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Polycyclic aromatic hydrocarbon quinone-mediated oxidation reduction cycling catalyzed by a human placental NADPH-linked carbonyl reductase
ARCHIVES
OF BIOCHEMISTRY
AND
BIOPHYSICS
Vol. 291, No. 2, December, pp. 334-338,199l
Polycyclic Aromatic Hydrocarbon Quinone-Mediated Oxidation Reduction Cycling Catalyzed by a Human Placental NADPH-Linked Carbonyl Reductase’ Joseph Jarabak Department
of Medicine,
University
of Chicago, Box 435,584l
South Maryland
Avenue, Chicago, Illinois 60637
Received May 21, 1991, and in revised form August 9, 1991
Polycyclic aromatic hydrocarbon quinones, hydroquinones, and glutathionyl adducts of quinones undergo oxidation-reduction (redox) cycling in the presence of NADPH and the NADPH-linked human placental carbony1 reductase. K-region and non-K-region o-quinones and their glutathione adducts are the best substrates of this enzyme; they are reduced to hydroquinones. Under aerobic conditions, the hydroquinones are autoxidized with the formation of potentially hazardous semiquinones and the superoxide anion. Because of these reactions it is unlikely that polycyclic aromatic hydrocarbon quinones or their glutathione adducts are inert products of detoxication in tissues that contain the carbonyl reductase or another enzyme with similar substrate specificity. If superoxide dismutase is added to reaction mixtures containing the carbonyl reductase and quinones, it inhibits redox cycling. Presumably this results from destruction of the superoxide anion which acts as a chain Q iosi Academic PAM, I~C. propagator in these reactions.
Human placental tissue contains a NADP-linked oxidoreductase which was initially thought to be a Shydroxyprostaglandin dehydrogenase/g-ketoprostaglandin reductase (1). Subsequent studies have shown this enzyme to have such broad substrate specificity that it is more aptly called a carbonyl reductase (2). Various K-region and non-K-region polycyclic aromatic hydrocarbon oquinones and their glutathione adducts are its best substrates (3). When these quinones or their glutathione adducts are present in limiting concentrations, an excess of NADPH is completely oxidized under aerobic conditions, suggesting that the quinone substrate has undergone ox1 This work was supported by American 701. 334
Cancer Society Grant BC-
idation-reduction (redox) cycling (3). During the course of redox cycling a quinone is reduced to a semiquinone or hydroquinone depending on whether the reduction involves the transfer of one or two electrons. In the presence of oxygen the semiquinone or hydroquinone is autoxidized to regenerate the quinone while the oxygen is reduced to yield a variety of products which may include the superoxide anion radical (O;), Hz02 , the hydroxyl radical (OH’), and singlet oxygen (4,5) depending on the setting. In view of these characteristics, the reaction between the carbonyl reductase and its quinone substrates has been examined in more detail. The influence of the enzyme superoxide dismutase on this reaction has also been examined since superoxide dismutase has proved useful in investigating the role of 0; in certain quinone-mediated reactions (6, 7). EXPERIMENTAL
PROCEDURES
Materials. NADP and NADPH were obtained from Pharmacia Biotechnology, Inc., phenol and XAD-2 from Mallinckrodt, and silica gel and Polygram Cel 400 TLC plates from Brinkman Instruments, Inc. Acetonitrile was obtained from Burdick and Jackson, and trifluoroacetic acid from Pierce Chemical Co.. Glutathione, 4aminoantipyrine, superoxide dismutase, horse heart cytochrome c (Type VI), and horseradish peroxidase were obtained from Sigma, and 9,10-phenanthrenequinone, 2-methyl-1,4benxoquinone, and 2-methyl-1,4-naphthoquinone from Eastman (these were recrystallixed prior to their use). Compounds synthesized by methods reported previously include 1,2-naphthoquinone (8), 1,2naphthohydroquinone (9), 1,2naphthodihydrodiol(9), 2-methyl1,4-naphthohydroquinone (lo), 2-methyl-5-glutathionyl-l,l-benxoquinone (3, ll), and 2-methyl-3-glutathionyl-1,4-naphthoquinone (3, 11). The compounds 9,10-phenanthrenedihydrodiol, 4,5- and 7,6benxo[a]pyrenequinone, and 5,6-chrysenequinone were generously provided by Professor R. G. Harvey. Volumes of 14 ml Synthesis of 4-glutathionyl-1,2-naphthoquinone. of 2 mM 1,2-naphthoquinone in acetonitrile and 14 ml of 3.3 mM glutathione in water were mixed and allowed to stand overnight at room temperature. The mixture was extracted with three 25-ml portions of ethyl acetate, and aliquots of the aqueous phase were applied to an Altex ooo3-9861/91$3.00
Copyright 0 1991 by Academic Press, Inc. All rightg of reproduction in any form reserved.
QUINONE-MEDIATED Ultrasphere ODS C1s HPLC’ column (4.6 X 250 mm, 5-pm particle size). The product was eluted using a linear gradient from 0.075% trifluoroacetic acid in water to 0.075% trifluoroacetic acid in 100% acetonitrile. The retention time of the glutathione adduct was 28 min; it gave a single ninhydrin positive spot (Rf = 0.35) on TLC performed on a cellulose plate with a solvent containing ethanol and water (77~23). Enzyme. Human placental NADP-linked 15-hydroxyprostaglandin dehydrogenase/g-ketoprostaglandin reductase was purified as described previously (1). It migrates as a single protein on disc electrophoresis, and the preparation used had a specific activity of 980 mU/mg protein. Preliminary studies were performed with enzyme purified through step 4 of the purification (specific activity = 50 mU/mg protein). These showed similar results to those obtained with the more highly purified enzyme. Assays. The reduction of quinones by the placental enzyme was measured as described previously (3) with the exception that 280 pmol of potassium phosphate, pH 7.0, was used instead of Tris-HCl. The formation of superoxide was assayed by measuring the reduction of cytochrome c at 550 nm (12). The molar extinction coefficient used for this reaction was 18,500 M-‘cm+ (13). Hydrogen peroxide was determined with a horseradish-peroxidase-coupled assay (14).
RESULTS
NADPH oxidation and Hz02 production during the reo?oxcycling of quinones and hydroquinones. The amounts of NADPH oxidized and HzOz produced during the redox cycling of quinones and hydroquinones and the effect of superoxide dismutase on these values are shown in Table I. For each reaction, the concentration of the quinone or hydroquinone is limiting. Despite this, most reactions proceed until the NADPH is completely oxidized.3 Hydrogen peroxide is formed during the course of most of these reactions in an amount that is somewhat less than the NADPH oxidized.4 After the oxidation of NADPH has gone to completion, the addition of more NADPH results in more NADPH oxidation and more HzOz formation (data not shown). This and HPLC or TLC studies (data not shown) reveal that the quinone is still present after a large excess of NADPH has been completely oxidized. The results with hydroquinones are similar to those with quinones.5 If NADPH is replaced by NADP, hydro* Abbreviations used: HPLC, high-performance liquid chromatography; TLC, thin-layer chromatography; SOD, superoxide dismutase; EDTA, ethylenediaminetetraacetate; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid. 3 Exceptions are 2-methyl-1,4-benzoquinone, 2-methyl-5-glutathionyl1,4-benzoquinone, the trans-1,2dihydrodiol of naphthalene and the trans-9,10-dihydrodiol of phenanthrene. The reactions with the two benzoquinones proceed until about half of the quinone is reduced. Neither of the dihydrodiols is a substrate in the presence of NADPH or NADH (not shown) or in the presence of NADP (not shown) or NAD (not shown). 4 Again 2-methyl-1,4-benzoquinone and 2-methyl-5-glutathionyl-1,4benzoquinone are exceptions. No HzOz is formed during the reduction of either of these compounds. ’ These observations indicate that the quinones and hydroquinones are undergoing redox cycling. They are markedly different from those when 9-keto- or 15-ketoprostaglandins are reduced by the carbonyl reductase (data not shown). When these ketoprostaglandins are substrates, the quantities of NADPH oxidized, ketoprostaglandin reduced, and hydroxyprostaglandin generated are equimolar and the reaction continues until an equilibrium has been reached.
REDOX
CYCLING
335
quinones do not undergo redox cycling. If glutathione adducts of quinones are substituted for quinones as substrates, the rates of NADPH oxidation and H202 production are comparable to, or more rapid than, those for the parent quinones. In the presence of superoxide dismutase, there is a striking inhibition of both NADPH oxidation and HzOz production. Data relating to this inhibition will be presented in a subsequent section. Superoxide formation during redox cycling. A sensitive indirect method for detecting the superoxide anion involves measuring the reduction of cytochrome c by this radical in the presence and absence of superoxide dismutase (12). Superoxide dismutase inhibits that portion of cytochrome c reduction due to the superoxide anion (12). In order to estimate the superoxide generated during redox cycling of quinones and hydroquinones, cytochrome c was added to reaction mixtures that were in other respects identical to those in Table I. Table II compares the rate of cytochrome c reduction in the presence and absence of superoxide dismutase. All of the quinones and hydroquinones that were examined mediate a rapid reduction of cytochrome c. In the presence of superoxide dismutase the initial rates of these reductions are inhibited by less than 50%. The reduction of cytochrome c that occurs as a consequence of the reduction of 7,8benzo[a]pyrenequinone provides a particularly striking example of this phenomenon. It is inhibited only 11% by superoxide dismutase. When 1,2-naphthohydroquinone or 2-methyl-1,4-naphthohydroquinone are added to reaction mixtures which contain no carbonyl reductase, the inhibition by dismutase is 23 and 9%, respectively. The results obtained when xanthine oxidase is used to generate the superoxide radical are quite different. Under such conditions the reduction of cytochrome c is inhibited by 94% in the presence of superoxide dismutase. Thus although the reduction of quinones gives rise to superoxide anions, most of the reduction of cytochrome c is due to substances that are not destroyed by superoxide dismutase. Similar results are obtained with the autoxidation of hydroquinones. These findings suggest that the reducing substances are hydroquinones (7), semiquinones (15), or both. Effectof superoxide dismutase on the time course of redox cycling. The time course of g,lO-phenanthrenequinone-mediated redox cycling (Fig. 1) shows that the rate of NADPH oxidation is the same in the absence and presence of superoxide dismutase until all of the quinone substrate has been reduced. Then the inhibitory effect of the superoxide dismutase becomes apparent. This suggests that the effect of the dismutase is to inhibit autoxidation of the hydroquinone to the quinone. Factors that influence the superoxide-dismutase-mediated inhibition of redox cycling. The inhibition of redox cycling by superoxide dismutase is influenced by a
336
JOSEPH
JARABAK
TABLE Redox
Cycling
Mediated
I
by Quinones
and Hydroquinones
NADPH oxidized” (nmol) Substrate (nmol)
Substrate 2-Methyl-1,4benzoquinone 2-Methyl-&glutathionyl-1,4-benzoquinone 1,2-Naphthoquinone 1,2-Naphthohydroquinone trun.s-1,2-Dihydroxy-1,2dihydronaphthalene 4-Glutathionyl-1,2-naphthoquinone 2-Methyl-1,4-naphthoquinone 2-Methyl-1,4-naphthohydroquinone 2-Methyl-3-glutathionyl-1,4-naphthoquinone 9,10-Phenanthrenequinone truns-9,10-Dihydroxy-9,10-dihydrophenanthrene 4,5Benzo[a]pyrenequinone 7,8-Benzo[a]pyrenequinone 5,6Chrysenequinone
-SOD
HsOz produced (nmol) Inhibition by SOD (W)
+SOD*
-SOD
+SOD
Inhibition by SOD (W)
0 0
40 40 10 10 10 10
27 21 92 114 0 140
40 40 38 3.0 47 7.0 7.0 7.8
140 176 150 170
55
196
0
0
196
92 100
0 95
80 95
32
0
166
60
37
67 13
39 90
0 0 0
100 166 166
147 33 78
166
38
110
130
0
140
15
93
0 10
180
89 100 94
99
78 145
’ The amount of NADPH added to the reaction cuvettes varied from 150 to 190 nmol and the amount of enzyme varied from 1.5 to 6 mU in a total reaction volume of 3 ml. Blank cuvettes were identical to the reaction cuvettes but contained no enzyme. The reactions were started with the addition of enzyme and were continued until the oxidation of NADPH ceased in cuvettes which did not contain superoxide dismutase. At that time the contents of all cuvettes were transferred to the reaction mixture used for the HzOz assay. The amount of NADPH oxidized and HsOs produced were determined by subtracting blanks from the appropriate reaction cuvettes. The reactions with and without superoxide dismutase were run at the same time for each substrate. * Thirty micrograms of superoxide dismutase was added.
of factors including the substrate, the buffer, and whether chelating agents or metal salts have been added to the reaction mixture. A summary of some of these effects on the redox cycling of 9,10-phenanthrenumber
TABLE Cytochrome
c Reduction
Mediated
nequinone is presented in Table III. Control studies (not shown) indicate that none of the parameters being varied influence the activity of the carbonyl reductase.
II by Quinones
and Hydroquinones Cytochrome
Substrate 2-Methyl-1,4-benzoquinone 2-Methyl-5-glutathionyl-1,4-benzoquinone 1,2-Naphthoquinqne 1,2-Naphthohydroquinone trons-1,2-Dihydroxy-1,2dihydronaphthalene 4-Glutathionyl-1,2-naphthoquinone 2-Methyl-1,4-naphthoquinone 2-Methyl-1,4-naphthohydroquinone 2-Methyl-3-glutathionyl-1,4-naphthoquinone 9,10-Phenanthrenequinone trans-9,10-Dihydroxy-9,lOdihydrophenanthrene 4,5-Benzo[a]pyrenequinone 7,8-Benzo[a]pyrenequinone 5,6-Chrysenequinone
Substrate (nmol)
40 40 10 10 10 10
40 10
38 3.0 47 7.0 7.0 7.8
-SOD
c reduced/l0 (nmol)
min”
+SOD*
7.1 4.7 6.2 430
3.7 2.4 3.7 330
0
0 19
22 12 172 29
75
7.8
Inhibition by SOD (%)
48 49
40 23 14
35
157
9
23 58
21 23
0
0
22
13
19 64
17
11
46
28
41
’ The contents of the reaction cuvettes are the same as those described in Table I except that 60 nmol of cytochrome c was present in each cuvette and 0.2 mU of enzyme was added to start most reactions. The initial rate of cytochrome c reduction was determined by measuring the change in absorbance at 550 nm. When 1,2-naphthohydroquinone and P-methyl-1,4naphthohydroquinone were substrates no enzyme was added. * Thirty micrograms of superoxide dismutase was added.
QUINONE-MEDIATED
BEDOX
331
CYCLING
DISCUSSION
TABLE
Although the human placental carbonyl reductase has a broad substrate specificity, K-region and non-K-region o-quinones of certain polycyclic aromatic hydrocarbons and their glutathione adducts are its best substrates. An analysis of the stoichiometry of this reaction reveals that all of the NADPH in the reaction mixture is oxidized in the presence of only catalytic amounts of these quinones or their hydroquinones. Furthermore, superoxide and Hz02 are formed in the process. These findings indicate that certain o-quinones undergo oxidation-reduction cycling in the presence of NADPH and the carbonyl reductase. The catalysis of redox cycling by the placental carbonyl reductase is noteworthy for three reasons. First, the placental enzyme occurs in the cytosol and it is not a flavoprotein, while most enzymes which catalyze redox cycling are microsomal flavoproteins (16). Second, both Kregion and non-K-region o-quinones and their glutathione adducts are the best substrates for the placental enzyme. Although not studied in detail (7, 17, 18), that does not appear to be the case with other enzymes catalyzing redox cycling. Third, the metabolic consequences of polycyclic
--
r
Carbonyl Reductase
Added
----___ 0.40
0.35
0.30
0.25
o.20 L-t+-10
50
30
Time
(min)
FIG. 1. The effect of superoxide dismutase on S,lO-phenanthrenequinone-mediated redox cycling. The assay cuvettes contained in a volume of 3 ml, 280 Fmol of potassium phosphate, pH 7.0, 225 nmol of NADPH, 15 nmol of 9,10-phenanthrenequinone, 0.6 mU of carbonyl reductase, and no superoxide dismutase (-) or 30 tg superoxide dismutase (- - -). The plot shows absorbance at 340 nm as a function of time. The recorder offset was adjusted so that the two traces would not overlap initially.
Inhibition
III
of 9,10-Phenanthrenequinone-Mediated Redox Cycling” Inhibition
Buffer 0.01 M PO1, pH 7.0 0.1 M PO,, pH 7.0 0.01 M Pod, pH 7.5 0.1 M PO,, pH 7.5 0.01 M Hepes, pH 7.0 0.1 M Hepes, pH 7.0 0.01 M Tris, pH 7.0 0.1 M Tris, pH 7.0
30 PLg SOD 88 54 89 30 93 88 90 92
30 pg SOD + 3 PM EDTA 90 85 93 81 95 91 94 96
(%) 30 gg SOD +lpMCuSO,
0 0 0 0 0 0 0 0
’ In addition to the components shown in the table, the cuvettes contained 225 nmol of NADPH, 3 nmol of 9,10-phenanthrenequinone, and 0.6 mU of enzyme in 3 ml of solution. The reactions were started with enzyme and the reaction rates were calculated from the slopes of the inhibited portion of the reaction. The reaction rates also were determined in cuvettes which did not contain dismutase, EDTA, or CuSO, but were in other respects identical to those above. These uninhibited rates were used to calculate percentage inhibition.
aromatic hydrocarbon quinones and their glutathione adducts may be considerably different from those proposed by Penning (19-21). In recent studies of a dihydrodiol dehydrogenase, Penning and his collaborators noted that this enzyme oxidized truns-dihydrodiols of polycyclic aromatic hydrocarbons to o-quinones (19,20). Glutathione and cysteine reacted nonenzymatically with these quinones, “resulting in their inactivation as potential electrophiles” (20). Because of the carcinogenicity of the 7,8dihydrodiol-9,10-epoxide of benzo[a]pyrene, the oxidation of the tran-s-7,8dihydrodiol of benzo[a]pyrene to 7,8benzo[u]pyrenequinone is particularly interesting. Penning and his co-workers suggested that oxidation of transdihydrodiols might be the first step in a two-step mechanism of polycyclic aromatic hydrocarbon detoxication. The second step involved the quinone being inactivated by reacting with a cellular nucleophile such as glutathione. Subsequent studies by these investigators showed that certain o-quinones (1,2-naphthoquinone and 7,8-benzo[ulpyrenequinone) acted as enzyme-generated irreversible inhibitors of the dihydrodiol dehydrogenase, presumably by reacting with an essential sulfhydryl group of the enzyme (21). The results of the present study demonstrate that polycyclic aromatic hydrocarbon quinones and their glutathione adducts are not necessarily products of detoxication. In tissues containing an enzyme with substrate specificity like the human placental carbonyl reductase,6 these compounds could undergo redox cycling to yield 6 These include carbonyl and human liver (23).
reductases present in the human brain (22)
338
JOSEPH
potentially hazardous substances (i.e., semiquinones and 0;). Furthermore the enzyme catalyzing this cycling is not inactivated in the process. Thus the carbonyl reductase, as well as a number of flavoproteins,’ catalyzes reactions which might account for the toxicity of certain quinones. In a recent study superoxide dismutase was observed to inhibit the redox cycling of certain quinones catalyzed by DT-diaphorase (17). The authors of that study suggested that these findings resulted from the dismutase acting as a superoxide: semiquinone oxidoreductase. Both earlier (28-30) and more recent (31-33) studies indicate that the inhibitory effect of superoxide dismutase on the autoxidation of a variety of compounds may be explained without having to postulate that it acts as a superoxide: semiquinone oxidoreductase. These studies demonstrate that the autoxidation results from a free-radical chain reaction. Since superoxide acts as a chain propagator, its destruction by superoxide dismutase inhibits the reaction. The addition of EDTA has been shown to enhance this inhibition (28, 30, 31, 33) while the addition of Cu2+ reverses it (28). Increasing the concentration of phosphate in the assay buffer from 0.01 to 0.1 M decreases dismutase activity, but changing the concentration of Hepes or Tris in the assay buffer has no effect (34). The results shown in Table III are consistent with these observations. In summary, the placental carbonyl reductase catalyzes the redox cycling of polycyclic aromatic hydrocarbon quinones, hydroquinones, and glutathione adducts of quinones. Although the physiologic significance of this observation is still unknown, the generation of semiquinones and 05 during redox cycling might play a role in the toxicity of these compounds.
3. Chung, H., Harvey, R. G., Armstrong, R. N., and Jarabak, J. (1987) J. Bill. Chem. 262, 12,44812,451. 4. Lorentzen, R. J., and Ts’o, P. 0. P. (1977) Biochemistry 16, 14671473. 5. Kappus, H., and Sies, H. (1981) Experientia
1. Lin, Y.-M., and Jarabak, J. (1978) B&hem.
Biophys. Res. Commun.
81,1227-1234. A., and Berkowitz,
D. (1983) Prostaglandins
‘Flavoprotein quinone reductases which transfer one electron are thought to be responsible for the toxicity of quinones (5,7), while those that transfer two electrons have generally been associated with protection against this toxicity, presumably because hydroquinones may be inactivated by undergoing glucuronidation (24) or other conjugation reactions (sometimes called Phase II reactions (25)). Whether enzymes that transfer two electrons actually protect against quinone-mediated toxicity may depend upon a number of factors, including the stability of the hydroquinone and which of a variety of conjugation reactions is predominant. For example, the DT-diaphorase has been shown to catalyze not only the redox cycling of quinones (4) but also the cycling of their glutathione adducts (26, 27).
37, 1233-1241.
6. Thor, H., Smith, M. T., Hartzell, P., Bellomo, G., Jewell, S. A., and Orrenius, S. (1982) J. Biol. Chem. 257, 12,419-12,425. 7. Chesis, P. L., Levin, D. E., Smith, M. T., Em&r, L., and Ames, B. N. (1984) Proc. Natl. Acad. Sci. USA 81,1696-1700. 8. Fieser, L. F. (1943) Org. Synth. 2,43S-432. 9. Platt, K. L., and Oesch, F. (1983) J. Org. Chem. 48, 265-268. 10. Fieser, L. F. (1940) J. Biol. Chem. 133,391-396. 11. Nickerson, 2,537-543.
W. J., Falcone, G., and Strauss, G. (1963) Biochemistry
12. McCord, J. M., and Fridovich, 6055. 13. Margoliash,
I. (1969) J. Bial. Chem. 244,6S49-
E. (1954) Biochem. J. S&535-543.
14. Frew, J. E., Jones, P., and Scholes, G. (1983) Anal. Chim. Acta 166,
139-150. 15. Winterbourn,
C. C. (1981) Arch. B&hem.
Biophys. 209,159-167.
16. Kappus, H. (1986) B&hem. Pharmacol35, l-6. 17. Cadenas, E., Mira, D., Brunmark, A., Lind, C., Segura-Aguilar, J., and Ernster, L. (1988) Free Radical Biol. Med. 5, 71-79. 18. Prochaska, H. J., and Talalay, P. (1987) Chcm. Ser. A 27,43-47. 19. Smithgall, T. E., Harvey, R. G., and Penning, T. M. (1986) J. Bial. Chem. 261,6184-6191. 20. Smithgall, T. E., Harvey, R. G., and Penning, T. M. (1988) J. Bial. Chem. 263,1814-1820. 21. Penning, T. M., Sharp, R. B., and Smithgall, T. E. (1989) Biachemistly 28,4505-4511. 22. Wermuth, B. (1981) J. Biol. Chem. 256, 1206-1213. 23. Wermuth, B., Platt, K. L., Seidel, A., and Oesch, F. (1986) B&hem. Pharmacol. 35.1277-1282. 24. Lind, C., Vadi, H., and Ernster,
L. (1978) Arch. Biochem. Biaphys.
190.97-108. 25. Williams, 1039.
REFERENCES
2. Jarabak, J., Luncsford, 26,849-868.
JARABAK
R. T. (1967) Fed. Proc. Fed. Am. Sot. Exp. Biol. 26,1029-
26. Wefers, H., and Sies, H. (1983) Arch. Biachem. Biaphys. 224,568578. 27. Bufhnton, G. D., Ollinger, K., Brunmark, Biachem. J. 267,561-571.
A., and Cadenas, E. (1989)
28. Mirsa, H. P., and Fridovich, I. (1972) J. Bial. Chem. 247, 31703175. 29. Cohen, G., and Heikkila, R. E. (1974) J. Bial. Chem. 249, 24472452. 30. Marklund, S., and Marklund, G. (1974) Eur. J. Biachem. 47,469474. 31. Ishii, T., and Fridovich,
I. (1990) Free Radical Biol. Med. 8, 21-24.
32. Ollinger, K., Buffinton, G. D., Ernster, L., and Cadenas, E. (1990) Chem. Bial. Interact. 73.53-76. 33. Bandy, B., Moon, J., and Davison, A. J. (1990) Free Radical Bial. Med. 9,143-148. 34. Cudd, A., and Fridovich,
I. (1982) J. Bial. C/rem. 257,11,443-11,447.
OF BIOCHEMISTRY
AND
BIOPHYSICS
Vol. 291, No. 2, December, pp. 334-338,199l
Polycyclic Aromatic Hydrocarbon Quinone-Mediated Oxidation Reduction Cycling Catalyzed by a Human Placental NADPH-Linked Carbonyl Reductase’ Joseph Jarabak Department
of Medicine,
University
of Chicago, Box 435,584l
South Maryland
Avenue, Chicago, Illinois 60637
Received May 21, 1991, and in revised form August 9, 1991
Polycyclic aromatic hydrocarbon quinones, hydroquinones, and glutathionyl adducts of quinones undergo oxidation-reduction (redox) cycling in the presence of NADPH and the NADPH-linked human placental carbony1 reductase. K-region and non-K-region o-quinones and their glutathione adducts are the best substrates of this enzyme; they are reduced to hydroquinones. Under aerobic conditions, the hydroquinones are autoxidized with the formation of potentially hazardous semiquinones and the superoxide anion. Because of these reactions it is unlikely that polycyclic aromatic hydrocarbon quinones or their glutathione adducts are inert products of detoxication in tissues that contain the carbonyl reductase or another enzyme with similar substrate specificity. If superoxide dismutase is added to reaction mixtures containing the carbonyl reductase and quinones, it inhibits redox cycling. Presumably this results from destruction of the superoxide anion which acts as a chain Q iosi Academic PAM, I~C. propagator in these reactions.
Human placental tissue contains a NADP-linked oxidoreductase which was initially thought to be a Shydroxyprostaglandin dehydrogenase/g-ketoprostaglandin reductase (1). Subsequent studies have shown this enzyme to have such broad substrate specificity that it is more aptly called a carbonyl reductase (2). Various K-region and non-K-region polycyclic aromatic hydrocarbon oquinones and their glutathione adducts are its best substrates (3). When these quinones or their glutathione adducts are present in limiting concentrations, an excess of NADPH is completely oxidized under aerobic conditions, suggesting that the quinone substrate has undergone ox1 This work was supported by American 701. 334
Cancer Society Grant BC-
idation-reduction (redox) cycling (3). During the course of redox cycling a quinone is reduced to a semiquinone or hydroquinone depending on whether the reduction involves the transfer of one or two electrons. In the presence of oxygen the semiquinone or hydroquinone is autoxidized to regenerate the quinone while the oxygen is reduced to yield a variety of products which may include the superoxide anion radical (O;), Hz02 , the hydroxyl radical (OH’), and singlet oxygen (4,5) depending on the setting. In view of these characteristics, the reaction between the carbonyl reductase and its quinone substrates has been examined in more detail. The influence of the enzyme superoxide dismutase on this reaction has also been examined since superoxide dismutase has proved useful in investigating the role of 0; in certain quinone-mediated reactions (6, 7). EXPERIMENTAL
PROCEDURES
Materials. NADP and NADPH were obtained from Pharmacia Biotechnology, Inc., phenol and XAD-2 from Mallinckrodt, and silica gel and Polygram Cel 400 TLC plates from Brinkman Instruments, Inc. Acetonitrile was obtained from Burdick and Jackson, and trifluoroacetic acid from Pierce Chemical Co.. Glutathione, 4aminoantipyrine, superoxide dismutase, horse heart cytochrome c (Type VI), and horseradish peroxidase were obtained from Sigma, and 9,10-phenanthrenequinone, 2-methyl-1,4benxoquinone, and 2-methyl-1,4-naphthoquinone from Eastman (these were recrystallixed prior to their use). Compounds synthesized by methods reported previously include 1,2-naphthoquinone (8), 1,2naphthohydroquinone (9), 1,2naphthodihydrodiol(9), 2-methyl1,4-naphthohydroquinone (lo), 2-methyl-5-glutathionyl-l,l-benxoquinone (3, ll), and 2-methyl-3-glutathionyl-1,4-naphthoquinone (3, 11). The compounds 9,10-phenanthrenedihydrodiol, 4,5- and 7,6benxo[a]pyrenequinone, and 5,6-chrysenequinone were generously provided by Professor R. G. Harvey. Volumes of 14 ml Synthesis of 4-glutathionyl-1,2-naphthoquinone. of 2 mM 1,2-naphthoquinone in acetonitrile and 14 ml of 3.3 mM glutathione in water were mixed and allowed to stand overnight at room temperature. The mixture was extracted with three 25-ml portions of ethyl acetate, and aliquots of the aqueous phase were applied to an Altex ooo3-9861/91$3.00
Copyright 0 1991 by Academic Press, Inc. All rightg of reproduction in any form reserved.
QUINONE-MEDIATED Ultrasphere ODS C1s HPLC’ column (4.6 X 250 mm, 5-pm particle size). The product was eluted using a linear gradient from 0.075% trifluoroacetic acid in water to 0.075% trifluoroacetic acid in 100% acetonitrile. The retention time of the glutathione adduct was 28 min; it gave a single ninhydrin positive spot (Rf = 0.35) on TLC performed on a cellulose plate with a solvent containing ethanol and water (77~23). Enzyme. Human placental NADP-linked 15-hydroxyprostaglandin dehydrogenase/g-ketoprostaglandin reductase was purified as described previously (1). It migrates as a single protein on disc electrophoresis, and the preparation used had a specific activity of 980 mU/mg protein. Preliminary studies were performed with enzyme purified through step 4 of the purification (specific activity = 50 mU/mg protein). These showed similar results to those obtained with the more highly purified enzyme. Assays. The reduction of quinones by the placental enzyme was measured as described previously (3) with the exception that 280 pmol of potassium phosphate, pH 7.0, was used instead of Tris-HCl. The formation of superoxide was assayed by measuring the reduction of cytochrome c at 550 nm (12). The molar extinction coefficient used for this reaction was 18,500 M-‘cm+ (13). Hydrogen peroxide was determined with a horseradish-peroxidase-coupled assay (14).
RESULTS
NADPH oxidation and Hz02 production during the reo?oxcycling of quinones and hydroquinones. The amounts of NADPH oxidized and HzOz produced during the redox cycling of quinones and hydroquinones and the effect of superoxide dismutase on these values are shown in Table I. For each reaction, the concentration of the quinone or hydroquinone is limiting. Despite this, most reactions proceed until the NADPH is completely oxidized.3 Hydrogen peroxide is formed during the course of most of these reactions in an amount that is somewhat less than the NADPH oxidized.4 After the oxidation of NADPH has gone to completion, the addition of more NADPH results in more NADPH oxidation and more HzOz formation (data not shown). This and HPLC or TLC studies (data not shown) reveal that the quinone is still present after a large excess of NADPH has been completely oxidized. The results with hydroquinones are similar to those with quinones.5 If NADPH is replaced by NADP, hydro* Abbreviations used: HPLC, high-performance liquid chromatography; TLC, thin-layer chromatography; SOD, superoxide dismutase; EDTA, ethylenediaminetetraacetate; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid. 3 Exceptions are 2-methyl-1,4-benzoquinone, 2-methyl-5-glutathionyl1,4-benzoquinone, the trans-1,2dihydrodiol of naphthalene and the trans-9,10-dihydrodiol of phenanthrene. The reactions with the two benzoquinones proceed until about half of the quinone is reduced. Neither of the dihydrodiols is a substrate in the presence of NADPH or NADH (not shown) or in the presence of NADP (not shown) or NAD (not shown). 4 Again 2-methyl-1,4-benzoquinone and 2-methyl-5-glutathionyl-1,4benzoquinone are exceptions. No HzOz is formed during the reduction of either of these compounds. ’ These observations indicate that the quinones and hydroquinones are undergoing redox cycling. They are markedly different from those when 9-keto- or 15-ketoprostaglandins are reduced by the carbonyl reductase (data not shown). When these ketoprostaglandins are substrates, the quantities of NADPH oxidized, ketoprostaglandin reduced, and hydroxyprostaglandin generated are equimolar and the reaction continues until an equilibrium has been reached.
REDOX
CYCLING
335
quinones do not undergo redox cycling. If glutathione adducts of quinones are substituted for quinones as substrates, the rates of NADPH oxidation and H202 production are comparable to, or more rapid than, those for the parent quinones. In the presence of superoxide dismutase, there is a striking inhibition of both NADPH oxidation and HzOz production. Data relating to this inhibition will be presented in a subsequent section. Superoxide formation during redox cycling. A sensitive indirect method for detecting the superoxide anion involves measuring the reduction of cytochrome c by this radical in the presence and absence of superoxide dismutase (12). Superoxide dismutase inhibits that portion of cytochrome c reduction due to the superoxide anion (12). In order to estimate the superoxide generated during redox cycling of quinones and hydroquinones, cytochrome c was added to reaction mixtures that were in other respects identical to those in Table I. Table II compares the rate of cytochrome c reduction in the presence and absence of superoxide dismutase. All of the quinones and hydroquinones that were examined mediate a rapid reduction of cytochrome c. In the presence of superoxide dismutase the initial rates of these reductions are inhibited by less than 50%. The reduction of cytochrome c that occurs as a consequence of the reduction of 7,8benzo[a]pyrenequinone provides a particularly striking example of this phenomenon. It is inhibited only 11% by superoxide dismutase. When 1,2-naphthohydroquinone or 2-methyl-1,4-naphthohydroquinone are added to reaction mixtures which contain no carbonyl reductase, the inhibition by dismutase is 23 and 9%, respectively. The results obtained when xanthine oxidase is used to generate the superoxide radical are quite different. Under such conditions the reduction of cytochrome c is inhibited by 94% in the presence of superoxide dismutase. Thus although the reduction of quinones gives rise to superoxide anions, most of the reduction of cytochrome c is due to substances that are not destroyed by superoxide dismutase. Similar results are obtained with the autoxidation of hydroquinones. These findings suggest that the reducing substances are hydroquinones (7), semiquinones (15), or both. Effectof superoxide dismutase on the time course of redox cycling. The time course of g,lO-phenanthrenequinone-mediated redox cycling (Fig. 1) shows that the rate of NADPH oxidation is the same in the absence and presence of superoxide dismutase until all of the quinone substrate has been reduced. Then the inhibitory effect of the superoxide dismutase becomes apparent. This suggests that the effect of the dismutase is to inhibit autoxidation of the hydroquinone to the quinone. Factors that influence the superoxide-dismutase-mediated inhibition of redox cycling. The inhibition of redox cycling by superoxide dismutase is influenced by a
336
JOSEPH
JARABAK
TABLE Redox
Cycling
Mediated
I
by Quinones
and Hydroquinones
NADPH oxidized” (nmol) Substrate (nmol)
Substrate 2-Methyl-1,4benzoquinone 2-Methyl-&glutathionyl-1,4-benzoquinone 1,2-Naphthoquinone 1,2-Naphthohydroquinone trun.s-1,2-Dihydroxy-1,2dihydronaphthalene 4-Glutathionyl-1,2-naphthoquinone 2-Methyl-1,4-naphthoquinone 2-Methyl-1,4-naphthohydroquinone 2-Methyl-3-glutathionyl-1,4-naphthoquinone 9,10-Phenanthrenequinone truns-9,10-Dihydroxy-9,10-dihydrophenanthrene 4,5Benzo[a]pyrenequinone 7,8-Benzo[a]pyrenequinone 5,6Chrysenequinone
-SOD
HsOz produced (nmol) Inhibition by SOD (W)
+SOD*
-SOD
+SOD
Inhibition by SOD (W)
0 0
40 40 10 10 10 10
27 21 92 114 0 140
40 40 38 3.0 47 7.0 7.0 7.8
140 176 150 170
55
196
0
0
196
92 100
0 95
80 95
32
0
166
60
37
67 13
39 90
0 0 0
100 166 166
147 33 78
166
38
110
130
0
140
15
93
0 10
180
89 100 94
99
78 145
’ The amount of NADPH added to the reaction cuvettes varied from 150 to 190 nmol and the amount of enzyme varied from 1.5 to 6 mU in a total reaction volume of 3 ml. Blank cuvettes were identical to the reaction cuvettes but contained no enzyme. The reactions were started with the addition of enzyme and were continued until the oxidation of NADPH ceased in cuvettes which did not contain superoxide dismutase. At that time the contents of all cuvettes were transferred to the reaction mixture used for the HzOz assay. The amount of NADPH oxidized and HsOs produced were determined by subtracting blanks from the appropriate reaction cuvettes. The reactions with and without superoxide dismutase were run at the same time for each substrate. * Thirty micrograms of superoxide dismutase was added.
of factors including the substrate, the buffer, and whether chelating agents or metal salts have been added to the reaction mixture. A summary of some of these effects on the redox cycling of 9,10-phenanthrenumber
TABLE Cytochrome
c Reduction
Mediated
nequinone is presented in Table III. Control studies (not shown) indicate that none of the parameters being varied influence the activity of the carbonyl reductase.
II by Quinones
and Hydroquinones Cytochrome
Substrate 2-Methyl-1,4-benzoquinone 2-Methyl-5-glutathionyl-1,4-benzoquinone 1,2-Naphthoquinqne 1,2-Naphthohydroquinone trons-1,2-Dihydroxy-1,2dihydronaphthalene 4-Glutathionyl-1,2-naphthoquinone 2-Methyl-1,4-naphthoquinone 2-Methyl-1,4-naphthohydroquinone 2-Methyl-3-glutathionyl-1,4-naphthoquinone 9,10-Phenanthrenequinone trans-9,10-Dihydroxy-9,lOdihydrophenanthrene 4,5-Benzo[a]pyrenequinone 7,8-Benzo[a]pyrenequinone 5,6-Chrysenequinone
Substrate (nmol)
40 40 10 10 10 10
40 10
38 3.0 47 7.0 7.0 7.8
-SOD
c reduced/l0 (nmol)
min”
+SOD*
7.1 4.7 6.2 430
3.7 2.4 3.7 330
0
0 19
22 12 172 29
75
7.8
Inhibition by SOD (%)
48 49
40 23 14
35
157
9
23 58
21 23
0
0
22
13
19 64
17
11
46
28
41
’ The contents of the reaction cuvettes are the same as those described in Table I except that 60 nmol of cytochrome c was present in each cuvette and 0.2 mU of enzyme was added to start most reactions. The initial rate of cytochrome c reduction was determined by measuring the change in absorbance at 550 nm. When 1,2-naphthohydroquinone and P-methyl-1,4naphthohydroquinone were substrates no enzyme was added. * Thirty micrograms of superoxide dismutase was added.
QUINONE-MEDIATED
BEDOX
331
CYCLING
DISCUSSION
TABLE
Although the human placental carbonyl reductase has a broad substrate specificity, K-region and non-K-region o-quinones of certain polycyclic aromatic hydrocarbons and their glutathione adducts are its best substrates. An analysis of the stoichiometry of this reaction reveals that all of the NADPH in the reaction mixture is oxidized in the presence of only catalytic amounts of these quinones or their hydroquinones. Furthermore, superoxide and Hz02 are formed in the process. These findings indicate that certain o-quinones undergo oxidation-reduction cycling in the presence of NADPH and the carbonyl reductase. The catalysis of redox cycling by the placental carbonyl reductase is noteworthy for three reasons. First, the placental enzyme occurs in the cytosol and it is not a flavoprotein, while most enzymes which catalyze redox cycling are microsomal flavoproteins (16). Second, both Kregion and non-K-region o-quinones and their glutathione adducts are the best substrates for the placental enzyme. Although not studied in detail (7, 17, 18), that does not appear to be the case with other enzymes catalyzing redox cycling. Third, the metabolic consequences of polycyclic
--
r
Carbonyl Reductase
Added
----___ 0.40
0.35
0.30
0.25
o.20 L-t+-10
50
30
Time
(min)
FIG. 1. The effect of superoxide dismutase on S,lO-phenanthrenequinone-mediated redox cycling. The assay cuvettes contained in a volume of 3 ml, 280 Fmol of potassium phosphate, pH 7.0, 225 nmol of NADPH, 15 nmol of 9,10-phenanthrenequinone, 0.6 mU of carbonyl reductase, and no superoxide dismutase (-) or 30 tg superoxide dismutase (- - -). The plot shows absorbance at 340 nm as a function of time. The recorder offset was adjusted so that the two traces would not overlap initially.
Inhibition
III
of 9,10-Phenanthrenequinone-Mediated Redox Cycling” Inhibition
Buffer 0.01 M PO1, pH 7.0 0.1 M PO,, pH 7.0 0.01 M Pod, pH 7.5 0.1 M PO,, pH 7.5 0.01 M Hepes, pH 7.0 0.1 M Hepes, pH 7.0 0.01 M Tris, pH 7.0 0.1 M Tris, pH 7.0
30 PLg SOD 88 54 89 30 93 88 90 92
30 pg SOD + 3 PM EDTA 90 85 93 81 95 91 94 96
(%) 30 gg SOD +lpMCuSO,
0 0 0 0 0 0 0 0
’ In addition to the components shown in the table, the cuvettes contained 225 nmol of NADPH, 3 nmol of 9,10-phenanthrenequinone, and 0.6 mU of enzyme in 3 ml of solution. The reactions were started with enzyme and the reaction rates were calculated from the slopes of the inhibited portion of the reaction. The reaction rates also were determined in cuvettes which did not contain dismutase, EDTA, or CuSO, but were in other respects identical to those above. These uninhibited rates were used to calculate percentage inhibition.
aromatic hydrocarbon quinones and their glutathione adducts may be considerably different from those proposed by Penning (19-21). In recent studies of a dihydrodiol dehydrogenase, Penning and his collaborators noted that this enzyme oxidized truns-dihydrodiols of polycyclic aromatic hydrocarbons to o-quinones (19,20). Glutathione and cysteine reacted nonenzymatically with these quinones, “resulting in their inactivation as potential electrophiles” (20). Because of the carcinogenicity of the 7,8dihydrodiol-9,10-epoxide of benzo[a]pyrene, the oxidation of the tran-s-7,8dihydrodiol of benzo[a]pyrene to 7,8benzo[u]pyrenequinone is particularly interesting. Penning and his co-workers suggested that oxidation of transdihydrodiols might be the first step in a two-step mechanism of polycyclic aromatic hydrocarbon detoxication. The second step involved the quinone being inactivated by reacting with a cellular nucleophile such as glutathione. Subsequent studies by these investigators showed that certain o-quinones (1,2-naphthoquinone and 7,8-benzo[ulpyrenequinone) acted as enzyme-generated irreversible inhibitors of the dihydrodiol dehydrogenase, presumably by reacting with an essential sulfhydryl group of the enzyme (21). The results of the present study demonstrate that polycyclic aromatic hydrocarbon quinones and their glutathione adducts are not necessarily products of detoxication. In tissues containing an enzyme with substrate specificity like the human placental carbonyl reductase,6 these compounds could undergo redox cycling to yield 6 These include carbonyl and human liver (23).
reductases present in the human brain (22)
338
JOSEPH
potentially hazardous substances (i.e., semiquinones and 0;). Furthermore the enzyme catalyzing this cycling is not inactivated in the process. Thus the carbonyl reductase, as well as a number of flavoproteins,’ catalyzes reactions which might account for the toxicity of certain quinones. In a recent study superoxide dismutase was observed to inhibit the redox cycling of certain quinones catalyzed by DT-diaphorase (17). The authors of that study suggested that these findings resulted from the dismutase acting as a superoxide: semiquinone oxidoreductase. Both earlier (28-30) and more recent (31-33) studies indicate that the inhibitory effect of superoxide dismutase on the autoxidation of a variety of compounds may be explained without having to postulate that it acts as a superoxide: semiquinone oxidoreductase. These studies demonstrate that the autoxidation results from a free-radical chain reaction. Since superoxide acts as a chain propagator, its destruction by superoxide dismutase inhibits the reaction. The addition of EDTA has been shown to enhance this inhibition (28, 30, 31, 33) while the addition of Cu2+ reverses it (28). Increasing the concentration of phosphate in the assay buffer from 0.01 to 0.1 M decreases dismutase activity, but changing the concentration of Hepes or Tris in the assay buffer has no effect (34). The results shown in Table III are consistent with these observations. In summary, the placental carbonyl reductase catalyzes the redox cycling of polycyclic aromatic hydrocarbon quinones, hydroquinones, and glutathione adducts of quinones. Although the physiologic significance of this observation is still unknown, the generation of semiquinones and 05 during redox cycling might play a role in the toxicity of these compounds.
3. Chung, H., Harvey, R. G., Armstrong, R. N., and Jarabak, J. (1987) J. Bill. Chem. 262, 12,44812,451. 4. Lorentzen, R. J., and Ts’o, P. 0. P. (1977) Biochemistry 16, 14671473. 5. Kappus, H., and Sies, H. (1981) Experientia
1. Lin, Y.-M., and Jarabak, J. (1978) B&hem.
Biophys. Res. Commun.
81,1227-1234. A., and Berkowitz,
D. (1983) Prostaglandins
‘Flavoprotein quinone reductases which transfer one electron are thought to be responsible for the toxicity of quinones (5,7), while those that transfer two electrons have generally been associated with protection against this toxicity, presumably because hydroquinones may be inactivated by undergoing glucuronidation (24) or other conjugation reactions (sometimes called Phase II reactions (25)). Whether enzymes that transfer two electrons actually protect against quinone-mediated toxicity may depend upon a number of factors, including the stability of the hydroquinone and which of a variety of conjugation reactions is predominant. For example, the DT-diaphorase has been shown to catalyze not only the redox cycling of quinones (4) but also the cycling of their glutathione adducts (26, 27).
37, 1233-1241.
6. Thor, H., Smith, M. T., Hartzell, P., Bellomo, G., Jewell, S. A., and Orrenius, S. (1982) J. Biol. Chem. 257, 12,419-12,425. 7. Chesis, P. L., Levin, D. E., Smith, M. T., Em&r, L., and Ames, B. N. (1984) Proc. Natl. Acad. Sci. USA 81,1696-1700. 8. Fieser, L. F. (1943) Org. Synth. 2,43S-432. 9. Platt, K. L., and Oesch, F. (1983) J. Org. Chem. 48, 265-268. 10. Fieser, L. F. (1940) J. Biol. Chem. 133,391-396. 11. Nickerson, 2,537-543.
W. J., Falcone, G., and Strauss, G. (1963) Biochemistry
12. McCord, J. M., and Fridovich, 6055. 13. Margoliash,
I. (1969) J. Bial. Chem. 244,6S49-
E. (1954) Biochem. J. S&535-543.
14. Frew, J. E., Jones, P., and Scholes, G. (1983) Anal. Chim. Acta 166,
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C. C. (1981) Arch. B&hem.
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