Cellular Biochemical Determinants Modulating the Metabolism of

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Chem. Res. Toxicol. 1994, 7, 609-613

609

Cellular Biochemical Determinants Modulating the Metabolism of Estrone 3,4-Quinone1 Louise M. Nutter,*?tBin Zhou,? Esteban E. Sierra,? Yu-Ying Wu,ty2 Margaret M. RummelJp* Peter Gutierrez,§ and Yusuf Abul-Hajj$ Departments of Pharmacology and Medicinal Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, and University of Maryland Cancer Center, Department of Biological Chemistry, University of Maryland Medical School, Baltimore, Maryland 21201 Received November 17, 1993@

The metabolism of the o-quinone derivative of estrone, 3,4-estrone quinone (3,4-EQ), has been investigated in human breast cancer cells. Unlike the p-quinone, diethylstilbestrol 4',4"quinone, 3,4-EQ was not a substrate for the two-electron reduction catalyzed by the putative detoxifying enzyme, NAD(P)H:quinone reductase (DT diaphorase; DT D). Accordingly, the DNA damage induced by 3,4-EQ in human MCF-7 cells was not affected by a n inhibitor of DT D. Although 3,4-EQ was not an apparent substrate for the two-electron reduction catalyzed by DT D, this o-quinone was a substrate for the one-electron reduction catalyzed by cytochrome P450 reductase. The one-electron reduction of 3,4-EQ catalyzed by cytochrome P450 reductase occurred in the face of a significant and potentially physiologically relevant spontaneous reduction of 3,4-EQ by NADPH. The impact of purified superoxide dismutase (SOD) upon the production of hydrogen peroxide produced as a consequence of 3,4-EQ metabolism was evaluated; surprisingly, SOD inhibited the hydrogen peroxide produced by this o-quinone. Possible reasons for the SOD-mediated inhibition of redox cycling of 3,4-EQ are discussed. In summary, important differences in the metabolism of 3,4-EQ vis-a-vis o- and p- quinones have been observed, and the implications of these differences are discussed.

Introduction The carcinogenicity of estrogens has been extensively documented in rodent models (1)and has recently been recognized in humans (2-4). The carcinogenic action of estrogens is not correlated with their hormonal potencies ( 5 , 6 ) ;rather, it has been proposed that the metabolism of estrogens to reactive species with the potential to damage macromolecules is an important feature of their carcinogenicity (5). The metabolism of the estrogens (e.g., estradiol and estrone) has been proposed to proceed via a reduction-oxidation cycling (redox cycling) process wherein the production of reactive oxygen species (ROS),3 such as the semiquinone of the estrogens and the Fenton-Haber-Weiss-derived hydroxyl radical, have been proposed. Recently, the production of ROS, including hydrogen peroxide, estrone 3,4-semiquinone, and the hydroxyl radical, was demonstrated in human breast cancer cells treated with the estrogen, estrone 3,4quinone (3,4-EQ;37). The production of ROS in 3,4-EQtreated cells was correlated with the DNA damage engendered by this estrogen (7, 8).

* Correspondence should be addressed to this author.

Department of Pharmacology, University of Minnesota. t Department of Medicinal Chemistry, University of Minnesota. 8 University of Maryland Cancer Center. @Abstractpublished in Advance ACS Abstracts, July 15, 1994. "his work was supported by the National Institutes of Health, National Cancer Institute Grants 1R2952618, ROlCA57615, and ROlCA53491; L.M.N. is the recipient of a Junior Faculty Research Award JFRA 423 from the American Cancer Society. In partial fulfillment for the degree of Doctor of Philosophy. Abbreviations: 3,4-estrone quinone (3,4-EQ); NADPH:quinone oxidoreductase(DT D); superoxide dismutase (SOD); reactive oxygen species (ROS); diaziquone(AZQ);phenanthrene quinone (PQ);5,6,7,8tetrahydro-l,2-naphthoquinone (TNQ); 1,2-naphthoquinone (NQ); 2-methyl-l,4-napthoquinoneor menadione (MD); diethylstilbestrol (DES); diethylstilbestrol 4',4"-quinone (DES Q); dichlorophenolindophenol (DCPIP). +

The enzyme-catalyzed metabolism of the synthetic estrogen diethylstilbestrol (DES)3via various enzymes involved in the redox cycling process has been investigated previously, including quinone oxidoreductase (DT diaphorase; DT D),3 superoxide dismutase (SOD),3and cytochrome P450 reductase (9). Indeed, ROS production has been documented for DES and its quinone, diethylstilbestrol 4',4"-quinone (DES Q;39). However, there is no information regarding the metabolism of the estrogen quinone, 3,4-EQ, by the former enzymes. Elucidation of the role of these enzymes in the metabolism of 3,4-EQ is essential in order to formulate possible mechanisms of carcinogenesis relevant to oxidative stress. In particular, understanding the role of these enzymes in the metabolism of 3,4-EQ to a DNA-damagingspecies could be very informative vis-a-vis the tissue specificity of estrogen carcinogenesis. In this report, we present findings which point to significant differences in the metabolism of 3,4EQ as compared to DES Q and other o- and p-quinones; of particular note is the inability of DT D to reduce 3,4EQ and the potent inhibition of 3,4-EQ redox cycling by SOD.

Materials and Methods Materials. Caution: All quinones used in this study were handled under strict safety guidelines due to their potential as carcinogens. 3,4-EQ and tetrahydro-1,2-napthoquinone(TNQ)3 were synthesized as described previously (8, IO), and their authenticity was verified by NMR, IR, and UV-VIS spectrophotometry. Since these quinones are unstable, new compounds were synthesized monthly as needed and only used once after dissolution. Diaziquone (AZQ)3 was supplied by the Drug Synthesis and Chemistry Branch, Division of Cancer Treatment, NCI (Bethesda, MD). NADPH cytochrome P450 reductase [Le., specific activity of 848 pmol of cytochrome c reduced4minamg of protein)] was purified by the method of Omura and Takeshue (11). Phenanthrene quinone (PQ)3 and 1,2-naphthoquinone

Q893-228x/94I27Q7-Q609$04.5Ql~0 1994 American Chemical Society

Nutter et al.

610 Chem. Res. Toxicol., Vol. 7, No. 5, 1994 (NQ)3were obtained from the Aldrich Chemical Co. (Milwaukee,

WI). Menadione (MD),3 dichlorophenolindophenol (DCPIP),3 NADPH, cytochrome c, dicoumarol, and bovine erythrocyte SOD were obtained from the Sigma Chemical Co. (St. Louis, MO). [2-W]Thymidine (57 mCi/mmol) used in DNA damage studies was purchased from the ICN Corp. (Costa Mesa, CAI. MCF-7 human breast cancer cells were maintained in a humidified atmosphere a t 37 "C and 5% COZ in Roswell Park Memorial Institute 1640 medium containing 10% fetal calf serum. DNA Damage. The induction of single-strand DNA breaks in 3,4-EQ-treated MCF-7 cells was measured by the alkaline elution method a s described previously (8, 12). Purification of Rat Liver SOD. Rat liver C d Z n SOD was purified by ammonium sulfate fractionation followed by chromatography using gel filtration on a G-150 column, a DEAE column, and a phenyl-Sepharose column. Its identity was verified by microsequencing of a CnBr fragment (i.e., the amino terminus was blocked) of the purified SOD on PVDF membranes (Millipore Corp., Bedford, MA); this was performed a t the University of Minnesota, Institute of Human Genetics Sequencing Facility. SOD enzyme activity was measured using the cytochrome clxanthine-xanthine oxidase assay (13). Purification of DT Diaphorase (DT D). Rat liver homogenate (10 mM Tris, pH 7.5) from 500 mg of r a t liver was centrifuged a t 105 OOOg to obtain the cytosol fraction. The cytosol was fractionated using a 30-60% ammonium sulfate precipitation step prior to application on a G-150 gel filtration column (94-cm height, 2.5-cm diameter). Fractions containing high DT D activity were pooled and applied directly to a 1-mL dicoumarol affinity column prepared as described by others (14). DT D was eluted with 20 mM NADH in 10 mM Tris (pH 7.5) and 0.1 mM EDTA. Excess NADH was removed using a Centricon 3 microconcentrator. The purified protein in two separate preparations exhibited specific activities of 131 i 8.3 and 877 pmoY(minmg) using DCPIP a s a substrate. Enzyme Assays. DT D activity was measured spectrophotometrically (600 nm) using DCPIP a s a n electron acceptor and NADH a s reductant a s described by others (15).Authentic DT D activity was that activity which was inhibited by dicoumarol (10 pM). NADPH-cytochrome P450 reductase activity was measured spectrophotometrically (550 nm) using cytochrome c as a substrate (16). SubcellularFractionationand HzOz Assay. Isolations of mitochondria, microsomes, cytosol, and nuclei from MCF-7 cells were performed as described previously (7)according to the original protocol by others (17). Fractions were freshly prepared weekly, and their fidelity was verified using MD-induced HzOz production. H202 was measured using a colorimetric assay which employed xylenol orange as an indicator a s described previously (18).

02

I 1

I

I

5

7

FRACTON NUM8ER

Figure 1. Effect of dicoumarol on induction of single-strand DNA breaks by 3,4-EQ in MCF-7 cells. Cells were incubated with different concentrations of 3,4-EQ in the presence or absence of the DT D inhibitor, dicoumarol. Control (0);100 pM dicoumarol (M);50 pM 3,4-EQ (A); and 50 pM 3,4-EQ 100 pM dicoumarol (0).

+

Chart 1. Structures of Quinones Used in This Study: 3,4-EQ (11, PQ (2), TNQ (31, NQ (41, MD (51, and AZQ (6)

3.4-EO

Pa

63

d

0

NO TNO

Results In this study we sought to elucidate the mechanisms by which human cells metabolize estrogen quinones such as 3,4-EQ, culminating with the production of ROS. We selected several model quinones in addition to 3,4-EQ (1) as depicted in Chart 1; included in this group are two well-characterized p-quinones, menadione (MD; 5 ) and the antineoplastic agent diaziquone (AZQ; 6). Since the induction of single-strand (ss) DNA breaks by 3,4-EQ had been demonstrated (8) and such damage has been proposed to play a possible role in estrogen-induced mutagenesis (51,we investigated the possible role of DT diaphorase in this process. DT D has been shown to play an important role in the two-electron reduction of AZQ and MD to the corresponding dihydro derivatives; the subsequent oxidation back to the semiquinones and quinones culminates in the redox cycling of these agents and contributes to their DNA-damaging abilities and cytotoxicity (12, 19,201. As a first step toward investigating the role of DT D in 3,4-EQ metabolism, we evaluated the impact of inhibition of intracellular DT D

I

I

3

d

-

MD

8

3

AZO

on 3,4-EQ-induced DNA damage in the MCF-7 breast cancer cell line. As depicted in Figure 1,3,4-EQ (50 pM) induced significant ss DNA breaks in MCF-7 cells, and this damage was not affected by dicoumarol, an inhibitor of DT D. The concentration of dicoumarol used in this study (100pM) has been shown to significantly attenuate MD-induced DNA damage in MCF-7 cells (12);accordingly, MD is a good substrate for the two-electron reduction catalyzed by DT D as shown below (Table 1). Although the data depicted in Figure 1 were not suggestive of a role for DT D in cellular 3,4-EQ metabolism, unequivocal assignment of the role of DT D required purified enzyme. To this end, DT D was purified from rat liver and used in assays to measure NADPH oxidation in the presence of 3,4-EQ. As shown in Table 1, signifi-

Metabolism of Quinone Forms of Estrogen

Chem. Res. Toxicol., Vol. 7, No. 5, 1994 611

Table 1. Evaluation of 3,4-EQand Menadione as Substrates for Purified DT Diaphorasea condition

activity

NADPH (100 pM) NADPH (100 pM) DTD NADPH (100 pM) MD NADPH (100 pM) DTD MD NADPH (100 pM) DTD MD dicoumarol NADPH (10 pM) MD NADPH (10 pM) MD DTD 3,4-EQ 3,4-EQ NADPH (100 pM) 3,4-EQ NADPH (100 +M) DTD 3,4-EQ NADPH (10 pM) 3,4-EQ NADPH (10 pM) DTD 3,4-EQ NADPH (10pM) DTD dicoumarol

+ + + + + + + + +

+ + + + +

+ + +

NDb ND ND 14.1 x ND

+

ND 3.49 x ND 7.46 x 7.78 x 6.67 x 6.98 x 6.51 x

+

Ha02 Production by o-Quinonesin the

(5.2) (2.9) (4.8) (7.0) (2.4) (5.1)

Presence

of Purified DT Diaphorasea quinone AZQ (5) AZQ (5) AZQ (2) 3,4-EQ (4) 3,4-EQ (4) 3,4-EQ (2)

reducing agent NADPH NADPH NADPH NADPH NADPH NADPH

DTD

Yes boiled

-

Yes boiled

3,4-EQ @MI

HZOZ(UM)

3,4-EQ (UM)

HZOZ(UM)

1 5 10 25

NDb ND ND 2.8 f 0.35

75 200 500

5.8 f 1.16 14.7 f 1.22 59.3 f 4.95

(2.4)

The activity is expressed as mol of NADPH consumedmin; the values shown are the average of two measurements; the number in parentheses represents the range of values from the mean (in %). The final concentrations of MD (menadione) and 3,4-EQ were 25 pM; dicoumarol was present a t 10 pM; the specific activity of the purified DT diaphorase (10 ng) was 877 pmol of DCPIP reduced(min.mg of protein). ND denotes not detectable.

Table 2.

Table 3. Spontaneous Reduction of 3,4-EQ by NADPH Production of Hydrogen Peroxidea

HzOz OcM) 4.2 f 0.45 19.1 f 1.8 4 (0%) 13.88 f 1.4 14.3 f 1.5 12.3 (8.6%)

a AZQ and 3,4-EQ were present at 50 pM; NADPH and DTD were present at 500 pM and 15 ng, respectively. The specific activity of DT D was 131 pmoY(minmg of protein). The numbers in parentheses denote the number of times the measurements were performed. For experiments involving boiled DTD, the average and range (%) for two measurements are given. All other values represent the average f standard error.

cant oxidation of NADPH occurred in the presence of MD (i.e., MD was reduced) and purified DT D; that this activity was indeed DT D was verified by its complete inhibition by dicoumarol (Table 1). There was no detectable spontaneous reduction of MD by NADPH. In contrast to the results from studies of MD, significant spontaneous reduction of 3,4-EQ occurred in the presence of NADPH (Table 1). However, purified DT D was without effect on the NADPH oxidation rate; accordingly, dicoumarol had no impact upon the NADPH consumed during the reduction of 3,4-EQ (Table 1). The results presented in Table 1 indicate that 3,4-EQ is not a good substrate for DT D and that 3,4-EQ is spontaneously reduced by NADPH. It was important to corroborate the data depicted in Table 1 and to examine whether NADH was sufficient as a hydrogen source for the reduction of 3,4-EQ. We examined the ability of 3,4-EQ and the p-quinone AZQ to support H2Oz production using purified DT D and NADPH. As shown in Table 2, AZQ was not significantly reduced by NADPH in the absence of purified DT D as evidenced by very low levels of detectable HzOZ. Addition of 15 ng of purified DT D to AZQ and NADPH increased the levels of HzOz 4.5-fold, commensurate with previous findings by others that AZQ is indeed a substrate for DT D (19, 21). In contrast, incubation of 3,4-EQ with NADPH resulted in relatively high levels of H202 production (Le., more than %fold that from incubations of AZQ with NADPH), suggesting that an extensive spontaneous reduction of 3,4-EQ occurs in the absence of enzyme

a The data shown are averages f standard errors from triplicate measurements; NADPH was present at a final concentration of 2 mM. Incubations were for 30 min a t 37 "C. Denotes not detectable.

Table 4. Ha02 Production by Quinones and Purified NADPH-Cytochrome P450 Reductasea

-

quinone

condition control NADPH NADPH NADPH NADPH NADPH NADPH NADPH NADPH NADPH NADPH NADPH NADPH NADPH NADPH NADPH

HzOz (UM) NDb ND 61.7 f 4.9 6.0 16 f 5.3 37.3 f 3.2 28.0 30.0 f 2.7 77.0 f 4.4 35 16.3 f 10.1 97.0 f 2.6 19 48.7 f 2.1 94 f 1.7 55

50 pM AZQ P450 reductase 50 pM AZQ boiled P450 reductase 50 pM AZQ 50 pM 3,4-EQ P450 reductase 50 pM 3,4-EQ boiled P450 reductase 50 pM 3,4-EQ 50 mM TNQ P450 reductase 50 pM TNQ boiled P450 reductase 50 pM TNQ 5 pM PQ P450 reductase 5pM PQ boiled P450 reductase 5 PM PQ 5 PM NQ P450 reductase 5 pM NQ boiled P450 red jictase 5 W NQ The values shown represent averages f standard errors from three measurements except experiments involving boiled enzyme (n= 1).The amount of P450 reductase used was 5 pL of a purified preparation [Le., specific activity = 848 pmol of cytochrome c reduced(minng of protein)] as described under Materials and Methods. NADPH was present at a final concentration of 500 pM. ND denotes not detectable.

+ + + + + + + + + +

catalysis. Furthermore, 3,4-EQ was not a good substrate for DT D as indicated by the small increase in H202 production in the presence of DT D as compared to that detected in reactions containing only 3,4-EQ and NADPH (Table 2). We examined the dose response of the spontaneous reduction of 3,4-EQ by NADPH. As shown in Table 3, significant levels of spontaneous reduction of 3,4EQ commenced at approximately 74 pM 3,4-EQ in the presence of 2 mM NADPH. Collectively, the data presented in Figure 1and Tables 1 and 2 are not consistent with a role for DT D in the metabolism of 3,4-EQ; these data suggest that 3,4-EQ does not undergo a direct two-electron reduction to the catechol via DT D. The one-electron reduction of DES quinone by cytochrome reductases, such as cytochrome P450 reductase, has been documented previously (9). Thus, it was of central interest to determine whether 3,4EQ could serve as a substrate for this enzyme; oneelectron reductions of quinones could result in generation of the semiquinone and in turn the catechol forms of 3,4EQ, which could contribute to the redox cycling process culminating in ROS. Such ROS production would be evidenced by the production of H2Oz. In order to examine this possibility, we incubated purified cytochrome P450 reductase with NADPH and 3,4-EQ or other quinones and measured H2Oz production. As shown in Table 4, all of the five quinones tested were substrates for cytochrome P450 reductase. The two quinones AZQ and PQ (i.e., p-and o-quinones, respectively) produced the highest levels of Hz02 when incubated with cytochrome P450 reductase; however, it should not be inferred that these two quinones are the best substrates for the reductase since the facility of their oxidations back to the

612 Chem. Res. Toxicol., Vol. 7, No. 5, 1994

Table 5. Effect of Superoxide Dismutase on Ha02 Produced by 3,4-EQ in Subcellular Fractions treatment conditiona mitochondria mitochondria 50 pM 3,4-EQ mitochondria 50 pM 3,4-EQ SOD (C) mitochondria 50 pM 3,4-EQ SOD (R) mitochondria 100 pM 3,4-EQ mitochondria 100 pM 3,4-EQ SOD (C) mitochondria 100 pM 3,4-EQ SOD (R) microsomes microsomes 50 pM 3,4-EQ microsomes 50 ,uM 3,4-EQ SOD (C) microsomes 50 pM 3,4-EQ SOD (R) microsomes 100 pM 3,4-EQ microsomes 100 pM 3,4-EQ SOD (C) microsomes + 100 pM 3,4-EQ SOD (R)

+

+ + + + + + + + + +

+ + + +

+

+

+ +

H2Oz @MI 4.5 f 0.58 57.5 f 5.07 3.75 f 0.96 9 f 1.15 72.5 f 5.32 6.25 f 0.96 11.75 f 0.96 9.75 f 0.96 85.75 f 1.5 5.75 f 0.96 10 f 1.15 129 f 4.76 7.25 f 0.5 11.25 f 0.96

SOD (C)denotes commercially obtained bovine erythrocyte SOD and SOD (R)denotes SOD purified from rat liver; 100 ng of SOD and 5 pg of mitochondria or microsomes were used in these experiments. NADPH was present at a final concentration of 2 mM; values are average f standard errors from four measurements.

semiquinone and quinone (after reduction) species could, theoretically, influence the production of H202. That is, the relative facility of the oxidization of the dihydro- and semiquinone forms of these agents to the semiquinones and quinones, respectively, will contribute to the ultimate levels of H202 produced in this assay since it is a redox cycling process. Hence, we conclude that all of the quinones tested are substrates for cytochrome P450 reductase, albeit to differing degrees. Since 3,4-EQ was spontaneously reduced by NADPH, obtaining a meaningvalue for the reaction of 3,4-EQ with cytochrome ful K,,, P450 reductase by measuring NADPH oxidation was not feasible due to nonlinear kinetic^.^ Having addressed the one- and two-electron reductions of 3,4-EQ, we initiated studies to elucidate the role of SOD in the production of ROS by 3,4-EQ. We reasoned that, a priori, the action of SOD would increase the levels of H202 produced by 3,4-EQ metabolism due to the dismutation of superoxide. In order to address this issue, we purified SOD from rat liver and evaluated the impact of SOD enzyme upon the H202 produced by 3,4-EQ when the latter was incubated with mitochondrial and microsomal fractions isolated from MCF-7 cells. Contrary to an expected increase in the level of H202 due to superoxide dismutation, we observed a virtually complete abrogation of H202 in 3,4-EQ-treatedcells in the presence of rat liver SOD (Table 5). Similar results were obtained when bovine erythrocyte SOD from commercial sources was used in these studies (Table 5). These seemingly enigmatic data with respect to the role of SOD in 3,4EQ metabolism are discussed ahead.

Discussion Hormonal carcinogenesis vis-a-vis estrogen has been documented in rodents in a laboratory setting and is a potentially important oncological issue in humans. Clearly, it is essential to understand the metabolism of the estrogens in order to elucidate the mechanism(s) operating behind their carcinogenic potential. In this study, the metabolism of estrogen quinones has been investigated, and the data reveal some unexpected findings. First, the results from several experiments suggest that 3,4-EQ is not a substrate for DT D. Dicoumarol, an inhibitor of DT D, had no impact upon 3,kEQ-induced Nutter et al., unpublished results.

Nutter et al.

single-strand DNA breaks in MCF-7 cells (Figure 1). Furthermore, although purified DT D did enhance the oxidation of NADPH in the presence of menadione, it was without effect when incubated with 3,4-EQ (Table 1). Accordingly, dicoumarol had no impact upon the spontaneous oxidation of NADPH in the presence of 3,4-EQ (Table 1). Corroborative data demonstrating that 3,4-EQ is not a substrate for DT D were obtained in H202 experiments where purified DT D had no effect upon 3,4-EQ-induced peroxide production; under similar conditions, a 4.5-fold enhancement of the H202 produced by the quinone AZQ was observed in the presence of purified DT D (Table 2). The latter results are consistent with findings by others where AZQ-induced DNA damage was examined in the presence and absence of dicoumarol (20). Collectively, these studies clearly show that 3,4-EQ is not a substrate for the two-electron reduction catalyzed by DT D. Similar findings were recently reported for the metabolism of polycyclic aromatic hydrocarbon o-quinones by DT D (22); DT D offered no protection against the cytotoxicity of these carcinogens. In contrast, thep-quinone DES Q has been convincingly shown to be reduced by DT D (9); it has been suggested that DT D provides a protective role vis-a-vis DES Q by preventing the ROS formed from DES Q redox cycling (9). Our data show that 3,4-EQ is reduced equally well by either NADPH or NADH.4 As suggested previously (71, the spontaneous reduction of 3,4-EQ by NADPH could be physiologicallyrelevant since the concentrations of NADPH used in our studies are commensurate with those reported previously for MCF-7 cells (23). The one-electron reduction of DES Q by cytochrome P450 reductase has been postulated to result in DES semiquinone formation; indeed, inhibition of cytochrome P450 reductase has been shown to engender a decrease in superoxide production in microsomal fractions incubated with this p-quinone (9). The role of the oneelectron reduction of the polycyclic aromatic hydrocarbon o-quinones has been inferred but not demonstrated (22). Using purified cytochrome P450 reductase and 3,4-EQ, we demonstrated an increase in the oxidation of NADPH over that measured in extracts of 3,4-EQ and NADPH alone; however, attempts to determine a K, value for this reduction were not successful due to nonlinear kinetics at higher 3,4-EQ concentrations resulting from a significant spontaneous reduction of 3,4-EQby NADPH (Table 3). We examined the ability of 3,4-EQ and several other quinones to generate H202 due to their redox cycling in the presence of NADPH with or without purified cytochrome P450 reductase. Of all the quinones examined, thep-quinone AZQ (i.e., 6 in Chart 1)produced the most peroxide in the presence of purified reductase as compared to boiled reductase controls (i.e., -10-fold versus boiled controls; Table 4); the p-quinone AZQ had a very low level of spontaneously produced peroxide in the presence of NADPH (Tables 2 and 4). In contrast, the o-quinones 3,4-EQ, TNQ, PQ, and NQ (i.e., 1,3,2,and 4 in Chart 1)produced significant levels of peroxide from spontaneous reductions by NADPH; the levels of peroxide were enhanced 2-6-fold in the presence of purified reductase (Table 4). The highest levels of reductasemediated peroxide production were observed with PQ (Le., 2, in Chart 1). The differences in peroxide production produced by the model o-quinones in comparison to 3,4-EQ are enigmatic, and studies are underway to address these differences with respect to the type of DNA damage they incur in human cells. In summary, this

Metabolism of Quinone Forms of Estrogen

aspect of the present study indicates that all of the o-quinones are substrates for cytochrome P450 reductase as evidenced by enhanced NADPH oxidation and hydrogen peroxide production in the presence of purified enzyme. During the course of a previous study we detected the presence of a potent inhibitor of the hydrogen peroxide produced by redox cycling of 3,4-EQ in the cytosol of MCF-7 cells (7). We purified this protein from rat liver and performed microsequencing of tryptic digests of the protein. A search of the NBRF Protein Database (new PIR) library revealed 75% homology (12 amino acids) to Cu-Zn SOD of rat and mouse. This result was initially surprising since SOD has been conventionally thought to convert superoxide to hydrogen peroxide; thus, a priori, one would expect to see increased levels of peroxide in the presence of SOD and a redox cycling quinone such as 3,4-EQ. However, as shown in Table 5, this is clearly not the case; purified rat liver SOD and commercially available bovine SOD virtually abrogated the 3,4-EQinstigated peroxide production in mitochondria and microsomes (Table 5). Furthermore, we have observed that SOD can attenuate the peroxide production by the nonenzymatic reduction of 3,4-EQ by NADPH.4 This apparently paradoxical role of SOD has been noted by others (24-28); one group of investigators have suggested that the ability of SOD to inhibit or accelerate autoxidation depends in part upon the reduction potential of the quinone in question and its accessibility for metal coordination (28). Others have suggested that SOD may act as a super0xide:semiquinone oxidoreductase, which could account, under some circumstances, for the inhibition of hydrogen peroxide production by redox cycling quinones (24). Further studies will be necessary to address this issue vis-a-vis 3,4-EQ. In summary, the ability of 3,4-EQ or its metabolites to damage DNA by a redox cycling process has been documented in an acute situation (i.e., refs 7 and 8 and the present paper). Some of the biochemical determinants modulating the metabolism of 3,4-EQ, including cytochrome P450 reductase and SOD, have been characterized. It is not known whether these entities play a role in humans treated chronically with the estrogens; accordingly, further studies are necessary. Other potential cellular determinants in addition to those examined in this study, such as glutathione and catechol omethyltransferase, deserve attention in this respect and are currently under study. Furthermore, the roles of metals in the 3,4-EQ-mediated formation of reactive oxygen species such as the hydroxyl radical (7) could be very important with respect to understanding 3,4-EQinduced DNA damage and mutagenicity and are under investigation.

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