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[CANCER RESEARCH 39, 2971 -2977, August 1979]0008-5472/79/0039-0000$02.00

Elevation of Extrahepatic Glutathione S-Transferase and EpoxideHydratase Activities by 2(3)- tert-Butyl-4-hydroxyanisole'

Ann M. Benson, Voung-Nam Cha, Ernest Bueding, Henry S. Heine, and Paul Talalay2

Department of Pharmacology and Experimental Therapeutics, The Johns Hopkins University School of Medicine (A. M. B., E. B., P. T.j, and Department ofPathobiology, The Johns Hopkins University School of Hygiene and Public Health (Y-N. C., E. B., H. S. H.], Baltimore, Maryland 21205

ABSTRACT

Investigations in these and other laboratories have established that administration of 2(3)-tert-butyl-4-hydroxyanisole(BHA) to rodents: (a) protects a variety of target tissues againstthe production of tumors by a wide range of chemical carcinogens; (b) reduces the levels of mutagenic metabolites produced from benzo(a)pyrene and numerous therapeutic agentsin viva; (c) elevates the hepatic activities of microsomal epoxidehydratase and cytosol glutathione S-transferase; (d) alters theactivities of other hepatic enzymes and affects the levels ofsome hepatic catalytic constituents; and (e) increases theconcentrations of nonprotein thiol compounds in liver andseveral other tissues. Addition of BHA to the diet resulted inelevated glutathione S-transferase and epoxide hydratase activities in multiple extrahepatic tissues of female CD-i mice andmale Sprague-Dawley rats. In mice, glutathione S-transferasespecific activities in cytosol doubled in lung and stomach andincreased to 15 times control levels (with i ,2-dichloro-4-nitrobenzene as substrate) in small intestine. Microsomal epoxidehydratase specific activity toward styrene oxide doubled inmouse colon and stomach and increased to nearly 6 timescontrol levels in small intestine. Enhanced activities of theseenzymes were observed in several other mouse tissues and inrat small intestine, kidney, and lung. Dietary administration ofBHA to mice led to elevations of the concentrations of nonprotein sulfhydryl compounds in the mucosa of several digestivetract tissues as well as in the urinary bladder. Epoxide hydratase is a detoxifying enzyme which inactivates numerous mutagenic epoxides. However, elevation of epoxide hydrataseactivity may not necessarily exert a protective function in thecase of all arene oxides since at least some forms of thisenzyme catalyze an essential step in the formation of carcinogenic diol-epoxides of benzo(a)pyrene. The enhancement byBHA of extrahepatic glutathione S-transferase activities andnonprotein sulfhydryl levels, and possibly also the enhancement of extrahepatic epoxide hydratase activity, may be important factors in the mechanisms by which this antioxidantprotects against chemical carcinogenesis.

INTRODUCTION

Reactive forms of chemical carcinogens are, in general,electrophilic derivatives resulting from the metabolic activitiesof the tissues of the host on the parent procarcinogens (41).The ultimate fate of these reactive intermediates depends notonly upon their ability to reach and to react with critical sites

1 Supported by NIH Grants GM 1 6492, AM 07422, and CA 1 8251 . A prelim

mary account of this work has been published (8).2 To whom requests for reprints should be addressed, at Department of

Pharmacology and Experimental Therapeutics, The Johns Hopkins UniversitySchool of Medicine, 725 N. Wolfe Street, Baltimore, Md. 21205.

Received November 22, 1978; accepted April 24, 1979.

on informational macromolecules, but also upon the rates ofinactivation by competing enzymatic reactions. Our studies areconcerned with 2 major types of host enzymes which metabolize electrophilic species: the soluble glutathione S-transferases (EC 2.5.1 .18) (25) and the microsomal membrane-boundepoxide hydratase (EC 4.2.1 .63) (46). We have demonstrated(6, 7, 14, 15) that the specific activities of these enzymes aredramatically elevated in the livers of mice and to a lesserdegree in rats receiving diets supplemented with BHA3 underconditions where the activities of the hepatic mixed-functionoxidase activation enzymes are relatively unchanged (13, 14).Furthermore, these elevations in enzyme levels are accompanied by increases in acid-soluble sulfhydryl compounds in mice(4). These observations may be of special interest since theadministration of BHA and certain other antioxidants effectivelysuppresses the production of tumors in a variety of rodenttissues by chemical carcinogens of quite diverse structures(63). The latter phenomenon, which was first systematicallyinvestigated by Wattenberg (61 , 62), is potentially of considerable theoretical and practical importance since BHA is oneof a class of antioxidants widely used as food additives and isnoted for its low toxicity (11, 23).

The inference that elevations of glutathione S-transferaseand epoxide hydratase activities may play a role in thesuppression of carcinogenicity by antioxidants is furtherstrengthened by the recent findings in these laboratories (3, 4)that the administration of dietary BHA or 1,2-dihydro-6-ethoxy2,2,4-trimethylquinoline (ethoxyquin) to mice resulted in amarked reduction of the levels of mutagenic metabolites ofbenzo(a)pyrene (and of a number of heterocyclic therapeuticagents) in the urine and in the peritoneal cavities of micetreated with these antioxidants. Moreover, the direct additionof glutathione and purified rat liver glutathione S-transferase Aor B to the mutagenesis assay system of Ames (1) depressedthe mutagenic activity of urinary benzo(a)pyrene metabolites(6, 7). Other investigators have shown that purified epoxidehydratase preparations obliterated the mutagenicity of severalbenzo(a)pyrene oxides (16, 18, 33, 47—49,69—71)but thatcertain of the resulting dihydrodiols yielded highly potent mutagens upon further oxidative activation (69, 70).

The glutathione S-transferases are a family of highly versatilecatalytic and binding proteins which are present in the cytosolsof many tissues and species (25, 50). They are characterizedby overlapping yet distinct substrate specificities and havebeen purified from human and rat livers where they constituteunder basal conditions an extraordinarily high proportion (estimated variously as 3 to i 0%) of the cytosolic proteins andplay a major role in the detoxification of foreign compounds(25). The transferases enhance the nucleophilicity of glutathi

3 The abbreviation used is: BHA, 2(3)-tert-butyl-4-hydroxyanisole.

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A. M. Benson et a!.

one and, among other reactions, promote thioether formationfrom hydrophobic compounds as a first step in mercapturicacid formation (10, 25). These enzymes conjugate a variety ofarene oxides with glutathione (5, 10, 24, 26, 43—45)and undersome conditions may also play a sacrificial role by reactingcovalently with specifically bound alkylating agents such asmetabolites of 3-methylcholanthrene (35) and 4—dimethylaminoazobenzene (31). Some of the relationships between theglutathione S-transferases and chemical carcinogenesis havebeen the subject of a recent review (54).

The microsomal membrane-bound epoxide hydratases havebroad specificity in the cleavage of hydrophobic oxiranes (alkylepoxides or arene oxides) which are converted to the corresponding trans dihydrodiols (46). Homogeneous preparationsof epoxide hydratase have been obtained from rat liver microsomes (37, 48). In such preparations, the activity with styreneoxide, which is commonly used as a substrate for epoxidehydratase, appeared to reside in the same catalytic protein asthat acting on polycyclic hydrocarbon derivatives, including theepoxides of benzo(a)pyrene (37, 48). Levin et a!. (34) haveshown that the effects of modifiers of epoxide hydratase activitydepend on the substrate studied. Epoxide hydratase activitiesare elevated to a modest extent by administration of phenobarbital and hydrocarbon inducers to the host animals (26,46). Evidence for the existence of multiple forms of epoxide

hydratase in rat liver microsomes includes differential inductionof activities toward several substrates (12) as well as chromatographic resolution of immunologically distinct proteins withhydratase activity toward styrene oxide (21).

Although the liver is quantitatively the most important organfor the metabolic activation and detoxification of various chemicals in viva, chemical carcinogens are often highly selectivewith respect to their target organs and tissues. There is mounting evidence that tissue susceptibility to carcinogens may bein part controlled by the metabolic patterns of specific targettissues (2). BHA and other antioxidants have been found toinhibit chemical carcinogenesis in a variety of tissues (63), andthe influence of these compounds on the detoxification capacities of extrahepatic tissues may be relevant to the protectiveeffects. This report is concerned with the effects of dietaryadministration of BHA on the levels of glutathione 5-transferaseand microsomal epoxide hydratase activities and on the concentrations of nonprotein thiol compounds in a number ofextrahepatic tissues of rodents.

MATERIALS AND METHODS

Treatment of Animals. Seven-week-old female CD-i mice(Charles River Breeding Laboratories, Wilmington, Mass.) and7-week-old male Sprague-Dawley rats (Madison, Wis.) werehoused in stainless steel wire cages (4 mice or 1 rat per cage)and were fed ad ilbitum either a powdered diet of Purinalaboratory chow (Ralston-Purina Co., St. Louis, Mo.) or theBHA diet. The BHA (Sigma Chemical Co., St. Louis, Mo.) was

incorporated into the powdered laboratory chow at a finalconcentration of 7.5 g/kg by mixing in a Liquid-Solids Blender(Patterson-Kelley Co., East Stroudsburg, Pa.). Mice and ratswere sacrificed by cervical dislocation and decapitation, respectively.

Collection and Processingof Tissues for Enzyme Studies.

Mouse tissues were perfused in situ. The portal vein wassevered, and cold 0.i 5 M KCI containing 2 mM ethylenediaminetetraacetic acid (final pH 7.5) was injected through the leftventricle. When necessary, the livers were perfused again afterexcision. Rat and mouse small intestines and mouse colonswere slit longitudinally, and the mucosal surface was scrapedwith a blunt-edged spatula. The material so obtained wasshown by histological examination to consist largely of mucosaand submucosa and to contain rather small amounts of muscular elements. The forestomach and glandular stomach wereanalyzed as a single organ. All tissues were excised, chilled,weighed, and frozen in liquid nitrogen within minutes of sacrifice, and were stored at —80°until further processing.

Preparation of Cytosol Fractions and Microsomes. Allsteps were carried out at 0 to 4°.Tissues were homogenizedin 3 ml of 0.25 M sucrose per g of tissue, with the exception ofrat intestine (2 ml/g) and mouse thymuses and bladders (5 mug). Some of the smaller mouse tissues were pooled beforehomogenization; thus, there were 4 bladders or 2 uteri, thymuses, or spleens per homogenate. The procedure for thepreparation of microsomes was based on that of Schenkmanand Cinti (52). To the supernatant fluid obtained by centrifugation at 9,000 x g for 15 mm was added 0.2 volume of 0.1M CaCI2. Centrifugation at 20,000 x g for 1 5 mm or at 9,000

x g for 30 mm then yielded the microsomal pellet and a clearcytosol fraction. The microsomes were resuspended in a volume of 0.25 Msucrose equivalent to one-half the volume of thehomogenate from which they were derived and resedimentedby centrifugation for 15 mm at 37,000 x g. The washedmicrosomes were resuspended in 0.25 M sucrose at a proteinconcentration of 1 to 5 mg/mI, frozen in liquid nitrogen, andstored at —80°.

Determinations of Enzyme Activities. GlutathioneS-transferase activities of the cytosol fractions were determined with1-chloro-2,4-dinitrobenzene, 1,2-dichloro-4-nitrobenzene, andp-nitrobenzylchloride, all of which were obtained from EastmanOrganic Chemicals (Rochester, N. V.), and glutathione (SigmaChemical Co.) as substrates. The initial velocities of glutathioneconjugate formation were measured spectrophotometrically at25°according to the procedures of Habig et al. (22). Epoxidehydratase activity of microsomes was assayed at 37°with [8-14C]styreneoxide (New England Nuclear Corporation, BostonMass.) essentially according to the thin-layer chromatographymicromethod of Jerina et al. (27). Precise details of the assayprocedures have been given elsewhere (15). The glutathioneS-transferase and epoxide hydratase specific activities arebased upon the protein concentrations of cytosol fractions andmicrosome suspensions, respectively, determined by themethod of Lowry et a!. (36) with crystalline bovine serumalbumin as a standard.

Determination of the Concentrations of Acid-solubleThiolCompounds in Mouse Tissues. Tissues were excised andplaced on a glass plate over wet ice. Gastrointestinal tissueswere slit longitudinally. Residual food and fecal matter wereremoved, and the mucosa and submucosa (see above) wascollected by gentle scraping with a blunt-edged spatula. Tissues were homogenized in 5% trichloroacetic acid (20 ml/g oftissue). After centrifugation of the homogenates, unprecipitatedthiols were determined by the spectrophotometric procedureof Grassetti et al. (20) as previously described (4). The resultsare expressed in terms of @moIof thiol per g of wet tissue.

2972 CANCERRESEARCHVOL. 39

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Microsomal epoxide hydratase and cytosol glutathione S-transferase specific activities of tissues from mice fed control and BHAdietsThetissueswereobtainedfromfemaleCD-i micewhichhadreceivedthe powdereddietor the samediet containing0.75%BHAfor 14days.Glutathione

S-transterase specific activitIes in cytosolfractions(nmol/mln/mg)

Epoxide hydratase spacific activity In micro- 1-chloro-2,44inItro- 1,2'.dlchloro-4-nltro- p-nltrobenzylchlor

Tissue Diet N somes (nmol/mln/mg) benzene benzeneideUver

BHA 8 11.72 ±0,52a, b i 3840 ±417L@ 440 ±14b 982 ±3l@'Control8 1.06 ±0.05 1669 ±87 40.2 ±2.3 124 ±6

Kidney BHA 8 0.82 ±0.04C 856 ±44b 19.3 ±i.? 66.5 ±43dControl8 0.65 ±0.05 528 ±13 11.2 ±0.3 45.9 ±2.2

Lung BHA 8 2.57±0.12 i664±2i&'466±54bControl8 2.20 ±0.20 774 ±51 20.9 ±1.5

Stomach BHA 8 1.67 ±0,16b 1292 ±35b 32.3 ±l,2@ 72.6±Control8 0.83 ±0.06 788 ±38 14.7 ±1.2 42.9 ±2.5

Small intestine BHA 8 1.94 ±0,2? 2285 ±236k 65.5 ±7,?Control8 0.33 ±0.04 521 ±23 4.4 ±0.2

Colon BHA 4, 8' 1.66 ±0.13d@@ 17@' 13.3 ±08dControl4, 8 0.81 ±0.02 517 ±20 9.1 ±0.7

Uterus@ BHA 4 Not measurable 424 ±30 11.5 ±o.? 32.6±Control4 Not measura@ple 295 ±10 6.4 ±0.3 20.4 ±0.8

Thymus@ BHA 4 1.42 ±0.12 195 ±26Control4 0.55 ±0.12 175 ±10Spleen0

BHA 4 Not measurable 198 ±4Control4 Not measurable 176 ±8

Bladder― BHA 2 9.02, 7.83 7092, 7402 218, 231 408,450Control2 4.00, 9. 13 5356, 6014 171•I 94 337, 400

G!utathione S-Transferase and Epoxide Hydratase

in colon. Both enzymes increased in kidney, although thedegree of elevation of the glutathione S-transferase activitieswas more substantial (45 to 72%) than that of epoxide hydratase (26%). More selective effects on these enzyme activitieswere observed in 2 tissues. In lung, glutathione S-transferaseactivity doubled in response to the BHA treatment, whereasepoxide hydratase activity increased only 17%. Conversely, inthe thymus, epoxide hydratase activity increased to 258% ofcontrol levels, whereas glutathione S-transferase activitiesshowed no response to BHA. Not enough data were obtainedfor statistical evaluation of the effects of BHA on the levels ofthese enzyme activities in mouse bladder. However, it is apparent that even in untreated mice the specific activities ofepoxide hydratase and of the glutathione S-transferases areextremely high in bladder in comparison to the activities in theother tissues examined and are at least several times those inliver.

Rat tissues. Of the 6 extrahepatic rat tissues examined, 3exhibited elevations of enzyme specific activities in responseto dietary BHA administration (Table 2). The tissue responsewas qualitatively identical for epoxide hydratase and glutathione S-transferase activities. We previously reported that inresponse to BHA, rat liver epoxide hydratase activity increasedto 277% of control levels, and glutathione S-transferase activities rose to about 200% of control levels. The data on thesehepatic enzyme activities have been published elsewhere (7,I 5) but are included in Table 2 to facilitate comparison withthe values for other tissues. The increases in epoxide hydrataseand glutathione S-transferase specific activities in the smallintestine to 255 and I 60% of controls, respectively, approached those observed in liver, whereas more modest butstatistically significant increases were observed in kidney and

Table1

a Mean ±SE.b signrncanny different from controls (p < 0.001).C Significantly different from controls (p < 0.05).

d Signrncanny different from controls (p < 0.01).a Not significantly different from controls (p > 0.05).

I Colon cytosols from 8 IndIvidual mice (N —8) and microsomes from 4 groups of 2 mice (N —4) were examined.g Uteri, thymuses, and spleens were pooled In pairs.

h Four bladders were pooled for each homogenate, resulting In only 2 preparations for each group of mice. The IndivIdual experimental values are given.

2973AUGUST1979

RESULTS

Glutathione S-Transferase and Epoxide Hydratase Levelsin Extrahepatlc Tissues of Rodents Receiving BHA. Dietaryadministration of BHA to mice and rats resulted in significantelevations of the specific activities of microsomal epoxide hydratase and cytosol glutathione S-transferases in most of thetissues examined.

MouseTissus. Two groups of mice were examined.Eightmice received the BHA diet for I 4 days, and 8 mice receivedthe control diet. The specific activities of epoxide hydratase(with styrene oxide) in the microsomal fractions and of theglutathione S-transferases (with 1-chloro-2,4-dinitrobenzene,1,2-dichloro-4-nitrobenzene, and p-nitrobenzylchloride) in thecytosol fractions from 10 tissues of these mice are shown inTable 1. The effects of BHA on the hepatic enzyme levels weresimilar to those which we reported previously (6, 7, 14, 15); inthe BHA group, the epoxide hydratase specific activity was 11times as great as that in the control group, and glutathione Stransferase specific activities with the various substrates were8 to I I times those of the controls. Among the extrahepatictissues examined, the most dramatic response to BHA administration was observed in the small intestine. The microsomalepoxide hydratase specific activity was elevated to nearly 6times control levels, and increases in glutathione S-transferaseactivities to 4.4 times control levels with 1-chloro-2,4-dinitrobenzene and to 14.9 times control levels with 1,2-dichloro-4-nitrobenzene, were observed. Sizable increases in these enzyme activities were also found in other tissues of the digestivetract. BHA treatment led to doubling of epoxide hydratasespecific activities in stomach and colon; glutathione S-transferass activities increased 60 to 120% in stomach, but only 40%

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Microsomal epoxide hydratase and cytosol glutathione S-transferase specific activities of tissues from rats fed control and BHAdietsThe

tissues were obtained from male Sprague-Dawley rats which had received the powdered diet or the same diet containing 0.75% BHAfor8days. Microsomes and cytosols were prepared by the calcium precipitation procedure.Glutathione

S-transferase specific activities in cytosol fractions(nmol/Epoxidehydratase mm/mg)

specific activity in microsomes (nmol/min/ 1-chloro-2,4-dinitro- 1,2-dichloro-4-nitro

Tissue Diet N mg) benzene benzenep-nitrobenzylchlorideLivera

BHA 5 5.52 ±0.02―C@ 800 ±84b@@ 2•0b 346±Control6 1.99 ±0.10 918 ±18 46.0 ±1.5 193 ±5

Small intestine BHA 5 1.89 ±016b 427 ±48dControl6 0.74 ±0.04 267 ±15Kidney

BHA 5 1.20 ±003b 248 ± 2b 25.6 ±0.8°Control6 0.91 ±0.03 210 ± 4 20.0 ±0.9

Lung BHA 4 0.41 ±0.02° 117±Control6 0.29 ±0.02 101 ± 4

Testis BHA 5 1.12 ±O.O6@ 971 ±26' 25.7 ±2.0' 130 ±3'Control6 1.13 ±0.03 960 ±15 25.9 ±1.0 129 ±1

Brain BHA 5 0.21 ±0.01@ 176 ± 5' 9.2 ±0.3'Control6 0.22 ±0.03 170 ± 2 9.1 ±0.1

Spleen BHA 5 0.34 ±0.02' 55 ±3'Control6 0.27 ±0.02 53 ± 1

Effects of feeding a BHA diet on the acid-soluble sulfhydryl levelsinvariousmousetissuesThe

tissues were obtained from 8-week-old female CD-i micethathadbeen fed a powdered diet or the same diet containing 0.75%BHAfor

10 days.Sulfhydryl

levels (@@mol/gwettissues)Tissue

Control diet BHAdietStomach

3.2 ±0.1 (9)° 4.1 ±0.4(6)p=0.02Upper

half of small Intes- 7.8 ±0.3 (14) 18.6 ±0.9(12)tine(duodenum and je- p <0.001junum)Lower

half of small intes- 6.6 ±0.2 (14) 8.2 ±0.5 (11)tine(ileum) p <0.01Colon

3.4±0.2(11) 4.5±0.3(9)p<0.01Urinary

bladder 2.5 ±0.1 (11) 3.1 ±0.1(8)p<0.01a

Numbers in parentheses, number of samples examined from separate ani

A. M. Benson et al.

Table 2

a These data for liver have been published previously (7, 15).b Significantly different from controls, (p < 0.001).

C Mean ± SE.

d Significantly different from controls (p < 0.02).aSignificantlydifferentfromcontrols(p < 0.01).â€Not significantly different from controls (p > 0.2).

Table3 DISCUSSION

These studies are part of a continuing effort to elucidate thebiochemical mechanisms by which several antioxidants protectagainst chemical carcinogenesis.

The anticarcinogenic effects of antioxidants are not restricted to a single strain, sex, species, or route of administration (63). Administration (p.o.) of BHA or the structurally relatedantioxidant, 3,5-di-tert-butyl-4-hydroxytoluene, has beenshown to inhibit carcinogen-induced neoplasia in female ratsof the Sprague-Dawley and CD SPF strains (57, 6i ) and malerats of CD SPF, Fischer, and F344 strains (57, 66). Protectionhas been achieved by administering BHA p.o. to female HalICR mice (61 , 62), p.o. and i.p. to female A/HeJ mice (60—62),and topically to female CD-i mice (53). The use of female CDi mice in the present experiments has its origin in the extensiveexperience with this strain in the chemotherapy of schistosomiasis. The carcinogenicity of a chemotherapeutic nitrovinylfuran (SQ i 8,506; trans-5-amino-3-[2-(5-nitro-2-furyl)vinyl]-i ,2,4-oxadiazole) is markedly reduced in female CD-i micereceiving dietary BHA.4 Furthermore, administration of BHA tothese mice reduced or eliminated the mutagenic activitiesresulting from the metabolism of several antischistosomal campounds as well as of benzo(a)pyrene and other promutagens(4).

Further studies have disclosed that administration of BHA toCD-i mice led to increased activities and/or levels of a numberof hepatic enzymes and catalytic components directly or mdirectly concerned with the metabolism of carcinogens (7, i 3—i 5). Extension of these studies to male Sprague-Dawley ratsrevealed that enhancement of hepatic enzyme activities byBHA was not restricted to a single strain, sex, or species (7,i 5).

The effects of antioxidants administered in viva on the activities of enzymes, some of which are involved in the metabolismof carcinogens, have been the subject of a number of studies

4 H. Dunsford, P. M. Dolan, and E. Bueding, unpublished observations.

mals.

lung. No increase in these enzyme activities was observed inrat spleen, testis, or brain.

Concentrations of Acid-soluble Thiol Compounds in Tissues of Mice Receiving BHA. Examinationof the effects ofBHA on the concentrations of acid-soluble (nonprotein) thiol

compounds in mouse tissues (4) was extended to a number ofadditional extrahepatic tissues. Mice received either the controldiet or the BHA diet for 10 days, and tissues were collectedand assayed as described. As shown in Table 3, the mostdramatic response to BHA was observed in the mucosa of theupper half of the small intestine, where nonprotein thiol levelsin the BHA group were elevated to 238% of control levels. Thisincrease represented higher thiol levels in both the duodenaland the jejunal mucosa of the BHA group. More modest increases (by 24 to 32%) were observed in other digestivetissues and in the urinary bladder (Table 3), whereas theconcentrations of acid-soluble thiol compounds in skeletalmuscle, heart, spleen, and diaphragm were unaffected bydietary BHA (data not presented).

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Glutathione S-Transferase and Epoxide Hydratase

(1 1, 17, 23, 29, 32, 39, 53, 55, 56, 64). In particular, Lam andWattenberg (32) and Speier et a!. (55, 56) observed that p.o.administration of BHA to mice resulted in alterations of patternsof metabolites produced from benzo(a)pyrene by hepatic microsomes in vitro with concomitant decrease in the binding toDNA and that these effects were observable within a few hoursafter a single dose of BHA. We reported recently (7, 15) thatdietary administration of 2 antioxidants, BHA and ethoxyquin,to rodents resulted in dramatic elevations of hepatic glutathioneS-transferase and epoxide hydratase activities, suggesting thatsome of the anticarcinogenic effects of these antioxidants maybe attributable to enhanced capacity for enzymatic inactivationof reactive metabolites.

Although the liver is the principal site of tumor production byrelatively specific carcinogens, such as azo-dyes (41) andaflatoxins (67), extrahepatic tissues are in general more susceptible to the action of a wide variety of carcinogens (9).Since BHA and other antioxidants protect not only the liver butat least several other target tissues from the effects of numerous chemically dissimilar carcinogens (63), the ability of BHAto elevate glutathione 5-transferase and epoxide hydrataseactivities in a number of extrahepatic tissues suggests thatincreased enzymatic inactivation of carcinogens may be involved in the mechanism of protection by this antioxidant.

Lung, stomach, and colon are among the major target tissuesfor carcinogenesis in which protection by BHA or the structurally related antioxidant, 3,5-di-tert-butyl-4-hydroxytoluene, hasbeen demonstrated (63). Administration of BHA to mice led todoubling of epoxide hydratase specific activities in stomachand colon microsomes and of some of the glutathione 5-transferase specific activities in lung and stomach cytosols. Theseincreases in enzyme activities appear to represent significantenhancement of the ability of these tissues to inactivate a widerange of xenobiotics. The enhancement by BHA of glutathione5-transferase levels, as well as of the concentrations of nonprotein thiols (4), in mouse lung may be of particular interestsince administration of BHA protects this tissue against theneoplastic effects of several carcinogens, includingbenzo(a)pyrene and other polycyclic aromatic hydrocarbons,diethylnitrosamine, nitroquinoline N-oxide, uracil mustard, andurethan (60, 62).

The most impressive elevations of enzyme activities in response to dietary BHA were observed in mouse liver (7, 15)and small intestine. In general, greater increases in the levelsof these enzyme activities occurred in the mouse tissues thanin the rat tissues examined. However, the experimental animaisdiffered not only in species but in sex and in duration of BHAtreatment. It is apparent that dietary administration of BHAresults in elevation of glutathione S-transferase and epoxidehydratase activities in multiple tissues of both species and thatthis response occurs in both female and male rodents. Speieret al. (55) recently reported that administration of BHA by p.o.intubation 4 hr prior to each of 3 p.o. doses of benzo(a)pyrene(at 2-week intervals) yielded substantial protection against theformation of pulmonary adenomas in mice. Thus, it would be ofinterest to investigate the effects of intermittent doses of BHAon the levels of detoxification enzymes in the digestive tissues(since both BHA and the carcinogen were administered by p.o.intubation) as well as in liver and lung.

Although the activities of these enzymes generally lead tothe formation of innocuous metabolites, recent studies have

shown that epoxide hydratase catalyzes the formation ofbenzo(a)pyrene 7,8-dihydrodiol, a precursor of bay region diolepoxides which are believed to be ultimate carcinogenic formsof benzo(a)pyrene (28, 30). Similarly, the conversion of 1,2-dichloroethane to mutagenic metabolites was reported to bedependent upon its conjugation with glutathione (51 ). Styreneoxide and benzo(a)pyrene 4,5-oxide, both of which have beenshown to be mutagenic (1, 19, 38, 42, 48, 68) and may beweakly carcinogenic (58, 59, 69), are converted into nonmutagenic dihydrodiols and glutathione conjugates by epoxidehydratase and glutathione S-transferases, respectively (5, 24,26, 45, 48, 69, 70). Suggested explanations for the relativeineffectiveness of benzo(a)pyrene 4,5-oxide in initiating carcinogenesis include the possibility that this arene oxide may besubject to efficient enzymatic inactivation (65, 69). The widespread tissue distribution of epoxide hydratase and glutathioneS-transferase activities, as well as their enhancement by BHA,may be significant in this respect.

Glutathione is normally the most abundant thiol compound inmammalian cells (40). Thus, the observations that administration of BHA to mice leads to increases in nonprotein thiol levelsin liver, kidney, lung, and duodenum (4) as well as in themucosa of stomach, jejunum, ileum, and colon, and in theurinary bladder, suggest enhanced conjugational capacity ofthe glutathione 5-transferases as well as increased availabilityof noncritical nucleophiles for nonenzymatic inactivation ofreactive electrophilic species in these tissues. The elevation byBHA of the levels of glutathione 5-transferase and epoxidehydratase activities and of the concentrations of nonproteinthiol compounds, not only in liver but also in extrahepatictissues, is a further indication that enhancement of detoxification of carcinogens and their metabolites may constitute partof the biochemical mechanisms by which this antioxidant protects against chemical carcinogenesis.

ACKNOWLEDGMENTS

We wish to thank Patrick M. Dolan for valuable technical assistance.

REFERENCES

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2. Bakshi, K., Brusick, D., Bullock, L., and Bardin, C. W. Hormonal regulationof carcinogen metabolism in mouse kidney. In: H. H. Hiatt, J. D. Watson,and J. A. Winsten (eds.), Origins of Human Cancer, Book B, pp. 683—695.Cold Spring Harbor. N. Y.: Cold Spring Harbor Laboratory, 1977.

3. Batzinger. R. P., Ou, S.-Y. L., and Bueding, E. Tert-butyl-hydroxyanisoleand antimicrobial agents decrease levels of mutagenic metabolites. Fed.Proc., 37: 596, 1978.

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G!utathione S-Transferase and Epoxide Hydratase

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1979;39:2971-2977. Cancer Res   Ann M. Benson, Young-Nam Cha, Ernest Bueding, et al.   -Butyl-4-hydroxyanisole

tertEpoxide Hydratase Activities by 2(3)--Transferase andSElevation of Extrahepatic Glutathione

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