- Email: [email protected]
Polycyclic Aromatic Hydrocarbon Quinone-Mediated Oxidation Reduction Cycling Catalyzed by a Human Placental 17β-Hydroxysteroid Dehydrogenase
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
Vol. 327, No. 1, March 1, pp. 174–180, 1996 Article No. 0106
Polycyclic Aromatic Hydrocarbon Quinone-Mediated Oxidation Reduction Cycling Catalyzed by a Human Placental 17b-Hydroxysteroid Dehydrogenase1 Rebecca Jarabak, Ronald G. Harvey,* and Joseph Jarabak2 Department of Medicine and the *Ben May Institute, The University of Chicago, Chicago, Illinois 60637
Received September 19, 1995, and in revised form December 15, 1995
The human placental 17b-hydroxysteroid dehydrogenase reduces a number of polycyclic aromatic hydrocarbon (PAH) o-quinones; some of the quinones undergo redox cycling at rates that approach or exceed the rate of reduction of estrone by the enzyme. The non-K-region o-quinone, 7,8-benzo[a]pyrenequinone, is the best o-quinone substrate tested. Cycling of all the quinone substrates is inhibited by superoxide dismutase; cycling is also inhibited by 17b-estradiol and other estrogens. Since 17a-estradiol is a competitive inhibitor of both the oxidation of 17b-estradiol and the cycling of 9,10-phenanthrenequinone by the 17b-hydroxysteroid dehydrogenase, it is likely that both reactions occur at the same active site on the enzyme. In the presence of the 17b-hydroxysteroid dehydrogenase, the equilibrium between 17b-estradiol, estrone, NADP, and NADPH is shifted by 7,8-benzo[a]pyrenequinone because the rapid redox cycling of this quinone results in the oxidation of NADPH. Unlike a number of hydroxysteroid dehydrogenases, the placental 17b-hydroxysteroid dehydrogenase does not oxidize any of the six PAH trans-dihydrodiols tested. q 1996 Academic Press, Inc.
Key Words: Redox cycling; 17b-hydroxysteroid dehydrogenase; PAH quinones; superoxide; superoxide dismutase; estrogens; dihydrodiol dehydrogenase.
The toxicity of some quinones results from their ability to alkylate nucleic acids, proteins, or lipids, or from the reactive oxygen species formed during their oxidation-reduction (redox) cycling (1). The one-electron re-
duction of quinones leads to their redox cycling, and one-electron reductases, such as the NADPH:cytochrome P450 oxidoreductase, have a broad substrate specificity for polycyclic aromatic hydrocarbon (PAH)3 quinones (2). Recent studies have shown that two-electron reduction of a number of naphthoquinones and PAH o-quinones leads to the redox cycling of these compounds. The quinone reductases which catalyze these reactions include DT-diaphorase (3–9), carbonyl reductase (8, 10–12), NAD-linked 15-hydroxyprostaglandin dehydrogenase (13), and 3a-hydroxysteroid dehydrogenase (14, 15). Unlike the one-electron reductases, these twoelectron reductases show a more limited substrate specificity. Of the quinones examined, PAH o-quinones are the best substrates for these enzymes. PAH quinones that are not o-quinones (e.g., 3,6-benzo[a]pyrenequinone, 3,6-pyrenequinone, 9,10-anthracenequinone, and 7,12-benz[a]anthracenequinone) are poor substrates or are not substrates for these enzymes (8). Both 3a-hydroxysteroid dehydrogenases (16–18) and 17b-hydroxysteroid dehydrogenases (19, 20) oxidize benzene dihydrodiol. As a consequence, these enzymes are also called dihydrodiol dehydrogenases. The rat liver 3a-hydroxysteroid dehydrogenase, in addition to oxidizing benzene dihydrodiol, has been shown to oxidize PAH trans-dihydrodiols. The importance of this observation is that this enzyme, and perhaps other hydroxysteroid dehydrogenases, may alter the toxicity of these proximate carcinogens by converting them to oquinones. In view of the observations noted here, the present Abbreviations used: HSD, hydroxysteroid dehydrogenase; O2•0, superoxide radical; SOD, superoxide dismutase; EDTA, ethylenediaminetetraacetate; 17a-E2 , 17a-estradiol; 17b-E2 , 17b-estradiol; 17a-EE2 , 17a-ethinyl estradiol; DES, diethylstilbestrol; PAH, polycyclic aromatic hydrocarbon; Q, quinone; Q•0, semiquinone; QH0, hydroquinone. 3
1
This work was supported by NIH Grants ES 06797 (J.J.) and ES 04732 (R.G.H.) and American Cancer Society Grant CN-22 (R.G.H.). 2 To whom correspondence and reprint requests should be addressed at Department of Medicine, M.C. 1027, 5841 S. Maryland Avenue, Chicago, IL 60637. Fax: (312) 702-9194. 174
0003-9861/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
/ 6b13$$9326
01-29-96 19:25:02
arca
AP: Archives
175
QUINONE-MEDIATED REDOX CYCLING
study was undertaken to determine whether either PAH o-quinones or PAH trans-dihydrodiols are substrates for the human placental 17b-hydroxysteroid dehydrogenase. Two major human 17b-hydroxysteroid dehydrogenases have been described. These differ in both their location within the cell and their substrate specificities (21). The 17b-hydroxysteroid dehydrogenase used in the present study is called type I; it is cytoplasmic and catalyzes the interconversion of 17b-estradiol and estrone much better than that of testosterone and androstenedione. The 1.3-kb mRNA for the type I enzyme has been identified in placenta, ovary, breast (normal and malignant), endometrium, skin, prostate (normal and malignant), and adipose tissue (22–24). The type I 17b-hydroxysteroid dehydrogenase used in this study has been purified to apparent homogeneity from human placenta (25). It has dual pyridine nucleotide specificity; 17b-estradiol and estrone are its best steroid substrates (25). This report describes the two-electron reduction and redox cycling of PAH o-quinones by this enzyme. Among other observations reported here are the unusual substrate specificity of this enzyme and the effect of the redox cycling of o-quinones on the 17b-estradiol _ estrone equilibrium controlled by the enzyme.
path, and 3-ml reaction mixtures were used. Assays were performed in duplicate or triplicate and rates are reported as the mean { the standard deviation. The cuvettes used for assays of redox cycling contained 0.2 ml dimethyl sulfoxide to which quinones and estrogens (dissolved in either 95% ethanol or DMSO) were added. This was followed by additions of water, 10 mmol of potassium phosphate, pH 7.0, 10 nmol of EDTA and 225 nmol of NADPH. Enzyme was omitted from the blank. The reactions were started by the addition of enzyme, and the rate of NADPH oxidation was determined by measuring the decrease in absorbance at 340 nm. The rate of redox cycling is the rate of NADPH oxidation that follows the initial reduction of the quinone. The cuvettes used for the dehydrogenase assays contained 0.2 ml dimethyl sulfoxide to which potential substrates (dissolved in either 95% ethanol or DMSO) were added. This was followed by additions of water, 270 mmol of Tris, pH 9.0, 10 nmol of EDTA, and either 1.36 mmol of NAD or 1.1 mmol of NADP. Enzyme was omitted from the blank. The reactions were started by the addition of enzyme, and the rate of pyridine nucleotide reduction was measured by the increase in absorbance at 340 nm.
RESULTS AND DISCUSSION
The two-electron reduction of a quinone by a quinone reductase produces the corresponding hydroquinone. The hydroquinone product may react to give the semiquinone and the superoxide radical, O•20, by several nonenzymatic mechanisms (35), including autoxidation: QH0 / O2 _ Q•0 / H/ / O•0 2
EXPERIMENTAL PROCEDURES Materials. Superoxide dismutase, NADPH, NADH, (S)-(/)-1indanol, 1,3,5(10)-estratrien-3,17a-diol (17a-estradiol, 17a-E2), and 5a-androstan-17b-ol-3-one were purchased from Sigma, EDTA from J.T. Baker, and Chelex 100 (100 – 200 mesh, sodium form) from Bio-Rad. NAD and NADP were obtained from Pharmacia Biotechnology, Inc., 4-androsten-17b-ol-3-one (testosterone), 5a-androstan-3a-ol-17-one, 5a-androstan-3b-ol-17-one, 1,3,5 (10)-estratrien-3, 17b-diol (17b-estradiol, 17b-E2), 1,3,5(10)-estratrien-17a-ethinyl-3,17b-diol (17a-ethinyl estradiol, 17a-EE2), and 3,4-bis-(4-hydroxyphenyl)-3-hexene (diethylstilbestrol, DES) from Steraloids, 9,10-phenanthrenequinone from Eastman, and 1-acenaphthenol from Aldrich. The remaining polycyclic aromatic hydrocarbon quinones (26, 27) and trans-dihydrodiols (26, 28 – 32) used in this study were synthesized by methods reported previously. Inorganic salts and dimethyl sulfoxide were the best commercially available analytic reagents. Solutions were prepared in laboratory distilled water which had been deionized with an Ionpure mixed bed resin. Stock solutions of phosphate buffer were treated batchwise with Chelex 100 resin to remove trace metal ions (33). Enzymes. The human placental 17b-hydroxysteroid dehydrogenase was prepared as described previously (25). It had an activity of 5.1 U/mg of protein when assayed with 17b-estradiol and NAD as described below. The human placental carbonyl reductase (34) and the rat liver DT-diaphorase (8) were purified as described previously. Their activities were 62 and 30 U/mg of protein, respectively, when assayed as described (8). One unit of enzyme catalyzes either the reduction of 1 mmol of NAD/min (17b-HSD) or the oxidation of 1 mmol of NADPH/min (carbonyl reductase and DT-diaphorase) under the assay conditions. Enzyme assays. The assays were performed in a Gilford recording spectrophotometer at 25 { 0.57C. Reaction cuvettes had a 1-cm light
/ 6b13$$9326
01-29-96 19:25:02
arca
[1]
The superoxide initiates a free radical chain reaction which regenerates the quinone (36): •0 H/ / QH0 / O•0 / H2O2 2 _ Q
Q
•0
•0 2
/ O2 _ Q / O
[2] [3]
The quinone is then reduced again by the enzyme; this alternate reduction and oxidation has been called redox cycling. The rate of reaction [1] is greatly influenced by the reactivity of the hydroquinone (9). For some hydroquinones this reaction is sufficiently rapid that twoelectron reduction of the quinone is followed by redox cycling. Redox cycling is of particular interest since it generates reactive oxygen species, e.g., O•0 2 and hydrogen peroxide, and thus contributes to oxidative stress. Lorentzen and Ts’o were the first to suggest that the toxicity of PAH metabolites might be related to the reduction of PAH quinones to hydroquinones followed by their redox cycling (37). Studies in this laboratory and by others have shown that two-electron reduction of a number of naphthoquinones and PAH o-quinones by several highly purified quinone reductases including DT-diaphorase (3–9), carbonyl reductase (8, 11, 12), NAD-linked 15-hydroxyprostaglandin dehydrogenase (13), and 3a-hydroxysteroid dehydrogenase (14, 15) causes the redox cycling of these quinones and the formation of reactive oxygen species. These studies have
AP: Archives
176
JARABAK, HARVEY, AND JARABAK TABLE I
Redox Cycling of PAH Quinones by 17b-Hydroxysteroid Dehydrogenase, Carbonyl Reductase, and DT-diaphorasea Rate of Cycling (nmol NADPH oxidized/min) 17b-HSDb
Quinone 9,10-Phenanthrenequinone 5,6-Chrysenequinone 3,4-Benz[a]anthracenequinone 5,6-Benz[a]anthracenequinone 4,5-Benzo[a]pyrenequinone 7,8-Benzo[a]pyrenequinone
0.97 0.72 0.53 2.4 1.9 6.2
{ { { { { {
0.029 0 0.058 0.029 0.029 0.058
Carbonyl reductase 5.4 4.2 0 2.6 1.6 0.86
{ 0.082 { 0.17 { 0.14 { 0.082 { 0.058
DT-diaphorase 3.8 3.4 0 2.8 1.6 0
{ 0.14 { 0.029 { 0.11 { 0.072
a
The contents of the reaction mixtures are the same as described under Experimental Procedures; 15 nmol of each quinone was assayed. The reactions were started with the addition of 14 mU of 17b-hydroxysteroid dehydrogenase, 27 mU of carbonyl reductase, or 24 mU of DT-diaphorase. For purposes of comparison, if 150 nmol of estrone is added rather than a PAH o-quinone, the rate of NADPH oxidation by 14 mU of 17b-hydroxysteroid dehydrogenase is 4.3 nmol/min. b None of the following quinones is a substrate for the 17b-HSD: 3,6-benzo[a]pyrenequinone, 3,6-pyrenequinone, 9,10-anthracenequinone, or 7,12-benz[a]anthracenequinone. Fifteen nmol of each quinone was assayed.
demonstrated that the enzyme-catalyzed cycling exhibits the following characteristics: (1) in the presence of catalytic quantities of the quinone, a very large initial excess of the reduced pyridine nucleotide is completely oxidized (11, 13, 15, 37); (2) there is O2 uptake (1, 4, 14) and the production of O•0 and H2O2 during the 2 cycling reaction (5, 6, 12, 13, 37); and (3) the addition of superoxide dismutase (SOD) inhibits the pyridine nucleotide oxidation, O2 uptake, and H2O2 production (6–8, 12, 13). The cytoplasmic human 17b-hydroxysteroid dehydrogenase catalyzes the following reaction: 17b-estradiol / NAD(P)/ _ estrone / NAD(P)H / H/ [4] The enzyme plays an important role in estrogen metabolism in a number of different tissues (22, 23, 38), and the enzyme from human placenta is well characterized (25, 39–43). The work presented here shows that this steroid dehydrogenase is also an efficient reductase of certain PAH o-quinones, such as 9,10-phenanthrenequinone: 9,10-phenanthrenequinone / NAD(P)H / H/r 9,10-phenanthrenehydroquinone / NAD(P)/ [5] Although the oxido-reduction of 17b-estradiol and estrone by this enzyme is more rapid in the presence of NAD and NADH than NADP and NADPH (39), the reduction of PAH o-quinones is more rapid in the presence of NADPH than NADH (data not shown). Table I compares the rates of redox cycling of six PAH o-quinones catalyzed by the 17b-hydroxysteroid dehydrogenase, carbonyl reductase, and DT-diapho-
/ 6b13$$9326
01-29-96 19:25:02
arca
rase. The relative rates of cycling vary considerably depending on the enzyme and quinone substrate used. The results in Table I suggest that the carbonyl reductase and DT-diaphorase cycle K-region o-quinones (e.g., 9,10-phenanthrenequinone) more rapidly than non-Kregion o-quinones (e.g., 7,8-benzo[a]pyrenequinone). The specificity of the 17b-hydroxysteroid dehydrogenase does not fit this pattern; in fact, 7,8-benzo[a]pyrenequinone is the best o-quinone substrate tested for this enzyme. The rate of cycling of some of the quinones by the 17b-hydroxysteroid dehydrogenase is not very different from the rate of reduction for a saturating concentration of estrone, one of the putative natural substrates for the enzyme (see Table I, footnote a). As shown in the Table, the rate of cycling for 7,8-benzo[a]pyrenequinone by the enzyme exceeds the rate of estrone reduction. In addition to the oxidation of NADPH which occurs during redox cycling there is also O2 consumption and H2O2 production. Although O2 consumption and H2O2 production were not measured in this study, H2O2 production was measured previously with the carbonyl reductase and four of the quinones tested here (12). The H2O2 produced/NADPH oxidized was 0.76, 0.81, 0.71 and 0.84 for 9,10-phenanthrenequinone, 5,6-chrysenequinone, 4,5-benzo[a]pyrenequinone, and 7,8-benzo[a]pyrenequinone, respectively. Recently Flowers-Geary et al. (44) have concluded that PAH o-quinones are poor substrates for enzymic two-electron reduction in rat liver cytosol. This conclusion was based on experiments using a particular enzyme (DT-diaphorase) and a particular set of PAH oquinones (non-K-region). The data presented in Table I indicate that this conclusion may not apply to various other enzyme-substrate pairs in other tissues.
AP: Archives
QUINONE-MEDIATED REDOX CYCLING
177
The enzyme superoxide dismutase destroys O•0 by 2 catalyzing the following reaction (45): 2 H/ / 2 O•/ 2 r H2O2 / O2
FIG. 1. Redox cycling of 9,10-phenanthrenequinone and 7,8-benzo[a]pyrenequinone by the 17b-hydroxysteroid dehydrogenase. (a) Data from experiments in which 9,10-phenanthrenequinone was the substrate. The contents of the assay cuvettes are the same as described under Experimental Procedures; 15 nmol of 9,10-phenanthrenequinone were added. The reaction mixtures contain no SOD (—) or 0.3 mM SOD (---). Arrowheads indicate the addition of 70 mU of 17 b-hydroxysteroid dehydrogenase. The plots show absorbance at 340 nm as a function of time. The recorder offset was adjusted so that traces would not overlap. (b) Data from experiments in which 7,8-benzo[a]pyrenequinone was the substrate. The reaction mixtures in b are the same as for a except that 15 nmol of 7,8-benzo[a]pyrenequinone was added instead of 9,10-phenanthrenequinone. Arrowheads indicate the addition of 14 mU of 17b-hydroxysteroid dehydrogenase.
Figure 1 shows the time course of NADPH oxidation during the redox cycling of 9,10-phenanthrenequinone (a) and 7,8-benzo[a]pyrenequinone (b) by the 17bhydroxysteroid dehydrogenase. Figures 1a (—) and 1b (—) show traces for reaction mixtures containing quinone, NADPH, and enzyme. In each case initial reduction of the quinone is followed by its redox cycling. When 9,10-phenanthrenequinone is the quinone substrate (Fig. 1a,—) oxidation of NADPH is linear with time, i.e., the rate of cycling is as rapid as the initial reduction of the quinone to the hydroquinone. In the case of 7,8-benzo[a]pyrenequinone (Fig. 1b,—) the initial rate of NADPH oxidation (the rate of quinone reduction) is more rapid than the rate of cycling; therefore a change of slope is observed after the initial reduction of the quinone. Thus the reduction of the quinone by the enzyme is the rate-limiting step in the redox cycling of 9,10-phenanthrenequinone while autoxidation of the hydroquinone is rate limiting for 7,8benzo[a]pyrenequinone. For both quinones, cycling continues until all of the NADPH is oxidized (not shown) despite the fact that the initial concentration of the NADPH greatly exceeds that of the quinone.
/ 6b13$$9326
01-29-96 19:25:02
arca
[6]
SOD inhibits the cycling of 9,10-phenanthrenequinone (Fig. 1a, ---) and 7,8-benzo[a]pyrenequinone (Fig. 1b, ---). As seen most clearly with 9,10-phenanthrenequinone (by comparing — and --- in Fig. 1a), SOD does not affect the enzymatic reduction of the quinone; instead, it inhibits autoxidation of the hydroquinone. There is a slow but measurable rate of NADPH oxidation following the initial reduction of both quinones. Presumably this results from autoxidation of the hydroquinones (reaction [1]) or from other nonenzymatic reactions of the hydroquinones to yield the corresponding semiquinones (35). The striking effect of SOD indicates that O•0 plays an important role in the redox 2 cycling of these quinones. The results obtained for the K-region o-quinones 5,6chrysenequinone, 5,6-benz[a]anthracenequinone, and 4,5-benzo[a]pyrenequinone (not shown) are similar to those shown for 9,10-phenanthrenequinone in Fig. 1a. The enzymic reduction of these quinones is the ratelimiting step in their cycling, and cycling is markedly inhibited by SOD. Table II shows the effect of 17b-estradiol and several other estrogens on the rate of cycling of PAH o-quinones catalyzed by the 17b-hydroxysteroid dehydrogenase. With only one exception each of these compounds inhibits the cycling of all of the quinones listed. When cycling of the quinones is catalyzed by the carbonyl reductase, none of these estrogens is an inhibitor (data not shown). While the results in Table II show that a number of estrogens inhibit the cycling of PAH o-quinones catalyzed by the 17b-hydroxysteroid dehydrogenase, they do not establish the nature of the inhibition. In order to do this, the effect of one estrogen on the cycling of one quinone was examined in more detail. Figure 2 shows that 17a-estradiol is a competitive inhibitor of the redox cycling of 9,10-phenanthrenequinone catalyzed by the 17b-hydroxysteroid dehydrogenase. A Dixon plot (not shown) gave a Ki of 2.8 mM. In an earlier study it was reported that 17a-estradiol is also a competitive inhibitor (Ki 4.5 mM) of the oxidation of 17bestradiol by this enzyme (reaction [4]) (46). The simplest explanation for the observation that 17a-estradiol is a competitive inhibitor of both the cycling of 9,10phenanthrenequinone and the oxidation of 17b-estradiol is that both reactions occur at the same active site on the enzyme. Since the 17b-hydroxysteroid dehydrogenase is thought to have a role in regulating the tissue concentrations of 17b-estradiol (47), the presence of sub-
AP: Archives
178
JARABAK, HARVEY, AND JARABAK TABLE II
Inhibition by Estrogens of Quinone-Mediated Redox Cyclinga,b (17b-Hydroxysteroid Dehydrogenase) % Inhibition of Cycling Quinone
17b-Ec2
17a-E2
17a-EE2
DES
9,10-Phenanthrenequinone 5,6-Chrysenequinone 5,6-Benz[a]anthracenequinone 4,5-Benzo[a]pyrenequinone 7,8-Benzo[a]pyrenequinone
77 60 60 78 48
82 81 80 88 22
66 41 50 63 0
58 56 31 78 38
a The contents of the reaction cuvettes were the same as described under Experimental Procedures. The control mixtures contained 15 nmol of quinone and no estrogen; the test mixtures contained 15 nmol of quinone and 60 nmol of estrogen. The reactions containing 7,8benzo[a]pyrenequinone were started with the addition of 7 mU of 17b-hydroxysteroid dehydrogenase while 70 mU of enzyme were used to start the reactions of the other quinones. b 17a-estradiol is only weakly estrogenic. It was included in these studies because it is an epimer of 17b-estradiol and a competitive inhibitor of the oxidation of 17b-estradiol by the 17b-hydroxysteroid dehydrogenase. c Because the rate of redox cycling is measured by the oxidation of NADPH, it is likely that 17b-estradiol is a less effective inhibitor than these data suggest. Since 17b-estradiol is a substrate for the enzyme, NADP formed during cycling will be reduced to NADPH in the presence of this steroid. Thus the net rate of NADPH oxidation will be slower than that of cycling.
stances that shift the equilibrium of the reaction it catalyzes may have important physiologic consequences. The following set of experiments was done to examine the effect of PAH o-quinones on this equilibrium. Figure 3a shows the time course of NADPH produc-
FIG. 2. Double reciprocal plots showing inhibition of the redox cycling of 9,10-phenanthrenequinone by 17 a-estradiol. The contents of the assay cuvettes are the same as described under Experimental Procedures except that the concentration of 9,10-phenanthrenequinone (9,10-PQ) was varied, and some cuvettes contained no 17aestradiol (l) while others contained 5 mM 17a-estradiol (s). Reactions were started with the addition of 70 mU of 17b-hydroxysteroid dehydrogenase. The velocity is DA340/10 min. The intersection of the plots on the ordinate shows that 17a-estradiol is a competitive inhibitor of the cycling.
/ 6b13$$9326
01-29-96 19:25:02
arca
tion after the addition of 17b-hydroxysteroid dehydrogenase at t Å 0 to a buffered solution of 17b-estradiol and NADP. The absorbance of NADPH at 340 nm increases as the 17b-estradiol is oxidized until an equilibrium is reached between the oxidized and reduced forms of both the steroid and the pyridine nucleotide. The Keq for reaction [4] at pH 7 is 0.18 (25). Figure 3b shows the effect of adding 7,8-benzo[a]pyrenequinone to the reaction mixture. The absorbance at 340 nm increases after the addition of enzyme until it reaches a maximum and then decreases to the baseline. Presumably the NADPH formed by oxidation of 17b-estradiol is used by the enzyme to reduce the 7,8benzo[a]pyrenequinone, and the quinone begins to cycle. This rapid cycling has a marked effect on the trace of A340 vs time. The cycling continues until all of the nucleotide is present as NADP. At this point the concentrations of 17b-estradiol and estrone will not be the same as they are in the equilibrium mixture of Fig. 3a; instead, the 17b-estradiol concentration will be lower and the estrone concentration higher. If SOD is present in the mixture containing 7,8benzo[a]pyrenequinone, 17b-estradiol, NADP, and enzyme (Fig. 3c), redox cycling of the quinone is inhibited. As a result, the trace of A340 vs time is only slightly different from that shown in Fig. 3a. When 9,10-phenanthrenequinone is present in the reaction cuvette with 17b-estradiol, NADP, and enzyme (Fig. 3d), again, the trace of A340 vs time is not very different from that in Fig. 3a. From the data in Tables I and II it is likely that the cycling of 9,10phenanthrenequinone is much slower than that of 7,8benzo[a]pyrenequinone in these reaction mixtures. Thus, the plots in Figure 3 suggest that the rapid
AP: Archives
179
QUINONE-MEDIATED REDOX CYCLING
FIG. 3. Influence of 7,8-benzo[a]pyrenequinone and 9,10-phenanthrenequinone on NADPH concentration during enzymic oxidation of 17b-estradiol. Each cuvette contained water, 0.2 ml of dimethyl sulfoxide, 10 mmol of potassium phosphate, pH 7.0, 10 nmol of EDTA, 150 nmol of 17b-estradiol and 1.1 mmol of NADP. In addition the samples contained: (a) no other additions, (b) 15 nmol of 7,8-benzo[a]pyrenequinone, (c) 15 nmol of 7,8-benzo[a]pyrenequinone and 0.9 nmol of SOD, and (d) 15 nmol of 9,10-phenanthrenequinone. The total volume of the reaction mixtures was 3.0 ml. The reactions were started at zero time by the addition of 70 mU of 17b-hydroxysteroid dehydrogenase, and the absorbance of NADPH was followed at 340 nm.
cycling of a PAH o-quinone may shift the equilibrium of reaction [4] by altering cofactor availability. Table III shows data from experiments which were done to test the activity of the placental 17b-hydroxysteroid dehydrogenase as a dihydrodiol dehydrogenase. Although the trans-dihydrodiols 7,8-dihydroxy-7,8dihydro-5-methylchrysene, 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene, and 3,4-dihydroxy-3,4-dihydrobenz[a]anthracene are oxidized by the rat liver 3a-hydroxysteroid dehydrogenase (dihydrodiol dehydrogenase) (48,
49), neither these nor other PAH trans-dihydrodiols tested in this study are substrates of the human placental 17b-hydroxysteroid dehydrogenase. Indanol, 1acenaphthenol, 5a-androstan-17b-ol-3-one, and 4-androsten-17b-ol-3-one are oxidized by the 17b-hydroxysteroid dehydrogenase and by various dihydrodiol dehydrogenases (17–20), but these compounds are poor substrates for the placental enzyme compared to 17bestradiol. While the placental 17b-hydroxysteroid dehydroge-
TABLE III
Oxidation of Potential Substrates by 17b-Hydroxysteroid Dehydrogenasea NADP Compound
Concentration (mM)
17b-Estradiol (S)-(/)-1-Indanol 1-Acenaphthenol 5a-Androstan-17b-ol-3-one 4-Androsten-17b-ol-3-one 5a-Androstan-3a-ol-17-one 5a-Androstan-3b-ol-17-one 9,10-Dihydroxy-9,10-dihydrophenanthrene 5,6-Dihydroxy-5,6-dihydrochrysene 7,8-dihydroxy-7,8-dihydro-5-methylchrysene 3,4-Dihydroxy-3,4-dihydro-benz[a]anthracene 4,5-Dihydroxy-4,5-dihydro-benzo[a]pyrene 7,8-Dihydroxy-7,8-dihydro-benzo[a]pyrene
50 1000 1500 75 50 75 75 50 20 50 20 20 20
NAD nmol oxidized/min
89 0.43 0.22 0.13 0.068 0 0 0 0 0 0 0 0
{ { { { {
2.9 0.0068 0 0.0068 0.0068
140 0.22 0.11 0.058 0.022 0 0 0 0 0 0 0 0
{ { { { {
0 0 0 0 0.0034
a The contents of the reaction cuvettes are the same as described under Experimental Procedures. One hundred-forty mU of enzyme were added to start all reactions except the oxidation of 17b-estradiol. Because of the rapid rate of 17b-estradiol oxidation, less enzyme was used in this reaction mixture but the results were calculated as though 140 mU of enzyme had been used.
/ 6b13$$9326
01-29-96 19:25:02
arca
AP: Archives
180
JARABAK, HARVEY, AND JARABAK
nase is not a PAH trans-dihydrodiol dehydrogenase, it is an efficient PAH o-quinone reductase. Thus, it may play a role in carcinogenesis by catalyzing the redox cycling of PAH o-quinones generated by other enzymes having dihydrodiol dehydrogenase activity, e.g., the 3ahydroxysteroid dehydrogenase (50). In summary, the 17b-hydroxysteroid dehydrogenase catalyzes the reduction of a number of PAH o-quinones; this initiates the redox cycling of these compounds. The specificity of this enzyme is unusual for a two-electron quinone reductase in that a non-K-region o-quinone is its best substrate. It is likely that 17b-estradiol oxidation and quinone reduction occur at the same active site on the enzyme since 17a-estradiol is a competitive inhibitor of both reactions. Finally, the rapid cycling of a PAH o-quinone may shift the 17b-estradiol _ estrone equilibrium by altering cofactor availability. While this observation could have important physiologic consequences, extensive work will be required to characterize its full significance.
20. 21.
22.
23.
24. 25. 26. 27. 28. 29. 30.
REFERENCES 1. O’Brien, P. J. (1991) Chem. -Biol. Interact. 80, 1–41. 2. Chesis, P. L., Levin, D. E., Smith, M. T., Ernster, L., and Ames, B. N. (1984) Proc. Natl. Acad. Sci. USA 81, 1696–1700. 3. Brunmark, A., Cadenas, E., Lind, C., Segura-Aguilar, J., and Ernster, L. (1987) Free Radical Biol. Med. 3, 181–188. 4. Brunmark, A., Cadenas, E., Segura-Aguilar, J., Lind, C., and Ernster, L. (1988) Free Radical Biol. Med. 5, 133–143. 5. Buffinton, G. D., Ollinger, K., Brunmark, A., and Cadenas, E. (1989) Biochem. J. 257, 561–571. 6. Ollinger, K., Buffinton, G. D., Ernster, L., and Cadenas, E. (1990) Chem. -Biol. Interact. 73, 53–76. 7. Cadenas, E., Hochstein, P., and Ernster, L. (1992) Adv. Enzymol. 65, 97–146. 8. Jarabak, J., and Harvey, R. G. (1993) Arch. Biochem. Biophys. 303, 394–401. 9. Cadenas, E. (1995) Biochem. Pharmacol. 49, 127–140. 10. Wermuth, B., Platts, K. L., Seidel, A., and Oesch, F. (1986) Biochem. Pharmacol. 35, 1277–1282. 11. Chung, H., Harvey, R. G., Armstrong, R. N., and Jarabak, J. (1987) J. Biol. Chem. 262, 12448–12451. 12. Jarabak, J. (1991) Arch. Biochem. Biophys. 291, 334–338. 13. Jarabak, J. (1992) Arch. Biochem. Biophys. 292, 239–243. 14. Klein, J., Post, K., Seidel, A., Frank, H., Oesch, F., and Platt, K. L. (1992) Biochem. Pharmacol. 44, 341–349. 15. Post, K., Seidel, A., Platt, K. L., Oesch, F., and Klein, J. (1994) Chem. -Biol. Interactions 90, 157–168. 16. Vogel, K., Bentley, P., Platt, K. L., and Oesch, F. (1980) J. Biol. Chem. 255, 9621–9625. 17. Penning, T. M., Mukharji, I., Barrows, S., and Talalay, P. (1984) Biochem. J. 222, 601–611. 18. Deyashiki, Y., Taniguchi, H., Amano, T., Nakayama, T., Hara, A., and Sawada, H. (1992) Biochem. J. 282, 741–746. 19. Sawada, H., Hara, A., Nakayama, T., Nakagawa, M., Inoue, Y.,
/ 6b13$$9326
01-29-96 19:25:02
arca
31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50.
Hasebe, K., and Zhang, Y.-P. (1988) Biochem. Pharmacol. 37, 453–458. Klein, J., Thomas, H., Post, K., Wo¨rner, W., and Oesch, F. (1992) Eur. J. Biochem. 205, 1155–1162. Wu, L., Einstein, M., Geissler, W. M., Chan, H. K., Ellison, K. O., and Andersson, S. (1993) J. Biol. Chem. 268, 12964– 12969. Martel, C., Rhe´aume, E., Takahashi, M., Trudel, C., Coue¨t, J., Luu-The, V., Simard, J., and Labrie, F. (1992) J. Steroid Biochem. Mol. Biol. 41, 597–603. Isomaa, V. V., Ghersevich, S. A., Ma¨entausta, O. K., Peltoketo, E. H., Poutanen, M. H., and Vihko, R. K. (1993) Ann. Med. 25, 91–97. Poutanen, M., Isomaa, V., Lehto, V.-P., and Vihko, R. (1992) Int. J. Cancer 50, 386–390. Jarabak, J. (1969) Methods Enzymol. 15, 746–752. Harvey, R. G., Goh, S. H., and Cortez, C. (1975) J. Am. Chem. Soc. 97, 3468–3479. Cho, H., and Harvey, R. G. (1976) J. Chem. Soc. Perkin Trans. I, 836–839. Booth, J., Boyland, E., and Turner, E. E. (1950) J. Chem. Soc. 1188–1190. Cortez, C., and Harvey, R. G. (1978) Org. Synth. 58, 12–16. Fu, P. P., and Harvey, R. G. (1977) Tetrahedron Lett. 24, 2059– 2062. Harvey, R. G., Pataki, J., and Lee, H. (1986) J. Org. Chem. 51, 1407–1412. Harvey, R. G. (1985) ACS Monogr. 283, 35–62. Buettner, G. R. (1988) J. Biochem. Biophys. Methods 16, 27–40. Lin, Y.-M., and Jarabak, J. (1978) Biochem. Biophys. Res. Commun. 76, 943–949. Brunmark, A., and Cadenas, E. (1989) Free Radical Biol. Med. 7, 435–477. Ishii, T., and Fridovich, I. (1990) Free Radical Biol. Med. 8, 21– 24. Lorentzen, R. J., and Ts’o, P. O. P. (1977) Biochemistry 16, 1467– 1473. Williamson, D. G. (1979) Steroid Biochem. 1, 84–110. Jarabak, J., Adams, J. A., Williams-Ashman, H. G., and Talalay, P. (1962) J. Biol. Chem. 237, 345–357. Karavolas, H. J., Baedecker, M. L., and Engel, L. L. (1970) J. Biol. Chem. 245, 4948–4952. Strickler, R. C., Tobias, B., and Covey, D. F. (1981) J. Biol. Chem. 256, 316–321. Murdock, G. L., Chin, C. C., and Warren, J. C. (1986) Biochemistry 25, 641–646. Lin, S.-X., Yang, F., Jin, J.-Z., Breton, R., Zhu, D.-W., Luu-The, V., and Labrie, F. (1992) J. Biol. Chem. 267, 16182–16187. Flowers-Geary, L., Harvey, R. G., and Penning, T. M. (1992) Biochem. (Life Sci. Adv.) 11, 49–58. McCord, J., and Fridovich, I. (1968) J. Biol. Chem. 243, 5753– 5760. Jarabak, J., and Sack, G. H. (1969) Biochemistry 8, 2203–2212. Reed, M. J. (1991) J. Endocrinol. 129, 163–165. Smithgall, T. E., Harvey, R. G., and Penning, T. M. (1986) J. Biol. Chem. 261, 6184–6191. Klein, J., Seidel, A., Frank, H., Oesch, F., and Platt, K. L. (1991) Chem.-Biol. Interact. 79, 287–303. Smithgall, T. E., Harvey, R. G., and Penning, T. M. (1988) J. Biol. Chem. 263, 1814–1820.
AP: Archives
Vol. 327, No. 1, March 1, pp. 174–180, 1996 Article No. 0106
Polycyclic Aromatic Hydrocarbon Quinone-Mediated Oxidation Reduction Cycling Catalyzed by a Human Placental 17b-Hydroxysteroid Dehydrogenase1 Rebecca Jarabak, Ronald G. Harvey,* and Joseph Jarabak2 Department of Medicine and the *Ben May Institute, The University of Chicago, Chicago, Illinois 60637
Received September 19, 1995, and in revised form December 15, 1995
The human placental 17b-hydroxysteroid dehydrogenase reduces a number of polycyclic aromatic hydrocarbon (PAH) o-quinones; some of the quinones undergo redox cycling at rates that approach or exceed the rate of reduction of estrone by the enzyme. The non-K-region o-quinone, 7,8-benzo[a]pyrenequinone, is the best o-quinone substrate tested. Cycling of all the quinone substrates is inhibited by superoxide dismutase; cycling is also inhibited by 17b-estradiol and other estrogens. Since 17a-estradiol is a competitive inhibitor of both the oxidation of 17b-estradiol and the cycling of 9,10-phenanthrenequinone by the 17b-hydroxysteroid dehydrogenase, it is likely that both reactions occur at the same active site on the enzyme. In the presence of the 17b-hydroxysteroid dehydrogenase, the equilibrium between 17b-estradiol, estrone, NADP, and NADPH is shifted by 7,8-benzo[a]pyrenequinone because the rapid redox cycling of this quinone results in the oxidation of NADPH. Unlike a number of hydroxysteroid dehydrogenases, the placental 17b-hydroxysteroid dehydrogenase does not oxidize any of the six PAH trans-dihydrodiols tested. q 1996 Academic Press, Inc.
Key Words: Redox cycling; 17b-hydroxysteroid dehydrogenase; PAH quinones; superoxide; superoxide dismutase; estrogens; dihydrodiol dehydrogenase.
The toxicity of some quinones results from their ability to alkylate nucleic acids, proteins, or lipids, or from the reactive oxygen species formed during their oxidation-reduction (redox) cycling (1). The one-electron re-
duction of quinones leads to their redox cycling, and one-electron reductases, such as the NADPH:cytochrome P450 oxidoreductase, have a broad substrate specificity for polycyclic aromatic hydrocarbon (PAH)3 quinones (2). Recent studies have shown that two-electron reduction of a number of naphthoquinones and PAH o-quinones leads to the redox cycling of these compounds. The quinone reductases which catalyze these reactions include DT-diaphorase (3–9), carbonyl reductase (8, 10–12), NAD-linked 15-hydroxyprostaglandin dehydrogenase (13), and 3a-hydroxysteroid dehydrogenase (14, 15). Unlike the one-electron reductases, these twoelectron reductases show a more limited substrate specificity. Of the quinones examined, PAH o-quinones are the best substrates for these enzymes. PAH quinones that are not o-quinones (e.g., 3,6-benzo[a]pyrenequinone, 3,6-pyrenequinone, 9,10-anthracenequinone, and 7,12-benz[a]anthracenequinone) are poor substrates or are not substrates for these enzymes (8). Both 3a-hydroxysteroid dehydrogenases (16–18) and 17b-hydroxysteroid dehydrogenases (19, 20) oxidize benzene dihydrodiol. As a consequence, these enzymes are also called dihydrodiol dehydrogenases. The rat liver 3a-hydroxysteroid dehydrogenase, in addition to oxidizing benzene dihydrodiol, has been shown to oxidize PAH trans-dihydrodiols. The importance of this observation is that this enzyme, and perhaps other hydroxysteroid dehydrogenases, may alter the toxicity of these proximate carcinogens by converting them to oquinones. In view of the observations noted here, the present Abbreviations used: HSD, hydroxysteroid dehydrogenase; O2•0, superoxide radical; SOD, superoxide dismutase; EDTA, ethylenediaminetetraacetate; 17a-E2 , 17a-estradiol; 17b-E2 , 17b-estradiol; 17a-EE2 , 17a-ethinyl estradiol; DES, diethylstilbestrol; PAH, polycyclic aromatic hydrocarbon; Q, quinone; Q•0, semiquinone; QH0, hydroquinone. 3
1
This work was supported by NIH Grants ES 06797 (J.J.) and ES 04732 (R.G.H.) and American Cancer Society Grant CN-22 (R.G.H.). 2 To whom correspondence and reprint requests should be addressed at Department of Medicine, M.C. 1027, 5841 S. Maryland Avenue, Chicago, IL 60637. Fax: (312) 702-9194. 174
0003-9861/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
/ 6b13$$9326
01-29-96 19:25:02
arca
AP: Archives
175
QUINONE-MEDIATED REDOX CYCLING
study was undertaken to determine whether either PAH o-quinones or PAH trans-dihydrodiols are substrates for the human placental 17b-hydroxysteroid dehydrogenase. Two major human 17b-hydroxysteroid dehydrogenases have been described. These differ in both their location within the cell and their substrate specificities (21). The 17b-hydroxysteroid dehydrogenase used in the present study is called type I; it is cytoplasmic and catalyzes the interconversion of 17b-estradiol and estrone much better than that of testosterone and androstenedione. The 1.3-kb mRNA for the type I enzyme has been identified in placenta, ovary, breast (normal and malignant), endometrium, skin, prostate (normal and malignant), and adipose tissue (22–24). The type I 17b-hydroxysteroid dehydrogenase used in this study has been purified to apparent homogeneity from human placenta (25). It has dual pyridine nucleotide specificity; 17b-estradiol and estrone are its best steroid substrates (25). This report describes the two-electron reduction and redox cycling of PAH o-quinones by this enzyme. Among other observations reported here are the unusual substrate specificity of this enzyme and the effect of the redox cycling of o-quinones on the 17b-estradiol _ estrone equilibrium controlled by the enzyme.
path, and 3-ml reaction mixtures were used. Assays were performed in duplicate or triplicate and rates are reported as the mean { the standard deviation. The cuvettes used for assays of redox cycling contained 0.2 ml dimethyl sulfoxide to which quinones and estrogens (dissolved in either 95% ethanol or DMSO) were added. This was followed by additions of water, 10 mmol of potassium phosphate, pH 7.0, 10 nmol of EDTA and 225 nmol of NADPH. Enzyme was omitted from the blank. The reactions were started by the addition of enzyme, and the rate of NADPH oxidation was determined by measuring the decrease in absorbance at 340 nm. The rate of redox cycling is the rate of NADPH oxidation that follows the initial reduction of the quinone. The cuvettes used for the dehydrogenase assays contained 0.2 ml dimethyl sulfoxide to which potential substrates (dissolved in either 95% ethanol or DMSO) were added. This was followed by additions of water, 270 mmol of Tris, pH 9.0, 10 nmol of EDTA, and either 1.36 mmol of NAD or 1.1 mmol of NADP. Enzyme was omitted from the blank. The reactions were started by the addition of enzyme, and the rate of pyridine nucleotide reduction was measured by the increase in absorbance at 340 nm.
RESULTS AND DISCUSSION
The two-electron reduction of a quinone by a quinone reductase produces the corresponding hydroquinone. The hydroquinone product may react to give the semiquinone and the superoxide radical, O•20, by several nonenzymatic mechanisms (35), including autoxidation: QH0 / O2 _ Q•0 / H/ / O•0 2
EXPERIMENTAL PROCEDURES Materials. Superoxide dismutase, NADPH, NADH, (S)-(/)-1indanol, 1,3,5(10)-estratrien-3,17a-diol (17a-estradiol, 17a-E2), and 5a-androstan-17b-ol-3-one were purchased from Sigma, EDTA from J.T. Baker, and Chelex 100 (100 – 200 mesh, sodium form) from Bio-Rad. NAD and NADP were obtained from Pharmacia Biotechnology, Inc., 4-androsten-17b-ol-3-one (testosterone), 5a-androstan-3a-ol-17-one, 5a-androstan-3b-ol-17-one, 1,3,5 (10)-estratrien-3, 17b-diol (17b-estradiol, 17b-E2), 1,3,5(10)-estratrien-17a-ethinyl-3,17b-diol (17a-ethinyl estradiol, 17a-EE2), and 3,4-bis-(4-hydroxyphenyl)-3-hexene (diethylstilbestrol, DES) from Steraloids, 9,10-phenanthrenequinone from Eastman, and 1-acenaphthenol from Aldrich. The remaining polycyclic aromatic hydrocarbon quinones (26, 27) and trans-dihydrodiols (26, 28 – 32) used in this study were synthesized by methods reported previously. Inorganic salts and dimethyl sulfoxide were the best commercially available analytic reagents. Solutions were prepared in laboratory distilled water which had been deionized with an Ionpure mixed bed resin. Stock solutions of phosphate buffer were treated batchwise with Chelex 100 resin to remove trace metal ions (33). Enzymes. The human placental 17b-hydroxysteroid dehydrogenase was prepared as described previously (25). It had an activity of 5.1 U/mg of protein when assayed with 17b-estradiol and NAD as described below. The human placental carbonyl reductase (34) and the rat liver DT-diaphorase (8) were purified as described previously. Their activities were 62 and 30 U/mg of protein, respectively, when assayed as described (8). One unit of enzyme catalyzes either the reduction of 1 mmol of NAD/min (17b-HSD) or the oxidation of 1 mmol of NADPH/min (carbonyl reductase and DT-diaphorase) under the assay conditions. Enzyme assays. The assays were performed in a Gilford recording spectrophotometer at 25 { 0.57C. Reaction cuvettes had a 1-cm light
/ 6b13$$9326
01-29-96 19:25:02
arca
[1]
The superoxide initiates a free radical chain reaction which regenerates the quinone (36): •0 H/ / QH0 / O•0 / H2O2 2 _ Q
Q
•0
•0 2
/ O2 _ Q / O
[2] [3]
The quinone is then reduced again by the enzyme; this alternate reduction and oxidation has been called redox cycling. The rate of reaction [1] is greatly influenced by the reactivity of the hydroquinone (9). For some hydroquinones this reaction is sufficiently rapid that twoelectron reduction of the quinone is followed by redox cycling. Redox cycling is of particular interest since it generates reactive oxygen species, e.g., O•0 2 and hydrogen peroxide, and thus contributes to oxidative stress. Lorentzen and Ts’o were the first to suggest that the toxicity of PAH metabolites might be related to the reduction of PAH quinones to hydroquinones followed by their redox cycling (37). Studies in this laboratory and by others have shown that two-electron reduction of a number of naphthoquinones and PAH o-quinones by several highly purified quinone reductases including DT-diaphorase (3–9), carbonyl reductase (8, 11, 12), NAD-linked 15-hydroxyprostaglandin dehydrogenase (13), and 3a-hydroxysteroid dehydrogenase (14, 15) causes the redox cycling of these quinones and the formation of reactive oxygen species. These studies have
AP: Archives
176
JARABAK, HARVEY, AND JARABAK TABLE I
Redox Cycling of PAH Quinones by 17b-Hydroxysteroid Dehydrogenase, Carbonyl Reductase, and DT-diaphorasea Rate of Cycling (nmol NADPH oxidized/min) 17b-HSDb
Quinone 9,10-Phenanthrenequinone 5,6-Chrysenequinone 3,4-Benz[a]anthracenequinone 5,6-Benz[a]anthracenequinone 4,5-Benzo[a]pyrenequinone 7,8-Benzo[a]pyrenequinone
0.97 0.72 0.53 2.4 1.9 6.2
{ { { { { {
0.029 0 0.058 0.029 0.029 0.058
Carbonyl reductase 5.4 4.2 0 2.6 1.6 0.86
{ 0.082 { 0.17 { 0.14 { 0.082 { 0.058
DT-diaphorase 3.8 3.4 0 2.8 1.6 0
{ 0.14 { 0.029 { 0.11 { 0.072
a
The contents of the reaction mixtures are the same as described under Experimental Procedures; 15 nmol of each quinone was assayed. The reactions were started with the addition of 14 mU of 17b-hydroxysteroid dehydrogenase, 27 mU of carbonyl reductase, or 24 mU of DT-diaphorase. For purposes of comparison, if 150 nmol of estrone is added rather than a PAH o-quinone, the rate of NADPH oxidation by 14 mU of 17b-hydroxysteroid dehydrogenase is 4.3 nmol/min. b None of the following quinones is a substrate for the 17b-HSD: 3,6-benzo[a]pyrenequinone, 3,6-pyrenequinone, 9,10-anthracenequinone, or 7,12-benz[a]anthracenequinone. Fifteen nmol of each quinone was assayed.
demonstrated that the enzyme-catalyzed cycling exhibits the following characteristics: (1) in the presence of catalytic quantities of the quinone, a very large initial excess of the reduced pyridine nucleotide is completely oxidized (11, 13, 15, 37); (2) there is O2 uptake (1, 4, 14) and the production of O•0 and H2O2 during the 2 cycling reaction (5, 6, 12, 13, 37); and (3) the addition of superoxide dismutase (SOD) inhibits the pyridine nucleotide oxidation, O2 uptake, and H2O2 production (6–8, 12, 13). The cytoplasmic human 17b-hydroxysteroid dehydrogenase catalyzes the following reaction: 17b-estradiol / NAD(P)/ _ estrone / NAD(P)H / H/ [4] The enzyme plays an important role in estrogen metabolism in a number of different tissues (22, 23, 38), and the enzyme from human placenta is well characterized (25, 39–43). The work presented here shows that this steroid dehydrogenase is also an efficient reductase of certain PAH o-quinones, such as 9,10-phenanthrenequinone: 9,10-phenanthrenequinone / NAD(P)H / H/r 9,10-phenanthrenehydroquinone / NAD(P)/ [5] Although the oxido-reduction of 17b-estradiol and estrone by this enzyme is more rapid in the presence of NAD and NADH than NADP and NADPH (39), the reduction of PAH o-quinones is more rapid in the presence of NADPH than NADH (data not shown). Table I compares the rates of redox cycling of six PAH o-quinones catalyzed by the 17b-hydroxysteroid dehydrogenase, carbonyl reductase, and DT-diapho-
/ 6b13$$9326
01-29-96 19:25:02
arca
rase. The relative rates of cycling vary considerably depending on the enzyme and quinone substrate used. The results in Table I suggest that the carbonyl reductase and DT-diaphorase cycle K-region o-quinones (e.g., 9,10-phenanthrenequinone) more rapidly than non-Kregion o-quinones (e.g., 7,8-benzo[a]pyrenequinone). The specificity of the 17b-hydroxysteroid dehydrogenase does not fit this pattern; in fact, 7,8-benzo[a]pyrenequinone is the best o-quinone substrate tested for this enzyme. The rate of cycling of some of the quinones by the 17b-hydroxysteroid dehydrogenase is not very different from the rate of reduction for a saturating concentration of estrone, one of the putative natural substrates for the enzyme (see Table I, footnote a). As shown in the Table, the rate of cycling for 7,8-benzo[a]pyrenequinone by the enzyme exceeds the rate of estrone reduction. In addition to the oxidation of NADPH which occurs during redox cycling there is also O2 consumption and H2O2 production. Although O2 consumption and H2O2 production were not measured in this study, H2O2 production was measured previously with the carbonyl reductase and four of the quinones tested here (12). The H2O2 produced/NADPH oxidized was 0.76, 0.81, 0.71 and 0.84 for 9,10-phenanthrenequinone, 5,6-chrysenequinone, 4,5-benzo[a]pyrenequinone, and 7,8-benzo[a]pyrenequinone, respectively. Recently Flowers-Geary et al. (44) have concluded that PAH o-quinones are poor substrates for enzymic two-electron reduction in rat liver cytosol. This conclusion was based on experiments using a particular enzyme (DT-diaphorase) and a particular set of PAH oquinones (non-K-region). The data presented in Table I indicate that this conclusion may not apply to various other enzyme-substrate pairs in other tissues.
AP: Archives
QUINONE-MEDIATED REDOX CYCLING
177
The enzyme superoxide dismutase destroys O•0 by 2 catalyzing the following reaction (45): 2 H/ / 2 O•/ 2 r H2O2 / O2
FIG. 1. Redox cycling of 9,10-phenanthrenequinone and 7,8-benzo[a]pyrenequinone by the 17b-hydroxysteroid dehydrogenase. (a) Data from experiments in which 9,10-phenanthrenequinone was the substrate. The contents of the assay cuvettes are the same as described under Experimental Procedures; 15 nmol of 9,10-phenanthrenequinone were added. The reaction mixtures contain no SOD (—) or 0.3 mM SOD (---). Arrowheads indicate the addition of 70 mU of 17 b-hydroxysteroid dehydrogenase. The plots show absorbance at 340 nm as a function of time. The recorder offset was adjusted so that traces would not overlap. (b) Data from experiments in which 7,8-benzo[a]pyrenequinone was the substrate. The reaction mixtures in b are the same as for a except that 15 nmol of 7,8-benzo[a]pyrenequinone was added instead of 9,10-phenanthrenequinone. Arrowheads indicate the addition of 14 mU of 17b-hydroxysteroid dehydrogenase.
Figure 1 shows the time course of NADPH oxidation during the redox cycling of 9,10-phenanthrenequinone (a) and 7,8-benzo[a]pyrenequinone (b) by the 17bhydroxysteroid dehydrogenase. Figures 1a (—) and 1b (—) show traces for reaction mixtures containing quinone, NADPH, and enzyme. In each case initial reduction of the quinone is followed by its redox cycling. When 9,10-phenanthrenequinone is the quinone substrate (Fig. 1a,—) oxidation of NADPH is linear with time, i.e., the rate of cycling is as rapid as the initial reduction of the quinone to the hydroquinone. In the case of 7,8-benzo[a]pyrenequinone (Fig. 1b,—) the initial rate of NADPH oxidation (the rate of quinone reduction) is more rapid than the rate of cycling; therefore a change of slope is observed after the initial reduction of the quinone. Thus the reduction of the quinone by the enzyme is the rate-limiting step in the redox cycling of 9,10-phenanthrenequinone while autoxidation of the hydroquinone is rate limiting for 7,8benzo[a]pyrenequinone. For both quinones, cycling continues until all of the NADPH is oxidized (not shown) despite the fact that the initial concentration of the NADPH greatly exceeds that of the quinone.
/ 6b13$$9326
01-29-96 19:25:02
arca
[6]
SOD inhibits the cycling of 9,10-phenanthrenequinone (Fig. 1a, ---) and 7,8-benzo[a]pyrenequinone (Fig. 1b, ---). As seen most clearly with 9,10-phenanthrenequinone (by comparing — and --- in Fig. 1a), SOD does not affect the enzymatic reduction of the quinone; instead, it inhibits autoxidation of the hydroquinone. There is a slow but measurable rate of NADPH oxidation following the initial reduction of both quinones. Presumably this results from autoxidation of the hydroquinones (reaction [1]) or from other nonenzymatic reactions of the hydroquinones to yield the corresponding semiquinones (35). The striking effect of SOD indicates that O•0 plays an important role in the redox 2 cycling of these quinones. The results obtained for the K-region o-quinones 5,6chrysenequinone, 5,6-benz[a]anthracenequinone, and 4,5-benzo[a]pyrenequinone (not shown) are similar to those shown for 9,10-phenanthrenequinone in Fig. 1a. The enzymic reduction of these quinones is the ratelimiting step in their cycling, and cycling is markedly inhibited by SOD. Table II shows the effect of 17b-estradiol and several other estrogens on the rate of cycling of PAH o-quinones catalyzed by the 17b-hydroxysteroid dehydrogenase. With only one exception each of these compounds inhibits the cycling of all of the quinones listed. When cycling of the quinones is catalyzed by the carbonyl reductase, none of these estrogens is an inhibitor (data not shown). While the results in Table II show that a number of estrogens inhibit the cycling of PAH o-quinones catalyzed by the 17b-hydroxysteroid dehydrogenase, they do not establish the nature of the inhibition. In order to do this, the effect of one estrogen on the cycling of one quinone was examined in more detail. Figure 2 shows that 17a-estradiol is a competitive inhibitor of the redox cycling of 9,10-phenanthrenequinone catalyzed by the 17b-hydroxysteroid dehydrogenase. A Dixon plot (not shown) gave a Ki of 2.8 mM. In an earlier study it was reported that 17a-estradiol is also a competitive inhibitor (Ki 4.5 mM) of the oxidation of 17bestradiol by this enzyme (reaction [4]) (46). The simplest explanation for the observation that 17a-estradiol is a competitive inhibitor of both the cycling of 9,10phenanthrenequinone and the oxidation of 17b-estradiol is that both reactions occur at the same active site on the enzyme. Since the 17b-hydroxysteroid dehydrogenase is thought to have a role in regulating the tissue concentrations of 17b-estradiol (47), the presence of sub-
AP: Archives
178
JARABAK, HARVEY, AND JARABAK TABLE II
Inhibition by Estrogens of Quinone-Mediated Redox Cyclinga,b (17b-Hydroxysteroid Dehydrogenase) % Inhibition of Cycling Quinone
17b-Ec2
17a-E2
17a-EE2
DES
9,10-Phenanthrenequinone 5,6-Chrysenequinone 5,6-Benz[a]anthracenequinone 4,5-Benzo[a]pyrenequinone 7,8-Benzo[a]pyrenequinone
77 60 60 78 48
82 81 80 88 22
66 41 50 63 0
58 56 31 78 38
a The contents of the reaction cuvettes were the same as described under Experimental Procedures. The control mixtures contained 15 nmol of quinone and no estrogen; the test mixtures contained 15 nmol of quinone and 60 nmol of estrogen. The reactions containing 7,8benzo[a]pyrenequinone were started with the addition of 7 mU of 17b-hydroxysteroid dehydrogenase while 70 mU of enzyme were used to start the reactions of the other quinones. b 17a-estradiol is only weakly estrogenic. It was included in these studies because it is an epimer of 17b-estradiol and a competitive inhibitor of the oxidation of 17b-estradiol by the 17b-hydroxysteroid dehydrogenase. c Because the rate of redox cycling is measured by the oxidation of NADPH, it is likely that 17b-estradiol is a less effective inhibitor than these data suggest. Since 17b-estradiol is a substrate for the enzyme, NADP formed during cycling will be reduced to NADPH in the presence of this steroid. Thus the net rate of NADPH oxidation will be slower than that of cycling.
stances that shift the equilibrium of the reaction it catalyzes may have important physiologic consequences. The following set of experiments was done to examine the effect of PAH o-quinones on this equilibrium. Figure 3a shows the time course of NADPH produc-
FIG. 2. Double reciprocal plots showing inhibition of the redox cycling of 9,10-phenanthrenequinone by 17 a-estradiol. The contents of the assay cuvettes are the same as described under Experimental Procedures except that the concentration of 9,10-phenanthrenequinone (9,10-PQ) was varied, and some cuvettes contained no 17aestradiol (l) while others contained 5 mM 17a-estradiol (s). Reactions were started with the addition of 70 mU of 17b-hydroxysteroid dehydrogenase. The velocity is DA340/10 min. The intersection of the plots on the ordinate shows that 17a-estradiol is a competitive inhibitor of the cycling.
/ 6b13$$9326
01-29-96 19:25:02
arca
tion after the addition of 17b-hydroxysteroid dehydrogenase at t Å 0 to a buffered solution of 17b-estradiol and NADP. The absorbance of NADPH at 340 nm increases as the 17b-estradiol is oxidized until an equilibrium is reached between the oxidized and reduced forms of both the steroid and the pyridine nucleotide. The Keq for reaction [4] at pH 7 is 0.18 (25). Figure 3b shows the effect of adding 7,8-benzo[a]pyrenequinone to the reaction mixture. The absorbance at 340 nm increases after the addition of enzyme until it reaches a maximum and then decreases to the baseline. Presumably the NADPH formed by oxidation of 17b-estradiol is used by the enzyme to reduce the 7,8benzo[a]pyrenequinone, and the quinone begins to cycle. This rapid cycling has a marked effect on the trace of A340 vs time. The cycling continues until all of the nucleotide is present as NADP. At this point the concentrations of 17b-estradiol and estrone will not be the same as they are in the equilibrium mixture of Fig. 3a; instead, the 17b-estradiol concentration will be lower and the estrone concentration higher. If SOD is present in the mixture containing 7,8benzo[a]pyrenequinone, 17b-estradiol, NADP, and enzyme (Fig. 3c), redox cycling of the quinone is inhibited. As a result, the trace of A340 vs time is only slightly different from that shown in Fig. 3a. When 9,10-phenanthrenequinone is present in the reaction cuvette with 17b-estradiol, NADP, and enzyme (Fig. 3d), again, the trace of A340 vs time is not very different from that in Fig. 3a. From the data in Tables I and II it is likely that the cycling of 9,10phenanthrenequinone is much slower than that of 7,8benzo[a]pyrenequinone in these reaction mixtures. Thus, the plots in Figure 3 suggest that the rapid
AP: Archives
179
QUINONE-MEDIATED REDOX CYCLING
FIG. 3. Influence of 7,8-benzo[a]pyrenequinone and 9,10-phenanthrenequinone on NADPH concentration during enzymic oxidation of 17b-estradiol. Each cuvette contained water, 0.2 ml of dimethyl sulfoxide, 10 mmol of potassium phosphate, pH 7.0, 10 nmol of EDTA, 150 nmol of 17b-estradiol and 1.1 mmol of NADP. In addition the samples contained: (a) no other additions, (b) 15 nmol of 7,8-benzo[a]pyrenequinone, (c) 15 nmol of 7,8-benzo[a]pyrenequinone and 0.9 nmol of SOD, and (d) 15 nmol of 9,10-phenanthrenequinone. The total volume of the reaction mixtures was 3.0 ml. The reactions were started at zero time by the addition of 70 mU of 17b-hydroxysteroid dehydrogenase, and the absorbance of NADPH was followed at 340 nm.
cycling of a PAH o-quinone may shift the equilibrium of reaction [4] by altering cofactor availability. Table III shows data from experiments which were done to test the activity of the placental 17b-hydroxysteroid dehydrogenase as a dihydrodiol dehydrogenase. Although the trans-dihydrodiols 7,8-dihydroxy-7,8dihydro-5-methylchrysene, 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene, and 3,4-dihydroxy-3,4-dihydrobenz[a]anthracene are oxidized by the rat liver 3a-hydroxysteroid dehydrogenase (dihydrodiol dehydrogenase) (48,
49), neither these nor other PAH trans-dihydrodiols tested in this study are substrates of the human placental 17b-hydroxysteroid dehydrogenase. Indanol, 1acenaphthenol, 5a-androstan-17b-ol-3-one, and 4-androsten-17b-ol-3-one are oxidized by the 17b-hydroxysteroid dehydrogenase and by various dihydrodiol dehydrogenases (17–20), but these compounds are poor substrates for the placental enzyme compared to 17bestradiol. While the placental 17b-hydroxysteroid dehydroge-
TABLE III
Oxidation of Potential Substrates by 17b-Hydroxysteroid Dehydrogenasea NADP Compound
Concentration (mM)
17b-Estradiol (S)-(/)-1-Indanol 1-Acenaphthenol 5a-Androstan-17b-ol-3-one 4-Androsten-17b-ol-3-one 5a-Androstan-3a-ol-17-one 5a-Androstan-3b-ol-17-one 9,10-Dihydroxy-9,10-dihydrophenanthrene 5,6-Dihydroxy-5,6-dihydrochrysene 7,8-dihydroxy-7,8-dihydro-5-methylchrysene 3,4-Dihydroxy-3,4-dihydro-benz[a]anthracene 4,5-Dihydroxy-4,5-dihydro-benzo[a]pyrene 7,8-Dihydroxy-7,8-dihydro-benzo[a]pyrene
50 1000 1500 75 50 75 75 50 20 50 20 20 20
NAD nmol oxidized/min
89 0.43 0.22 0.13 0.068 0 0 0 0 0 0 0 0
{ { { { {
2.9 0.0068 0 0.0068 0.0068
140 0.22 0.11 0.058 0.022 0 0 0 0 0 0 0 0
{ { { { {
0 0 0 0 0.0034
a The contents of the reaction cuvettes are the same as described under Experimental Procedures. One hundred-forty mU of enzyme were added to start all reactions except the oxidation of 17b-estradiol. Because of the rapid rate of 17b-estradiol oxidation, less enzyme was used in this reaction mixture but the results were calculated as though 140 mU of enzyme had been used.
/ 6b13$$9326
01-29-96 19:25:02
arca
AP: Archives
180
JARABAK, HARVEY, AND JARABAK
nase is not a PAH trans-dihydrodiol dehydrogenase, it is an efficient PAH o-quinone reductase. Thus, it may play a role in carcinogenesis by catalyzing the redox cycling of PAH o-quinones generated by other enzymes having dihydrodiol dehydrogenase activity, e.g., the 3ahydroxysteroid dehydrogenase (50). In summary, the 17b-hydroxysteroid dehydrogenase catalyzes the reduction of a number of PAH o-quinones; this initiates the redox cycling of these compounds. The specificity of this enzyme is unusual for a two-electron quinone reductase in that a non-K-region o-quinone is its best substrate. It is likely that 17b-estradiol oxidation and quinone reduction occur at the same active site on the enzyme since 17a-estradiol is a competitive inhibitor of both reactions. Finally, the rapid cycling of a PAH o-quinone may shift the 17b-estradiol _ estrone equilibrium by altering cofactor availability. While this observation could have important physiologic consequences, extensive work will be required to characterize its full significance.
20. 21.
22.
23.
24. 25. 26. 27. 28. 29. 30.
REFERENCES 1. O’Brien, P. J. (1991) Chem. -Biol. Interact. 80, 1–41. 2. Chesis, P. L., Levin, D. E., Smith, M. T., Ernster, L., and Ames, B. N. (1984) Proc. Natl. Acad. Sci. USA 81, 1696–1700. 3. Brunmark, A., Cadenas, E., Lind, C., Segura-Aguilar, J., and Ernster, L. (1987) Free Radical Biol. Med. 3, 181–188. 4. Brunmark, A., Cadenas, E., Segura-Aguilar, J., Lind, C., and Ernster, L. (1988) Free Radical Biol. Med. 5, 133–143. 5. Buffinton, G. D., Ollinger, K., Brunmark, A., and Cadenas, E. (1989) Biochem. J. 257, 561–571. 6. Ollinger, K., Buffinton, G. D., Ernster, L., and Cadenas, E. (1990) Chem. -Biol. Interact. 73, 53–76. 7. Cadenas, E., Hochstein, P., and Ernster, L. (1992) Adv. Enzymol. 65, 97–146. 8. Jarabak, J., and Harvey, R. G. (1993) Arch. Biochem. Biophys. 303, 394–401. 9. Cadenas, E. (1995) Biochem. Pharmacol. 49, 127–140. 10. Wermuth, B., Platts, K. L., Seidel, A., and Oesch, F. (1986) Biochem. Pharmacol. 35, 1277–1282. 11. Chung, H., Harvey, R. G., Armstrong, R. N., and Jarabak, J. (1987) J. Biol. Chem. 262, 12448–12451. 12. Jarabak, J. (1991) Arch. Biochem. Biophys. 291, 334–338. 13. Jarabak, J. (1992) Arch. Biochem. Biophys. 292, 239–243. 14. Klein, J., Post, K., Seidel, A., Frank, H., Oesch, F., and Platt, K. L. (1992) Biochem. Pharmacol. 44, 341–349. 15. Post, K., Seidel, A., Platt, K. L., Oesch, F., and Klein, J. (1994) Chem. -Biol. Interactions 90, 157–168. 16. Vogel, K., Bentley, P., Platt, K. L., and Oesch, F. (1980) J. Biol. Chem. 255, 9621–9625. 17. Penning, T. M., Mukharji, I., Barrows, S., and Talalay, P. (1984) Biochem. J. 222, 601–611. 18. Deyashiki, Y., Taniguchi, H., Amano, T., Nakayama, T., Hara, A., and Sawada, H. (1992) Biochem. J. 282, 741–746. 19. Sawada, H., Hara, A., Nakayama, T., Nakagawa, M., Inoue, Y.,
/ 6b13$$9326
01-29-96 19:25:02
arca
31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50.
Hasebe, K., and Zhang, Y.-P. (1988) Biochem. Pharmacol. 37, 453–458. Klein, J., Thomas, H., Post, K., Wo¨rner, W., and Oesch, F. (1992) Eur. J. Biochem. 205, 1155–1162. Wu, L., Einstein, M., Geissler, W. M., Chan, H. K., Ellison, K. O., and Andersson, S. (1993) J. Biol. Chem. 268, 12964– 12969. Martel, C., Rhe´aume, E., Takahashi, M., Trudel, C., Coue¨t, J., Luu-The, V., Simard, J., and Labrie, F. (1992) J. Steroid Biochem. Mol. Biol. 41, 597–603. Isomaa, V. V., Ghersevich, S. A., Ma¨entausta, O. K., Peltoketo, E. H., Poutanen, M. H., and Vihko, R. K. (1993) Ann. Med. 25, 91–97. Poutanen, M., Isomaa, V., Lehto, V.-P., and Vihko, R. (1992) Int. J. Cancer 50, 386–390. Jarabak, J. (1969) Methods Enzymol. 15, 746–752. Harvey, R. G., Goh, S. H., and Cortez, C. (1975) J. Am. Chem. Soc. 97, 3468–3479. Cho, H., and Harvey, R. G. (1976) J. Chem. Soc. Perkin Trans. I, 836–839. Booth, J., Boyland, E., and Turner, E. E. (1950) J. Chem. Soc. 1188–1190. Cortez, C., and Harvey, R. G. (1978) Org. Synth. 58, 12–16. Fu, P. P., and Harvey, R. G. (1977) Tetrahedron Lett. 24, 2059– 2062. Harvey, R. G., Pataki, J., and Lee, H. (1986) J. Org. Chem. 51, 1407–1412. Harvey, R. G. (1985) ACS Monogr. 283, 35–62. Buettner, G. R. (1988) J. Biochem. Biophys. Methods 16, 27–40. Lin, Y.-M., and Jarabak, J. (1978) Biochem. Biophys. Res. Commun. 76, 943–949. Brunmark, A., and Cadenas, E. (1989) Free Radical Biol. Med. 7, 435–477. Ishii, T., and Fridovich, I. (1990) Free Radical Biol. Med. 8, 21– 24. Lorentzen, R. J., and Ts’o, P. O. P. (1977) Biochemistry 16, 1467– 1473. Williamson, D. G. (1979) Steroid Biochem. 1, 84–110. Jarabak, J., Adams, J. A., Williams-Ashman, H. G., and Talalay, P. (1962) J. Biol. Chem. 237, 345–357. Karavolas, H. J., Baedecker, M. L., and Engel, L. L. (1970) J. Biol. Chem. 245, 4948–4952. Strickler, R. C., Tobias, B., and Covey, D. F. (1981) J. Biol. Chem. 256, 316–321. Murdock, G. L., Chin, C. C., and Warren, J. C. (1986) Biochemistry 25, 641–646. Lin, S.-X., Yang, F., Jin, J.-Z., Breton, R., Zhu, D.-W., Luu-The, V., and Labrie, F. (1992) J. Biol. Chem. 267, 16182–16187. Flowers-Geary, L., Harvey, R. G., and Penning, T. M. (1992) Biochem. (Life Sci. Adv.) 11, 49–58. McCord, J., and Fridovich, I. (1968) J. Biol. Chem. 243, 5753– 5760. Jarabak, J., and Sack, G. H. (1969) Biochemistry 8, 2203–2212. Reed, M. J. (1991) J. Endocrinol. 129, 163–165. Smithgall, T. E., Harvey, R. G., and Penning, T. M. (1986) J. Biol. Chem. 261, 6184–6191. Klein, J., Seidel, A., Frank, H., Oesch, F., and Platt, K. L. (1991) Chem.-Biol. Interact. 79, 287–303. Smithgall, T. E., Harvey, R. G., and Penning, T. M. (1988) J. Biol. Chem. 263, 1814–1820.
AP: Archives