Determinants of specificity for aflatoxin B1-8,9-epoxide in Alpha-class glutathione S-transferases

McDonagh, PD; Judah, DJ; Neal, G.E.; Roberts, G. C. K.; LIAN, L. Y.; Hayes, John D.; Wolf, C. Roland

doi:10.1042/0264-6021:3390095

Cited by 7 publications

(5 citation statements)

References 31 publications

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“…The resistance of adult mice to AFB1 has been suggested to be due to constitutive expression in mouse liver of the A3 subunit of GST (mGSTA3; also known as Yc or Ya 3 ), which exhibits high catalytic activity toward AFB1 (Buetler and Eaton, 1992; Hayes et al, 1992; McDonagh et al 1999). In support of this suggestion are the findings that mGSTA3 confers protection against 8,9-epoxide when transfected into hamster cells (Fields et al, 1999) and that substitution of the five critical mGSTA3 amino acid residues into the rat GSTA3 sequence increases the conjugation activity over 200-fold (van Ness et al, 1998).…”

Section: Introductionmentioning

confidence: 99%

Glutathione-S-transferase A3 knockout mice are sensitive to acute cytotoxic and genotoxic effects of aflatoxin B1

Ilić

Crawford

Egner

et al. 2010

Toxicology and Applied Pharmacology

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Aflatoxin B1 (AFB1) is a major risk factor for hepatocellular carcinoma (HCC) in humans. However, mice, the major animal model for the study of AFB1 carcinogenesis, are resistant, due to high constitutive expression, in the mouse liver, of glutathione S-transferase A3 subunit (mGSTA3) that is lacking in humans. Our objective was to establish a mouse model for AFB1 toxicity could be used to study mechanisms of toxicity that are relevant for human disease, i.e., an mGSTA3 knockout (KO) mouse that responds to toxicants such as AFB1 in a manner similar to humans. Exons 3-6 of the mGSTA3 were replaced with a neomycin cassette by homologous recombination. Southern blotting, RT-PCR, Western blotting, and measurement of AFB1-N 7 -DNA adduct formation were used to evaluate the mGSTA3 KO mice. The KO mice have deletion of exons 3-6 of the mGSTA3 gene, as expected, as well as a lack of mGSTA3 expression at the mRNA and protein levels. Three hours after injection of 5 mg/kg AFB1, mGSTA3 KO mice have more than 100-fold more AFB1-N 7 -DNA adducts in their livers than do similarly treated wild-type (WT) mice. In addition, the mGSTA3 KO mice die of massive hepatic necrosis, at AFB1 doses that have minimal toxic effects in WT mice. We conclude that mGSTA3 KO mice are sensitive to the acute cytotoxic and genotoxic effects of AFB1, confirming the crucial role of GSTA3 subunit in protection of normal mice against AFB1 toxicity. We propose the mGSTA3 KO mouse as a useful model with which to study the interplay of risk factors leading to HCC development in humans, as well as for testing of additional possible functions of mGSTA3.

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Section: Introductionmentioning

confidence: 99%

Glutathione-S-transferase A3 knockout mice are sensitive to acute cytotoxic and genotoxic effects of aflatoxin B1

Ilić

Crawford

Egner

et al. 2010

Toxicology and Applied Pharmacology

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“…Therefore, the activation of AFB 1 can be measured by the formation of a dihydrodiol–Tris adduct [ 50 ]. Quail liver microsomes were used as a positive control as they were reported to be highly active in AFB 1 epoxidation [ 37 , 51 , 52 , 53 ]. The HPLC assays showed that the AFB 1 metabolite produced by quail microsomes had the same retention time as that formed during CYP3A29 reaction with AFB 1 , suggesting that CYP3A29 was capable of oxidizing AFB 1 into AFBO.…”

Section: Discussionmentioning

confidence: 99%

Bioactivation and Regioselectivity of Pig Cytochrome P450 3A29 towards Aflatoxin B1

Chen

Zhang

et al. 2016

Toxins

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Due to unavoidable contaminations in feedstuff, pigs are easily exposed to aflatoxin B1 (AFB1) and suffer from poisoning, thus the poisoned products potentially affect human health. Heretofore, the metabolic process of AFB1 in pigs remains to be clarified, especially the principal cytochrome P450 oxidases responsible for its activation. In this study, we cloned CYP3A29 from pig liver and expressed it in Escherichia coli, and its activity has been confirmed with the typical P450 CO-reduced spectral characteristic and nifedipine-oxidizing activity. The reconstituted membrane incubation proved that the recombinant CYP3A29 was able to oxidize AFB1 to form AFB1-exo-8,9-epoxide in vitro. The structural basis for the regioselective epoxidation of AFB1 by CYP3A29 was further addressed. The T309A mutation significantly decreased the production of AFBO, whereas F304A exhibited an enhanced activation towards AFB1. In agreement with the mutagenesis study, the molecular docking simulation suggested that Thr309 played a significant role in stabilization of AFB1 binding in the active center through a hydrogen bond. In addition, the bulk phenyl group of Phe304 potentially imposed steric hindrance on the binding of AFB1. Our study demonstrates the bioactivation of pig CYP3A29 towards AFB1 in vitro, and provides the insight for understanding regioselectivity of CYP3A29 to AFB1.

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“…That the 2,3-double bond is a critical structural requirement for carcinogenicity and mutagenicity has been pinpointed by structure-activity relationship studies on a variety of structural analogs of AFB 1 [183,184]. Moreover, quantum mechanical calculations have shown that the 2,3-double bond is the most reactive site in the molecule [185,186]. Although attempts to isolate the putative reactive intermediate, AFB 1 -2, 3oxide, have been unsuccessful because of instability, its formation can be deduced from its reaction products with cellular constituents and its hydrolysis products.…”

Section: Metabolism and Mechanism Of Action Of Aflatoxin Bmentioning

confidence: 99%

Drug-Metabolizing Enzymes Mechanisms and Functions

Sheweita

2000

CDM

213

128

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Drug-metabolizing enzymes are called mixed-function oxidase or monooxygenase and containing many enzymes including cytochrome P450, cytochrome b5, and NADPH-cytochrome P450 reductase and other components. The hepatic cytochrome P450s (Cyp) are a multigene family of enzymes that play a critical role in the metabolism of many drugs and xenobiotics with each cytochrome isozyme responding differently to exogenous chemicals in terms of its induction and inhibition. For example, Cyp 1A1 is particularly active towards polycyclic aromatic hydrocarbons (PAHs), activating them into reactive intermediates those covalently bind to DNA, a key event in the initiation of carcinogenesis. Likewise, Cyp 1A2 activates a variety of bladder carcinogens, such as aromatic amines and amides. Also, some forms of cytochrome P450 isozymes such as Cyp 3A and 2E1 activate the naturally occurring carcinogens (e.g. aflatoxin B1) and N-nitrosamines respectively into highly mutagenic and carcinogenic agents. The carcinogenic potency of PAHs, and other carcinogens and the extent of binding of their ultimate metabolites to DNA and proteins are correlated with the induction of cytochrome P450 isozymes. Phase II drug-metabolizing enzymes such as glutathione S-transferase, aryl sulfatase and UDP-glucuronyl transferase inactivate chemical carcinogens into less toxic or inactive metabolites. Many drugs change the rate of activation or detoxification of carcinogens by changing the activities of phases I and II drug-metabolizing enzymes. The balance of detoxification and activation reactions depends on the chemical structure of the agents, and is subjected to many variables that are a function of this structure, or genetic background, sex, endocrine status, age, diet, and the presence of other chemicals. It is important to realize that the enzymes involved in carcinogen metabolism are also involved in the metabolism of a variety of substrates, and thus the introduction of specific xenobiotics may change the operating level and the existence of other chemicals. The mechanisms of modification of drug-metabolizing enzyme activities and their role in the activation and detoxification of xenobiotics and carcinogens have been discussed in the text.

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Determinants of specificity for aflatoxin B1-8,9-epoxide in Alpha-class glutathione S-transferases

Cited by 7 publications

References 31 publications

Glutathione-S-transferase A3 knockout mice are sensitive to acute cytotoxic and genotoxic effects of aflatoxin B1

Glutathione-S-transferase A3 knockout mice are sensitive to acute cytotoxic and genotoxic effects of aflatoxin B1

Bioactivation and Regioselectivity of Pig Cytochrome P450 3A29 towards Aflatoxin B1

Drug-Metabolizing Enzymes Mechanisms and Functions

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