Resistance to Aflatoxin Bj Is Associated with the Expression of A

[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 Â¡T.D. H.Â¡;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 AFBÂ¡.Aflatoxin-protein adduct formation takes place following ylated products aflatoxin MÂ¡,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 AHÃ•, 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 (AFBÂ¡-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 reducÃaseactivity was determined at 30Â°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 Ãaseis 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 reducÃasewithactivity towards AFB, patite fraction (Fig. 2) the bicinchoninic acid method (Sigma) was used. (AFB,-AR) was undertaken lo allow Ihis detoxificalion step to be The various chromatography steps used for the purification of AFB,-AR are similar to those used to isolate GST (12). With the exception of FPLC on the better understood and lo demonstrate its independence from other Waters Protein-PAK Glass SP-8HR column, which was performed at 20Â°C,all resistance mechanisms. AR was isolated from the livers of rats fed an chromatography and dialysis steps were carried out at 4Â°C.SDS/PAGE was EQ-containing diet using, consecutively, DEAE-cellulose, CM-cellu- undertaken in 11% resolving gels as described by Laemmli (22). Laser des- lose, hydroxyapatite, and chromatography on a Protein-PAK Glass orption mass analysis of AFB,-AR was performed on a Finnigan Mat (Hemel SP-8HR column (Table 1). The hepatic 10,000 g supernatant was Hempstead, Hertfordshire, United Kingdom) Lasermat instrument according to charged immediately onto DEAE-cellulose, equilibrated with 20 ITIM the manufacturer's instructions. Tris/HCl (pH 7.5). The material that was not retarded by DEAE- Portions of purified protein (approximately 100 fig in 2 ml 10 mm sodium phosphate buffer; pH 7) were emulsified with an equal volume of Freund's cellulose was collected and dialyzed against 10 mw sodium phosphate (pH 6.7) before being applied to a column of CM-cellulose. Although complete adjuvant and injected s.c. at 8 separate sites on the back of female this column was found to achieve a 20-fold purification of AFB,-AR New Zealand White rabbits. Three rabbits were used for each of the immu- nogens (i.e., rat AFB,-aldehyde reducÃase, rat -rr-class GST Yf, and mouse activily, the fractions which were responsible for catalyzing the for a-class GST Yc). After 6 weeks each rabbit was reinoculated with 100 jug of mation of the hydroxylated AFB, metabolite also contained substan the original Â¡mmunogenin incomplete Freund's adjuvant and this was followed tial levels of GST activity towards AFB,-8,9-epoxide (Fig. 3). The by a final inoculation 2 weeks later, again with 100 /xg of immunogen in contaminating GST was resolved from AFB,-AR by chromatography incomplete Freund's adjuvant. After a further 2 weeks the animals were killed and bled. The scrum obtained was stored at -20Â°C, in the presence of 0.1% (w/v) sodium azide until required. Western blotting was performed as de 1.0 scribed previously (23) and the following antiserum dilutions were used: AFB,-aldehyde reducÃase, 1/10,000; GST Yc, 1/5,000; GST Yf, 1/500. It 0> 0.8 should be noted that one of the antisera obtained against murine GST Yc was U found to cross-react with all the major rat a-class GST (i.e.. Yd,, Ya2, Yc,, Yc2) ffi while another antiserum, raised against the same immunogen, was specific for 'Su 0.6 rat GST Yc2 (these differences arc apparent in Figs. 6 and 7). O Amino acid sequencing was carried out on an Applied Biosystems (War- rington, Cheshire, United Kingdom) 477A instrument with a 120A on-line 0> 0.4 phenyllhiohydantoin analyser (24). The sequences obtained were compared in the Swissprot and Genbank data bases using the FASTA and TFASTA pro O grams, respectively (25). 0.2

RESULTS Enzyme Purification. In an earlierstudywe reportedthe isolation O 2 4 6 8 10 of the GST Yc2 subunit from the livers of rats administered dietary Retention time (min) ethoxyquin (12). The purification of GST Ya,Yc2 and Yc,Yc2 was Fig. 2. Separation of AFB,-dialcohol and AFB,-dhd-Tris by HPLC. AFB, (c) metab monitored using an HPLC-based assay which allowed the simulta olized in the presence of Tris buffer by 5 /LI!hydroxyapatitc-purificd aldehyde reducÃase (19.5 fxg of protein from peak fraction; see Fig. 4), forming AFB]-diaIcohol (a) and neous analysis of several AFB, metabolites. While not documented in AFB|-dhd-Tris (b). Incubation conditions and HPLC analyses were performed as previ the earlier paper describing GST Yc2, an unidentified highly fluores ously reported (10). K' of AFB,-dhd and AFB.-dhd-Tris are essentially similar. 38X8

Table 1 Purification of the AFBÂ¡-metabolizing aldehyde redactase Characterization of Aflatoxin lÃ¬,-aldehyde ReducÃase. Calibra Enzyme purification was undertaken from approximately 170 g of liver from 8-week- old Fischer 344 rats which had been fed an ethoxyquin-containing diet (0.5%) for the 5 tion proteins which were included in the SDS/PAGE analysis shown days before being sacrificed. The percentage yield of the different activities during the in Fig. 5 indicate that AFB,-AR comprises subunits with an approx purification are shown in parentheses imate Afr of 40,000. AFB,-AR was estimated to have an Mr of 36,622 activity by laser desorption mass spectrometry. The elution position of the protein)AFB,-(CHO)2Â°0.018(100)(nmol/min/mg of protein purified enzyme from a Waters Protein PAK Glass 200SW FPLC stepRatPurification (mg)18590 filtration column indicated that AFB,-AR is a monomeric protein. liver 10 000 g supernatant (100) In addition to its ability to metabolize aflatoxin, purified AFBrAR DEAE-cellulose flowthrough 6588 0.069 (135) 54 (29) was found to exhibit activity towards the model substrate 4-nitroben CM-cellulose 238 1.45 (103) 565 (11) Hydroxyapatite 54 2.31 (37) 1973 (8.8) zaldehyde (Table 1). By contrast, the enzyme exhibited little capacity Protein-PAK Glass SP-8HRTotal 15Specific 8.3 (37)NBA651970(2.4) to reduce either glucuronic acid or menadione. AFBi-(CHO)2, aflatoxin B, dialdehyde phenolate ion; NBA, 4-nitrobenzaldehyde. It was found that rat AFBrAR is not directly amenable to amino acid sequencing and it is therefore concluded that it possesses a blocked NH2 terminus. However, following cleavage with CNBr the on columns of hydroxyapatite (Fig. 4). The final purification step, largest fragment which was recovered from reverse-phase HPLC was which involved FPLC on a Waters Protein-PAK SP-8HR cation ex subjected to Edman degradation and the first 10 cycles gave the changer, resulted in the removal of small amounts of additional pro tein which contaminated the AFB,-AR preparation. The major protein sequence SSLEQLEQNL. This decapeptide sequence was not found peak eluted from SP-8HR was found to migrate as a single eleclro- in any of the proteins contained in either the Swissprot or the Genbank phoretic band when subjected to SDS/PAGE, suggesting that the data bases. Most significantly, this peptide was not found in any of those members of the aldo-keto reducÃasesuperfamily that have been preparation was homogeneous (Fig. 5). The purity of the enzyme preparation was also supported by the fact that when examined by cloned to date, i.e., aldehyde reducÃase(26), aldose reducÃase(26), reverse-phase HPLC it eluted as a single symmetrical peak from a chlordecone reducÃase(27), 3a-hydroxysteroid dehydrogenase (28- p,-Bondapak C-18 column which was developed with a 40-70% ac- 30), proslaglandin F synlhase (31) and p-cryslallin (32). Furthermore, etonitrile gradient. Ihe peplide was noi presenl in carbonyl reducÃase,a member of Ihe

Ã• i

f u.

> 1 Fig. 3. Elution of AFB|-aldehyde reducÃaseac ce tivity from CM-cellulose. The hepatic extract from ethoxyquin-treated rats was charged onto DEAE- cellulose as described in the text. The material which failed to adsorb to the DEAE-matrix was dialyzed against 5 changes, each of 4 liters, of 10 rriMsodium phosphate/1 mM dithiothreitol (pH 6.7) before being applied to a 3.2- x 82-cm column of CM-cellulose. This column, which was eluted at 35.5 ml/h, was developed with a 0-120 mM gradi ent of NaCI (â€¢)formed in the same 10 mM sodium phosphate buffer. Fractions of 11.8 ml were col .,150 lected and glutathione 5-transferase activities to ward both l-chloro-2,4-dinitrobenzene (A) and AFBi-8,9-epoxide (A) were measured. ReducÃase activity with the aflatoxin B, dialdehydic phenolate ion (â€¢)was determined and the fractions with high est activity were combined (horizontal bar). - 100_

- 50 Ã•

90 110 Fraction no. 3889

Fig. 4. Resolution of AFB,-AR from GST by hydroxyapatite chromalography. The fractions from CM-cellulose which contained the reducÃase activity towards the dialdehyde metabolite of AFB| were combined (see Fig. 3) and applied directly to a 1.6 x 23 cm column of hydroxyapatite. The col umn was eluted at 16 ml/h with 10 rriM sodium phosphate/1 mM dithiothreitol (pH 6.7) and devel oped with a 10-250 mM disodium phosphate gra dient; the gradient was followed by measuring the sodium concentration (â€¢)in the eluent. Fractions of 5.3 ml were collected, and the absorbancc at 280 nm (O), GST activity toward l-chloro-2,4-dini- trobenzenc (A). GST activity toward AFB,-8,9- 300 epoxide (A), and reducÃaseactivity with the aflatoxin B] dialdehyde phenolatc ion (â€¢)were mea sured. Horizontal bar, fractions which were com bined.

200 i

too

short-chain alcohol dehydrogenase family (33). It is therefore appar male rats, livers bearing preneoplastic nodules, and a resected afla- ent that AFB|-AR has not, to date, been characterized at a molecular toxin B |-induced hepatoma from a 1-year-old male rat. level and it is possible that it represents an addition to the family of Both Western blotting (Fig. 6) and enzyme assay (Table 2) showed aldo-keto reductases. low levels of AFB,-AR in fetal liver and in liver from 9-week-old Regulation of AFB|-AR in the Liver. Hepatic expression of male rats fed a normal diet. Comparison between the levels of AFB,-AR was studied to determine whether the levels of this enzyme AFB i-AR and GST Yc^ in fetal liver is particularly interesting. Both coincide with instances of relative sensitivity and resistance to AFB,. the enzyme assay and blots show significant amounts of GST Yci in Examples of tissues sensitive to AFB, were provided by liver speci fetal liver cytosol whereas, by contrast, AFB,-AR is detected only in mens from fetal rats and 9-week-old male rats while examples of low amounts in the same samples. Since fetal rat is sensitive to AFB, AFB i resistance were provided by livers from EQ-treated 9-week-old (34-36) our data suggest that resistance to this mycotoxin may require

Table 2 Lc\vls ofAFB/-AK and GST activities in hepatic samples from Fischer 344 nils Cytosols (100 (X)O-gsupernatants) were prepared at 4Â°Cfrom various liver samples. Enzyme activities were determined as described in "Materials and Methods." The results represent means Â±SD for four determinations and are expressed as nmol/min/mg. SDS/PAGE and Western blotting of these samples are shown in Fig. 6 activitiesLiver Enzyme

reducÃase sampleControl ofrat9 AFB,-(CHOb0.004 AFB,-8.9-epoxide0.077 weeks AFB i Elhoxyquin 9 weeks Resis ant to AFB, 0.115 0.483 0.035 Fetal 17 days gestation Sensi ive to AFB, 0.002 Â±0.002 0.225 Â±0.013 Nodule-bearing 1 year Resis antanttoto AFB, 0.026 Â±0.001 0.220 Â±0.004 HepatomaAge 1 yearPhcnotype"Sensi Resisive to AFE,Aldehyde 0.093Â±0.001Â±0.010Â±0.008Glulathione-.V-transferase0.657Â±Â±0.035 " Comments on resistance phenotype are based on data from Refs. 2, 10, 16, 34, 35, 36, and 37. The hepatoma sample was obtained from 3 carcinomas. Nodule-bearing sample was liver containing multiple nodules identified histologically as preneoplastic and neoplastic, < 3 mm in diameter. 3890

1234 5678 9 10 â€¢¿â€¢*

(a) 123456789 10

'1â€” anti AFB,-AR

6 7 8 9 10 (b)

anti GST-Yc- ; Fig. 6. Pathophysiological control of AFB]-AR in rat liver. Cytosols were prepared from the livers of fetal rats (17 days of gestation), from the livers of male rats (9 weeks old) administered either control or ethoxyquin-containing diets, from the liver of a male rat (~1 year old) that contained AFB|-ÃŒnducedpreneoplastic nodules, and from resected hepatomas obtained from a rat (â€”¿1year old) that had been subjected to chronic exposure to AFB,. a. SDS/PAGE of the following samples: tracks 1, 7, and 10, 2 /Â¿gAFB,-AR; Fig. 5. Purification of AFB|-metabolizing aldehyde reducÃase.The enzyme-containing track 2, 30 /^.g of hepatic cytosol from rats fed control diet; track 3, 30 fig of hepatic fractions that were obtained from the hydroxyapatite column were combined (see Fig. 4) cytosol from rats fed an ethoxyquin-containing diet; track 4, 30 Â¿igof fetal rat liver and dialyzed against 10 HIMsodium phosphate/1 mM dithiothreitol (pH 6.4). Portions cytosol; track 5, 30 fig of cytosol from a preneoplastic nodule-bearing liver; track 6, 30 (5-ml) of the dialyzed material were injected onto a Protein-PAK Glass SP-8HR column Â¿igof cytosol from an individual hepatoma; track 8, Mr standards, phosphorylase ÃŸ(MT (1 x 10 cm) equilibrated with the phosphate buffer. Aldehyde reducÃasewas eluted at 60 97,400), bovine serum albumin (A/r, 66,200), ovalbumin (A/r, 45,000), carbonic anhydrase ml/h from this cation exchanger, which was controlled using a Waters Advanced (650E) (MT 31,000), soybean trypsin inhibitor (M, 21,500), and lysozyme (Mr 14,400); track 9, 2 Protein Purification System, by a 0-67 m\i NaCI gradient formed in the running buffer; /j-g GST YciYc2. /?, an immunoblot analysis of AFBrAR and GST Yc2 expression in the same samples as described in "Materials and Methods." In the immunoblot for GST Yc2, the gradient, which was formed in two stages (0-67 HIMNaCI and 67-200 mM NaCI), is shown by the simigli! lines. The elualc was monitored continuously at 280 nm and a the antiserum shows some cross-reactivity with the Ya subunits (lower arrow) as well as typical elution profile is shown in a. The material which eluted between 43 and 47 min with the Vf] subunit (upper arrow); the Yc2 subunit (larges! arrow) has an electrophoretic was collected and examined by SDS/PAGE. The gel in b shows an SDS/PAGE analysis mobility that is between that of Ya and Yc|. Also, in this blot, track I contains 2 fig GST of protein recovered from the various Chromatographie steps used to purify AFB[-AR YC|Yc2 as a positive control (tracks 2-10 were loaded as shown in panel a). (Lanes 4-8). The samples subjected to clectrophoresis are as follows: tracks 1 and 10, Mr standards, bovine scrum albumin (M, 66,200), ovalbumin (M, 45,000). trypsinogen (MT from DEAE-cellulose; track 6, fractions combined from CM-celluIose; track 7, GST- 24,000). ÃŸ-lactoglobulin(M( 17,000), lysozyme (MT 14,400); track 2, hepatic cytosol from depleted pool from hydroxyapatite; track 8, purified aldehyde reducÃasethat eluted from rats fed control diet; track 3, hepatic cytosol from rats fed an ethoxyquin-containing diet: the Protein-PAK Glass SP-8HR column between 43 and 47 min; track 9, purified GST track 4, 10,000 g liver supernatant from rats fed ethoxyquin; track 5, flowthrough material Yc,Yc2. 3891

elevated expression of both GST Yc2 and AFBrAR. Rat liver that 1 2 3 4 5 6 7 8 9 10 11 12 contained preneoplastic nodules was found to possess moderately increased levels of the reducÃase.The highest levels of AFB,-AR were observed in the resistant EQ-treated rats and the resected rat hepatoma, both of which express substantial amounts of GST Yc2 and are resistant to AFB, (37). Induction of AFB t-metabolizing Enzymes by Ethoxyquin. The expression of AFB,-AR and GST Yc2 was examined in brain, kidney, liver and lung from rats fed EQ-containing diet and control diet to determine (a) the constitutive levels of these enzymes in organs which have been reported to contain aldehyde reducÃaseactivity (38) and (h) the abilily of EQ to serve as an enzyme inducer in extrahcpatic tissue. The hepalic samples also included a nodule-bearing liver cytosol to allow comparison wilh dala in Fig. 6. Among the organs subjected to immunoblotling, the kidney was found to express the highest constilutive levels of AFB,-AR (Fig. 7; Table 3). Expression of renal AFB,-AR was increased approximately 3-fold by Irealmenl wilh EQ. In 10-week-old rals fed a control diet the hepatic levels of AFB,-AR were substanlially lower lhan the renal levels. However, following administration of EQ the renal and hepatic levels of AFB,-AR were found to be comparable. Only trace amounts of ihis enzyme were observed in lung cytosol and none was detected in brain. EQ failed to increase the levels of the reducÃase in either brain or lung. The GST a-class Yc2 subunit was found to be overexpressed in liver following treatment with EQ (Fig. 7; Table 3). Trace amounts of Yc2 were observed in renal cytosol from EQ-treated rats but it was not detecled in renal cytosol from rats fed on the control diet. Brain was found to contain low levels of GST Yc2 as was lung. Intereslingly, b Weslern blotling also showed that the GST Tr-class Yf subunit, like 1 2 3 4 5 6 7 8 9 10 11 12 AFB i-AR, is also overexpressed in liver and kidney from EQ-treated (Q) rats.

onti AFB|-AR DISCUSSION

Chemoprotection against AFB, has been advocated as a valuable therapeulic strategy to help reduce the incidence of liver cancer in those regions of the world where individuals are commonly exposed 1 2 3 5 6 7 8 9 10 11 12 to this mycotoxin. Although a large number of chemicals that inhibit (b) carcinogenesis have been identified, the resistance mechanisms in volved are not well understood. Phenolic antioxidants such as EQ, ant i GST butylated hydroxyanisole, and butylated hydroxytoluene represent an important group of chemoprotectors (39-41) which, when adminis tered to experimental animals, serve to increase the efficiency of carcinogen detoxification pathways. In previous studies (12, 16) our laboratories have shown that EQ induces cytochrome P-450 and GST 12 345678 9 10 11 12 isoenzymes involved in the deactivation of AFB,. The present article (c) describes a hitherto unrecognized detoxification route for AFB, that is increased at least 15-fold by EQ. This step, which is catalyzed by anti GST Yf AFB|-AR, involves reduction of the AFB, dialdehyde phenolate form of AFB,-dihydrodiol to a dialcohol compound (see Fig. 1). This Fig. 7. Constitutive expression of AFBrmetabolizing enzymes and their induction by hydroxylated AFB, metabolite is itself likely to serve as a substrate for ethoxyquin in various rat organs. Cytosols were obtained from liver, kidney, brain, and UDP-glucuronosyl transferase or sulphotransferase. lung of rats (10 weeks old) fed on either ethoxyquin-containing or control diets. For comparison, additional hepatic cytosols were prepared from two rats (~75 weeks old) Swenson et al. (42) have shown that following exposure of rats to which had been fed on a control diet but had received four i.p. injections of AFBi- A AFB,, binding of the carcinogen to protein accounts for more than further hepatic cylosol was included in this series that had been prepared from a nodule- bearing liver of a rat (~ 1 year old) following chronic feeding with an AFB|-contaminated 50% of the macromolecular-bound toxin in the liver. It is therefore diet. a. SDS/PAGE analysis of these samples which were applied as follows: track 1, 2 /ig reasonable to assume that conversion of AFB, to the dihydrodiol AFB|-AR; track 2, 30 fig ethoxyquin-treated rat liver cytosol; track 3, 30 /ig control rat represents a significant (if not major) metabolic route for this hepa- liver cytosol; tracks 4 and 5, 30 ng of rat liver cytosol from animals that had received four separate doses of AFBi; track 6, 30 /ig preneoplastic nodule-bearing rat liver; track 7, 30 tocarcinogen in the rat (8). The parallel between the relative abilities /ig ethoxyquin-treated kidney cytosol; track 8, 30 /ig control kidney cytosol: track 9, 30 of microsomes isolated from various species to catalyze the formation /ig ethoxyquin-treated brain cytosol; track 10. 30 /ig control brain cytosol; track 11. 30 /ig ethoxyquin-treated lung cytosol; track 12, 30 /ig control lung cytosol. b, immunoblot analyses of these samples using antibodies raised against AFBrAR, GST a-class Yc2 and GST TT-class Yf; in each of the immunoblotting experiments track 1 contained a 2-/ig displayed little cross-reactivity toward the other a-class GST subunits (see also Fig. ft). aliquot of the purified immunogen. In this experiment the antiserum against GST Yen Enzyme analyses of the cytosol samples examined are presented in Table 3. 3892

Table 3 Enzyme levels in liver bearing preneoplastic nodules and in tissues from rats fed either control or ethaxyquin-containing diets The 100,000-g supernatant fraction (cytosol) was prepared from various tissues of Fischer 344 rats fed on control or ethoxyquin-containing diets. Hepatic cytosol was also obtained from two separate animals which had received four i.p. injections of aflatoxin Bt while being fed a control diet [i.e., liver specimens AFB, (l)andAFB| (2)] and from a nodule-bearing liver produced by chronic exposure to AFBt (PN). Enzyme activities were determined as described in the text and results are expressed as means Â±SD for four determinations. SDS/PAGE and Western blotting of these samples is shown in Fig. 7 Specific activities in cytosols"

Aldehyde reductase Glutathione-S-transferase Peroxidase

OrganLiverLiverLiverLiverLiverKidneyKidneyBrainBrainLungLungTreatmenl/specimenEthoxyquinControlAFB,(nmol/min/mg)0.092 (nmol/min/mg)56.9 Â±0.0020.0(12 Â±1.226.9 Â±0.0070.089 +936 Â±19716 Â±0.0000.005 Â±0.549.1 110.088Â±0.0 Â±246 +221387 (1)AFB, Â±0.0020.004 Â±1.246.3 Â±0.0070.083 +240 +301357+ (2)PNEthoxvquinControlEthoxyquinControlEthoxyquinControlAFB,-(CHO)2Â±0.0000.1 Â±1.196.4 Â±0.0050.534 Â±1135 14670 14Â±0.0040.045 Â±2.098.8 Â±0.0210.142 Â±1470 +9529 Â±0.0020.012 Â±3.379.2 Â±0.0050.069 Â±642 +7597 Â±0.0020.0000.0000.0000.000NBA+4.110.8 +0.0100.019 +224 Â±992 Â±0.810.8 Â±0.0010.016 Â±133 +288 Â±0.69.6 +0.0010.027 +327 +340 Â±0.86.7 Â±0.0020.017 Â±220 Â±731 Â±0.5AFB,-E(nmol/min/mg)0.431Â±0.001CDNB(nmol/min/mg)3280124719681710465242317748555114414g2171616515132952EA(nmol/min/mg)80Â±1CuOOH(units/g)65773714291383100156259995983342718172836531352653H,02(units/g)461+3 " AFB|-(CHO)2 aflatoxin B, dialdehyde phenolate ion: AFB,-E, aflatoxin B,-8,9-epoxide; NBA, 4-nitrobcnzaldehyde; CDNB, l-chloro-2,4-dinitrohenzene; EA, ethacrynic acid: CuOOH, eumene hydroperoxide. of AFB,-dhd and the in vivo susceptibilities of those species to AFB, ment and in hepatoma suggests that coordinated regulation of these acute toxicity suggests that protein binding plays a significant role in two enzymes may occur in adult liver. Regulation may be transcrip- AFB, toxicity (9). It is therefore probable that the reaction catalyzed tional, in which case it is possible that the 5' flanking regions of the by AFB,-AR represents a pivotal detoxification step which, by pre genes encoding these two proteins contain similar regulatory ele venting the reaction of AFB-dhd with lysyl residues in protein (8), ments. Unfortunately nothing is known about the molecular events limits the formation of AFB, protein adducts and inhibits the acutely involved in induction by EQ but it is reasonable to suppose that an toxic response resulting from this metabolic pathway. The increase in antioxidant responsive element, similar to that present in the rat GST expression of AFB,-AR produced by dietary consumption of EQ is Ya2 subunit gene and rat quinone reductase gene (46, 47), may be therefore likely to be of considerable importance in limiting the cy- present in the promotors of both GST Yc2 and AFB,-AR. However, totoxicity of AFB,. Prevention of the cytotoxic effects of AFB, will developmental control of these two genes is clearly distinct since GST also serve to inhibit carcinogenesis since the compensatory hyperpla- Yc2 is active in fetal liver (Fig. 6) while significantly lower levels of sia which occurs following cell necrosis makes it more probable that AFB,-AR are found in the same tissue. It is also intriguing that in the AFB,-DNA adducts are converted into mutations before DNA repair adult rat the levels of AFB,-AR closely mirror those of the GST can take place (41, 43, 44). It is therefore envisaged that protection TT-classYf subunit (Fig. 7). conferred by expression of AFB,-AR operates by inhibiting the pro Future studies are required to determine whether humans also pos motion and progression of carcinogenesis rather than its initiation. sess an enzyme related to the rat AFB,-AR. Human aldo-keto reduc During the present study we have shown that the sensitivity of rat tases have been reported but their ability to detoxify AFB, metabolites liver to AFB, closely reflects the levels of expression of AFB,-AR. has not been described. Clearly this avenue of research merits inves Low levels of AFB,-AR occur in fetal liver and tissue from 10- tigation since the rat enzyme appears to play a unique role in com week-old rats, both of which are sensitive to the mycotoxin, while bating the cytotoxic effects of aflatoxin. high levels of this enzyme are found in situations such as EQ treat ment, nodule-bearing livers, and hepatoma, where resistance to ACKNOWLEDGMENTS AFB, is observed. It will be interesting to establish whether AFB,-AR is expressed constitutively in species such as mouse and We thank Dr. E. M. Ellis for critically reading the manuscript and Dr. T. J. hamster (2), that are intrinsically resistant to aflatoxin Bj. In all spe Mantle for his valuable advice about AR enzyme assays. We are greatly cies, resistance to AFB, is likely to be multifactorial and it would be indebted to E. Ward and B. E. Craggs for excellent secretarial assistance. unwise to attribute it solely to AFB,-AR. Cytochrome P-450, GST Yc2, P-glycoprotein, and DNA repair enzymes also contribute to the REFERENCES resistant phenotype (37). 1. Groopman, J. D., Cain, L. G., and Kensler, T. W. Aflatoxin exposure in human The enzyme which catalyzes the reduction of the AFB, dialdehyde populations: measurements and relationship to cancer. CRC Crit. Rev. Toxicol, 19: 113-145, 1988. phenolate ion has been purified and shown to be a monomeric protein 2. Newberne, P. M., and Butler, W. H. Acute and chronic effects of aflatoxin on the liver of M, 36,600. This enzyme also exhibits catalytic activity towards of domestic and laboratory animals: a review. Cancer Res., 29: 236-250, 1969. 4-nitrobenzaldehyde indicating that it is an aldo-keto reductase. 3. Peers, F., Bosch, X., Kaldor, J., Linciseli, A., and Pluijmen, M. Aflatoxin exposure, hepatitis B virus infection and liver cancer in Swaziland. Int. J. Cancer, 39: 545-553, Amino acid sequencing experiments indicate that the aflatoxin-me- 1987. tabolizing enzyme is distinct from those members of the aldo-keto 4. Ross, R. K., Yuan, J-M., Yu, M. C, Wogan, G. N., Qian, G-S., Tu, J-T., Groopman. J. D., Gao, Y-T., and Henderson, B. E. Urinary aflatoxin biomarkers and risk of reductase superfamily the primary structures of which have been hepatocellular carcinoma. Lancet, 339: 943-946, 1992. reported previously (26-32) and therefore it has been designated 5. Hsu, I. C., Metcalf, R. A., Sun, T., Welsh, J. A., Wang, N. J., and Harris, C. C. AFB,-AR. The fact that the expression of AFB,-AR is increased by Mutational hotspot in the p53 gene in human hepatocellular carcinomas. Nature (Lond.) 350: 427-^28, 1991. EQ treatment is particularly noteworthy since aldo-keto reductases are 6. Bressac, B., Kew, M., Wands, J., and Ozturk, M. Selective G to T mutations of p53 seldom induced by xenobiotics (45). Among these enzymes, chlorde- gene in hepatocellular carcinoma from Southern Africa. Nature (Lond.) 550; 429- cone reductase has been shown to be inducible by its own substrate 431, 1991. 7. Ozturk, M., Bressac, B., Puisieux, A., Kew, M., Volkmann, M., Bozcall, S., Mura, J. (27) but it is not known whether it is also regulated by phenolic B., de la Monte, S., Carlson, R., Blum, H., Wands, J., Takahashi, H., von WeizsÃ¤cker, antioxidants. F., Galun, E., Kar, S., Carr, B. I., Schroder, C. H., Erkcn, E., Varinli, S., Rustgi, V. K., Prat, J., Toda, G., Koch, H. K., Liang, X. H., Tang, Z-Y, Shouval, Dâ€žLee,H-S., Vyas, The fact that the expression of GST Yc2, which protects against the G. N., and Sarosi, I. p53 mutation in hepatocellular carcinoma after aflatoxin expo genotoxic AFB,-8,9-epoxide, is also increased following EQ treat sure. Lancet, 338: 1356-1359, 1991. 3893

8. Neal, G. E. Influences of metabolism: aflatoxin metabolism and Â¡Ispossible relation superfamily: cDNAs and deduced amino acid sequences of human aldehyde and ships with disease. In: D. H. Watson (ed.). Natural Toxieants in Food. Progress and aldose reductases. J. Biol. Chem., 264: 9547-9551, 1989. Prospects., pp. 125-168. Chichester, England: Ellis Horwood, 1987. 27. Winters, C. J., Molowa, D. T.. and Guzclian. P. S. Isolation and characterization of 9. Sabbioni, G., and Wild, C. P. Identification of an aflatoxin G[-serum albumin adduci cloned cDNAs encoding human liver chlordecone reducÃase.Biochemistry. 2Â°:1080- and its relevance to the measurement of human exposure to aflatoxins. Carcinogenesis 1087, 1990. (Land.), 72:97-103, 1991. 28. Pawlowski, J. E., Huizinga, M.. and Penning, T. M. Cloning and sequencing of Ihe 10. Neal, G. E., Judah, D. J., Stirpe, F., and Patterson, D. S. P. The formation of cDNA for ral liver 3a-hydroxysteroid/dihydrodiol dehydrogenase. J. Biol. Chcm., 2,3-dihydroxy-2,3-dihydroaflatoxin Bt by the metabolism of aflatoxin BI by liver 266: 8820-8825, 1991. microsomes isolated from certain avian and mammalian species. Toxicol. Appi. 29. Stolz, A., Rahimi-Kiani. M.. AmÃ©is.D., Chan, E., Ronk. M., and Shively, J. E. Pharmacol., 5Â«:431-437, 1981. Molecular structure of rat hepatic 3a-hydroxysteroid dehydrogenase, a member of Ihe 11. Judah, D. J., Hayes, J. D., Yang, J-C. Lian, L-Y., Roberts, G. C. K., Farmer. P. B.. oxidoreductase gene family. J. Biol. Chem., 266: 15253-15257. 1991. Lamb. J. H., and Neal, G. E. A novel aldehyde reducÃasewith activity towards a 30. Cheng. K.-C., White. P. C.. and Qin. K-N. Molecular cloning and expression of rat metabolite of aflatoxin B, is expressed in rat liver during Carcinogenesis and follow liver 3a-hydroxysteroid dehydrogenase. Mol. Endocrinol., 5: 823â€”828,1991. ing Ihe administration of an anti-oxidant. Biochem. J., 292: 13â€”18,1993. 31. Watanabe, K., Fujii, Y, Nakayama, K., Ohkubo, H.. Kuramitsu, S., Kagamiyama. H., 12. Hayes, J. D., Judah, D. J., McLellan, L. I.. Kerr, L. A., Peacock, S. D., and Neal, G. Nakanishi, S., and Hayaishi, O. Structural similarity of bovine lung prostaglandin F E. Ethoxyquin-induced resistance to aflatoxin B, in the ral is associated with the synthase to lens p-crystallin of the European common frog. Proc. Nati. Acad. Sci. expression of a novel a-class glulathione .V-transferase subunit. Yc2. which possesses USA, 85: 11-15, 1988. high catalytic activity for aflatoxin B,-8,9-epoxide. Biochem. J., 279: 385-398, 1991. 32. Carper, D., Nishimura, C., Shinohara, T. Dietzchold, B., Wistow, G., Craft, C., Kador, 13. Cabrai, J. R. P., and Neal, G. E. The inhibitory effects of ethoxyquin on the carci P., and Kinoshita. J. H. Aldose reducÃaseand p-crystallin belong to the same protein nogenic action of aflatoxin BI in rats. Cancer Lett., 79: 125-132, 1983. superfamily as aldehyde reducÃase.FEES Lett., 220: 209-213, 1987. 14. Williams, G. M., Tanaka, T., and Maeura, Y. Dose-dependent inhibition of aflatoxin 33. Wermuth, B., Bohren. K. M., Heincmann, G., von Wartburg, J-P.. and Gabbay, K. H. B, Â¡nduced-hcpatocarcinogenesis by the phenolic antioxidants butylated hydroxytoluene and butylated hydroxyanisole. Carcinogenesis (Lond.), 7: 1043â€”1050,1986. Human carbonyl reducÃase.Nuclcotide sequence analysis of a cDNA and amino acid sequence of the encoded protein. J. Biol. Chem., 263: 16185-16188, 1988. 15. Kensler, T. W., Egner, P. A., Davidson, N. E., Roebuck, B. D., Pikul, A., and Groopman, J. D. Modulation of aflatoxin metabolism, aflatoxin Â¿V^guanineformation 34. Grice, H. C.. Moodie. C. A., and Smith, D. C. The carcinogenic potential of aflatoxin or its metabolites in rats from dams fed aflatoxin pre- and post-partum. Cancer Res., and hepatic tumorigenesis in rats fed elhoxyquin: role of induction of glutathione 33: 262-268, 1973. S-transferase. Cancer Res., 46: 3924-3931, 1986. 35. Goerttler, K.. Lohrke, H., Schweizer, H-J., and Hesse, B. Effects of aflatoxin B, on 16. Mandel, H. G., Manson. M. M.. Judah. D. J.. Simpson, J. L., Green, J. A., Forrester, pregnant inbred Sprague-Dawlcy rats and their FI generation. A contribution to L. M., Wolf, C. R., and Neal, G. E. Metabolic basis for the protective effect of the transplacental Carcinogenesis. J. Nati. Cancer Inst-, 64: 1349-1354. 1980. antioxidant ethoxyquin on aflatoxin B, hepatocarcinogenesis in the rat. Cancer Res., 47: 5218-5223, 1987. 36. Geissler, F., and Faustman, E. M. Developmental toxicity of aflatoxin B, in Ihe rodent embryo in \itro: contribution of exogenous biotransformation systems to toxicity. 17. Monroe. D. H.. and Eaton. D. L. Comparative effects of butylated hydroxyanisole on Teratology, 37: 101-111. 1988. hepatic in vivo DNA binding and in virm biotransformation of aflatoxin BI in the rat and mouse. Toxicol. Appi. Pharmacol.. 90: 401-109. 1987. 37. Hayes, J. D.. Judah, D. J., McLellan. L. L, and Neal, G. E. Contribution of the 18. Jhee, E-C., Ho, L. L., and Lotlikar, P. D. Effect of butylated hydroxyanisole pretreat glutathione .V-transferases to Ihe mechanisms of resistance to aflatoxin B,. Pharmacol. ment on in vitro hepatic aflatoxin B,-DNA binding and aflatoxin B,-glutathione Ther., 50: 443-472, 1991. conjugation in rats. Cancer Res., 48: 2688-2692, 1988. 38. Von Wartburg, J-P, and Wermuth, B. Aldehyde reducÃase,Â¡n:W.B. Jakoby (ed.). Enzymatic Basis of Detoxication. Vol. 1. pp. 249-260. New York: Academic Press, 19. Neal, G. E., Nielsch. U., Judah, D. J.. and Hulberl, P. B. Conjugation of model substrates or microsomally-activated aflatoxin BI with reduced glutathione, catalysed 1981. 39. Benson, A. M.. Batzinger. R. P.. Ou, S-Y. L.. Bueding, E., Cha. Y-N., and Talalay, P. by cytosolic glutathione 5-transferases in livers of rats, mice and guinea pigs. Bio Elevation of hepatic glutathione -S'-transferase activities and protection against mu- chem. Pharmacol., .?6: 4269-1276, 1987. 20. Moss, E. J., Judah, D. J., Przbylski, M., and Neal, G. E. Some mass-spectral and tagenic metabolites of benzo(Â«)pvrene by dietary antioxidants. Cancer Res., 38: 4486-1495, 1978. N.M.R. analytical studies of a glutathione conjugate of aflatoxin B,. Biochem. J., 270: 227-233, 1983. 40. Wattenberg. L. W. Inhibitors of chemical Carcinogenesis. Adv. Cancer Res.. 26: 21. Bradford, M. M. A rapid and sensitive method for the quantitation of microgram 197-226, 1978. quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 41. Wattenberg, L. W. Chemoprevention of cancer. Cancer Res.. 45: 1-8, 1985. 72: 248-254, 1976. 42. Swenson, D. H., Lin, J. K., Miller, E. C, and Miller, J. A. Aflatoxin B,-2,3-oxide as 22. I .miniili. U. K. Cleavage of structural proteins during the assembly of the head of a possible intermediate in Ihe covalenl binding of aflatoxins B, and 82 to rat liver bacteriophage T4. Nature (Lond.) 227: 680-685, 1970. DNA and ribosomal RNA in vivo. Cancer Res., 37: 172-181, 1977. 23. Hayes, J. D., and Mantle, T. J. Use of immuno-hlot techniques to discriminate 43. Ames, B. N., and Gold, L. S. Chemical Carcinogenesis: too many rodent carcinogens. between the glutathione .S'-translcrase Yf, Yk, Ya. Yn/Yb and Yc subunits and to study Proc. Nati. Acad. Sci. USA, 87: 7772-7776, 1990. their distribution in extra-hepatic tissues. Evidence for three immunochemicallv- 44. Cohen, S. M., and Ellwein, L. B. Cell proliferation in Carcinogenesis. Science (Wash distinct groups of mammalian glutathione .S'-transferase isoenzymes. Biochem. J.. ington DC), 249: 1007-1011, 1990. 233: 779-788, 1986. 45. Bachur, N. R. Cytoplasmic aldo-keto-reduclases: a class of drug metabolising en 24. Hayes, J. D., Kerr, L. A., and Cronshaw, A. D. Evidence that glutathione 5-trans zymes. Science (Washington DC), 193: 595-597. 1976. ferases B|B| and 6262 arc the products of separate genes and that their expression in 46. Rushmore. T. H.. Morton, M. R-. and Pickett. C. B. The antioxidant responsive human liver is subject to inter-individual variation. Molecular relationships between element. Activation by oxidative stress and identification of the DNA consensus the BI and 62 subunits and other a-class glutathione .V-transferases. Biochem. J., 264: sequence required for functional activity. J. Biol. Chem.. 266: 11632-11639. 1991. 437-445, 1989. 47. Favreau, L. V, and Pickett, C. B. Transcriptional regulation of the rat NAD(P)H: 25. Pearson, W. R.. and Lipman, D. J. Improved tools for biological sequence compari quinone reducÃasegene. Identification of regulatory elements controlling basal level son. Proc. Nati. Acad. Sci. USA, 85: 2444-2448, 1988. expression and inducible expression by planar aromatic compounds and phenolic 26. Bohren. K. M., Bullock. B.. Wermuth, B., and Gabbay. K. H. The aldo-keto reducÃase antioxidants. J. Biol. Chem., 266: 4556-4561, 1991.

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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1993 American Association for Cancer Research. Resistance to Aflatoxin B1 Is Associated with the Expression of a Novel Aldo-Keto Reductase Which Has Catalytic Activity towards a Cytotoxic Aldehyde-containing Metabolite of the Toxin

John D. Hayes, David J. Judah and Gordon E. Neal

Cancer Res 1993;53:3887-3894.

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