AFB1-N7-Gua), the putative 2,3-(N5-formyl-2-2', 5',6'-triamino-4-oxo-N5-pyrimidyl)-3-hydroxyaflatoxin B1 (AFB1-FAPyr), 2,3-dihydro-2,3-dihydroxyaflatoxin B1 (AFB1-diol), AFB1-N7-Gua-modified DNA, and AFB1-FAPyr-modified DNA were prepared by in vitro incubation or chemical methods and subjected to competitive AFB2a ELISA. The antiserum showed significant reactivity with all five compounds, indicating that it had a high degree of specificity for both the cyclopentenone and the methoxy group of the parent aflatoxin molecule. Sensitivity for AFB-N7-Gua-modified DNA, AFB1-FAPyr-modified DNA, and AFB1-diol by the ELISA method was 0.1 pmol per assay. To test the applicability of immunological detection of covalent binding of AFB1 to DNA, the ELISA was compared with a conventional radioisotopic assay in two in vitro studies. The results showed that estimates of the kinetics and substrate dependence of covalent binding to calf thymus DNA in rat microsomal incubation mixtures by both methods were comparable. The broad specificity AFB2a antibody might be of considerable value in the detection of AFB1 macromolecular adducts and related metabolites in epidemiological investigations or in the diagnosis of aflatoxicosis.     

Intrahepatic conversion of a glutathione conjugate to its mercapturic acid. Metabolism of 1-chloro-2,4-dinitrobenzene in isolated perfused rat and guinea pig livers

Because of the low hepatic activity of gamma-glutamyl-transferase in the rat, the liver is generally considered to play only a minor role in the degradation of glutathione conjugates, a limiting step in mercapturic acid formation. Recent findings indicate, however, that the liver has a prominent role in glutathione catabolism, particularly in species other than rat. To examine the contributions of liver to mercapturic acid biosynthesis, mercapturate formation was compared in isolated perfused livers from rats and guinea pigs dosed with either 0.3 or 3.0 mumol of 1-chloro-2,4-dinitrobenzene (CDNB). Chemically synthesized glutathione conjugate, mercapturic acid, and intermediary metabolites of CDNB were used as standards in the high performance liquid chromatography analysis of bile and perfusate samples. Biliary excretion accounted for almost all of the recovered metabolites. A marked species difference was observed in the pattern of CDNB metabolism. Rat livers dosed with 0.3 mumol of CDNB excreted 55% of total biliary metabolites as the glutathione conjugate and 8.2% as the mercapturic acid, whereas guinea pig livers excreted only 4.8% as the glutathione conjugate and 47% as the mercapturate. Mercapturic formation was also dose-dependent, with a larger fraction formed at the 0.3- versus the 3.0-mumol dose (8.2 versus 3.7% in the rat; 47 versus 19% in the guinea pig). Hepatic conversion of the glutathione conjugate to the mercapturic acid was markedly inhibited in both species after retrograde intrabiliary infusion of acivicin, an inhibitor of gamma-glutamyltransferase activity. These findings provide direct evidence for intrahepatic biosynthesis of mercapturic acids. Thus, glutathione conjugates synthesized within hepatocytes are secreted into bile and broken down to cysteine conjugates; the latter are then presumably reabsorbed by the liver, N-acetylated to form the mercapturic acid and re-excreted into bile.     

The requirement for glutathione S-transferase in the conjugation of activated aflatoxin B1 during aflatoxin hepatocarcinogenesis in the rat

The formation of an aflatoxin B1-reduced glutathione (AFB1-GSH) conjugate in in vitro systems has been examined. AFB1 was activated by a chicken liver microsomal system and factors affecting the subsequent conversion to the AFB1-dihydrodiol or conjugation with GSH were investigated by HPLC. A requirement for glutathione S-transferase in the formation of the AFB1-GSH conjugate was observed. Studies using CM-cellulose columns showed the fractions containing glutathione S-transferase B activity were the most effective in catalysing the formation of the AFB1-GSH conjugate. The possibility of changes in the level of AFB1-GSH conjugate production in the liver during carcinogenesis by AFB1 has been examined. It has been found, using freshly isolated rat hepatocytes, that low level feeding with AFB1 in vivo increases the production of the conjugate in vitro. Further increases in the production of the conjugate by hepatocytes in vitro, accompanying increases in the preneoplastic lesions, are achieved by partially hepatectomising the AFB1-fed animals. Partial hepatectomy of control-fed animals yielded no similar changes. The AFB1/partial hepatectomy treatment resulted in increased levels of all the glutathione S-transferase activities fractionated on CM-cellulose. Macromolecular binding of AFB1 and/or of its metabolites was detected in the fractions containing glutathione S-transferase activity, but there was no evidence for a greater binding in the glutathione S-transferase B/ligandin containing fractions. Furthermore fractionation on Sephadex G-75 indicated a predominance of binding of AFB1 to proteins of a higher molecular weight than the glutathione S-transferases, although some binding in the molecular weight range of the latter was observed.     

Epoxidation of aflatoxin B1 by Aspergillus flavus microsomes in vitro: interaction with DNA and formation of aflatoxin B1-glutathione conjugate

Metabolism of aflatoxin B1 (AFB1) by subcellular preparations of Aspergillus flavus is least understood. The results reported here have demonstrated for the first time the epoxidation of AFB1 and subsequent conjugation with glutathione (GSH). Microsomes prepared from toxigenic mycelia catalysed [3H]AFB1 to calf thymus DNA to a greater extent (approximately 2-fold) as compared to that of non-toxigenic. The binding of [3H]AFB1 to exogenous and A. flavus nuclear DNA catalyzed by A. flavus microsomes was found to be comparable with that of mammalian extrahepatic tissue such as lung. Addition of phenobarbitone to the growing cultures resulted in 1.5-fold increase in [3H]AFB1-DNA binding mediated by microsomes prepared from either of the two strains. Tolnaftate, an inhibitor of aflatoxin synthesis enhanced the epoxidation rate in a dose-related manner. The binding of [3H]AFB1 to DNA catalyzed by A. flavus microsomes was significantly reduced (50% of control) upon addition of hamster liver cytosol, thereby substantiating the formation of the carcinogen adduct with DNA as reported in mammalian tissues. The metabolite formed by subcellular preparation of A. flavus was found to be AFB1-GSH having Rf value (6.5) similar to that obtained for mammalian liver preparations.     

Biotransformation of aflatoxin B1 and its conjugated metabolites by rat gastrointestinal microfloras

Rat cecal microflora from high- and low-fiber-fed animals hydrolyzed aflatoxin conjugates to metabolites indistinguishable from aflatoxin B1 and aflatoxin P1, but aflatoxicol was not a transformation product.     

On the binding of aflatoxin B 1 and its metabolites to hepatic microsomes

The metabolism of aflatoxin B₁ was studied using the cytochrome P450-dependent mixed function oxidase system of rat liver microsomes. An aflatoxin metabolite produced in the presence of microsomes and NADPH and not produced in the presence of SKF-525A seems to become covalently bound to microsomes. The bound metabolite is observed as a spectral peak at 412 nm by means of difference spectroscopy. This metabolite appears to be related to either aflatoxin B_2a or its precursor.     

Biotransformation of aflatoxin B1 and its conjugated metabolites by rat gastrointestinal microfloras.

Rat cecal microflora from high- and low-fiber-fed animals hydrolyzed aflatoxin conjugates to metabolites indistinguishable from aflatoxin B1 and aflatoxin P1, but aflatoxicol was not a transformation product.     

Vacuolar transport of the glutathione conjugate of trans-cinnamic acid

Red beet (Beta vulgaris L.) tonoplast membrane vesicles and [14C]trans-cinnamic acid-glutatione were used to study the vacuolar transport of phynylpropanoid-glutathione conjugates which are formed in peroxidase-mediated reactions. It was determined that the uptake of [14C]trans-cinnamic acid-glutathione into the tonoplast membrane vesicles was MgATP dependent and was 10-fold faster than the uptake of non-conjugated [14C]trans-cinnamic acid. Uptake of the conjugate in the presence of MgATP was not dependent on a trans-tonoblast H+-electrochemical gradient, because uptake was not affected by the addition of NH4Cl (1 mM; 0% inhibition) and was only slightly affected by gramicidin-D (5 microM; 14% inhibition). Uptake of the conjugate was inhibited 92% by the addition of vanadate (1 mM) and 71% by the addition of the model substrate S-(2,4-dinitrophenyl) glutathione (500 microM). Uptake did not occur when a nonhydrolyzable analog of ATP was used in place of MgATP. The calculated Km and Vmax values for uptake were 142 microM amd 5.95 nmol mg(-1) min(-1), respectively. Based on these results, phenylpropanoid-glutation conjugates formed in peroxidase-mediated reactions appear to be transported into the vacuole by the glutathione S-conjugate pump(s) located in the tonoplast membrane.     

The effect of aflatoxin B1 on normal and cortisol-stimulated rat liver ribonucleic acid synthesis

1. Aflatoxin B(1), administered in vivo, inhibits the incorporation of [(14)C]orotic acid in vivo into rat liver nuclei, and also inhibits both Mg(2+)- and Mn(2+)-dependent RNA polymerase activities in nuclei assayed in vitro. 2. Aflatoxin B(1) inhibits the cortisol-induced increase in incorporation of [(14)C]leucine in vivo, but does not affect the control value of this activity. 3. Aflatoxin B(1) administered in vivo inhibits the increase in nuclear Mg(2+)-dependent RNA polymerase activity, assayed in vitro, which results from the treatment with cortisol. 4. Adrenalectomy causes a decrease in Mg(2+)-dependent RNA polymerase activity. The effect on this enzymic activity of adrenalectomy plus treatment with aflatoxin B(1) is no greater than that of treatment with aflatoxin B(1) alone. 5. These results suggest that the inhibition of cortisol-stimulated biochemical pathways by aflatoxin B(1) is due to an inhibition of cortisol-stimulated RNA synthesis. 6. The cytoplasmic action of aflatoxin is thought to be due to a competition for receptor sites on the endoplasmic reticulum between steroid hormones and aflatoxin B(1). No evidence was obtained for a similar competition for nuclear receptor sites between [(3)H]cortisol and aflatoxin B(1). 7. No differences were observed between the activities of RNA polymerase preparations solubilized from control or aflatoxin-inhibited nuclei. 8. No differences in ;melting' profiles were observed between DNA and chromatin preparations isolated from control nuclei or from aflatoxin-inhibited nuclei. 9. It is suggested that aflatoxin B(1) exerts its effect on RNA polymerase by decreasing the template capacity of the chromatin and that the aflatoxin ;target' area of the chromatin includes that region which is stimulated by cortisol. This process, however, does not involve inhibiting the movement of cortisol from the outside of the hepatic cell to the nuclear chromatin.     

The interaction of aflatoxin B1 with polynucleotides and its effect on ribonucleic acid polymerase

1. The interaction of aflatoxin B(1) with different polynucleotides was studied spectrophotometrically. Equations were derived that enable the degree of binding to be determined without first determining the extinction coefficient of the bound form. 2. The interaction with calf thymus DNA obeys first-order relationships with an association constant of 0.40mm(-1), but there is some evidence for a secondary binding process from results obtained at 390nm. 3. The spectral shifts decreased in the order polyadenylic acid+polyuridylic acid>DNA>polyadenylic acid>polyadenylic acid+polyinosinic acid. Polycytidylic acid, polyuridylic acid, polyinosinic acid (both single- and triple-stranded), AMP, CMP, GMP and UMP did not interact with aflatoxin. It was concluded that there is a requirement for the amino group of adenine (or possibly guanine) for binding of aflatoxin to polynucleotides to occur. 4. Binding is reversed by increasing ionic strength, and by Mn(2+) and Mg(2+) in the concentration range studied (0-5mm). The effect of the Mn(2+) or Mg(2+) was far greater than would be expected on the basis of their ionic strength. With both the bivalent cations and sodium chloride the reversal is greatest with double-stranded polynucleotides. 5. Inhibition in vitro of the DNA-dependent RNA polymerase of Escherichia coli by aflatoxin B(1) was detected only in the absence of Mg(2+) and at concentrations of Mn(2+) below the optimum for RNA synthesis in vitro. 6. The degree of inhibition (maximally 30%) was dependent on the concentration of Mn(2+) and decreased during incubation.     

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.     

Alterations of phosphatidylinositol signal transduction pathway in hepatic mitochondria following aflatoxin B1 administration

A single dose of aflatoxin B1 (7 mg/kg body wt) to male rats significantly stimulated the turnover of mitochondrial phosphoinositides 1-7 hr following its administration. The elevation of phosphatidylinositol 3,4,5-trisphosphate was most pronounced whose level continued to be moderately high even at 17 hr period. The level of diacylglycerol showed a marked increase from 4 hr till 7 hr after carcinogen treatment, whereas that of inositol 1,4,5-trisphosphate recorded an increase with a maximum at 7 hr followed by a gradual decrease to near normal level at 24 hr period. The activation of phosphatidylinositol cycle together with an activation of PI 3-kinase, whose product PIP3 is known to be involved in apoptosis might contribute to the early step in the manifestation of toxicity and/or carcinogenicity.     

真菌对黄曲霉毒素B_1污染的防治研究

黄曲霉毒素是由黄曲霉、寄生曲霉等曲霉属菌株所分泌的毒性次级代谢产物,其中,尤以黄曲霉毒素B1(Aflatoxin B1,AFB1)的毒性、致突变性、致癌性最强,而且其在农作物的栽培、收获、贮藏和加工过程中污染严重,受到全世界的广泛关注。因此,为保证食品的安全性,各国研究人员一直都在寻求安全、高效、经济、环保的方法来控制食品和饲料中AFB1的污染。近年来真菌在AFB1的生物防治方面已取得很大进展,并有部分菌株已应用于生产中。本研究就真菌对AFB1的防治机制及其前景展望进行综述。     

Effect of glutathione and uridine 5'-diphosphoglucuronic acid on mutagenesis by benzo[a]pyrene and aflatoxin B1 in the Salmonella/microsome assay

In the Salmonella/microsome plate or liquid assay, the addition of glutathione (GSH) and uridine 5'-diphosphoglucuronic acid (UDPGA), both cofactors for GSH-S-transferases or UDPGA-transferases, altered the rat-liver microsome-mediated mutagenesis of benzo[a]pyrene (BP) and aflatoxin B1 (AFB). With either BP or AFB, an increased, unchanged or decreased number of revertant colonies of S. typhimurium was observed, depending on the substrate concentration, the source of rat-liver 9000 X g supernatant (S9), the time of incubation and the type of mutagenicity test (liquid or plate assay). Several factors responsible for quantitative changes in the pattern of BP and AFB metabolites under various assay conditions in vitro, which alter the overall mutagenic activity of the parent compound, are discussed.