Dennis P. H. Hsieh

Maintenance of cytochrome P-450 and metabolism of aflatoxin B1 in primary hepatocyte cultures

The cytochrome P-450 content of primary hepatocyte cultures was maintained at levels close to those found in vivo by using a defined medium containing testosterone, thyroxine, hydrocortisone, estradiol, glucagon, insulin, linoleic acid and oleic acid. Using these cultures, [14C]aflatoxin B1, a potent liver carcinogen, was metabolized primarily to water-soluble metabolites. In agreement with in vivo results, aflatoxin M1 was the only nonpolar metabolite detected. In addition, a significant portion of radioactivity was covalently bound to cell constituents. These results suggest that primary hepatocyte cultures may be a good model of the liver for studying the metabolism and mechanism of action of toxic chemicals.

The comparative metabolism and toxicokinetics of aflatoxin B1 in the monkey, rat, and mouse.

Abstract The in vivo metabolism and toxicokinetics of aflatoxin B 1 (AFB 1 ) were compared among the monkey, rat, and mouse, each representing a different model of sensitivity to the acute and carcinogenic effects of AFB 1 . Species relatively sensitive to the acute effect of AFB 1 showed a higher volume of distribution, a higher equilibrium transfer rate constant, higher levels of total aflatoxins in liver and plasma, and a longer plasma biological half-life of AFB 1 . Species relatively resistant to the carcinogenic effect of AFB 1 were more active in the in vivo conversion of AFB 1 to aflatoxin P 1 and water-soluble metabolites. In addition, the recovery of aflatoxicol from rat plasma but not from the plasma of similarly treated resistant species, the monkey and mouse, supported and proposed correlation between species sensitivity to the carcinogenic effect of AFB 1 and aflatoxicol-forming activity. The results obtained support previously proposed metabolic models for the prediction of human sensitivity to AFB 1 toxicity.

Airborne mutagens produced by frying beef, pork and a soy-based food

Airborne cooking by-products from frying beef (hamburgers), pork (bacon strips) and soybean-based food (tempeh burgers) were collected, extracted, tested for mutagenicity and chemically analysed. The fumes generated by frying pork and beef were mutagenic, with 4900 and 1300 revertants/g of food cooked, respectively. No mutagenicity was detected in fumes from frying tempeh burgers. Bacon fried to a well-done but non-charred state was eight times more mutagenic in a microsuspension Ames/Salmonella test (TA98 with S-9) than hamburgers and about 350 times more mutagenic than tempeh burgers. Among food samples cooked to a well-done, non-charred state, bacon strips had almost 15-fold more mass (109.5 ng/g) than that of the beef, whereas no heterocyclic amine (HCA) was detected in the fried tempeh burgers. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) was the most abundant HCA, followed by 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (DiMeIQx). No 2-amino-9H-pyrido[2,3-b]indole (A alpha C) was detected in the food samples fried at about 200 degrees C, although it was present in the collected airborne products. The total amounts of HCAs in the smoke condensates were 3 ng/g from fried bacon, 0.37 ng/g from fried beef and 0.177 ng/g from fried soy-based food. This study indicates that cooks are potentially exposed to relatively high levels of airborne mutagens and carcinogens and that long-term sampling inside restaurants and kitchens may be warranted in order to assess the potential risk of prolonged exposure.

Butylate hydroxytoluene pretreatment protects against cytotoxicity and reduces covalent binding of aflatoxin B1 in primary hepatocyte cultures

Primary cultures of adult rat hepatocytes were used to characterize the effect of butylated hydroxytoluene (BHT) pretreatment on the metabolic disposition and cytotoxicity of a single dose of aflatoxin B1 (AFB1). Four male Sprague-Dawley rats were fed a control diet, and five were fed a diet containing 0.5% BHT. After 10 days, hepatocytes were prepared and cultured in chemically defined, hormone-supplemented medium. After 20–22 hr in culture, 120–150 ng of [14C]aflatoxin B1 was added to dishes containing 2.5 × 106 cells. By 10 hr, control cells had converted 59% of the AFB1 to aqueous metabolites, while 15.5% was bound covalently. During the same interval, cells from BHT-fed rats produced 69% aqueous metabolites, and only 6.6% was bound covalently. The rate of AFB1 disappearance in the two groups was not statistically different. AFB1 produced marked cytotoxicity in hepatocyte cultures from control rats but had no apparent toxic effect on hepatocyte cultures from BHT-pretreated rats, as indicated by light microscopic examination and release of lactate dehydrogenase into the medium. These results suggest that reduction of cytotoxicity by BHT was associated with increased output of nontoxic, water-soluble metabolites and decreased binding of metabolites to macromolecules. These results also indicate that BHT may protect against the acute toxicity and carcinogenicity of aflatoxin B1 in vivo.

Accelerated parathion degradation in soil inoculated with acclimated bacteria under field conditions

The feasibility of decontaminating soil at parathion spillage or disposal sites by inoculation with a highly acclimated culture of parathion-degrading bacteria was demonstrated underin situ field conditions. The acclimated culture (AC), capable of utilizing parathion as a sole carbon and energy source, was inoculated into Yolo silt loam soil in which parathion was applied at rates up to 5000 kg/ha. The AC was shown to be capable of completely degrading parathion in soil containing up to 1250 kg/ha of parathion within 35 days. A slower rate of parathion degradation by the AC was observed when the pesticide was applied as the commercial 46.5% emulsifiable concentrate than when applied as the 98% technical grade. The ability of the AC to degrade parathion deteriorated at application rates greater than 1250 kg/ha. The AC may have been adversely affected by the accumulation of the parathion hydrolytic products,p-nitrophenol and ionic diethyl thiophosphate, which were tentatively identified in soil samples.

Metabolism of aflatoxin B1 in cultured mouse hepatocytes: Comparison with rat and effects of cyclohexene oxide and diethyl maleate☆

The metabolism of aflatoxin B1 was studied in cultured hepatocytes from adult male mice. After a 10-hr incubation, 89% of the dose of [14C]aflatoxin B1 was converted to aqueous metabolites, while 0.77% was covalently bound to cellular macromolecules. In contrast, it has been previously shown that hepatocytes cultured from the rat and incubated with aflatoxin B1 form 57.2% aqueous metabolites and covalently bind 12.2% to cellular macromolecules. The nearly 16-fold greater covalent binding in rat hepatocytes correlates with the greater susceptibility of the rat to the hepatocarcinogenic effect of aflatoxin B1. Diethyl maleate and cyclohexene oxide each was shown to significantly increase the amount of covalent binding in hepatocytes from both species. At 10−3 m, diethyl maleate and cyclohexene oxide, respectively, produced 805 and 61% increase in covalent binding in mouse hepatocytes, suggesting that both diol formation and glutathione conjugation are important detoxification pathways for aflatoxin B1. Both pathways are apparently more active in mouse hepatocytes than in rat hepatocytes.

Aflatoxin biosynthetic pathway: Elucidation by using blocked mutants of Aspergillus parasiticus

Abstract Two mutant strains of Aspergillus parasiticus , both deficient in aflatoxin production, were used to elucidate the biosynthetic pathway of this mycotoxin. One of the mutants, A. parasiticus ATCC 24551, was capable of accumulating large amounts of averufin, and the other, A. parasiticus 1-11-105 wh-1, accumulated versicolorin A. The averufin producing mutant efficiently converted 14 C-labeled versiconal acetate, versicolorin A, and sterigmatocystin into aflatoxin B 1 and G 1 , indicating that averufin preceded these compounds in the aflatoxin biosynthetic pathway. In the presence of dichlorvos (dimethyl 2,2-dichlorovinyl phosphate), a known inhibitor of aflatoxin biosynthesis, the conversion of versicolorin A and sterigmatocystin was unaffected, but the conversion of versiconal acetate was markedly inhibited. The mutant accumulating versicolorin A incorporated 14 C-labeled acetate, averufin, and versiconal acetate into versicolorin A. In the presence of dichlorvos, however, the major conversion product was versiconal acetate. This strongly suggested that dichlorvos inhibited the conversion step of versiconal acetate into versicolorin A. This mutant resumed production of aflatoxin B 1 if sterigmatocystin was added to the resting cell cultures, indicating that the mutant was blocked at the enzymatic step catalyzing the conversion of versicolorin A into sterigmatocystin, and as a result was incapable of aflatoxin production. The experimental evidence is thus provided for the involvement and interrelationship of three anthraquinones (averufin, versiconal acetate, and versicolorin A) and a xanthone (sterigmatocystin) in aflatoxin biosynthesis. A pathway for the biosynthesis of aflatoxin B 1 is proposed to be: acetate →→→ averufin → versiconal acetate → versicolorin A → sterigmatocystin → aflatoxin B 1 .

Comparison of multi-media transport and transformation models: Regional fugacity model vs. CalTOX

Two multimedia environmental transport and transformation computer models are summarized and compared. The regional fugacity model published by Mackay and Paterson (1991), termed Fug3ONT, is a four compartment steady-state model designed to simulate the relative distribution of nonionic organic chemicals in a multimedia system. CalTOX is a seven compartment multimedia total exposure model for hazardous waste sites. Both models are based on the principles of fugacity. CalTOX, however, separates the soil into three layers (surface, root, and vadose) and uses a different approach to estimate the diffusive mass transfer rate in soil. These differences result in lower estimates of the steady-state contaminant concentrations of six environmentally relevant chemicals in the root soil of CalTOX as compared to the bulk soil of Fug3ONT. The difference is greatest for compounds with low mobility in soil such as 2,3,7,8-Tetrachlorodibenzo-p-dioxin and Benzo(a)pyrene where estimates from CalTOX and Fug3ONT differ by more than 3 orders of magnitude. Otherwise, the models provide similar estimates for the distribution of the six chemicals among the air, water, sediment and surface soil.

Conversion of sterigmatocystin to aflatoxin B1 by Aspergillus parasiticus

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

Mutagenicity of fungal metabolites related to aflatoxin biosynthesis

Fungal metabolites identified as the intermediates in aflatoxin biosynthetic pathway were screened for their mutagenic activity to Salmonella typhimurium TA98. Norsolorinic acid, averufin, and versiconal acetate were found to possess questionable mutagenic activity, but versicolorin A, and sterigmatocystin were significant mutagens relative to aflatoxin B1. The mutagenic activity appears to be related to the bisfuran and not the anthraquinone moiety of the molecule, even though the latter is a key structure of such potent carcinogenic mycotoxin as luteoskyrin.