The Mechanism of Stimulation of NADPH Oxidation during the

Jul 29, 1993 - The 1-OH-NMC is further metabolized in an NADPH- dependent manner by the reconstituted system or by purified NADPH-cytochrome P450...
0 downloads 0 Views 1MB Size
Download PDF
Chem. Res. Toxicol. 1994, 7, 231-238

23 1

The Mechanism of Stimulation of NADPH Oxidation during the Mechanism-Based Inactivation of Cytochrome P450 2B1 by N-Methylcarbazole: Redox Cycling and DNA Scission+ Tingliang Shed and Paul F. Hollenberg' Department of Pharmacology, Wayne State University School of Medicine, 540 East Canfield, Detroit, Michigan 48201 Received July 29,1993"

The oxidation rate of NADPH is markedly stimulated during the mechanism-based inactivation of cytochrome P450 2B1 by N-methylcarbazole (NMC) in a reconstituted system consisting of NADPH-cytochrome P450 reductase, cytochrome P450, and phospholipid. The stimulation of NADPH oxidation in this system is due to l-hydroxy-N-methylcarbazole(1-OH-NMC), one of the major metabolites of NMC. The 1-OH-NMC is further metabolized in an NADPHdependent manner by the reconstituted system or by purified NADPH-cytochrome P450 reductase to give a more polar metabolite which has been isolated by HPLC. The conversion of 1-OH-NMC to this product was inhibited by superoxide dismutase (SOD), and incubation of the 1-OH-NMC with hypoxanthine-xanthine oxidase resulted in the formation of the same product, suggesting that the superoxide anion was involved in the metabolism of 1-OH-NMC by the reductase. Redox cycling activity during the metabolism of 1-OH-NMC by reductase has been demonstrated. The oxidation of NADPH by the reductase in the presence of 35 pM 1-OH-NMC was enhanced approximately 23-fold [240 nmol of NADPH oxidized/(min.nmol of reductase)] relative to control levels in the presence of 500 pM NMC [ 10.5 nmol/(min-nmol of reductase)]. 1-OH-NMC (35 pM) caused a40-fold increase in the rate of formation of superoxide during its metabolism by reductase. The rapid rates of NADPH oxidation and superoxide formation were inhibited by the addition of reduced glutathione (GSH) to the reaction mixture. Neither SOD nor GSH inhibited the reductase activity directly. These results suggest that a quinone-like compound may be formed from 1-OH-NMC and that this metabolic intermediate can redox cycle and consume NADPH. DNA damage caused during the metabolism of 1-OHNMC was demonstrated by monitoring 4X-174 DNA strand scission.

Introduction Carbazole and several of its alkylated derivatives have been detected as major components of cigarette smoke condensates, occurring in amounts equal to or greater than the polycyclic aromatic hydrocarbons such as benzol'alpyrene (1-3). Subfractions of tobacco smoke condensate containing methylcarbazoles as major constituents were shown to exhibit tumor-initiating activity (4,5). Although N-methylcarbazole (NMC)1 was inactive as a tumor initiator, it was active as a coinitiator or accelerator when applied on mouse skin with benzo[alpyrene (5). NMC has been shown to be mutagenic in Salmonella typhimurium TA 100 in the presence of rat liver homogenates (6). Mutagenic activity was also observed with the 2-, 3-, and 4-methyl derivatives of NMC (6). N-(Hydroxymethy1)carbazole (NHMC),a metabolite of NMC, is a very potent mutagen for S. typhimurium in the presence of rat f The data in this paper are taken from a thesis submitted by T.S. in partial fulfillment of the requirement for the Doctor of Philosophy in Pharmacology in the Graduate School of Wayne State University. Author to whom correspondence should be addressed. 8 Present address: Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI 48109. Abstract published in Aduance ACS Abstracts, March 1, 1994. 1 Abbreviations: P450, cytochrome P450; P450 2B1, the major form of P450 from liver microsomes of phenobarbital-treated rats, P450 2B4, the major form of P450 from liver microsomesof phenobarbital-treated rabbits, reductase, NADPH-cytochrome P450 reductase; DLPC, dilauroyl-L-a-phosphatidylcholine; NMC,N-methylcarbazole;1-OH-NMC, l-hydroxy-NMC;2-OH-NMC,2-hydroxy-NMC;3-OH-NMC,3-hydroxyNMC; NHMC, N-(hydroxymethy1)carbaole; NFC, N-formylcarbazole; GSH, reduced glutathione; SOD,superoxide dismutaae.

liver S-9and cofactors, exhibiting a mutagenicity comparable to that of benzo[a]pyrene (6, 7). The metabolism of NMC has been studied in a number of different mammalian species including rabbits (7, 8), rats (7, 9, IO), mice, guinea pigs, and hamsters (10). As shown in Figure 1, NMC is converted to four major metabolites: NHMC, l-hydroxy-N-methylcarbazole(1OH-NMC),2-hydroxy-N-methylcarbazole (2-OH-NMC), and 3-hydroxy-N-methylcarbazole(7, 8). In addition, carbazole, presumably formed due to the decomposition of NHMC, and N-formylcarbazole (NFC) have been detected (11). Metabolism of NMC by phenobarbitalpretreated rabbit liver microsomes or by purified cytochrome P450 2B1 or P450 2B4 in a reconstituted system consisting of cytochrome P450 (P450), NADPH-cytochrome P450 reductase (reductase), and dilauroyl-L-aphosphatidylcholine (DLPC)results in the same four major hydroxylated metabolites already indicated (7,8,12).NMC has also been shown to be a mechanism-based inactivator of purified P450 2B1 (12,13). During the inactivation of P450 2B1 by NMC, the rate of NADPH oxidation increased significantly while the catalytic activity of P450 2B1 for the metabolism of NMC and 7-ethoxycoumarin was destroyed (14). We have undertaken studies to determine why the NADPH oxidase activity was markedly increased during the mechanism-based inactivation of P450 2B1 by NMC. The studies described here show that the stimulation of NADPH consumption during P450 inactivation is due to

0893-228x/9412707-0231$04.50/00 1994 American Chemical Society

232 Chem. Res. Toxicol., Vol. 7, No. 2, 1994

I

V

I

I

V

I

, 7% on

Figure 1. Chemical structures of NMC and ita four major liver microsomal metabolites.

1-OH-NMC, one of the major metabolites of NMC. The 1-OH-NMC is further metabolized by the reductase to give a more polar product. Our results suggest that metabolism of 1-OH-NMC involves formation of a quinone-like intermediate which can then be involved in redox cycling and generate reactive oxygen species. The involvement of redox cycling, the production of oxygen free radicals, and the induction of DNA damage during the metabolism of 1-OH-NMC by reductase have been characterized in this report.

Experimental Procedures Materials. The following chemicals were purchased from Sigma Chemical Co. (St.Louis, MO): NADPH, DLPC, reduced glutathione (GSH),superoxide dismutase (SOD),hypoxanthine, xanthine oxidase, catalase, benzoate, deferoxamine mesylate, cytochrome c, and 4X-174 DNA. HPLC-grade hexane, 2-propanol, and acetonitrile were purchased from Burdick & Jackson (Muskegon, MI). Phenobarbital was obtained from Henry Schein, Inc. (Port Washington, NY). NMC was synthesized by the method of Stevens and Tucker (15). Preparation of Microsomes and Isolation of Enzymes. Microsomes were prepared from the livers of fasted male LongEvans rata (150-175 g, Harlan Sprague Dawley; Indianapolis, IN) pretreated with phenobarbital (0.1% in the drinking water for 12 days) as previously described (16),and they are stored at -70 "C until used. P450 2B1 was purified to electrophoretic homogeneity according to the procedure of Imai et al. (17)and exhibited a final specific content of 11-13 nmol/mg of protein. Reductase was purified to electrophoretic homogeneity by the method of Guengerich and Martin (18). Assay for NADPH Oxidation. The oxidation of NADPH in the reconstituted system was monitored in incubation mixtures containing either 0.05nmol of P460 2B1,O.l nmol of reductase, and 30 pM DLPC or 0.1 nmol of reductase only in 50 mM potassium phosphate buffer (pH 7.4) in a final volume of 1mL in the presence or absence of substrates. After preincubation for three min a t 30 OC, the reaction was initiated by the addition of 0.1 mM NADPH, and the oxidation of NADPH was monitored by following the decrease in absorbance at 340 nm in a Uvikon 860 spectrophotometer. An extinction coefficient of 6.2 mM-' cm-1 was used to convert absorbance changes to nanomoles of NADPH consumed (18). Preparation of Acetylated Cytochrome c. Acetylated with cytochrome c was prepared by the method of Azzi et al. (19), some modifications. Ferricytochrome c (100 mg) was added to

Shen and Hollenberg 10 mL of a half-saturated solution of sodium acetate a t 0 OC. A 10-fold excess of acetic anhydride (180 pL) with respect to the lysine residues of cytochromec (20mol of lysines/mol) was added with stirring. The reaction was allowed to proceed with stirring a t 0 "C for 30 min. The solution of acetylated cytochrome c was dialyzed for 24 h a t 4 "C against 300 mL of distilled water with four changes. The concentration of acetylated cytochromec was determined spectrally by measuring the absorbance at 548 nm using an extinction coefficient of 18.6 mM-l cm-l for the difference between the oxidized and reduced forms of the protein. The protein was reduced with sodium dithionite. Measurement of Superoxide Formation. The reaction mixtures contained 0.1 nmol of reductase, 0.1 mM EDTA, and 30 pM acetylated cytochrome c in 1.0 mL of 50 mM potassium phosphate buffer (pH 7.4) with either 35 pM 1-OH-NMC or 500 pM NMC. The reaction mixtures were preincubated at 30 "C for 3 min, and the reactions were initiated by the addition of 0.5 mM NADPH. Superoxide anion formation was determined spectrophotometrically by measuring the rate of reduction of acetylated cytochrome c at 548 nm during the first 3 min of incubation in the presence and absence of 800 units of superoxide dismutase (SOD). An extinction coefficient of 19.6 mM-I cm-' was used to quantitate cytochrome c reduction. The SODinhibitable reduction of acetylated cytochrome c was used to quantitate production of superoxide anion.

Detection and Isolation of the Metabolite of 1-OH-NMC. The 1-OH-NMC, obtained as a product of NMC metabolism by rat liver microsomes from phenobarbital-pretreated rats, was isolated using normal-phase HPLC as described by Koop and Hollenberg (7). Samples of the eluates were collected by hand, and peaks from several runs were pooled to obtain sufficient quantities of 1-OH-NMC. The eluates were evaporated to dryness under reduced pressure using a Cole Parmer vacuum pump, and the 1-OH-NMCwas redissolved in methanol. The concentration of 1-OH-NMC was determined spectrophotometrically using an extinction coefficient of 22.9 mM-l cm-1 at 254 nm as described by Novak et al. (8). 1-OH-NMC (35 pM) was incubated with 0.1 nmol of reductase and 2.5 mM NADPH in a final volume of 1.0 mL of 50mM potassium phosphate buffer (pH 7.4). The reaction mixtures were incubated a t 37 OC for 45 min, and the reactions were terminated by the addition of ethyl acetate (3 mL). The substrate and metabolites were recovered from the reaction mixture by three successive extractions with 3 mL of ethyl acetate. The extracts were combined, evaporated to about 1mL with Nn, loaded onto a 0.5 X 7 cm silica gel column (30-70 mesh), and eluted with 9 mL of hexane/2-propanol(85:15). The eluates were evaporated to dryness with Na. The metabolite of 1-OH-NMC was separated by normal-phase HPLC using a Glenco System I isocratic pump and a Whatman Partisi10.4 X 25 cm silica column. The eluate was monitored at 254 nm using a Varian 43001-00 UV detector. The column was eluted isocratically with a 90% hexane/lO% 2-propanol mobile phase and a flow rate of 1mL/ min. Metabolism of 1-OH-NMCby Xanthine Oxidase. The reaction mixtures contained 50mM potassium phosphate buffer (pH 7.4), 0.2 unit of xanthine oxidase, 2 mM hypoxanthine, and 35 pM 1-OH-NMC in a final volume of 1.0 mL. After a 3-min preincubation, the reaction was initiated by addition of hypoxanthine. The reaction mixture was incubated for 60 min a t 37 "C, and the metabolite of 1-OH-NMCwas extracted and detected by HPLC as described above. 4X-174 DNA Strand Scission Assay. The conversion of $X-174 DNA from the supercoiled form to the open circular form was used as an index of DNA damage. The assay was performed as described previously (20,21). The reaction mixtures (20 pL) contained 50 mM potassium phosphate buffer (pH 7.41, 0.2 pg of 4X-174 DNA, 0.01 nmol of reductase, 10 or 35 pM 1-OH-NMC as indicated, and 2 mM NADPH. Reactions were started by the addition of NADPH, and the reaction mixtures were incubated at 37 OC for 2 h. Controls contained only 4X-174 DNA in buffer or 4X-174 DNA plus the above componentaexcept NADPH was omitted. When present in the incubations, the

NMC Metabolism a n d Stimulation of N A D P H Oxidation

Chem. Res. Toxicol., Vol. 7,No. 2, 1994 233

I

0.8 I

A

. \ 0 1 0

I

0.1

B

c

1 .5

3

4.5

91

6

cy

a

Time (min)

Figure 2. NADPH oxidation rates during and after the mechanism-based inactivation of P450 2B1 by NMC. (A) The reaction mixture contained 0.05 nmol of P450 2B1,0.075 nmol of reductase, 30 pg of DLPC, 0.1 mM NMC, and 50 mM potassium phosphate buffer (pH 7.4) in a final volume of 1mL. The reaction was initiated by the addition of NADPH (0.1mM), and the change in absorbance at 340 nm was recorded on a Uvikon 860 spectrophotometer as described in the Experimental Procedures. (B) NADPH oxidation was measured after complete inactivation of P450 2B1 by NMC in the reaction mixture indicated in panel A. The reaction mixture was dialyzed a t 4 "C for 72 h against 50 mM potassium phosphate buffer (pH 7.4) containing 20% glycerol with six changes. NMC (0.1 mM) was added to this mixture, and then NADPH was added to the cuvette at 0 time to start the reaction. (C) NADPH oxidation was measured after addition to the solution indicated in panel B of a reaction mixture comparable to that in panel A and which had been heated to 70 "C for 5 min to inactivate the P450 and reductase. amounts of scavengers, deferoxamine mesylate or HzOz, were as indicated. When catalase was used, an equal amount of bovine albumin was added to controls in order to test for nonspecific protein effects on DNA damage. Reactions were terminated by addition of 3 p L of gel loading buffer (pH 8.0) containing 50% glycerol, 0.1 M Na*EDTA, 1.0% SDS, 0.25% bromophenol blue and 0.25% xylene cyanole. The supercoiled and open circular forms of 4X-174 DNA were separated by electrophoresis on a 1% agarose gel containing 0.05% ethidium bromide using a BRL Model H3 horizontal gel electrophoresis system with a Bio-Rad Model 1420 power supply. The electrophoresis was run at 65 V for 4 h. Following electrophoresis,the DNA bands were visualized and photographed using a UV transilluminator.

Results Stimulation of NADPH Oxidation by 1-OH-NMC. As shown in Figure 2, trace A, the rate of NADPH oxidation increased markedly during the metabolism of NMC by the reconstituted system. Under the conditions used for these studies, the P450 was more than 90% inactivated by the end of 3 min. The enhanced rate of NADPH oxidation could be seen when more NADPH was added (data not shown). However, as shown in Figure 2, trace B, the rate of NADPH oxidation decreased greatly after removal of NMC and ita metabolites from the reconstituted system by dialysis after complete inactivation of the P450. As shown in Figure 2, trace C, the enhanced rate of NADPH oxidation could be completely restored by adding back to the dialyzed reconstituted system containing inactive P450 a reaction mixture which had been inactivated and then heated to denature the P450 and reductase while retaining any heat-stable metabolites. These results suggested that one or more metabolites of NMC were responsible for

0

0.1

C

c

91 N

e

0 0

.

5

.-7

10

15

Time (min)

Figure 3. Normal-phase HPLC profile of the metabolite formed from 1-OH-NMC. The conditions for the reactions and HPLC analysis were described in the Experimental Procedures. (A) Metabolite profile following metabolism of 1-OH-NMC by reductase in the presence of NADPH. (B) Metabolite profile after incubation of 1-OH-NMC with hypoxanthine-xanthine oxidase. (C) Metabolite profile following metabolism of 1-OHNMC under the same conditions as in panel A with SOD (800 units) added. The new metabolite (met.) elutes a t approximately 8.9 min.

stimulating the oxidation of NADPH since NMC did not stimulate NADPH oxidation when added back by itself. The three major metabolites of NMC (1-OH-NMC,3-OHNMC, and NHMC) were purified from reaction mixtures by HPLC and added back to either the reconstituted system or the reductase alone. 1-OH-NMCwas active in stimulating NADPH oxidation in both systems, and while 3-OH-NMC also had a minor effect in stimulating the NADPH oxidation rate, it was muchless than that of 1-OHNMC, and NHMC had no effect (data not shown). Further Metabolism of 1-OH-NMC. As shown in Figure 3A, the 1-OH-NMC was further metabolized by the purified P450 reductase. The metabolism exhibited an absolute requirement for NADPH (data not shown). The metabolite was detected by normal-phase HPLC using an isocratic mobile phase of 90% hexane and 10% 2-propanol, and as shown in Figure 3A, the retention time of the metabolite (met.) was 8.9 min, while 1-OH-NMC eluted at 4.4 min, giving complete separation of the substrate and ita metabolite. Several different compounds have been reported to be oxidized or hydroxylated by the P450 reductase via a mechanism involving formation of superoxide anion as a reactive intermediate (22,231.The possible involvement of superoxide in the reductasecatalyzed metabolism of 1-OH-NMCwas investigated. As

234 Chem. Res. Toxicol., Vol. 7, No. 2, 1994 Table 1. Stimulation of NADPH Oxidation during the Metabolism of 1-OH-NMC. rate of NADPH oxidation [nmol of NADPH oxidized/ (min-nmol of reductase)] addition -GSH +GSH % inhibition by GSH NMC 10.5 f 2.5 12.0 f 1.8 0 1-OH-NMC 240 f 34 26.0 f 7.5 89 a The incubation mixture contained 50 mM potassium phosphate buffer (pH 7.4), 0.1 nmol of reductase, and 500 pM NMC or 35 pM 1-OH-NMC. When present, the GSH concentration was 1mM. The reaction mixtures were preincubated for 3 min at 30 OC. NADPH (0.1 mM) was then added, and the rate of NADPH oxidation at 30 OC was monitored by measuring the decrease in absorbance at 340 nm using an extinction coefficient of 6.22 mM-1 cm-l. Results represent the mean SD of four determinations.

shown in Figure 3C, addition of 800 units of superoxide dismutase (SOD) to the reaction mixture resulted in complete inhibition of the formation of the metabolite of 1-OH-NMC, suggesting involvement of the superoxide anion in the metabolism of 1-OH-NMC. The enzymatic generation of superoxide anion by the hypoxanthinexanthine oxidase system has been previously described (24). Incubation of 1-OH-NMC with hypoxanthinexanthine oxidase resulted in the formation of a product which eluted on normal-phase HPLC with a retention time identical to that of metabolite formed during the reductasecatalyzed metabolism of 1-OH-NMC (Figure 3B). Furthermore, product formation was inhibited by addition of SOD to the hypoxanthine-xanthine oxidase system (data not shown). These results suggest that superoxide anion may be involved in the metabolism of 1-OH-NMCby the reductase. Redox Cycling during Reductase-Catalyzed Metabolismof 1-OH-NMC. In order to examine the possible occurence of redox cyclingduring the reductase-catalyzed metabolism of 1-OH-NMC, the following studies were performed. First, the stimulation of NADPH oxidation during the reductase-catalyzed metabolism of 1-OH-NMC was examined. Upon addition of 1-OH-NMC, the rate of cofactor oxidation by the reductase increased by more than 23-fold [up to 240 nmol/(min.nmol of reductase)] compared with the level observed in the presence of NMC alone (Table 1). The addition of reduced glutathione (GSH) at a final concentration of 1mM resulted in almost complete inhibition (89%) of the rate of NADPH oxidation whereas it had no effect on the NMC-stimulated activity. The NADPH oxidase activity of the reductase was also determined over the substrate (1-OH-NMC)concentration range from 6.25 to 37.5 pM. Lineweaver-Burk analysis of the initial velocity data gave a K, value of 15.8 pM and a V,, of 360 nmol of NADPH oxidizedl(min.nmo1 of reductase) (Figure 4). As shown in Table 2, the formation of superoxide anion by the reductase during the metabolism of 1-OH-NMCwas much greater than observed with NMC (40-fold),and this was inhibited by approximately 85 % in the presence of 1mM GSH. These results support the hypothesis that the metabolism of 1-OH-NMC may involve a reductase-catalyzed redox cycle. The production of substantial amounts of superoxide and the marked enhancement of NADPH oxidation upon the addition of 1-OH-NMC suggest that a quinone-like compound may be produced during the metabolism of 1-OH-NMC and that this metabolic intermediate may then undergo oneelectron reduction by the reductase to give a semiquinone radical at the expense of NADPH. The semiquinone may

Shen and Hollenberg 0.012

,

/Y

0.002 O.Oo4I 0.oooi

-0.1

I

I

,’

I

1

0.0

0.1

0.2

[l-OH-NMC] -l(uM -l)

Figure 4. Lineweaver-Burk plot of velocity [nmol of NADPH oxidized/(min.nmol of reductase)l versus 1-OH-NMC concentrations. Incubations and measurement of rates of NADPH oxidation were performed as described in the Experimental Procedures. The concentrations of 1-OH-NMC were 6.25,9.37, 12.5, 18.75, 25, and 37.5 pM, respectively. Each determination was performed in duplicate. The K, value was determined from the intercept on the abscissa and the V,, from the intercept on the ordinate. Table 2. Superoxide Anion Production during the Metabolism of 1-OH-NMC. nmol/(min.nmol of reductase) acetylated superoxide % of cytochrome c anion control addition reduction formation (-SOD) NMC -SOD 30.7 f 4.5 +SOD 26.3 f 6.8 4.4 86 1-OH-NMC -SOD 241 i 21 +SOD 52.7 f 8.5 188.5 21 1-OH-NMC + GSH -SOD 67.0 f 5.2 28.8 (15)b 57 +SOD 38.2 f 5.9 a The reaction mixture contained 0.1 nmol of reductase, 0.1 mM EDTA, and 30 fiM acetylated cytochrome c in 1.0 mL of 50 mM potassium phosphate buffer (pH 7.4) with 25 fiM 1-OH-NMCor 500 mM NMC. When GSH was present, its concentration was 1 mM. The reaction mixtures were preincubated at 30 OC for 3 min, and the reactions were initiated by the addition of 0.5 mM NADPH. Superoxide anion formation was determined spectrophotometrically by measuring the rate of reduction of acetylated cytochrome c at 548 nm during the first 3 min in the presence and absence of 800 units of SOD. An extinction coefficient of 19.6 mM-1 cm-l was used to quantitate the cytochrome c reduction. The SOD-inhibitable reduction of acetylated cyrochrome c was usedto quantitate superoxide anion production. Results represent the mean f SD of four determinations. % of superoxide formation not inhibited by GSH.

then autoxidize to produce superoxide in the presence of molecular oxygen (25,26).The inhibition of both NADPH oxidation and superoxide production by GSH could be due to its ability to inhibit the reduction of the quinone or by trapping of the quinone by formation of a GSH adduct. Quinones are electrophilicspecieswhich may react directly with nucleophiles such as GSH. The marked decrease in the rate of NADPH oxidation and superoxide production as a consequence of GSH addition suggests that GSH may be quite effective in reacting with the intermediate quinone and preventing redox cycling. DNA Damage as a Consequence of RsductaseCatalyzed Metabolism of 1-OH-NMC. DNA strand scission during the reductase-catalyzed metabolism of 1-OH-NMC was determined by gel electrophoresis. The conversion of supercoiled 4X-174 DNA to the open circular form was demonstrated by a decrease in the intensity of

NMC Metabolism and Stimulation of NADPH Oxidation

Chem. Res. Toxicol., Vol. 7, No. 2, 1994 236

oc * sc *

sc I

1 2 3 4 5 Figure 5. Strand scission of 4 x 4 7 4 DNA during the metabolism of 1-OH-NMC by reductase. All samples contained 50 mM potassium phosphate buffer (pH 7.4) and 4X-174 DNA (0.2 pg). Incubations and electrophoretic analyses of the samples were performed as described under Experimental Procedures. Lane 1: Control. Lane 2: 1-OH-NMC (35 pM) was added. Lane 3: The metabolite of 1-OH-NMC was added. Lane 4: 1-OH-NMC (35 pM) + reductase (0.01 nmol) were added. Lane 5: 1-OHNMC (35 pM) + reductase (0.01 nmol) + NADPH (2 mM) were added. OC, open circular; SC supercoiled.

oc sc *

1 2 3 4 5 6 Figure 6. Effects of catalase and benzoate on 4X-174 DNA strand scission during reductase-catalyzed metabolism of 1-OHNMC, and the effect of H202 on DNA strand damage. Incubations and electrophoretic analyses of the samples were performed as described in the Experimental Procedures. All samples contained 50 mM potassium phosphate buffer (pH 7.4), 1-OH-NMC (10 pM),reductase (0.01 nmol), and 4X-174 DNA (0.2 pg). Lane 1: NADPH (2 mM) was added. Lane 2 Same as lane 1+ catalase (1.0 pg). Lane 3: Same as lane 1+ benzoate (10 mM). Lane 4 No further additions (control). Lane 5: Same as lane 4 + 500 pM H202. Lane 6: Same as lane 4 + 5 mM H202. OC, open circular, SC, supercoiled.

the supercoiledDNA band and an increase in the intensity of the band associated with the open circular form (Figure 5). During the metabolism of 1-OH-NMC by reductase in the presence of NADPH there was complete conversion of the supercoiled DNA to the open circular form (Figure 5, lane 5). Neither 1-OH-NMC nor its metabolite caused DNA damage in the absence of metabolism (Figure5, lanes 2 and 3). There was no DNA damage when NADPH was omitted (Figure 5, lane 4). Therefore, degradation of the DNA occurred as a result of the metabolism of 1-OHNMC. In order to investigate the mechanism of DNA damage, scavengers of H202 and free radicals were employed. Catalase almost completely inhibited DNA strand breakage (Figure 6, lane 2), while boiled catalase was inactive in protecting DNA from breakage (data not shown). Benzoate, a hydroxyl radical scavenger, also afforded protection against DNA scission (Figure 6, lane 3).

1 2 3 4 5 6 Figure 7. Effects of deferoxamine and DMSO on 4X-174 DNA strand scission during reductase-catalyzed metabolism of 1-OHNMC. All samples contained 50 mM potassium phosphate buffer (pH 7.4) and 4X-174 DNA (0.2 pg). Incubations and electrophoretic analyses were performed as desribed in the Experimental Procedures. Lane 1: No further additions. Lane 2: 1-OH-NMC (35 pM) + reductase (0.01) + NADPH (2 mM) were added. Lane 3: Same as lane 2 + deferoxamine (0.5 mM). Lane 4: Same as lane 2 + deferoxamine (0.75 mM). Lane 5: Same as lane 2 + DMSO (0.5% v/v). Lane 6 Same as lane 2 + DMSO (1% v/v). OC, open circular; SC, supercoiled.

Another hydroxyl radical trapping agent DMSO (27) showed a dose-dependent protective effect against DNA damage (Figure 7, lanes 5 and 6). Deferoxamine,a specific iron chelating agent, also protected DNA from breakage (Figure 7, lanes 3 and 4). The doses of the compounds used in these experimentsdidnot inhibit reductase activity as measured by the assay for cytochromec reduction (data now shown). In order to seewhether the protection against DNA scission by catalase was due to its ability to scavenge H202, H202 was added to the incubation mixture in the absence of NADPH. As shown in Figure 6, neither 500 pM H202 (lane5 ) nor 5 mM H202 (lane6)caused detectable DNA damage. These results suggest that DNA damage as a consequence of the metabolism of 1-OH-NMC is probably due to the generation of the hydroxyl radical, a very reactiveradical which can cause DNA scission without regard to sequence (28).

Discussion The rate of NADPH oxidation was stimulated with time during the metabolism of NMC by the reconstituted system containing cytochrome P450 2B1, reductase, and phospholipid. The increase in the rate of NADPH oxidation occurred concomitantly with the loss of P460 2B1 catalytic activity and the loss of spectrally detectable P450 (14), and with covalent binding of NMC to the apoprotein (13). This finding initially led us to suggest that covalent binding of NMC to P450 2B1 modified the enzyme in such a way as to stimulate its ability to serve as an NADPH oxidase while preventing its ability to serve as a monooxygenase. Thus, the covalent binding of NMC in the active site of P450 might facilitate the transfer of electrons from NADPH to molecular oxygen while preventing the binding of substrates which could undergo hydroxylation. However, when the unbound substrate and its metabolites were removed from the reaction mixture by dialysis, the NADPH oxidase activity remaining was diminished by 90%. This observation,in conjunction with

236 Chem. Res. Toxicol., Vol. 7, No. 2, 1994

the demonstration that the NADPH oxidase activity could be fully restored by adding back the metabolites of NMC to the dialyzed solution containing fully inactivated cytochrome P450, led us to conclude that it was a metabolite of NMC and the reductase that was responsible for stimulating NADPH consumption in this system rather than a change in the catalytic properties of the P450.1-OHNMC, one of the four major metabolites of NMC formed by the cytochrome P450 2B1 reconstituted system, was shown to be primarily responsible for stimulating NADPH oxidation in this system. A number of compounds have been shown to stimulate rates of NADPH oxidation by the reconstituted system by acting as uncouplers of electron transport and hydroxylation (29, 30). These uncouplers are compounds that bind reversibly to cytochrome P450 and cause increases in the rates of NADPH utilization and oxygen consumption without undergoing oxidationthemselves (31, 32). These uncouplers form reversible enzyme-substrate complexes with the P450 but, for chemical or steric reasons, cannot undergo oxidation to form products, yet they stimulate NADPH oxidation and the reduction of oxygen. However, 1-OH-NMC appears to stimulate NADPH oxidation by a different mechanism. First, the 1-OHNMC-dependent increase in the rate of NADPH oxidation did not require cytochrome P450, indicating that P450 is not involved in this process. Second, 1-OH-NMC was further metabolized by the reductase, and the marked increase in the rate of NADPH oxidation occurs primarily as a consequence of further oxidation of 1-OH-NMC.These results suggest that 1-OH-NMC is not acting as an uncoupler by binding to cytochrome P450. Several compounds have been reported to be oxidized or hydroxylated by reductase through a superoxide anion-dependent mechanism (22,23). The demonstration that a superoxidegenerating system containing hypoxanthine and xanthine oxidase catalyzed the oxidation of 1-OH-NMCsuggested the involvement of the superoxide anion in the metabolism of 1-OH-NMC. Superoxide dismutase completely inhibited the metabolism of 1-OH-NMC either by the hypoxanthine-xanthine oxidase system or by the reductase. These data provided further evidence indicating that superoxide is the primary oxidizing species for the oxidation of 1-OH-NMCby reductase. Reductase has been shown to be able to produce superoxide by reducing oxygen (33). Two possible mechanisms have been suggested for the oxidation of substrates by superoxide anion (34,351. One mechanism involves initial proton abstraction from the substrate to yield a substrate anion which is then oxidized by either 0 2 or H202; the other involveshydrogen atom transfer from the substrate to superoxide. The ability of the reductase to catalyze the further metabolism of 1-OH-NMC has additional implications for toxicity and/or mutagenicity (at least for in vitro studies). The reductase-catalyzed metabolism of 1-OH-NMCmay involve the formation of a quinone-likeintermediate,which may be mutagenic or carcinogenic (36). Therefore, the possible occurrence of redox cycling of the 1-OH-NMC during metabolism by the reductase was investigated. A substantial increase in the rate of NADPH oxidation and superoxide production as a consequence of product formation suggested the involvement of a quinonedependent redox cycle. Since the microsomal NADPHcytochrome P450 reductase catalyzes this redox cycling, it is possible for it to occur either via a one-electron

Shen and Hollenberg

J frm radical lormation

- NADPH 0

CH3

0

Figure 8. Scheme proposed for metabolism of 1-OH-NMCand redox cycling catalyzedby NADPH-cytochromeP450 reductase.

reduction of the quinone to a semiquinone free radical or via a two-electron reduction to the hydroquinone, which could then undergo autoxidation. The addition of reduced glutathione markedly inhibited (-90%) the 1-OH-NMCdependent stimulation of NADPH oxidation. Presumably, this is due to conjugation of the glutathione with the quinone, which would decrease the amount of quinone available for redox cycling and thereby decrease the rate of NADPH oxidation. Figure 8 shows a proposed sequence of steps in which 1-OH-NMCis first converted to a 1,4-dihydroquinone by superoxide formed as a result of reductase-catalyzed oxygen reduction. The dihydroquinone can then be oxidized by molecular oxygen to give the semiquinone and the quinone with the concomitant formation of superoxide and hydrogen peroxide. The semiquinone and quinone formed by these reactions can also undergo reduction by NADPH. Thus, the autoxidation of the 1,4-dihydroquinone of NMC coupled to the one-electron reduction of the NMC quinone and semiquinone provides for an efficient redox cycle which may serve as a major source for the generation of oxygen free radicals. The inhibition of this cycle by reaction of glutathione with the quinone is also indicated in this figure. It should be noted that the l-OH-NMC-stimulated production of superoxide was measured as the superoxide dismutase-inhibitable reduction of acetylated cytochrome c (monitored a t 548 nm). Although acetylation of cytochrome c preferentially decreases its rate of reduction by NADPH-cytochrome P450 reductase as compared to the rate of reduction by superoxide anion (33, acetylated cytochrome c can nevertheless be reduced by the reductase. In these experiments the acetylated cytochrome c was reduced by the reductase at approximately 4% of the rate of reduction of the native cytochrome c. The possibilitythat the metabolism of 1-OH-NMCcould result in DNA damage was investigated. DNA damage did occur during 1-OH-NMC redox cycling as a consequence of the substantial amounts of superoxide formed. No significant damage of DNA was observed in systems where either the 1-OH-NMCor NADPH was omitted. In an effort to modulate DNA damage and identify the oxygen

NMC Metabolism and Stimulatiolt of NADPH Oxidation

Chem. Res. Toxicol., Vol. 7, No. 2, 1994 237

species involved in the damage, the effects of catalase, SOD, benzoate, and DMSO were investigated. Catalase, benzoate, and DMSO inhibited DNA damage. Furthermore, the addition of 5 mM hydrogen peroxiclv to the incubation mixture did not cause any significant DNA damage. These findings suggest that DNA damage during 1-OH-NMC metabolism was due to the iron-catalyzed Haber-Weiss reaction (38) in which superoxide both reduces iron and dismutates to form H202. The HzOz could then interact with reduced iron to form the highly reactive hydroxyl radical, which can then cause DNA damage:

by Grant CA 16954 from the National Cancer Institute, USPHS.

-

0;-+ Fe3+ H,O,

+ Fez++ H+

sum:

02'-+ H,O,

-

+ H+

0, + Fez+

OH'

+ Fe3++ H,O

0, + OH'

+ H,O

Although iron was not added on purpose as part of the reaction mixture, it is possible that the potassium phosphate buffer or one of the other components used for these studies contained trace amounts of Fe3+as a contaminant, as has been reported by other investigators (39). In order to test this possibility, deferoxamine was included in the incubation mixture and DNA scission was analyzed. The protection by deferoxamine against DNA damage suggests that the iron-catalyzed Haber-Weiss reaction may be the major process responsible for DNA damage in this system and that the hydroxyl radical is the reactive intermediate involved. For reason(s) which we cannot explain, SOD did not afford any protective effects against DNA damage (data not shown). A similar result has also been reported by Kim and Novak (40). Although this finding appears to refute the role of superoxide or the hydroxyl radical generated from superoxide and peroxide by the HaberWeiss cycle in DNA damage, we cannot rule out the possibility that, under the assay conditions used, the SOD added could not intercept and decompose the reactive oxygen species prior to their reaction with DNA. Experiments aimed at identifying the reactive intermediate(s) responsible for DNA damage are in progress. In conclusion, evidence has been provided that during metabolism of NMC in the reconstituted system the increase in the rate of NADPH oxidation is due to the formation of 1-OH-NMC, which can be further metabolized by NADPH-cytochrome P450 reductase. The further metabolism of l-OH-NMC leads to redox cycling and thereby stimulates NADPH oxidation and superoxide production. The superoxide anion appears to be involved in the reductase-dependent meatabolism of 1-OH-NMC. Quinone-like metabolic intermediate(@ may be formed which may play an important role in redox cycling and cytotoxicity. Finally, DNA damage in vitro also results from the metabolism of 1-OH-NMC, presumably due to the formation of oxygen free radicals produced as a consequence of the redox cycling. Acknowledgment. We are grateful to David A. Putt for purifying cytochrome P450 2B1 and NADPH-cytochrome P450 reductase. We also thank Drs. Elizabeth Roberts and Kathy Yuh for their review of the manuscript and helpful suggestions. This work was supported in part

Rsferences (1) Hoffmann, D.,Rathkamp, G., and Woziwodeki, H. (118) Chemical studies on tobacco smoke. VI. The determination of carbazole in cigarette smoke. Beitr. Tabakforsch. 4, 253-263. (2) Hoffmann, D., Rathkamp, G., and Nesnow, S. (1969) Quantitative determination of 9-methylcarbazole in cigarette smoke. Anal. Chem. 41, 1256-1259. (3) Schmeltz, I., and Hoffmann, D. (1977) Nitrogen-containing compounds in tobacco and tobacco smoke. Chem. Rev. 77, 295-311. (4) Wynder, E. L., and Hoffmann, D. (1959) A study of tobacco carcinogenesis. VIII. The role of acidic fractions as promoters. Cancer 12, 1079-1086. (5) Hoffmann, D., and Wynder, E. L. (1971) A study of tobacco carcinogenesis. XI. Tumor initiators, tumor accelerators,and tumor promoting activity of condenaate fractions. Cancer 27, 848-864. (6) LaVoie, E. J., Briggs, G., Bedenko, V., and Hoffmann, D. (1982) Mutagenicityof substitutedcarbazolesinSalmonella typhimurium. Mutat. Res. 101, 141-150. (7) Koop, D. R., and Hollenberg, P. F. (1980) Liver microsomal metabolismof N-methylcarbazole: Aprobe for induction, inhibition, and species differences. Mol. Pharmacol. 17, 118-127. (8) Novak, R. F., Koop, D. R., and Hollenberg, P. F. (1980) Liver microsomal metabolism of N-methylcarbazole: Structural identification of the four major metabolites of N-methylcarbazole using 1HFourier transform NMRspectroscopy. Mol. Pharmacol. 17,128136. (9) Yang, W., Jiang, T., Davis, P. J., and Acosta, D. (1991) In vitro metabolismand toxicity aeeeasmentof N-methylcarbazolein primary cultured rat hepatmytea. Tozicology 68, 217-226. (10) Gorrod, J. W., and Temple, D. J. (1976) The formation of an N-hydroxymethyl intermediate in the N-demethylation of N-methylcarbazole in vivo and in vitro. Xenobiotica 6, 265-274. (11) Kedderis, G. L., Rickert, D. E., Pandey, R. N., and Hollenberg, P. F. (1980) 180 studies of the peroxidase-catalyzed oxidation of N-methylcarbazole: Mechanisms of carbinolamine and carbosaldehyde formation. J. Biol. Chem. 261, 15910-15914. (12) Kuemmerle, S., and Hollenberg, P. F. (1986) Suicide inactivation of liver microsomal cytochrome P45Ob and P450 LM2 by N-methylcarbazole. Fed. Proc. 46, 1508. Hollenberg, P. F., Kuemmerle, S., and Gurka, D. (1988) Suicide inactivation of cytochrome P-450b by N-methylcarbazole resulta in the covalent modification of P-450 apoprotein, FASEB J. 2, 563. Shen, T., and Hollenberg, P. F. (1990)The mechanism of NADPH oxidation during the metabolism of N-methylcarbazole (NMC) by cytochrome P450 IIBl (P-450b). FASEB J. 4, 2240. Stevens, T. S., and Tucker, S. H. (1923) The preparation of N-derivatives in the carbazole series. J. Chem. SOC.No. 123,21402147. Coon, M. J., Van der Hoeven, T. A., Dahl, S. B., and Haugen, D. A. (1978)Two forms of livermicrosomalcytochromeP-450, P-450LM2 and P-450LM4 (rabbit liver). Methods Enzymol. 62, 109-117. Imai, Y., Hashimoto-Yutaudo, C., Satake, H., Giradin, A., and Sato, R. (1980) Multiple forms of cytochrome P-450 purified from liver microsomes of phenobarbital- and 3-methylcholanthrenepretreated rabbita. I. Resolution, purification, and molecular properties. J. Biochem. (Tokyo) 88,489-503. Guengerich,F. P., and Martin, M. (1980)Purification of cytochrome P-450,NADPH-cytochrome P-450reductase, and epoxidehydrafrom a single preparation of rat liver microsomes. Arch. Biochem. Biophys. 205, 366379. Assi,A., Montecucco,C., and Richter, C. (1975)Theuse of acetylated ferricytochrome c for the detection of superoxide radicals produced in biologicalmembranes. Biochem.Biophys. Res. Commun.65,597603. Veda, K., Kobayashi, S., Morita, J., and Komano, T. (1985) Sitespecific DNA damage caused by lipid peroxidation producta. Biochim. Biophys. Acta 824, 341-348. Lewis, T. G., Stewart, W., and Adams, D. 0. (1988) Role of oxygen radicals in induction of DNA damage by metabolites of benzene. Cancer Res. 48, 4762-4765. Aust, S. D., Roering, D. L., and Pederson, T. C. (1972)Evidence for superoxide generation by NADPH-cytochrome c reductase of rat liver microsomes. Biochem. Biophys. Res. Commun. 47,1133-1137. Prema Kumar, R., Ravindranath, S. D., Vaidyanathan, C. S., and Appaji Rao, N. (1972) Mechanism of hydroxylation of aromatic compounds: Evidence for the involvement of superoxide anion in a model system. Biochem. Biophys. Res. Commun. 48,1049-1054. McCord,J. M., andFridovich, I. (1968)The reduction of cytochrome c by milk xanthine oxidase. J. Biol. Chem. 243, 5753-5758. Powis, G., Svingen, B. A., and Appel, P. (1981)Quinone-stimulated superoxide formation by subcellular fractions, isolated hepatocytes, and other cells. Mol.Pharmacol. 20, 387-394.

238 Chem. Res. Toxicol., Vol. 7, No.2, 1994 (26) Kappua, H., and Sies, H. (1981) Tosic drug effecta aswxiated with oxygen metabolism: Redox-cycling and lipid peroxidation. Experientia 37, 1233-1241. (27) Britigan, B. E., Coffman, T. J., and Buettner, G. R. (1990) &in trapping evidence for the lack of significant hydroxyl radical production during the respiration buret of human phagocyteswing a spin adduct resistant to superoxide-mediateddestruction. J.Biol. Chem. 265,26$0-2656. (28) McCord, J. M.,and Day, E. D. (1978) Superoxide-dependent production of hydroxyl radical catalyzed by iron-EDTA complex. FEBS Lett. 86, 139-142. (29) Duncan, J. D.,DiStefano,E. W., Miwa, G. T., and Cho, A. K. (1985) Role of superoxide in the N-oxidation of N-(2-methyl-l-phenyl-2propy1)hydroxylamine by the rat liver cytochrome P450 system. Biochemistry 24,4155-4161. (30) Parkinson,A., Thomas, P. E., Ryan, D. E., Gorsky, L. D., Shively, J. E., Sayer, J. M., Jerina, D.M., and Levin, W. (1986) Chemical modification and inactivation of rat liver micrmmd cytochrome P-45Oc by2-bromo-4’-nitrophenone. J.Biol. Chem.261,1148711495. (31) Ullrich, V., and Diehl, H. (1971) Uncoupling of monooxygenation and electron transport by fluorocarbonsin liver microsomes. Eur. J. Biochem. 20,509-512. (32) Nordblom, G. D., and Coon, M. J. (1977) Hydrogen peroxide formation and stoichiometry of hydroxylation reactions catalyzed by highly purified liver microsomal cytochrome P-450. Arch.

Shen and Hollenberg Biochem. Biophys. 180,343-347. (33) Fong, K. L., McCay, P. B., Poyer, J. L., Keele, B. B., and Misra, H. P. (1973) Evidence that peroxidation of lyeoeomal membranes is initiated by hydroxyl free radical produced during flavin enzyme activity. J. Biol. Chem. 248, 7792-7797. (34) Sawyer,D. T., and Valentine, J. S.(1981) How super is superoxide? Acc. Chew Res. 14,393-400. (35) Fukuto, J. M., DiStefano,E. W., Burstyn, J. N.,Valentine, J. S., and Cho, A. K. (1985)Mechanismof oxidation of N-hydroxyphetermine by superoxide. Biochemistry 24,4161467. (36) Chesis, P. L., Levin, D. E., Smith, M. T., Emeter, L., and Ames,B. N. (1984) Mutagenicity of quinones: Pathway of metabolic activation and detoxification. Roc. Natl. Acad. Sei. U.S.A. 81,16961700. (37) Guengerich,F. P. (1982) in Principles and Methods of Toxicology (Hayes, W., Ed.) pp 609-634, Raven Press, New York. (38) Haber, F., and Webs, J. (1934) The catalytic decomposition of hydrogen peroxide by iron salta. Roc. R. SOC.London Al47,332351. (39) Diguieeppi, J., and Fridovich, I. (1980) Ethylene from 2-keto-4thiomethylbutyricacid The Haber-Weissreaction. Arch.Biochem. Biophys. 205, 323-329. (40) Kim, S. G., and Novak, R. F. (1990) Role of P450IIEl in the metabolism of 3-hydroxypyridine,a constituent of tobacco emoke; Redox cycling and DNA strand scission by the metabolite 2,5dihydroxypyridine. Cancer Res. 50,5333-5339.