Comparative metabolic conversion of aflatoxin B1 to M1 and Q1 by monkey, rat and chicken liver

We studied the $\text{in vitro}$ metabolish of flatoxin B₁ by liver microsomal preparations from monkey, rat and chicken. With all these species, both the previously recognized metabolite aflatoxin M₁ as well as the newly identified aflatoxin Q₁ were produced from the aflatoxin B₁ substrate. Aflatoxin Q₁ is an isomer of aflatoxin M₁ (with the hydroxyl on the carbon β to the carbonyl of the cyclopentenone ring) which we discovered recently in rat and monkey liver incubations with aflatoxin B₁. In our incubations we did not detect aflatoxin P₁ which has been reported as a major metabolite of aflatoxin B₁$\text{in vivo}$ in the monkey.In general the conversion to aflatoxin M₁ was comparable among the different species (1–3% of the substrate) except in the chicken in which it was lower (0.1–0.3%). Also the conversion to Q₁ was comparable to or slightly higher than the conversion to M₁ with rat and chicken liver but the conversion to Q₁ with the monkey liver was outstandingly high, accounting for 19–52% of the substrate in three species of monkeys tested.     

Mutagenic activation of aflatoxin B1 by P-450 HFLa in human fetal livers

The genotoxic and mutagenic activation of promutagens by human fetal livers was measured by the induction of umu gene expression in Salmonella typhimurium TA1535/pSk1002. Liver homogenates of human fetuses were the most active for the mutagenic activation of aflatoxin B1 (AFB1), followed by 2-amino-3-methylimidazo(4,5-f)quinoline (IQ), and to a lesser extent by 2-amino-6-methyldipyrido(1,2-a:3',2'-d)imidazole (Glu-P-1). The amounts of P-450 HFLa immunochemically determined in human fetal livers correlated highly with the induction of umu gene expression by AFB1 (r = 0.98, n = 5). P-450 HFLa catalyzed the mutagenic activation of AFB1 in a reconstituted system: cytochrome b5 markedly stimulated the activation. Anti-P-450 HFLa antibodies inhibited the mutagenic activation of AFB1 in a dose-dependent manner. These results strongly support the idea that P-450 HFLa is responsible for the mutagenic activation of AFB1 in human fetal livers.     

Dual effects of phloretin on aflatoxin B1 metabolism: activation and detoxification of aflatoxin B1

Typically, chemopreventive agents involve either induction of phase II detoxifying enzymes and/or inhibition of cytochrome P450 enzymes (CYPs) that are required for the activation of procarcinogens. In this study, we investigated the protective effects of phloretin against aflatoxin B1 (AFB1) activation to the ultimate carcinogenic intermediate, AFB(1)-8, 9-epoxide (AFBO), and its subsequent detoxification. Phloretin markedly inhibited formation of the epoxide with human liver microsomes in a dose-dependent manner. Phloretin also inhibited the activities of nifedipine oxidation and ethoxyresorufin O-deethylase (EROD) in human liver microsomes. These data show that phloretin strongly inhibits CYP1A2 and CYP3A4 activities, which are involved in the activation of AFB1. Phloretin increased glutathione S-transferase (GST) activity of alpha mouse liver 12 (AML 12) cells in a dose-dependent manner. GST activity toward AFBO in cell lysates treated with 20 μM phloretin was 23-fold that of untreated control cell lysates. The expression of GSTA3, GSTA4, GSTM1, GSTP1 and GSTT1 was induced by phloretin in a dose-dependent manner in AML 12 cells. GSTP1, GSTM1, and GSTT1 were able to significantly increase the conjugation of AFBO with glutathione. Concurrently, induction of the GST isozyme genes was partially associated with the Nrf2/ARE pathway. Taken together, the results demonstrate that phloretin has a strong chemopreventive effect against AFB1 through its inhibitory effect on CYP1A2, CYP3A4, and its inductive effect on GST activity.     

Quantitation of aflatoxin B1 and aflatoxin B1 antibody by an enzyme-linked immunosorbent microassay.

A specific microtest plate enzyme immunoassay has been developed for the rapid quantitation of aflatoxin B1 at levels as low as 25 pg per assay. Multiple-site injection of rabbits with an aflatoxin B1 carboxymethyloxime-bovine serum albumin conjugate was used for the production of hyperimmune sera. Dilutions of the purified antibody were air dried onto microplates previously treated with bovine serum albumin and glutaraldehyde and then incubated with an aflatoxin B1 carboxymethyloxime-horseradish peroxidase conjugate. The amount of enzyme bound to antibody was determined by monitoring the change in absorbance at 414 nm after the addition of a substrate solution consisting of hydrogen peroxide and 2,2'-azino-di-3-ethyl-benzthiazoline-6-sulfonate. Antibody titers determined in this manner closely correlated with those determined by radioimmunoassay. Competition assays as performed by incubation of different aflatoxin analogs with the peroxidase conjugate showed that aflatoxins B1 and B2 and aflatoxicol caused the most inhibition of conjugate binding to antibody. Aflatoxins G1 and G2 inhibited the conjugate binding to a lesser degree, whereas aflatoxins M1 and B2a had no effect of the assay.     

Characterization of monosomes produced by aflatoxin B1.

A single injection of 1.5 mg aflatoxin B1 per kg body weight produced approx. 70% disaggregation of rat liver polysomes into monosomes within 18 h. Isolated monosomes dissociated into 40 S subunits during centrifugation in linear sucrose gradients containing 0.3 M KCI. The 4 S to 5 S molar RNA ratio of the monosomes was calculated to be 0.6, indicating 0.6 tRNA and/or aminoacyl tRNA molecule per ribosome; no peptidyl tRNA was present. These results suggest that a single injection of affatoxin B1 produces monosomes which resemble runoff ribosomes.     

Toxicity of aflatoxin B1 to penaeid shrimp.

Single intramuscular injections of aflatoxin B1 into the tail muscle of Penaeus stylirostris produced 24- and 96-h median lethal doses of 100.5 (78.3 to 129.0) and 49.5 (29.8 to 82.3) mg/kg, respectively. A toxicity curve showed no threshold at the levels tested. The mortality response in a feeding study with P. vannamei was not dose dependent, but tissue and organ damage were similar to that seen in injected animals.     

Modification of the cytotoxicity of aflatoxin B1 in rat liver cells by steroids

The addition of steroids with aflatoxin B1 (AFB1) to rat liver cells in culture has been shown to increase the toxin's inhibitory action on growth and protein synthesis. In contrast the inhibition of RNA synthesis by AFB1 was unaffected. The steroid potentiates the direct action of AFB1 at initiation of translation.     

Intracellular aflatoxin B1-binding proteins in rat liver

Intracellular aflatoxin B1 binding in rat liver was studied under both in vitro and in vivo conditions. Binding in vivo appeared similar to that observed in vitro except that some covalent adduct formation was detected. Participation of previously described carcinogen-binding proteins such as the Ah receptor, h2-5S protein, 4-5S receptor for 3-methylcholanthrene and the Z-protein fraction was discounted on the grounds of competition binding studies and gel-permeation chromatography. The molecular weight of 45,000 was estimated for the major aflatoxin B1-binding component. Aflatoxin B1 co-eluted with the glutathione S-transferases during gel-permeation and separation of the various isozymes by cation-exchange chromatography indicated interactions with the YaYa and YaYc-forms. These proteins, however, account for less than 20% of the total intracellular aflatoxin binding. A protein of apparent monomeric structure appears to form the major in vitro/in vivo complex with aflatoxin B1.     

Mutagenic activation of aflatoxin B1 by several forms of purified cytochrome P-450

Metabolic activation by several forms of purified cytochrome P-450 of aflatoxin B1 to a product(s) mutagenic to Salmonella typhimurium TA100 was examined. Of the 5 forms of cytochrome P-450 purified from liver microsomes of untreated and PCB-treated male rats, a constitutive form purified from untreated male rats, P-450-male, and a high-spin form of cytochrome P-450, P-448-H, from PCB-treated rats were highly active.     

Conversion of sterigmatocystin to aflatoxin B 1 by Aspergillus parasiticus

¹⁴C-Sterigmatocystin isolated from cultures of $\text{Aspergillus}$$\text{versicolor}$ supplemented with (1-¹⁴C)acetate was shown to be efficiently converted to aflatoxin B₁ by the resting mycelium of $\text{A. parasiticus}$. The experimental results may indicate a biosynthetic pathway leading from 5-hydroxysterigmatocystin to sterigmatocystin and then to aflatoxin B₁.     

Effect of aflatoxin B1 on human platelet protein kinase C.

1. Aflatoxin B1 serves as an activator for protein kinase C (PKC). 2. Following activation, the enzyme translocates from the cytosol to the particulate fraction. 3. Aflatoxin B1 leads to rapid and extensive phosphorylation of the known substrate of the enzyme. 4. Aflatoxin B1 causes rapid hydrolysis of phosphatidylinositol-4,5-biphosphate to diacylglycerol and inositol-1,4,5-triphosphate.     

Genetic analysis of aflatoxin B1 activation in rat hepatoma cells

Summary We present a strategy to elucidate the rate-limiting steps in activation of carcinogenic compounds by cytochromes P450. The principle was to select Reuber rat hepatoma cells for resistance to a procarcinogen. The hypothesis was that resistant cells should be systematically deficient in the P450 enzyme(s) involved in the activation process. Here we present an example of the use of this approach using aflatoxin B1 (AFB1), a potent hepatocarcinogen, as the selective agent. Parental cells as well as individual and pooled colonies selected for AFB1 resistance from three independent rat hepatoma lines were characterized for their content of 1) mRNA hybridizing to cDNA and/or oligonucleotide probes for cytochromes P450IIB1, P450IIB2 and albumin; and 2) aldrin epoxidase activity. Parental aflatoxin B1-sensitive cells were shown to express P450IIB1 but not P450IIB2. The majority of the aflatoxin B1-resistant clones failed to accumulate cytochrome P450IIB1 mRNA and expressed no or only very low aldrin epoxidase activity. Albumin mRNA levels remained unchanged, demonstrating that loss of expression of cytochrome P450IIB1 was not a consequence of a general dedifferentiation event. A revertant population showing restoration of both cytochrome P450IIB1 mRNA accumulation and aldrin epoxidase activity was fully sensitive to aflatoxin B1. The correlation between expression of cytochrome P450IIB1 and sensitivity to aflatoxin B1 in both parental cells and revertants strongly suggests that cytochrome P450IIB1 is a major contributor to the activation of aflatoxin B1 in rat hepatoma cells. The kind of strategy described here could be applied to other compounds that become cytotoxic for hepatoma cells following activation by cytochromes P450.Abbreviations AFB1 aflatoxin B1 - AE aldrin epoxidase - AHH aryl hydrocarbon hydroxylase - PAH polycyclic aromatic hydrocarbons - PB phenobarbital