[CANCER RESEARCH 53. 3887-3894. September 1, 1993] Resistance to Aflatoxin Bj Is Associated with the Expression of a Novel Aldo-Keto Reductase Which Has Catalytic Activity towards a Cytotoxic Aldehyde-containing Metabolite of the Toxin1 John D. Hayes, David J. Judah, and Gordon E. Neal Biomedicai Research Centre, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, Scotland, United Kingdom ¡T.D. H.¡;and MRC Toxicology Unit, Hodgkin Building, University of Leicester, P. O. Box 138, Lancaster Road, Leicester, LEI 9HN United Kingdom [D. J. J., G. E. N./ ABSTRACT Aflatoxin B, is extensively metabolized in mammalian liver through a complex number of toxification and detoxification steps (1). Fischer 344 rats readily develop liver cancer when exposed to aflatoxin Initial metabolism of AFB, by the cytochrome P-450 monooxygena- BI (AFBi) but dietary administration of the antioxidant ethoxyquin (EQ) ses entails either activation to the ultimate carcinogen AFB]-8,9- provides protection against hepatocarcinogenesis. Chemoprotection by epoxide or, alternatively, deactivation to the less mutagenic hydrox- EQ is accompanied by the overexpression of enzymes which detoxify activated AFB¡.Aflatoxin-protein adduct formation takes place following ylated products aflatoxin M¡,aflatoxin Q,, and aflatoxin P,. The metabolism of AFB, to the dialdehydic form of AFB,-dihydrodiol. The activated metabolite, AFB,-8,9-epoxide, readily modifies DNA and dialdehyde can be detoxified by reduction to a dialcohol through the protein but its toxicity can be substantially reduced by conjugation catalytic actions of an enzyme present in the hepatic cytosol from rats fed with reduced GSH, a reaction catalyzed by GST. AFB,-8,9-epoxide is EQ-containing diets; this metabolite is essentially undetectable in reaction a short-lived electrophile and as an alternative to reacting with nucleic mixtures that use hepatic cytosol from rats fed control diets. The enzyme acids or GSH it can hydrolyze either spontaneously or via epoxide responsible for catalyzing the formation of dihydroxy-aflatoxin I!, has hydrolase to form the 8,9-dihydrodiol. In turn, the dihydrodiol under been purified from the livers of rats fed on diets supplemented with EQ. goes opening of both of the furan rings to yield a dialdehydic pheno- It is a soluble monomeric protein with an approximate Mr of 36,600. late ion (8) which forms Schiff bases with primary amine groups in Besides its activity toward AHÕ, this enzyme also catalyzes the reduction of the model substrate 4-nitrobenzaldehyde. Amino acid sequencing of proteins. Evidence has been obtained indicating that the majority of cyanogen bromide-derived peptides obtained from this reductase indi the binding of AFB, to serum albumin, and probably to other proteins, results from initial reaction between AFB,-dhd and primary amine cated that it has not been characterized hitherto, at least not at a molec ular level. Therefore, this inducible enzyme has been designated aflatoxin groups (9). The relative production of AFB,-dhd by microsomes from K,-aldehyde reductase (AFB¡-AR). The livers of adult rats administered various animal species parallels the in vivo susceptibilities to acute dietary EQ contain at least 15-fold greater levels of AFB,-AR than the AFB, toxicity which supports an important role for this metabolite in livers from rats fed control diets. Aflatoxin BrAR was also found to be the acutely toxic response to AFB, (10). Recently, we have isolated an present in increased amounts in livers bearing preneoplastic nodules and aflatoxin metabolite which we have characterized as AFB,-dialcohol in rat hepatoma, both of which are known to express increased resistance (11), formed by the reduction of the dialdehydic form of AFB,-8,9- to AFB|. Kidney contains high constitutive levels of VI I(,-AK and the dihydrodiol (see Fig. 1). Although previously nothing was known administration of EQ increases its concentration in renal cytosol about 3-fold. Although AFB,-AR is present in trace amounts in rat lung it was about the enzyme responsible for catalyzing this reaction, it is rea not detected in brain and in neither tissue was it found to be induced by sonable to suppose that it is mediated by AR. EQ. Evidence suggests that AFB,-AR is a previously unrecognized enzyme The consequences of exposure to AFB, depend primarily on the that could provide protection against the cytotoxic effects of aflatoxin K, relative hepatic levels of enzymes responsible for its activation and its resulting from the formation of protein adducts. The relative importance deactivation. In the rat the 8,9-epoxidation reaction is catalysed by of AFBi-AR and the glutathione-S-transferase Yc2 subunit in conferring CYP2C11,3 a male-specific cytochrome P-450, while deactivation of resistance to aflatoxin B, is discussed. AFB,-8,9-epoxide and the dialdehydic phenolate ion is catalyzed by GST isoenzymes containing Yc subunits (12) and the putative alde INTRODUCTION hyde reductase, respectively. Fischer 344 rats are highly sensitive to AFB i2 is a potent hepatocarcinogen that is produced by the mould AFB, but can resist its carcinogenic effects when fed diets containing AsperigiHiisflaviis. It is widely encountered as a contaminant of cereal ethoxyquin or other antioxidants (13, 14). The protection afforded by crops and other foodstuffs in regions of the world with high humidity phenolic antioxidants is a consequence of their ability to increase the (1). The carcinogenicity of AFB, has been demonstrated in various efficiency of the various detoxification pathways through induction of the enzymes involved (12, 15-18). GST-mediated resistance to AFB, species such as rat, turkey, duck, trout, and primates (2). Evidence suggests that AFB, is an important human hepatocarcinogen; not only has been studied by a number of research groups but the role of AR does an association exist between the level of exposure to AFB, and as a resistance mechanism has not received attention. the incidence of liver cancer (3, 4), but also recent data suggest that During the present study we describe the purification and charac this mycotoxin produces hepatocellular carcinoma through specific terization of a novel AR which metabolizes AFB,. This reductase is mutations in the p53 tumor suppressor gene at codon 249 (5-7). induced by dietary ethoxyquin and its expression is also increased in rat livers bearing preneoplastic nodules as well as in rat hepatoma. Received 2/1/93; accepted 6/10/93. The costs of publication of this article were defrayed in part by the payment of page MATERIALS AND METHODS charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1J. D. H. gratefully acknowledges the financial support of the Medical Research The chemicals used were from Sigma Chemical Co. (Poole, Dorset, United Council (Project Grant G9200034SA) as well as the Scottish Office Home and Health Kingdom) and BDH Merck (Thornliebank, Glasgow, Scotland, United Department (K/CSO/2/20/3/7). Kingdom). 2 The abbreviations used are: AFBi, aflatoxin BI; AR, aldehyde reductase; HPLC, high Male Fischer 344 rats were used throughout this investigation. All rats performance liquid chromatography; SDS, sodium dodecyl sulfate; PAGE, polyacrylam- (including the control animals) were fed on powdered MRC41B rat diet which ide gel electrophoresis; dhd, dialdehyde; EO, ethoxyquin; GST, glutathione S-transferase; GSH, glutathione; FPLC, fast protein liquid chromatography; DEAE-cellulose, dieth- ylaminoethyl-cellulose; CM-cellulose, carboxymethyl-cellulose. 3 G. E. Neal, D. J. Judah, and C. R. Wolf, unpublished results. 3887 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1993 American Association for Cancer Research. METABOLISM OF AFLATOX1N B, BY AN ALDEHYDE REDUCTASE AFB,-8,9-dihydrodiol dialdehydic phenolate proposed dialcohol structure "OCH, OCHj Fig. 1. Reduction of the dialdehyde form of AFB,-dihydrodiol. contained 2% arachis oil. Ethoxyquin-inducible enzymes were studied in rats cent metabolite was observed in AFB,-containing reaction mixtures (150-175 g) that had received 0.5% EQ for the 5 days immediately before examined by HPLC. This assay demonstrated that feeding rats on a sacrifice (16). Hepatic preneoplastic nodules or hepatoma were obtained from diet supplemented with 0.5% EQ results in a 5-fold increased capacity the livers of rats which had been fed MRC41B rat diet fortified with 2% arachis of hepatic cytosol to produce the AFB,-glutathione conjugate and an oil that contained AFB, (2 ppm.) for approximately 1 year (19); these animals increase of at least 15-fold in the ability of liver cytosol to form the were fed on the control diet for a month prior to sacrifice. Two rats (of about 75 weeks), that were fed on Ihe control diet but had received between 12 and unidentified AFB, product. While the role of GST in protection 19 weeks of age four i.p. injections (each 175 /ng) of AFB,, were also included against AFB, is well recognized, the substantially increased ability of in the study. the liver to produce another AFB, metabolite following the adminis The HPLC method used to analyze AFB, metabolites has been described tration of dietary EQ suggested the possible presence of another elsewhere (12, 20). Aldehyde reducíaseactivity was determined at 30°Cusing resistance mechanism. We have subsequently identified the novel 0.67 niM 4-nitrobenzaldehyde and 0.5 fXMNADPH in 100 ITIMsodium phos AFB, metabolite as a dialcohol and propose that an aldehyde reduc phate buffer (pH 7.0); the reaction was monitored at 340 nm. Glutathione íaseis responsible for its formation (11). The resolution of AFB,- 5-transferase and glutathione peroxidase activities were measured on a cen dialcohol from AFB-dhd [obtained as the Tris-AFB-dhd compound trifugal analyzer as described previously (12). Protein concentrations were (10)] by HPLC is shown in Fig. 2. determined by the method of Bradford (21), or in the assays of the hydroxya- Purification of the aldehyde reducíasewithactivity towards AFB, patite fraction (Fig.