Proc. Natl. Acad. Sci. USA Vol. 88, pp. 946-950, February 1991 Medical Sciences Free radical-derived quinone methide mediates skin tumor promotion by butylated hydroxytoluene hydroperoxide: Expanded role for electrophiles in multistage carcinogenesis (chemical carcinogenesis/phenoxyl radicals/reactive intermediates/metabolic switching/ornithine decarboxylase) KATHRYN Z. GUYTON*, PURSHOTAM BHANt, PERIANNAN KUPPUSAMYt, JAY L. ZWEIER , MICHAEL A. TRUSH*, AND THOMAS W. KENSLER*§ *Division of Toxicological Sciences, Department of Environmental Health Sciences, tDepartment of Biochemistry, and tElectron Paramagnetic Resonance Laboratories, Department of Medicine, Division of Cardiology, Johns Hopkins Medical Institutions, Baltimore, MD 21205 Communicated by Paul Talalay, October 23, 1990 (receivedfor review September 25, 1990)

ABSTRACT Free radical derivatives of peroxides, hydrop- tabolism of BHT in its actions as a promoter and toxin is eroxides, and anthrones are thought to mediate tumor promo- highlighted by the ability of several inhibitors of cytochrome tion by these compounds. Further, the promoting activity of P450 to suppress BHT toxicity (6, 7) and the observation that phorbol esters is attributed, in part, to their ability'to stimulate a hydroxylated metabolite is more effective than BHT as either the cellular generation of oxygen radicals. A hydroperoxide a tumor promoter or toxin in mouse lung (8, 9). Glutathione- metabolite of butylated hydroxytoluene, 2,6-di-tert-butyl4- depleting agents enhance liver and lung damage and elevate the hydroperoxyl4-methyl-2,5-cyclohexadienone (BHTOOH), has covalent binding of BHT in these tissues, suggesting that previously been shown to be a tumor promoter in mouse skin. toxicity may be mediated through an electrophilic intermediate (10, 11). Structure-activity studies of the 4-methyl position of BHTOOH is extensively metabolized by murine keratinocytes to BHT indicate that a quinone methide intermediate may be the several radical species. The primary radical generated from toxic electrophile (12). BHTOOH is a phenoxyl radical that can disproportionate to In a study of acute toxicities 2,6-di-tert-butyl-4-hydroper- form butylated hydroxytoluene quinone methide, a reactive oxyl-4-methyl-2,5-cyclohexadienone (BHTOOH), a hydro- electrophile. Since electrophilic species have not been previously peroxide metabolite of BHT, was demonstrated to be 18-fold postulated to mediate tumor promotion, the present study was more potent than the parent compound (13). Further, this undertaken to examine the role of this electrophile in the metabolite, in contrast with the parent compound, is a tumor promoting activity ofBHTOOH. The biological activities oftwo promoter in mouse skin (14), a tissue that apparently does not chemicalanalogs of BHTOOH, 4-trideuteromethyl-BHTOOH generate BHTOOH from BHT (unpublished observations). and 4-terl-butyl-BHTOOH, were compared with that of the BHTOOH is of particular interest because it undergoes parent compound. 4-Trideuteromethyl-BHTOOH and 4-tert- extensive metabolism in murine keratinocytes to form sev- butyl-BHTOOH have a reducedability or inability, respectively, eral free radical intermediates, including phenoxyl, peroxyl, to form a quinone methide; however, like the parent compound, alkoxyl, and alkyl radical derivatives (14). Considerable they both generate a phenoxyl radical when incubated with evidence suggests that free radicals and free radical-mediated keratinocyte cytosol. The potency of BHTOOH, 4-trideutero- processes are involved in the biochemical and biological methyl-BHTOOH, and 4-tert-butyl-BHTOOH as inducers of events of tumor promotion (15, 16), and the propensity of ornithine decarboxyiase, a marker of tumor promotion, was BHTOOH to generate free radicals may, therefore, be related commensurate with their capacity for generating butylated to its enhanced potency as a toxin and tumor promoter. hydroxytoluene quinone metbide. These initial results were The primary radical species generated from BHTOOH is confirmed in a two-stage tumor promotion protocol in female the BHT phenoxyl radical; this radical metabolite will readily SENCAR mice. Together, these data indicate that a quinone undergo disproportionation to regenerate the parent com- methide is mediating tumor promotion by BHTOOH, providing pound BHT with the concomitant production of 2,6-di-tert- direct evidence that an electrophilic intermediate can elicit this butyl-4-methylene-2,5-cyclohexadienone (BHT-QM), a reac- stage of carcinogenesis. tive electrophile (Fig. 1). Electrophiles have long been rec- ognized to play preeminent roles in the initiation of chemical Butylated hydroxytoluene (BHT; 2,6-di-tert-butyl4methyl- carcinogenesis through the covalent modification of nucleic phenol) has found wide commercial application because of its aci-ds (18). On the other hand, electrophilic species have not excellent antioxidant properties. Although BHT has attained previously been linked with tumor promotion. The present generally regarded as safe (GRAS) status as afood additive, this study was undertaken to probe the possible role ofBHT-QM, phenolic antioxidant has toxic as well as carcinogenicproperties a model electrophile, in this biological process. (1, 2). For example, BHT is toxic in both liver and lung and has been reported to increase tumorformation inthe progeny ofrats MATERIALS AND METHODS that had high lifetime feeding of BHT (3). BHT is also a weak Chemicals and Syntheses. BHT, 3,5-di-tert-butyl-hydroxy- hepatocarcinogen in male mice (4). The most notable carcino- benzoic acid, 2,4,6-tri-tert-butylphenol, LiAI[2Hh4, deuter- genic property of BHT, however, lies in its ability to act as a tumor promoter in a variety oftissues, including the liver, lung, Abbreviations: BHT, butylated hydroxytoluene (2,6-di-tert-butyl4 colon, bladder, and thyroid (5). BHT is known to be extensively methylphenol); [2H3]BHT, 2,6-di-tert-butyl-4-[aaa- 2H3]methyl- metabolized in its target tissues, and the toxic as well as phenol; BHTOOH, 2,6-di-tert-butyl-4-hydroperoxyl-4-methyl-2,5- tumor-promoting activities of BHT are thought to be mediated cyclohexadienone; [2H3]BHTOOH, 2,6-di-tert-butyl-44[a,aa-2H31- by metabolites of the parent compound. The role of the me- methyl-2,5-cyclohexadienone; t-Bu-BHTOOH, 2,4,6-tri-tert-butyl4 hydroperoxyl-2,5-cyclohexadienone; BHT-QM, 2,6,-di-tert-butyl4 methylene-2,5-cyclohexadienone; PMA, phorbol 12-myristate 13- The publication costs ofthis article were defrayed in part by page charge acetate; ODC, ornithine decarboxylase; EPR, electron paramagnetic payment. This article must therefore be hereby marked "advertisement" resonance. in accordance with 18 U.S.C. §1734 solely to indicate this fact. §To whom reprint requests should be addressed. 946 Downloaded by guest on September 30, 2021 Medical Sciences: Guyton et al. Proc. Natl. Acad. Sci. USA 88 (1991) 947 ated dioxane, and hematin as well as chemical solvents were treatment group of 1, 4, 8, 20, or 40 ,umol of hydroperoxide. obtained from Aldrich. 7,12-Dimethylbenz[a]anthracene was The compounds were dissolved in 200 ,Al of acetone and purchased from Sigma, and phorbol 12-myristate 13-acetate applied to the shaved dorsal skin ofmice in hair-growth-cycle (PMA) was supplied by LC Services (Woburn, MA). remission. At the time of maximal induction, 10 hr after L-[4CjOrnithine (56 uCi/mol; 1 Ci = 37 GBq) was from treatment, the animals were sacrificed, and the epidermis was Amersham/Searle. isolated. Tissue from two animals was pooled for generation 2,6-Di-tert-butyl-4-[a,a,a-2H3]methylphenol [2H3JBHT of the 12,000 x g epidermal supernatant. The ODC activity was synthesized by the method of Mizutani et al. (19). of this cytosol was subsequently determined in triplicate by 3,5-Di-tert-butyl-hydroxybenzoic acid was converted to the measuring the release of 14CO2 from L-[14C]ornithine by using methyl ester (melting point, 166-1670C), which was then an Eppendorf microvessel assay (24). Protein content was added to an excess of LiAl[2H14 in anhydrous ethyl ether. determined by using bovine serum albumin as a standard by After refluxing under nitrogen for 12 hr, the excess reagent the method of Bradford (25). was decomposed with sodium potassium tartrate (20). The Two-Stage Tumor Promotion Study. Two hundred SEN- precipitate was then removed, and the resulting solution w~s CAR female mice 7-9 weeks of age in the resting phase of washed, dried, and freed from solvent. Column chromatog- hair-growth cycle were initiated on the shaved dorsal skin raphy yielded [2H3]BHT, NMR (deuterated dioxane) 8 8.9 (s, with 20 nmol of7,12-dimethylbenz[a]anthracene dissolved in 2 H), 7.6 (s, 1 H), 3.4 (s, 18 H). BHT, [2H3]BHT, and 100 Al of acetone. The first stage of promotion was begun 10 2,4,6-tri-tert-butylphenol were oxidized by the method of days later and consisted of twice weekly treatments of 2 ,g Kharasch and Joshi (21), and the corresponding hydro- of PMA for 2 weeks. The mice were then randomly assigned peroxides were then recrystallized from hexane. NMR (deu- to eight second-stage promotion treatment groups of 25 mice terated dioxane) gave BHTOOH 6 12.1 (s, 1 H), 8.6 (s, 1 H), each. Twice weekly thereafter, the mice in each group were 3.3 (s, 3 H), 3.2 (s, 18 H); 2,6-di-tert-butyl-4-[a,a,a- exposed to their respective treatments dissolved in 200/4 of 2H3]methyl-2,5-cyclohexadieone ([2H3]BHTOOH) 6 12.1 (s, acetone and applied to the shaved dorsal skin. The treatment 1 H), 8.6 (s, 1 H), 3.2 (s, 18 H); and 2,4,6-tri-tert-butyl-4- groups included 8- and 20-Amol doses of each of the three hydroperoxyl-2,5-cyclohexadienone (t-Bu-BHTOOH) 812.1 hydroperoxides as well as an acetone-negative control and a (s, 1 H), 8.7 (s, 1 H), 3.2 (s, 18 H), 2.9 (s, 9 H). BHTOOH and PMA-positive control. Treatment continued for a total of 24 [2H3]BHTOOH were determined to be >99% pure from other weeks of first- and second-stage promotion. metabolites by the HPLC procedure ofWand and Thompson (22), whereas t-Bu-BHTOOH was further purified by pre- parative HPLC to be >98% free from its quinol derivative. RESULTS The isotopic purity of [2H3]BHTOOH was >98% as deter- Formation of BHT-QM from BHTOOH and Its Analogs. mined by high-resolution proton NMR (deuterated dioxane). The production of BHT-QM from BHTOOH is postulated to Preparation of Murine Keratinocyte Cytosol. Suspensions of occur through the initial formation of a quinol ether inter- isolated keratinocytes were prepared as described by Yuspa et mediate from phenoxyl radicals (17, 26, 27); this intermediate al. (23). Late-gestation SENCAR mice were obtained from then spontaneously dismutates to give BHT-QM through the Harlan-Sprague-Dawley. Detached whole skins of neonatal loss of one of the 4-a-hydrogens (Fig. 1). When these mice 3-4 days of age were blotted and then washed -sequen- 4-a-hydrogens are replaced with deuteriums, the cleavage of tially in distilled deionized water and Dulbecco's phosphate- this carbon-deuterium bond requires more energy and is thus buffered saline before floating dermis down on 0.25% trypsin less likely to occur. The formation of BHT-QM from overnight at 4°C. The following day the epidermis was re- I2H3JBHTOOH will, therefore, occur at a slower rate than moved from the dermis, minced, and stirred with Eagle's does the generation of BHT-QM from BHTOOH. Further, minimal essential medium for 1 hr. The cells were then filtered t-Bu-BHTOOH has no 4-a-hydrogens and is, therefore, in- through nylon gauze, spun at 300 x g, and washed twice in capable of metabolism to a quinone methide intermediate. phosphate-buffered saline before resuspension for lysis by The relative inherent capabilities of the three compounds to freeze-thawing. The 12,000 x g supernatant was used for form BHT-QM is shown in Fig. 2A. electron paramagnetic resonance (EPR) analyses. Hematin has been widely employed as a model for metab- EPR Studies. EPR spectra were recorded at room temper- olism of hydroperoxides by hemoproteins (28) and has been ature with a Bruker ESP300 series spectrometer operating at previously demonstrated as an excellent model ofepidermal- X band with 100 kHz modulation frequency and a TM110 mediated metabolism ofBHTOOH (14). As shown in Fig. 2B, cavity. An EIP (EIP Microwave, San Jose, CA) 575 micro- activation of BHTOOH by hematin results in the initial wave frequency counter and Bruker 035M NMR gaussmeter generation of BHT-QM (Amax = 285 nm). Because of its were used to precisely determine frequency and magnetic inherent reactivity, over time BHT-QM will readily dimerize field. EPR data acquisition, processing, and spectral simu- to form a stilbenequinone (Ama = 450 nm) (29). Fig. 2C shows lations and integrations were performed by using a personal this initial time-dependent increase in A285 when BHTOOH is computer with software developed in the EPR laboratories. incubated with hematin. Although [2H3]BHTOOH may oth- Spectral components were individually simulated and fit to erwise be metabolized by hematin similarly to BHTOOH, the experimental spectrum. there is a 2.4-fold kinetic isotope effect on the rate of Assay of Ornithine Decarboxylase (ODC) Activity. Eight BHT-QM generation. Further, hematin does not metabolize female SENCAR mice 7-9 weeks of age were used per t-Bu-BHTOOH to a quinone methide. Thus, spectrophoto-

0 0 0 0 *tx+

OOE H ° + CH3 62. El H DETOO} NET Pam~yl Rak Q-d Ew _me BUT.QM BET

FIG. 1. Formation of BHT-QM from BHTOOH, as adapted from Becker (17). Through a one-electron oxidation to the peroxyl radical and a subsequent loss of oxygen, BHTOOH is nrtabolized to BHT phenoxyl radical. Two molecules of this radical can then form a quinol ether intermediate, which spontaneously disproportionates to give BHT-QM. Downloaded by guest on September 30, 2021 948 Medical Sciences: Guyton et al. Proc. Natl. Acad. Sci. USA 88 (1991)

A 0<1)

CH3 OOH BHTOOH 0a

'u z 0 CD3O3 H 0 0 0 0

OOH t-Bu-BHTOOH R-Hor D 250 500 0 60 WAVELENGTH (nm) TIME (seconds) FIG. 2. (A) Relative inherent capabilities of the three hydroperoxides to form BHT-QM. (B) Spectrophotometric measurement of the formation of BHT-QM (A02) and, subsequently, the dimer stilbenequinone (A45o) over time from BHTOOH (2.5 AuM) incubated with hematin (0.75 AtM) in phosphate-buffered saline. The spectrophotometer was "blanked" against this mixture, and then successive scans were made'at 1 and 10 min. (C) Actual kinetics of BHT-QM formation, as measured by A2A5, from the three hydroperoxides incubated with hematin. 4-CD3-BIiTOOH, [2H3]BHTOOI.

metric determination of the relative rates of BHT-QM gen- equivalent methyl hydrogens. The splitting ofeach ofthe four eration from in vitro metabolism of the three hydroperoxides lines into a triplet of 1:2:1 intensity concurs with the inter- confirms their intrinsic abilities to form this intermediate. action of the electron with the 2 equivalent meta hydrogens. Phenoxyl Radical Formation from BHTOOH and Its Ana- Coupling constants derived from computer simulations ofthe ogs by Keraginocytes. An alkaline ethanol chemical system spectrum (Fig. 3 scan b) were identical with those reported by was initillly used to evaluate the activation of either BHT- Macomber (30) for the BHT phenoxyl radical (aiHP3 = 11.3 G OOH, [zIH3]BHTOOH, or t-Bu-BHTOOH to' an EPR- and alcta = 1.5 G). The [2H3]BHT phenoxyl radical gives rise detectable phenoxyl radical (Fig. 3 scans q, e, and i). These to a 9-line EPR spectrum of a 1:5:12:24:26:24:12:5:1 signal spectra were then simulated for characterization and deriva- intensity (Fig. 3 scan e), which is consistent with the com- tion of the coupling constants (Fig. 3 scans b, f, and j). The bined interactions of the electron with the two meta hydro- EPR signal for the BHT phenoxyl radical shown in Fig. 3 scan gens and the three deuteriums at the 4-methyl position. a is comprised of a quartet structure of a 1:3:3:1 signal Simulation of this EPR signal '(Fig. 3 scan f) gave approxi- intensity consistent with electron coupling with the three mately equal hyperfine coupling constants for the deuterium

BHTOOH 4-CD3-BHTOOH t-Bu-BHTOOH

alkaline ethanol

b simulation

25 SMM-- keratinocyte cytosol

hoat-inactivated d'V cytosol

FIG. 3. Formation of phenoxyl radical signals from BHTOOH, [2H31BHTOOH (4-CD3-BHTOOH), and t-Bu-BHTOOH. EPR spectra were recorded with the following instrument conditions: frequency, 9.77 GHz; microwave power, 20 mW; time constant, 0.16 s; and scan time, 60 s. The modulation amplitude was 0.5 G for scans a-h and 0.25 G for scans i-l. The gain was 1 x 106 for scans a, c, d, g, and h; for scan e it was 1 x 10 and for scans i, k, and I it was 1 x 104. All spectra repiesent averaged EPR signals of 10 scans except for scan g (one scan). All incubations were-bubbled with nitrogen before aspiration into the EPR flat cell. Scans a, e, and i show the chemical generation ofphenoxyl radical from 25 mg of compound dissolved in 0.4 ml of alkaline ethanol. Scans b, f, and j show the computer simulations of these signals; all spectra are centered at a g value of 2.004, and the coupling constants used were as follows: for the parent phenoxyl radical, am'et = 1.5 G, a = 11.3 G; for the'deuterated analog, aWeu = aD = 1.6 G; and for the tert-butyl analog, a~ea = 1.6 G. Scans c, g, and k are the spectra generated from incubations of keratinocyte cytosol and 20 mg of hydroperoxide in 70%' ethanol. Spectra d, h, and I are the EPR signals seen when 20 mg of compound is incubated in 70%o ethanol with cytosol that was heated in vacuo for 30 min. Downloaded by guest on September 30, 2021 Medical Sciences: Guyton et al. Proc. Natl. Acad. Sci. USA 88 (1991) 949 and hydrogen interactions, with aeta = aD = 1.6 G. Alkaline ethanol activates t-Bu-BHTOOH to a phenoxyl radical W LJ5 La IrOU-P I-'J whose EPR signal is shown in Fig. 3 scan i. The broad line ,AMIIUI 3 1.5 A-A 20 pmnol 4-CD3_BHTOOH spectrum is consistent with the couplings ofthe electron with 2 A-A 8 the two equivalent meta hydrogens (aHeta = 1.6 G) as jsnol 4-CD3-BHTOOH previously reported by Valoti et al. (31). 1.-0-s-s 20 imOl BHTOOH * EPR-detectable phenoxyl radicals were also produced o 0.50-0 8 jsmol BHTOOH upon incubation of the three compounds with cytosol pre- 0.5- pared from keratinocytes (Fig. 3 scans c, g, and k). The radicals generated from both BHTOOH and [2H3]BHTOOH 0.01 *A n * had hyperfine structure identical to the corresponding phe- noxyl radicals produced with alkaline ethanol. However, the spectra generated from the incubation of t-Bu-BHTOOH 50-- ~ ~ ~ ~ 5- with cytosol appears to consist of two separate radical signals; in addition to a signal identical to the chemically 250 generated phenoxyl radical, the spectrum contains a fine triplet structure identified by subtraction of the first signal from the cytosol-mediated spectrum. Although the identity of A~4 this second radical remains tentative, it is likely to be the semiquinone radical. It is well established that 4-alkyl hy- droperoxide analogs of BHTOOH can undergo univalent 0 5 10 1,5 20 25 reduction to produce alkoxyl radicals (32). These alkoxyl WEEKS OF PROMO1ION radicals subsequently undergo fragmentation through P-scis- sion by which alkyl radicals and quinone intermediates are FIG. 5. Time course of tumor induction by BHTOOH, [2H3]- BHTOOH (4-CD3-BHTOOH), and t-Bu-BHTOOH as expressed by generated. The BHT quinone can then be readily reduced to incidence and multiplicity of papillomas. Mice were initiated and the semiquinone radical, whose EPR signal is consistent with subsequently treated as described. At end of the study, .92% of the the fine triplet structure seen when t-Bu-BHTOOH is incu- mice in each group was alive. Not shown is data for the control bated with cytosol. Double integration of the keratinocyte- groups: first-stage tumor promotion followed by acetone treatment mediated spectra (corrected for differences in gain, number for 22 weeks did not induce tumor formation, whereas treatment with of scans, modulation amplitude, and sweep width) indicated 2 ,ug ofPMA for 24 weeks produced a 100%o incidence and an average a 2-fold increase in the amount ofphenoxyl radical produced of 5.5 papillomas per mouse. from [2H3]BHTOOH as compared with BHTOOH, whereas compound were chosen for examination in a long-term pro- t-Bu-BHTOOH generated 270-fold more of this radical than motion study. did an equivalent amount of the parent compound. Finally, Tumor-Promotion Study. The results of a 24-week, two- no phenoxyl radicals were generated when the cytosol was stage tumor-promotion study are shown in Fig. 5. Consistent heat inactivated (Fig. 3 scans d, h, and 1). with a previous study (34), repetitive treatments with 20 ,umol ODC Activity. The induction of ODC activity has been of BHTOOH caused a 64% incidence of papillomas with an widely used as a marker of tumor promotion, and although average tumor burden of 1.9 tumors per mouse at the end of not sufficient, it represents a characteristic effect of tumor the study. The use of a two-stage promotion protocol, promoters (33). BHTOOH has been previously demonstrated however, seemed to significantly reduce the tumor-latency to cause a rapid, transient, and dose-dependent induction of period as compared with the application of BHTOOH as a epidermal ODC activity (34). Fig. 4 shows the results of a complete promoter. By contrast to BHTOOH, treatment comparative dose-response study of BHTOOH and its with an equimolar dose of [2H3]BHTOOH resulted in only a 4-methyl-deuterated and 4-tert-butyl analogs. At correspond- 28% incidence ofpapillomas and a tumor burden of <1 tumor ing doses, [2H3]BHTOOH caused a lesser induction of ODC per mouse. The 8-,umol dose of the parent compound pro- activity as compared to the parent hydroperoxide, whereas duced a tumor incidence and multiplicity that was remarkably t-Bu-BHTOOH did not increase the basal activity of this similar to those of a 2.5-fold higher dose of the deuterated enzyme. From this study, the 8- and 20-I.mol doses of each analog. The low dose of [2H3]BHTOOH was inactive as a tumor promoter in this study, as were both doses of t-Bu- BHTOOH. No tumors formed in mice that were initiated but o e 1.2 *-* BHTOOH received only first-stage promoter treatment. a. A-A 4-CD3-BHTOOH W al 06 *-* t-Bu-BHTOOH o'.9 DISCUSSION ~:0.6 Interest in the peroxide, hydroperoxide, and anthrone tumor T promoters has centered on their capacity for generating free T radicals (16, 35, 36). Much indirect evidence suggests that z 0 0.3 radicals may play a prominent role in mediating tumor promotion (15, 16). However, the intracellular targets of 0.0 4- ~ these species are not known, and the actual free radical 1 10 100 species formed from promoters in target tissues have rarely DOSE (jsmoles) been characterized. The primary radical known to be gener- FIG. 4. Dose-response for the induction of ODC activity by ated from BHTOOH in keratinocytes is the phenoxyl radical BHTOOH, [2H3]BHTOOH (4-CD3-BHTOOH), and t-Bu-BHT- (14). Because this radical also serves as an intermediate in the OOH. Each point represents the mean ± SEM of triplicate deter- formation of BHT-QM, the production of the phenoxyl minations of ODC activity from four epidermal cytosolic prepara- radical was investigated using chemical analogs of BHT- tions, each of which consisted of tissue pooled from two separate OOH. Integration of the keratinocyte-mediated EPR signals animals. Enzyme activity was determined as described. indicated that, from the same amount of hydroperoxide, Downloaded by guest on September 30, 2021 950 Medical Sciences: Guyton et al. Proc. Natl. Acad. Sci. USA 88 (1991) [2H3]BHTOOH and t-Bu-BHTOOH generate =2 and 270 lular signaling pathways, the initial mechanism is probably times as much phenoxyl radical, respectively, as does BHT- different. Covalent interaction of the quinone methide with OOH. These increases in phenoxyl radical concentrations sulfhydryl groups or other nucleophiles in its target cell, the correlate with the reduced ability, and inability, respectively, keratinocyte, must somehow transmit the molecular signal of [2H3]BHTOOH and t-Bu-BHTOOH to eliminate this rad- for division and replication. Modification of proteins func- ical through quinone methide formation (Fig. 2A). Through tionally involved in the control of second-messenger signals the process of metabolic switching, these two analogs of (i.e., calcium ions) may provide an enhanced growth stimulus BHTOOH may also preferentially undergo alternative routes to initiated cells; a coordinate possibility provides that altered of metabolism, including other free radical pathways. A ion fluxes trigger a cytotoxic response in normal but not principle alternative pathway involves reduction and 1-scis- initiated cells. sion of the hydroperoxide to form alkoxyl, alkyl, and semi- quinone radicals. Metabolism of t-Bu-BHTOOH by keratin- We thank Chang-Sing Lee and Lou-Sing Kan for analyses per- ocyte cytosol yielded another directly detectable free radical formed at the nuclear magnetic resonance facility of Johns Hopkins in addition to the phenoxyl radical, which may be the University (established by National Institutes of Health Grant semiquinone radical generated through j-scission. Thus, GM27512). The electron paramagnetic resonance laboratories are [2H3JBHTOOH and t-Bu-BHTOOH may also generate higher supported by National Institutes of Health Grants HL17655-13 and yields of other free radical species than does the parent HL38324. This work was supported by grants CA44530, ES07141, hydroperoxide in addition to producing increased steady- and ES05131 from the National Institutes ofHealth. T.W. Kensler is state concentrations of phenoxyl radicals. recipient of National Institutes of Health Research Career Develop- The results of the tumor promotion study indicate, how- ment Award CA01230. ever, that tumor formation by BHTOOH was not solely 1. Kahl, R. (1984) Toxicology 33, 185-228. dependent on phenoxyl or other radical generation. The 2. Witschi, H., Malkinson, A. M. & Thompson, J. A. (1989) Pharmacol. analog that produces the highest level of phenoxyl radical Ther. 42, 89-113. (t-Bu-BHTOOH) did not act as a tumor promoter in this 3. Olsen, P., Meyer, O., Bille, N. & Wfrzten, G. (1986) Food Chem. study. Instead, the ability of the hydroperoxides to act as Toxicol. 24, 1-12. 4. Inai, K., Kobuke, T., Nambu, S., Takemoto, T., Kou, E., Nishina, H., tumor promoters was directly related to their ability to form Fujihara, M., Yonehara, S., Suehiro, S., Tsuya, T., Horiuchi, K. & BHT-QM. Thus, while the production of phenoxyl radical Tokuoka, S. (1988) Jpn. J. Cancer Res. 79, 49-58. represents an obligatory step in the generation ofthe ultimate 5. Ito, N., Fukushima, S. & Tsuda, H. (1985) CRCRev. Toxicol. 15, 109-150. reactive metabolite, it is not itself sufficient for tumor pro- 6. Malkinson, A. M. & Beer, D. S. (1984) J. Nat. Cancer Inst. 73, 925-933. motion. The 2.4-fold kinetic isotope effect on BHT-QM 7. Masuda, Y. & Nakayama, N. (1984) Toxicol. Appl. Pharmacol. 75,81-90. 8. Malkinson, A. M., Thaete, L. G., Blumenthal, E. J. & Thompson, J. A. generation of the deuterated analog determined in vitro with (1989) Toxicol. Appl. Pharmacol. 101, 196-204. hematin was borne out in the tumor promotion study. In this 9. Thompson, J. A., Schullek, K. M., Fernandez, C. A. & Malkinson, experiment, the tumor response of the high dose of A. M. (1989) Carcinogenesis 10, 773-775. [2H3JBHTOOH was equivalent to a 2.5-fold lower dose ofthe 10. Mizutani, T., Nomura, H., Yamamoto, K. & Tajima, K. (1984) Toxicol. Lett. 23, 327-331. parent compound; these doses presumably generate equimo- 11. Mizutani, T., Nomura, H., Nakanishi, K. & Fujita, S. (1987) Toxicol. lar amounts of BHT-QM in the epidermis. Collectively, these Appl. Pharmacol. 87, 166-176. results provide explicit evidence that reactive intermediates, 12. Mizutani, T., Yamamoto, K. & Tajima, K. (1983) Toxicol. Appl. Phar- in general, and electrophiles, in particular, are directly in- macol. 69, 283-290. volved in tumor promotion. This observation broadens the 13. Yamamoto, K., Tajima, K. & Mizutani, T. (1980) Toxicol. Lett. 6,173-175. 14. Taffe, B. G., Zweier, J. L., Pannell, L. K. & Kensler, T. W. (1989) postulated roles for electrophiles in carcinogenesis from Carcinogenesis 10, 1261-1268. participants in initiation and progression to include all stages 15. Cerutti, P. (1985) Science 227, 375-381. of carcinogenesis. 16. Kensler, T. W. & Taffe, B. G. (1986) Adv. Free Radical Biol. Med. 2, Tumor promotion involves the selection and clonal expan- 347-387. 17. Becker, H.-D. (1969) J. Org. Chem. 34, 1203-1209. sion ofinitiated but preneoplastic cells from their surrounding 18. Miller, J. A. (1970) Cancer Res. 30, 559-576. normal counterparts. This effect on initiated cells may occur 19. Mizutani,T., Yamamoto, K. & Tajima, K. (1983) Toxicol. Appl. Phar- through the direct stimulation of mitogenesis or by the macol. 69, 283-290. indirect induction of compensatory proliferation (37). Nor- 20. Bhan, P., Soman, R. & Dev, S. (1980) Agric. Biol. Chem. 44, 1483-1488. 21. Kharasch, M. S. & Joshi, B. S. (1957) J. Org. Chem. 22, 1439-1443. mal cells have been shown to be more sensitive to the 22. Wand, M. D. & Thompson, J. A. (1986) J. Biol. Chem. 261,14049-14056. cytotoxic effects of tumor promoters than initiated cells (38). 23. Yuspa, S. H., Hawley-Nelson, P., Stanley, J. R. & Hennings, H. (1976) Further, by contrast with initiated cells, normal keratin- Transplant. Proc. 12S, 114-122. ocytes will undergo terminal differentiation and migrate out 24. Kozumbo, W. J., Seed, J. L. & Kensler, T. W. (1983) Cancer Res. 43, 2555-2559. of the basal layer of the skin in response to a proliferative 25. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254. stimulus. By either mechanism, the removal of growth inhi- 26. Rilegge, D. & Fischer, H. (1988) J. Chem. Soc. Faraday Trans. 1 84, bition provided by contact with normal cells will stimulate 3187-3205. initiated cells to undergo compensatory proliferation and 27. Omura, K. (1984) J. Org. Chem. 49, 3046-3050. 28. Kalyanaraman, B., Mottley, C. & Mason, R. P. (1983) J. Biol. Chem. expansion. 258, 3855-3858. The mechanism through which electrophiles mediate ini- 29. Filar, L. J. & Weinstein, S. (1960) Tetrahedron Lett. 25, 9-16. tiation and progression involves a direct genetic effect 30. Macomber, R. S. (1982) J. Org. Chem. 47, 2481-2483. through covalent interactions with DNA. The changes in 31. Valoti, M., Sipe, H. J., Jr., Sgaragli, G. & Mason, R. P. (1989) Arch. Biochem. Biophys. 269, 423-432. gene expression characteristic oftumor promotion, however, 32. Yumibe, N. P. & Thompson, J. A. (1988) Chem. Res. Toxicol. 1, 385-390. appear epigenetic in origin. Molecular targets subject to 33. O'Brien, T. G., Simsiman, R. C. & Boutwell, R. K. (1975) Cancer Res. covalent modification that can mediate this stage of carcino- 35, 2426-2433. genesis and, thus, a mechanism through which electrophiles 34. Taffe, B. G. & Kensler, T. W. (1988) Res. Commun. Chem. Pathol. Pharmacol. 61, 291-303. act in tumor promotion remain to be elucidated. The widely 35. Taffe, B. G., Takahashi, N., Kensler, T. W. & Mason, R. (1987) J. Biol. studied phorbol ester tumor promoters are thought to act Chem. 262, 12143-12149. through a receptor-mediated stimulation of second- 36. DiGiovanni, J., Kruszewski, F. H., Coombs, M. M., Bhatt, T. S. & messenger pathways, which ultimately leads to altered cell Pezechk, A. (1988) Carcinogenesis 9, 1437-1443. 37. Cohen, S. M. & Eliwein, L. B. (1990) Science 249, 1007-1011. proliferation and differentiation patterns in both initiated and 38. Farber, E. (1984) Cancer Res. 44, 4217-4223. noninitiated cells (39). Although an electrophile may also 39. Perchellet, J. P. & Perchellet, E. M. (1988) ISI Atlas of Science: Phar- mediate tumor promotion through the alteration of intracel- macology 2, 325-333. Downloaded by guest on September 30, 2021