induction of glutathione transferases and nad(p)h:quinone

[CANCER RESEARCH 50, 7871-7875, December 15, 1990)

Induction of Glutathione Transferases and NAD(P)H:Quinone Reductase byFumarie Acid Derivatives in Rodent Cells and Tissues1

Sharon R. Spencer, Cynthia A. Wilczak, and Paul I alulay '

Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

ABSTRACT

Dimethyl fumarate and dimethyl malÃ©ateare potent inducers of cyto-solic NAD(P)H:(quinone acceptor) oxidoreductase (here designated uni-none reducÃase)activity in Hepa Iclc? murine hepatoma cells in culture,whereas fumarie and maleic acids are much less potent, in agreementwith the much greater reactivity of the esters as Michael reactionacceptors (P. Talalay, M. J. De Long, and H. J. Prochaska, Proc. Nati.Acad. Sci. USA, Â«5:8261-8265,1988). Dimethyl fumarate also inducedquinone reductase in mutants of the Hepa Iclc7 cell line that were eitherdefective in the Ah receptor or in cytochrome 1V45U activity, therebyestablishing that this compound is a monofunctional inducer (H. J.Prochaska and P. Talalay, Cancer Res., 48:4776-4782,1988). Additionof dimethyl fumarate to the diet of female CD-I mice and female Sprague-Dawley rats at 0.2-0.5% concentrations elevated cytosolic glutathionetransferases and quinone reductase activities in a variety of organs,whereas much higher concentrations of fumarie acid were only marginallyactive. The widespread induction of such detoxication enzymes by dimethyl fumarate suggests the potential value of this compound as aprotective agent against chemical carcinogenesis and other forms ofelectrophile toxicity. This proposal is supported by the finding that theconcentrations of dimethyl fumarate required to obtain substantial enzyme inductions were well tolerated by rodents. Furthermore, the parentfumarie acid has low chronic toxicity and is a naturally occurring metabolic intermediate that is already in the food chain as an additive, andfumarate salts and esters are used for therapeutic purposes in man.

INTRODUCTION

A variety of structurally dissimilar chemical agents block theneoplastic and toxic effects of chemical carcinogens, a processthat has been designated chemoprotection (1,2). Many chemo-protectors induce enzymes of xenobiotic metabolism in cells inculture and in the liver and peripheral organs of mice and rats(2). Such inducers are of two types (3): bifunctional and monofunctional. Bifunctional inducers elevate the activities of bothPhase 1 enzymes3 (such as cytochrome P,-450), which play a

central role in activating carcinogens, and Phase 2 enzymes[such as glutathione transferases (EC 2.5.1.18) andNAD(P)H:quinone oxidoreductase (EC 1.6.99.2)], which arelargely involved in detoxication reactions. In contrast, mono-functional inducers elevate the activities of Phase 2 enzymesselectively. Whereas the mechanism of cytochrome Pi-450 in-

Received7/31/90;accepted9/18/90.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1These studies were supported by Grant NIH PO1CA 44530 from the National

Cancer Institute, Department of Health and Human Services, and by SpecialInstitutional Grant SIG-3 from the American Cancer Society.

2To whom requests for reprints should be addressed, at Department of

Pharmacology and Molecular Sciences, The Johns Hopkins University School ofMedicine. 725 North Wolfe Street, Baltimore, MD 21205.

3The enzymes involved in the metabolism of xenobiotics have been classifiedinto two broad categories. Phase 1 enzymes (which include the cytochromes P-450) functionalize compounds by oxidation or reduction, whereas Phase 2 enzymes carry out the conjugations of functionalized compounds with endogenousligands (e.g., glucuronic acid, glutathione. and sulfate). Although quinone reductase does not promote a synthetic function, it is here classified as a Phase 2enzyme since it does not introduce new functional groups, is often inducedcoordinately with conjugation enzymes, and protects cells against the toxicitiesof quiÃ±onesor their precursors (4, 5).

duction by bifunctional inducers (polycyclic aromatics, azodyes, tetrachlorodibenzo-/Â»-dioxin) involves the binding of theseinducers to a complementary cytosolic Ah (Aryl Aydrocarbon)receptor (6-8), the inductive mechanism of Phase 2 enzymesby monofunctional inducers depends on the presence or acquisition by metabolism of electrophilic centers (9, 10). Thus,some monofunctional inducers are conventional Michael reaction acceptors, whereas others are subject to nucleophilic displacement or addition reactions. The electrophilic inductionsignal was identified by measuring QR4 in Hepa Iclc? murine

hepatoma cells in culture (9, 11, 12). With the help of thissystem several new classes of monofunctional inducers werecharacterized, including a,|8-unsaturated lactones and carbox-ylic acid derivatives, e.g., coumarins, fumarates, malÃ©ales,ac-rylates, cinnamates, and related compounds.

In developing chemoprotective enzyme inducers for use inman, attention must be accorded to three desirable characteristics of such agents: (a) capacity for monofunctional ratherthan bifunctional induction. This minimizes the potential complications of carcinogen activation through the induction ofcertain cytochromes P-450; (b) ability to raise Phase 2 enzymelevels in multiple tissues, thereby enhancing the potential forprotecting a variety of organs; (c) low toxicity, e.g., presence inthe food chain or in living matter, thereby minimizing the needfor extensive, long-term toxicity testing prior to clinical trials.Based on these desirable characteristics, we have selected fromamong the many known inducers of Phase 2 enzymes (9) afumarie acid derivative, DIMEFU, for detailed study in cellculture and in rodents. Fumarate (the parent compound) is oneof the few monofunctional inducers that is an endogenousconstituent and an essential metabolic intermediate of animaltissues. Fumarate has low toxicity, is used widely as a foodadditive, and is on the "Generally Recognized As Safe," list.

Chronic toxicological studies in several species have foundfumarate to be free of significant toxicity (13-15). Furthermore,oral ferrous fumarate is used widely to treat iron deficiency inman, and alkyl esters of fumarie acid (specifically DIMEFU)have been used successfully both topically and systemically inthe treatment of psoriasis in man (see Ref. 16 and referencestherein). Moreover, administration of fumarie acid to rodentsprotects against chemical carcinogenesis (17, 18). We show inthis paper that DIMEFU has many of the desirable characteristics of a useful chemoprotective agent and deserves furtherexploration as a protector against carcinogenesis and otherforms of electrophile toxicity.

MATERIALS AND METHODS

Materials. Esters of fumarate, malÃ©ate,and succinate were obtainedfrom Aldrich Chemical Co. (Milwaukee, WI). Crystalline dimethyl

4The abbreviations and trivial names used are: QR, quinone reductase, which

is officially designated NAD(P)H:(quinone acceptor) oxidoreductase (EC1.6.99.2), and also known as DT-diaphorase or menadione reductase; DIMEFU,dimethyl fumarate; GST. glutathione S-transferase (EC 2.5.1.18); CDNB, 1-chloro-2,4-dinitrobenzene; DCNB. 1.2-dichloro-4-nitrobenzene; CD, concentration of an inducer required to double the specific activity of quinone reductase;BHA, 2(3)-ferr-butyl-4-hydroxyanisole.

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INDUCTION OF PHASE 2 ENZYMES BY FUMARATES

fumarate was ground to a fine powder in a mortar prior to addition tothe diets. The powdered AIN 76A semipurified diet was from ICNBiomedica Is (Costa Mesa, CA), and the powdered Purina 5001 dietwas from Purina Ralston (St. Louis, MO). BHA and ethoxyquin (6-ethoxy-l,2-dihydro-2,2,4-trimethylquinoline) were from Sigma Chemical Co. (St. Louis, MO).

Measurement of Quinone ReducÃasein Hepa Iclc7 Murine HepatomaCells. The method for measuring the potency of inducers in raising QRspecific activities in these cells has been described (12). For the presentexperiments, Hepa Iclc? cells were grown in 96-well microtiter platesand QR was measured on cell lysates (19). This procedure was modifiedby use in the medium of fetal calf serum that had been heated for 90min at 55Â°Cin the presence of activated charcoal (1 g/100 ml; Sigma),

and filtered twice through 0.22-^m filters. This treatment increased theinduction ratios of QR by lowering the basal activities of the enzyme,and by removing inhibitor(s) of induction present in untreated serum.The Hepa Iclc7 cells were plated at 10,000 cells/microtiter plate well(150 n\ of medium/well) and grown for 24 h. Serial dilutions of inducerswere added in 0.1% (by volume) of acetonitrile in fresh medium, andthe QR specific activities were determined after a 48-h exposure to theinducers. A plot of the ratio of QR specific activities of treated cells tocontrol cells (which had received the solvent only) as a function ofinducer concentration (Fig. 1) permitted the determination of the CD(Concentration required to Oouble the specific activity). All sets ofdeterminations included plates induced with a series of concentrations(0.01-1.0 UM) of /3-naphthoflavone as a standard inducer. The meanCD value for fi-naphthoflavone was 35 n\i.

Two mutants of Hepa Iclc7 cells were also used. The cl mutant (agift from O. Hankinson, University of California, Los Angeles, CA)synthesizes an inactive (truncated) cytochrome P,-450 gene product(20), whereas the BP'cl mutant (a gift from James P. Whitlock, Jr.,

Stanford University, Palo Alto, CA) contains no detectable aryl hydrocarbon hydroxylase activity or its mRNA. It has a normal Ah receptor,but does not translocate the ligand-bound receptor to the nucleus (21).The mutants of Hepa Iclc7 cells were grown and induced under similarconditions to the wild-type cell line (12, 22).

Treatment of Animals and Collection of Tissues. Weanling femaleSprague-Dawley rats were fed a powdered AIN 76A diet supplementedwith 0.2, 0.4, or 0.5% (by weight) of DIMEFU for 14 days beginningat 25 days of age. Control groups were fed this diet without DIMEFU.A positive control group received 0.4% (by weight) ethoxyquin in thediet. The rats were housed in plastic cages and were weighed dailyduring the feeding of the experimental diets.

Female CD-I mice (Charles River Laboratories, Wilmington, MA)were fed a powdered Purina 5001 laboratory chow, supplemented with0.2 or 0.5% (by weight) of DIMEFU for 14 days beginning at 45 daysof age. A positive control group received a diet containing 0.75% 2(3)-fert-butyl-4-hydroxyanisole. The mice were housed in hanging cageswithout bedding, and were weighed daily during the feeding of theexperimental diets.

The animals were killed by C'Oi inhalation and the tissues wereremoved, frozen in liquid nitrogen, and stored at â€”80Â°Cuntil they were

processed for enzyme assays. The mouse livers were perfused via theportal vein with cold 0.15 M KC1 containing 2 ITIMEDTA, pH 7.5,prior to freezing. The rat livers were not perfused.

All animal experiments were in compliance with "Guide for the Careand Use of Laboratory Animals," NIH Publication 81-2385, United

States Department of Health and Human Services, Washington, DC.Preparation of Tissue Cytosols and Assay of Their Enzymatic Activi

ties. Cytosols were prepared from rodent tissues as described (23). QRactivities were determined on tissue cytosols by measuring the rate ofthe NADPH-dependent menadiol-mediated reduction of 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide at 610 nm, as described by Prochaska and SantamarÃa(19), with the following modifications. The assays were conducted at 25"C in 3.0-ml systems contain

ing: 25 mM Tris-Cl (pH 7.4), 50 MMmenadione in 10 ^1 of acetonitrile,0.75 mM 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, 30 tÃMNADP, 5 fiM flavin-adenine dinucleotide, 1 mM glucose6-phosphate, 5 units of yeast glucose-6-phosphate dehydrogenase,

0.07% bovine serum albumin, and 0.01% Tween 20. GST activitieswere measured with CDNB and DCNB (24).

Protein concentrations were determined according to the method ofBradford (25) with bovine serum albumin as standard. All enzyme-specific activities are expressed as nmol of product formed per min permg of protein.

Statistical Treatment. The mean enzyme specific activities for cytosols obtained from 4-6 animal tissues were calculated and the standarderrors of the means were computed. The ratios of the mean enzymespecific activities of tissue cytosols from animals treated with inducersto those receiving control diets were then calculated (see Figs. 1 and 2,and Table 1). The standard errors of these ratios were obtained asfollows. If the mean specific activity values for groups A and B are XA(Â±SEM^) and XB (Â±SEMâ€ž),respectively, the SEM^/Â«of the ratioAVA-Â«is:

SEM.,/,XA/XB

]2 + \SEMB/XB]2

RESULTS

Quinone Reductase Induction in Murine Hepatoma Cells. Themurine hepatoma Hepa Iclc? cell line responds to nearly allknown chemoprotectors by severalfold elevation of the specificactivities of QR (9, 11, 12). The concentration-dependent inductions of QR in these cells by fumarie and maleic acids andtheir methyl and ethyl esters are shown in Fig. 1. The estersare much more potent inducers than the acids, as would beexpected, because they are more efficient Michael reaction

Oi-ucfrl4SPECIFIC

ro

O

l

DimethylMALÃ‰ATEv

DlethylMALÃ‰ATE

DlethylFUMARATE

DimethylFUMARATE

FUMARICACID

Â«I MALEIC" ACID

DimethylSUCCINATE

10 25 50 100 200

CONCENTRATION,

Fig. 1. Induction of quinone reducÃasein Hepa Iclc? murine hepatoma cellsby fumarie and maleic acids and their dimethyl and diethyl esters, and by dimethylsuccinate. The cells were grown in duplicate 96-well microtiter plates for 24 h,and then induced for 48 h by addition of serial dilutions of acetonitrile solutionsof the specified compounds in fresh medium. The QR activity was determined incell lysates on one plate and related to the cell density determined on the secondof each pair of plates stained with crystal violet. The results are presented as theratios of specific activities of QR in treated cells to those of control cells that hadreceived equivalent volumes of the solvent only. The CD values were as follows:dimethyl fumarate (ÃranÃ-CH3O2CCH=CHCO2CH3),5.3 Â»IM;dimethyl malÃ©ate(cis-CH3O2CCH=CHCO2CH3), 6.8 /IM; diethyl malÃ©ate(c/s-C2H,O2CCH=CHCO2C2H5), 9.5 Ã•Ã•M;diethyl fumarate (frani-C2H5O2CCH=CHCO2C2H5),20 Â»M.Fumarie (/rons-HO2CCH=CHCO2H) and maleic (rw-HO2CCH=CHCO2H) acids were only weakly active, whereas dimethyl succinate(CH3O2CCH2CH2CO2CH3) was inactive.

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acceptors than the acids (26). The CD values were 5.3 and 6.8Ã•Â¿Mfor dimethyl fumarate and dimethyl malÃ©ate,respectively.Significant elevations of QR occurred only at concentrations ofthe free acids exceeding 50 Â¿tM.Dimethyl succinate was inactiveas an inducer, again as expected, since it is not a Michaelreaction acceptor. The diethyl esters were somewhat less potentthan the dimethyl esters. The CD values were 20 and 9.5 ^Mfor diethyl fumarate and diethyl malÃ©ate,respectively.5

As noted previously (22), the basal QR specific activities ofthe cytosols of the BPrcl mutant were much lower (26%) andof the cl mutant much higher (402%) than those of the wild-type Hepa Iclc7 line. Despite the large differences in basal QRactivities, however, the potencies of induction of QR of thedimethyl esters of fumarate and malÃ©atewere almost the samein these mutants as in the wild-type cell line (data not shown).In contrast, 0-naphthoflavone was not an inducer in the mutants, whereas it is a very potent inducer (CD = 35 HM) in thewild-type Hepa Iclc? cells. These findings establish that DI-MEFU is a monofunctional inducer (3,10) that does not dependon a functional Ah receptor or on cytochrome Pi-450 (arylhydrocarbon hydroxylase) activity for induction.

Enzyme Inductions in Mouse Organs. Addition of 1.0 or 2.0%fumarie acid to the diets of 6-week-old female CD-I mice for14 days resulted in normal weight gain and was well tolerated.Survey of tissue cytosols disclosed only minor, although insome tissues, significant elevations of the specific activities ofQR and GSTs (data not shown). Since fumarie acid is a relatively poor inducer compared to its esters (9), we next examinedthe effects of 0.2 and 0.5% DIMEFU under similar conditions.The mice weighed 25.9 Â±1.2 g at the beginning of DIMEFUfeeding. The weight gain of the control group was 12% duringthe 14-day feeding period. The animals receiving DIMEFUgained 10.0 and 1.3% of their initial weight for the groupsreceiving 0.2 and 0.5% DIMEFU, respectively, and 10.0% forthose that received 0.75% BHA.

As previously described in CD-I mice (27), the basal QRspecific activities were 10- to 60-fold higher in nearly all peripheral tissues (except spleen) than in the liver. In contrast,the basal GST specific activities were quite similar in all thetissues examined except for the very low levels in the spleen(28) (see Fig. 2 caption). DIMEFU feeding raised the specificactivities of QR and GST in a dose-dependent manner in nearly

all organs (Fig. 2). At the higher dose, the ratios of the specificactivities of these enzymes (treated/controls) were elevated atleast 2.1-fold and as much as 9.0-fold (Fig. 2) in several organsexamined, except for the spleen, where the GST activities withDCNB were not measurable, and the colonie mucosa, wherethis activity was not significantly increased. The greatest elevations of QR in response to DIMEFU were observed in theforestomach and small intestine (4-fold in each tissue), whereasthe GST activities in liver, forestomach, glandular stomach,and small intestine were increased 3.7- to 9.0-fold.

In contrast to the effects of feeding 0.75% BHA to femaleCD-I mice, which resulted in significant inductions of QR andGST primarily in liver and small intestine (27-29), DIMEFUraised these activities in many tissues and might therefore beexpected to exert more widespread protection.

Enzyme Inductions in Rat Organs. Female Sprague-Dawley

9 In a previous publication we reported somewhat higher CD values for these

compounds and larger differences between the potencies of the dimethyl anddiethyl esters (9). The experimental conditions of the present assays have beenmodified, i.e., 48-h induction and use of heat- and charcoal-treated fetal calfserum.

LIVER GS SM INTES COLON SPLEEN

Fig. 2. Effect of feeding 0.2% (D) and 0.5% (â€¢)DIMEFU for 14 days tofemale CD-I mice on the specific activities of cytosolic quinone reducÃaseandglutathione transferases [(measured with CDNB (GST-CDNB) and DCNB (GST-DCNB) as substrates] in liver, forestomach (FS), glandular stomach (GS), proximal small intestine (SM INTES), colon, and spleen. The results are expressed asthe ratios of the specific activities of organ cytosols from treated animals to thoseof controls receiving the unsupplemented diet. Five animals were studied in eachof the treated groups and six in the control group. The specific activities (nmol/min/mg of protein Â±SEM) of the organ cytosols of control animals receiving thePurina 5001 diet only were as follows. Liver: QR, 54.9 Â±6.0; GST with CDNB,1203 Â±93; GST with DCNB, 12.7 Â±1.2. Forestomach: QR, 1159 Â±34; CDNB,1104 Â±111; DCNB, 23.6 Â±4.67. Glandular stomach: QR, 3488 Â±131; CDNB,823 Â±69.8; DCNB, 7.21 Â±0.57. Proximal small intestine: QR. 592 Â±51; CDNB,1388 Â±166; DCNB, 15.2 Â±3.8. Colon: QR, 543 Â±59.8; CDNB, 940 Â±94.4;DCNB, 16.7 Â±1.0. Spleen: QR, 42.4 Â±2.9; CDNB, 13.5 Â±2.3; DCNB, notdetectable. The relative standard errors of the ratios [(SEM/mean) x 100] are asfollows: no designation, 0-5%; a, 5-10%; *, 10-15%, c, 15-20%.

rats were fed a diet supplemented with 0.2, 0.4, or 0.5%DIMEFU for 14 days beginning at 25 days of age. Controlgroups received this diet without supplement, and a positivecontrol group received 0.4% ethoxyquin, which is a knowninducer of GST in rodent tissues (30). The gains in body weightsof the rats at the end of the 14-day feeding period (from aninitial weight of 63.7 Â±4.4 g to a final weight of 135.0 Â±5.6 gin the control group) were unaffected by the feeding of 0.2%DIMEFU, but were reduced to 93 and 87% of control valueson the 0.4 and 0.5% DIMEFU diets, respectively. Liver weightswere similar in rats receiving 0.2% DIMEFU to those ofcontrols, but were lowered by the feeding of 0.4 and 0.5%DIMEFU to 96.3 and 86.9% of control values, respectively. Incontrast, whereas 0.4% ethoxyquin treatment decreased finalbody weights to 95.3% of controls, liver weights were raised to113% of controls.

The enzyme induction patterns evoked by ethoxyquin andDIMEFU were quite different among the various organs (compare Fig. 3 and Table 1). Thus, the most dramatic effects ofethoxyquin were in the liver (3- to 4-fold inductions of the three

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LIVER FS GS SM INTES COLON LUNG KIDNEY

Fig. 3. Effect of feeding 0.2% (D), 0.4% (Â®),or 0.5% (â€¢)DIMEFU for 14days to female Sprague-Dawley rats on the specific activities of cytosolic quinonereducÃaseand GSTs (measured with CDNB (GST-CDNB) and DCNB (GST-DCNB) as substrates] in liver, forestomach (FS), glandular stomach (GS), proximal small intestine (SM INTES), colon, lung, and kidney. The results areexpressed as the ratios of the specific activities of organ cytosols of treatedanimals to those of controls receiving the unsupplemented diet. Six animals werestudied per group except that the group receiving 0.5% DIMEFU contained fouranimals. The specific activities (nmol/min/mg of protein Â±SEM) of the organcytosols of control animals receiving the unsupplemented AIN 76A diet were asfollows. Liver: QR, 56.1 Â±2.8; GST with CDNB, 339 Â±8.1; GST with DCNB,9.60 Â±0.50. Forestomach: QR, 350 Â±24.9; CDNB, 92.6 Â±6.3; DCNB. 1.56 Â±0.29. Glandular stomach: QR, 531 Â±28; CDNB, 129 Â±4.7; DCNB, 4.87 Â±0.51.Proximal small intestine: QR. 71.9 Â±8.6; CDNB, 214 Â±26.1; DCNB, 0.82 Â±0.19. Colon: QR, 221 Â±39.7; CDNB, 103 Â±19.4; DCNB. not detectable. Lung:QR, 204 Â±13.6; CDNB, 75.0 Â±6.2; DCNB, 3.29 Â±0.53. Kidney: QR, 35.6 Â±1.8; CDNB, 73.1 Â±7.7; DCNB, 1.07 Â±0.12. The relative standard errors of theratios [(SEM/mean) x 100] are as follows: no designation, 0-5%; a, 5-10%; b10-15%; c 15-20%; d, 20-25%.

enzymes), whereas the highest doses of DIMEFU producedonly a 2.1-fold induction of QR and a 1.39 (CDNB)- to 1.53(DCNB)-fold increase in GST activities in this organ. In contrast, in the forestomach, 0.4% ethoxyquin raised the enzymeactivities 1.19- to 1.75-fold, whereas DIMEFU increased thesespecific activities 6.0- to 11.3-fold. In all of the organs studied,QR was increased by DIMEFU in a dose-dependent manner.Similarly, GSTs measured with CDNB and with DCNB wereraised by DIMEFU, except in the colon where GST activitywith CDNB was barely elevated, and GST activity with DCNBwas not measurable.

Since there are multiple GSTs in rat tissues, and the precisecomposition of isoenzymes in each tissue following inductionby DIMEFU is unknown, it is clear that the response toDIMEFU is complex, and that undoubtedly various types ofGSTs are induced differentially, as are the induction patternsevoked by BHA in the liver (31, 32). Nevertheless, the inductions of the Phase 2 enzymes evoked by DIMEFU were much

Table 1 Induction ofQR and GST activities by 0.4% dietary ethoxyquin incytosols of female Sprague-Dawley ral organs

The specific activities (nmol/min/mg of protein Â±SEM) of cytosols of controlanimal receiving the AIN 76A diet only were as follows. Liver: QR, 56.1 Â±2.8;GST with CDNB, 339 Â±8.1; GST with DCNB, 9.50 Â±0.50. Forestomach: QR,350 Â±24.9; CDNB, 92.6 Â±6.3; DCNB, 1.56 Â±0.29. Glandular stomach: QR,531 Â±28; CDNB, 129 Â±4.7; DCNB, 4.87 Â±0.51. Small intestine: QR, 71.9 Â±8.6; CDNB, 214 Â±26.1; DCNB, 0.82 Â±0.19. Colon: QR, 221 Â±39.7; CDNB,103 Â±19.4. Lung: QR, 204 Â±13.6; CDNB, 75.0 Â±6.2; DCNB: 3.29 Â±0.53.Kidney: QR. 35.6 Â±1.8; CDNB 73.1 Â±7.7; DCNB 1.07 Â±0.12.

Ratio of specific activities (treated/control)

GST

Organ QR CDNB DCNB

LiverForestomachGlandular

stomachSmallintestineColonLung

Kidney3.70

Â±0.111.75Â±0.741.390.102.950.203.680.231.78

0.132.37 0.114.

13Â±0.061.19+0.131.21

0.063.030.191.240.251.14

0.153.18 0.173.24

Â±0.101.45Â±0.331.00

+0.151.10Â±0.40NDÂ°1.21

Â±0.212.16 Â±0.21

" ND, not detectable.

less organ specific than those produced by the antioxidantethoxyquin, which raised enzymes principally in the liver andsmall intestinal mucosa (Table 1).

DISCUSSION

Well-tolerated levels (0.2-0.5%) of DIMEFU in the diet offemale CD-I mice and female Sprague-Dawley rats elevatedthe specific activities of chemoprotective enzymes (QR andGSTs) in the liver and several peripheral tissues. Such enzymeinduction patterns are characteristic consequences of the administration of many types of compounds (e.g., phenolic an-tioxidants, isothiocyanates, l,2-dithiole-3-thiones, thiocarba-mates, coumarins) that protect animals against the carcinogenicand other toxic effects of a wide variety of chemical agents (2).Therefore, it seems likely that DIMEFU will be found to be achemoprotective agent, especially since the chemoprotectivefunction against carcinogenesis has already been demonstratedfor fumarie acid (17, 18, 33, 34).

The following considerations make the possibility of developing DIMEFU as a chemoprotector especially attractive: (a)DIMEFU is a monofunctional inducer and it is therefore unlikely to induce Ah receptor-dependent cytochromes P-450 thatactivate many carcinogens to their ultimate reactive forms, (b)Fumarie acid, the parent acid of DIMEFU, is widely distributedin plants, is used as a food additive, and has very low acute andchronic toxicity (13-15). (e) Fumarie acid salts and esters havebeen used as therapeutic agents in man, e.g., ferrous fumaratefor iron deficiency and alkyl esters for psoriasis (see Ref. 16and references therein), (d) High doses of fumarie acid protectagainst the carcinogenic effects of a nitrofuran (l-methyl-7-[2-(5-nitro-2-furyl)vinyl]-2-oxo-l,4-dihydro-l,8-naphthyridine-3-carboxylate) on the forestomach and lung in mice (17), andagainst the hepatocarcinogenicity of 3-methyl-4'-(dimethyl-

amino)azobenzene in rats (18). (e) H. G. Crabtree (35, 36) madethe observation many years ago that fumarie acid, maleic anhydride, and similar a,/3-unsaturated dicarboxylic acids andanhydrides delay or prevent the induction of tumors in mouseskin by benzo(a)pyrene. (/) Fumarie acid protects animals andcells against the toxicity of mitomycin C apparently withoutabolishing its antineoplastic effects (34).

It is of interest that the pharmacological and therapeutic(diuretic, oxytoxic, antiinflammatory, antiulcer, and tumorgrowth inhibitory) effects of fumarie acid were uncovered by

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studies of the reputed medicinal properties of shepherd's purse

(Capsella bursa-pastoris), a cruciferous weed that has beenwidely used as a traditional herbal remedy (37-41). The activeprinciple of shepherd's purse responsible for these activities

was isolated and identified as fumarie acid (42). Fumarie acidwas then shown to inhibit the growth of solid Ehrlich tumor inmice (41), to inhibit gastric ulcÃ©rationin rats (42), and to reducethe toxicity of mitomycin C for mice (34) and ameliorate itstoxicity for cultured cells (34, 43). The finding that extracts ofshepherd's purse protect rats against the hepatocarcinogenesisof azo dyes in rats (40) and that fumarie acid is a chemoprotec-tor against carcinogenesis in rodents (17, 18) argue for thechemoprotective potential of fumarate esters. A clear-cut causalrelation between the protective effects of fumarie acid and thepotent induction of Phase 2 enzymes by fumarate esters hasnot been established. Since, however, chemical protectionagainst the toxic and neoplastic effects of chemical carcinogensis often associated with induction of Phase 2 enzymes, thisrelation mandates further analysis.

ACKNOWLEDGMENTS

The expert technical assistance of Annette B. SantamarÃais acknowledged with gratitude. We thank Patrick M. Dolan and Patricia A.Egner for their participation in some of the experiments. We are alsograteful to our colleague Thomas W. Kensler for much valuable advice.

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4. Ernster, L. DT Diaphorase: a historical review. Chem. Ser., 27A: 1-13,1987.5. Talalay, P., and Prochaska, H. J. Mechanisms of induction of NAD(P)H:

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1990;50:7871-7875. Cancer Res Sharon R. Spencer, Cynthia A. Wilczak and Paul Talalay TissuesReductase by Fumaric Acid Derivatives in Rodent Cells and Induction of Glutathione Transferases and NAD(P)H:Quinone

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induction of glutathione transferases and nad(p)h:quinone

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