Generation of Reactive Oxygen Species during the Enzymatic

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Generation of Reactive Oxygen Species during the Enzymatic Oxidation of Polycyclic Aromatic Hydrocarbon trans-Dihydrodiols Catalyzed by Dihydrodiol Dehydrogenase Trevor M. Penning,*,† S. Tsuyoshi Ohnishi,‡ Tomoki Ohnishi,† and Ronald G. Harvey§ Department of Pharmacology and Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, Philadelphia Biomedical Research Institute, King-of-Prussia, Pennsylvania 19102, and Ben May Institute, University of Chicago, Chicago, Illinois 60637 Received April 4, 1995X

Dihydrodiol dehydrogenase (DD; EC 1.3.1.20) catalyzes the oxidation of polycyclic aromatic hydrocarbon (PAH) trans-dihydrodiols (proximate carcinogens) to catechols which rapidly autoxidize to yield o-quinones (Smithgall, T. E., Harvey, R. G., and Penning, T. M. (1988) J. Biol. Chem 263, 1814-1820). Although this pathway suppresses the formation of the PAH anti- and syn-diol epoxides (ultimate carcinogens), the process of autoxidation is anticipated to yield reactive oxygen species (ROS). We now show that the NADP+ dependent oxidation of (()-trans-1,2-dihydroxy-1,2-dihydronaphthalene (Npdiol) and (()-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (Bpdiol) catalyzed by homogeneous DD is accompanied by the consumption of molecular oxygen and the production of H2O2. With both trans-dihydrodiol substrates, oxygen consumption was stoichiometric with H2O2 production consistent with the reaction: QH2 + O2 ) H2O2 + Q, where QH2 is the catechol and Q is the o-quinone. Using Npdiol or Bpdiol as substrates, a burst of superoxide anion production is catalyzed by DD which can be detected as the rate of cyt c reduction that is inhibited by superoxide dismutase. Using 5,5-dimethyl1-pyrroline N-oxide (DMPO) as spin-trapping agent, secondary spin adducts corresponding to DMPO-CH3 were formed during the enzymatic oxidation of Npdiol and Bpdiol. The formation of the CH3• radical arises from the OH• attack of DMSO, which was used as cosolvent. These spin adducts were attenuated by superoxide dismutase and catalase, implying that O2-• and H2O2 are obligatory for the formation of DMPO-CH3. It is proposed that O2-• is the radical that propagates autoxidation and that the resultant H2O2 undergoes Fenton chemistry to produce the OH• radical. Identical spin adducts were observed using a superoxide anion generating system (hypoxanthine/xanthine oxidase) and DMPO as spin-trapping agent in the presence of DMSO. The ability of DD to generate ROS during the oxidation of PAH transdihydrodiols (proximate carcinogens) may have important implications for tumor initiation and promotion.

Introduction Polycyclic aromatic hydrocarbons (PAH)1 are environmental pollutants and human carcinogens which require metabolic activation to exert their mutagenic, carcinogenic, and tumorigenic effects (1, 2). An accepted pathway of PAH activation involves formation of non-K region trans-dihydrodiols (proximate carcinogens) which are transformed to anti- and syn-diol epoxides which alkylate DNA (3, 4). Although this mechanism explains how PAH cause an initiation event, it is less clear how these * To whom correspondence and reprint requests should be addressed. † University of Pennsylvania. ‡ Philadelphia Biomedical Institute. § University of Chicago. X Abstract published in Advance ACS Abstracts, December 1, 1995. 1 Abbreviations: dihydrodiol dehydrogenase, trans-1,2-dihydrobenzene-1,2-diol dehydrogenase (DD; EC 1.3.1.20). PAH, polycyclic aromatic hydrocarbons; Npdiol, (()-trans-1,2-dihydroxy-1,2-dihydronaphthalene; Bpdiol, (()-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene; NPQ, naphthalene-1,2-dione; BPQ, benzo[a]pyrene-7,8-dione; ATBS, 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid); DMPO, 5,5-dimethyl-1-pyrroline N-oxide; SOD, superoxide dismutase; ROS, reactive oxygen species; XO, xanthine oxidase.

0893-228x/96/2709-0084$12.00/0

compounds can act as complete carcinogens and cause tumor promotion, progression, and metastasis. One possible mechanism is that diol epoxides alkylate hotspots on DNA that correspond to proto-oncogenes and tumor supressor genes (5). For example, exposure of either mouse papillomas or lungs of A/J mice to benzo[a]pyrene will lead to mutation of the 12th and 61st codons of the c-ras proto-oncogene (6, 7). Similarly, G f T transversions are induced by the anti-diol epoxide of benzo[a]pyrene in the p53 tumor supressor gene at sites most often found modified in patients with lung cancer (8). As an alternative to this mechanism, PAH may be activated to reactive species that could act as both initiators and promotors. Dihydrodiol dehydrogenase (DD) catalyzes the oxidation of non-K-region trans-dihydrodiols to yield catechols which rapidly autoxidize to yield reactive o-quinones (9, 10). In the case of benzo[a]pyrene, the reaction sequence is the NADP+ dependent oxidation of trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (Bpdiol) to yield 7,8dihyroxybenzo[a]pyrene, followed by air oxidation to yield benzo[a]pyrene-7,8-dione (BPQ) (Scheme 1). Recently, BPQ has been identified as an authentic metabolite of © 1996 American Chemical Society

Reactive Oxygen and Dihydrodiol Dehydrogenase

Chem. Res. Toxicol., Vol. 9, No. 1, 1996 85

Scheme 1. Formation of PAH o-Quinones Catalyzed by Dihydrodiol Dehydrogenase

Bpdiol in rat subcellular fractions (11) and in vitro in rat hepatocytes (12, 13). By removing the trans-dihydrodiols from the accepted pathway of PAH activation, DD can suppress the formation of the anti- and syn- diol epoxides. However, the autoxidation of catechols to reactive nonK-region o-quinones is anticipated to generate reactive oxygen species (ROS). Once formed, PAH o-quinones can be preferentially diverted down enzymatic 1e- reduction pathways and enter futile redox cycles to generate o-semiquinone radicals and ROS multiple times (14). This mechanism of free radical amplification may have important consequences for the carcinogenic potential of PAH. Roles for ROS in both tumor initiation and promotion have been proposed (15, 16). In the case of radiation induced carcinogenesis, ROS are believed to be the causative agents (17). Additionally, it has been proposed that ROS may act as selective mitogens and cause initiated cells to expand (18, 19). Further, ROS have been shown to cause the translocation and activation of protein kinase C (phorbol ester receptor) (20) and the induction of the cellular proto-oncogenes c-jun and c-fos in mouse fibroblasts (21). Thus, a connection between the biotransformation of PAH such as benzo[a]pyrene and the generation of ROS is of immediate interest. The generation of ROS may represent only one component of the bioactivation of PAH-trans-dihydrodiols by DD. The PAH o-quinones produced by the enzyme have cyto- and geno-toxic properties of their own. Treatment of rat and human hepatoma cells with PAH o-quinones results in cell death, and this has been attributed to either the production of superoxide anion and o-semiquinone radicals or the depletion of glutathione (22). PAH o-quinones also have the potential to be genotoxic. Model studies in which [3H]BPQ was reacted with calf thymus DNA indicated that the level of adduct formation was comparable to that observed in reactions containing the anti-diol epoxide of benzo[a]pyrene [(()-anti-7β,8R-dihydroxy-9R,10R-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (23)]. Based on addition chemistry, the BPQ-deoxyguanosine adducts that form are believed to be 1,4-Michael addition products (24, 25). Further, PAH o-quinones have been shown to be direct acting mutagens in the Ames test and cause predominantly frameshift mutations (26). In this paper we document the production of ROS (O2-•, H2O2, and OH•) during the DD catalyzed oxidation of PAH trans-dihydrodiols. The production of these radicals may contribute to the complete carcinogenic potential of PAH.

Experimental Procedures Materials. Androsterone was purchased from Steraloids (Wilton, NH). β-NAD+ and NADP+ were obtained from Boerhinger-Manheim (Indianapolis, IN). Superoxide dismutase (SOD; bovine erthrocytes; 3750 units/mg), catalase (bovine

liver; 58 000 units/mg), horseradish peroxidase (type II: 200 purpurogallin units/mg), cyt c, hypoxanthine, xanthine oxidase (grade I from buttermilk; 17.9 units/mL), 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ATBS), 3,3′,5,5′-tetramethylbenzidine, and 5,5-dimethyl-1-pyrroline N-oxide (DMPO) were purchased from Sigma Chemical Co. (St. Louis, MO). DMPO was further purified by charcoal chromatography (27). Npdiol and Bpdiol were synthesized as previously described (9, 28). Caution: All PAH are potentially hazardous and should be handled in accordance with “NIH Guidelines for the Laboratory Use of Chemical Carcinogens”. Source of Enzyme. DD was purified to homogeneity according to our published procedure to a final specific activity of 2.2 µmol of androsterone oxidized/(min‚mg) (29). Enzyme was stored in 20 mM potassium phosphate (pH 7.0), containing 1 mM 2-mercaptoethanol, 1 mM EDTA, and 20% glycerol. Enzymatic Oxidation of trans-Dihydrodiols. The oxidation of Npdiol was conducted in 1.0 mL systems containing the following: 100 mM potassium phosphate (pH 8.0), 2.3 mM NADP+, and 2.0 mM Npdiol in 8% DMSO. The oxidation of Bpdiol was conducted in 1.0 mL systems containing the following: 50 mM glycine/NaOH (pH 9.0), 2.3 mM NADP+, and 20 µM Bpdiol in 8% DMSO. Reactions were monitored on a DU-7 spectrophotometer at 340 nm at 25 °C assuming an ) 6270 M-1 cm-1 for NADPH. Reactions measuring Npdiol and Bpdiol oxidation were monitored over 120 and 360 min, respectively, and data points were taken every 20 s. Reactions were performed in triplicate. Individual experiments were replicated 2-4 times. The validity of the assay for Bpdiol oxidation has been established previously by monitoring the disappearence of [3H]Bpdiol by TLC and HPLC (30, 31). Oxygen Uptake during the Oxidation of trans-Dihydrodiols. The consumption of molecular oxygen was measured using a Clark-style oxygen electrode in 600 µL chambers containing the reaction components for enzymatic oxidation of trans-dihydrodiols. The output from the microelectrode was connected through an amplifier (Instech) which allowed the simultaneous display of oxygen concentration and the rate of oxygen uptake as a function of time. The data acquisition software allowed the recording of 1-1000 data points/min. Npdiol and Bpdiol oxidation were monitored over 120 and 360 min periods, respectively, and data points were taken every 6 s. Reactions were performed in duplicate. All oxygen uptake measurements were performed at 25 °C, assuming that the oxygen concentration is 0.137 µmol at 25 °C at 760 mmHg. Hydrogen Peroxide Formation during the Oxidation of trans-Dihydrodiols. Aliquots (200 µL) were removed over time from reaction mixtures (2.0 mL) containing the components for the enzymatic oxidation of trans-dihydrodiols and diluted directly into mixtures containing the following: 100 µL of 1.0 M potassium phosphate buffer (pH 6.0), 10 µL of 10 mM 3,3′,5,5′tetramethylbenzidine, and 185 µL of H2O to quench the reaction. Hydrogen peroxide was detected colorimetrically following the addition of 5 µL of horseradish peroxidase (2.0 mg/mL). Nanomoles of hydrogen peroxide produced were calculated from a standard curve which was linear over the range of 0-40 nmol of H2O2 (λmax 650 nm, ) 30 680 M-1 cm-1). The assay was able to detect the production of 1.0 nmol of hydrogen peroxide (32). All reactions were performed in duplicate. Superoxide Anion Production during the Oxidation of trans-Dihydrodiols. The NADP+ dependent oxidation of Npdiol or Bpdiol catalyzed by DD was linked to the reduction

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of cyt c that was followed at 550 nm. The rate of cyt c reduction that was inhibited by superoxide dismutase was taken as a measure of O2-• formation. Complete reactions contained the components for enzymatic trans-dihydrodiol oxidation plus cyt c (60 µM) with or without SOD (500 units/mL). No change in absorbance at 550 nm was detected in reactions that were devoid of NADP+, DD, or cyt c. Before each reaction was conducted, the ability of SOD to completely block the reduction of cyt c observed in the presence of 200 µM hypoxanthine plus xanthine oxidase (25 milliunits/mL) was established. EPR Measurements. Free radical production was measured during the oxidation of Npdiol in reaction systems (200 µL) containing the following: 0.5 mM Npdiol, 2.3 mM NADP+, 8% DMSO, 100 mM potassium phosphate buffer (pH 8.0), plus 50 mM DMPO. Reactions were initiated by the addition of 10 µg of DD. Free radical production was also measured during the oxidation of Bpdiol in reactions (200 µL) containing the following: 45 µM Bpdiol, 2.3 mM NADP+, 8% DMSO, 50 mM glycine buffer (pH 9.0), plus 50 mM DMPO. Reactions were initiated by the addition of 33.3 µg of DD. The concentration of DMPO used had no effect on DD activity. As a positive control, free radical production was measured in reactions (200 µL) containing the following: 800 µM hypoxanthine, 8% DMSO, 100 mM potassium phosphate buffer (pH 8.0), plus 50 mM DMPO. Reactions were initiated by the addition of 39 milliunits of xanthine oxidase. Additional samples contained either 200 units of SOD or 580 units of catalase. EPR spectra were recorded on a Varian E109 EPR spectrometer operating in the X-band (9.25 GHz), employing 0.1 M modulation amplitude and 200 mW microwave power with the receiver gain set at 5 × 104 at ambient temperature (unless otherwise stated).

Results Oxygen Consumption during the Enzymatic Oxidation of PAH trans-Dihydrodiols. Homogeneous DD from rat liver will catalyze the NADP+ dependent oxidation of Npdiol to form NPQ (10). When these reactions were monitored with an oxygen electrode at pH 8.0, molecular oxygen was consumed (Figure 1A). In parallel reactions the oxidation of Npdiol was accompanied by the formation of NADPH and H2O2. Salient features were that once Npdiol oxidation reached the equilibrium end point (at 40 min), O2 uptake and H2O2 formation continued. Second, the amount of O2 consumed and H2O2 formed exceeded the amount of diol oxidized. Third, the amount of O2 consumed and H2O2 formed were identical with each other. Homogeneous DD will also catalyze the NADP+ dependent oxidation of Bpdiol to yield BPQ (10). When these reactions were monitored at pH 9.0, O2 was again consumed and H2O2 was formed (Figure 1B). In these reactions the initial rate of H2O2 formation greatly exceeded the initial rate of oxygen consumption. Further, in contrast to the Npdiol reaction, the rate of oxygen consumption lagged significantly behind the rates of Bpdiol oxidation and H2O2 formation. At the end of the reaction, the amount of O2 consumed and H2O2 formed were almost identical and exceeded the amount of Bpdiol oxidized. Although differences exist in the oxygen metabolism observed during the enzymatic oxidation of Npdiol and Bpdiol, a common feature was that in an individual reaction the amount of O2 consumed and H2O2 formed were similar and that these amounts exceeded the amount of diol oxidized. The almost stoichiometric relationship that exists between O2 consumption and H2O2 formation indicates that the reactions in eqs 1-3 and Scheme 2 may be responsible for the autoxidation of the intermediate catechol (in eqs 1-3, QH2 ) catechol,

Penning et al.

Figure 1. Oxygen uptake and hydrogen peroxide formation during the enzymatic oxidation of Npdiol (panel A) and Bpdiol (panel B). Nanomoles of diol oxidized (9) and hydrogen peroxide formed (b) were measured spectrophotometrically, while nanomoles of oxygen consumed were measured in parallel reactions with an oxygen electrode (4). Oxygen uptake and diol oxidation were measured in triplicate, and the mean ( SD are shown. Where the error bar is absent, the bar is smaller than the symbol. Hydrogen peroxide formation was determined in duplicate, and the mean is shown.

SQ-• ) o-semiquinone radical, and Q ) quinone). This

QH2 + O2-• ) SQ-• + H2O2

(1)

SQ-• + O2 ) O2-• + Q

(2)

net:

QH2 + O2 ) Q + H2O2

(3)

sequence of autoxidation has been proposed to take place following the bioreductive alkylation of 1,4-naphthoquinone and its glutathionyl conjugates (33). However, these reactions do not explain why the amounts of O2 consumed and H2O2 formed exceed the amount of diol oxidized. The higher than anticipated changes in O2 consumption and H2O2 production may result from two events: the reactivity of the enzymatically generated o-quinones with prevailing nucleophiles and the redox cycling of PAH o-quinones with NADPH produced during the reaction. We have shown by extensive studies that PAH oquinones are highly reactive in the presence of nucleophiles (10, 24, 25). Indeed, it is this event that led to the difficulty in o-quinone isolation. We have reported rate constants for the formation of phosphate, Tris, glycine, mercapturyl, glutathionyl, and cysteinyl adducts of NPQ. Structures of thiol adducts of NPQ and BPQ correspond to 1,4-Michael addition products obtained at the level of the fully oxidized o-quinone. This implies that in each instance the resultant adduct is obtained from the the autoxidation of the corresponding catechol

Reactive Oxygen and Dihydrodiol Dehydrogenase Scheme 2. Production of ROS during the Oxidation of PAH trans-Dihydrodiols Catalyzed by Dihydrodiol Dehydrogenase

adduct. This autoxidation would consume additional molecular oxygen and produce more hydrogen peroxide. Similarly, redox cycling of the PAH o-quinone with NADPH will produce the catechol which will ultimately autoxidize again and contribute to the increased oxygen metabolism observed. This redox cycling has a minimal impact on the changes observed at 340 nm during transdihydrodiol oxidation. Thus our spectrophotometric assay for Bpdiol oxidation has been validated by measuring the disappearance of [3H]Bpdiol using HPLC and TLC methods (30, 31). It should be emphasized that in these experiments the oxidation of the trans-dihydrodiols was followed at different pH values. At pH 9.0, the oxidation of transdihydrodiols was optimal; however, if this pH was used for the Npdiol reaction, the total absorbance change at 340 nm was too large to measure accurately, and oxygen uptake was very rapid. The use of a lower pH slowed the Npdiol reaction, and an easily defined equilibrium end point was reached. In contrast, Bpdiol oxidation was followed at pH 9.0; at lower pH values, oxidation was also slower. However, since Bpdiol was used at the limit of its solubility (20-40 µM), changes in oxygen metabolism were more difficult to detect at the lower pH. Two different assays were used to measure H2O2 formation: one involved monitoring the formation of the radical cation that is produced from ATBS in the presence of H2O2 and horseradish peroxidase (34), and the other


was an end point colorimetric assay using 3,3′,5,5′tetramethylbenzidine as the chromophoric substrate (32). The reactions with ATBS were found to give a substantial underestimate of the amount of H2O2 produced, and this may be related to the instability of the radical cation (t1/2 ) 30 and 20 min, at pH 8.0 and 9.0, respectively). On this basis, tetramethylbenzidine was employed since it was the more reliable indicator of H2O2 formation. Detection of Superoxide Anion Radical during the Enzymatic Oxidation of PAH-trans-Dihydrodiols. The formation of superoxide anion during the oxidation of Npdiol and Bpdiol at pH 8.0 and 9.0, respectively, was initially determined by measuring the rate of cyt c reduction that was inhibited by SOD. In the case of Npdiol the rate of O2-• formation almost paralleled the initial rate of diol oxidation (Figure 2A). By 10 min all of the cyt c was reduced, but only 30% of the chromophore formed was abolished by SOD (Figure 2B). In the case of Bpdiol the rate of O2-• formation lagged behind the oxidation of the substrate (Figure 2C,D). It will be recalled that the rate of oxygen uptake also lagged behind the rate of Bpdiol oxidation, suggesting that the second one-electron oxidation, in which the o-semiquinone radical is oxidized by molecular oxygen to yield the quinone and O2-• (eqn 2 and Scheme 2), may be rate-limiting. By 20 min all of the cyt c was reduced, but only 20% of the chromophore was abolished by SOD. The noninhibited rate of cyt c reduction observed with both diol substrates implies that reductants other than O2-• may be responsible for the absorbance change. Candidates for these reductants include NADPH and o-semiquinone anion radicals. Semiquinone anion radicals predominate at physiological pH and have a pKa of 4.0 (35). Spin Trapping of Oxygen Free Radicals during the Enzymatic Oxidation of PAH-trans-Dihydrodiols. The ability of SOD to inhibit the reduction of cyt c during the enzymatic catalyzed oxidation of PAH transdihydrodiols is an indirect measurement of O2-• formation. In an attempt to measure O2-• directly, the spintrapping agent DMPO was used and EPR spectra were recorded. Using either Npdiol or Bpdiol as substrates for DD, an intense six-line hyperfine spectrum was observed (Figure 3A,B) which was fundamentally different from that reported for DMP-OOH, which would arise from spin trapping O2-• (36). Instead, the hyperfine splitting patterns were indistinguishable from that reported for the DMPO-CH3 adduct (AN ) 15.31 G and AH ) 22.0 G) (36). The generation of the DMPO-CH3 adduct has been attributed to the reaction of a hydroxyl radical with DMSO to yield a methyl radical and methanesulfinic acid (37-39). It should be noted that all the reactions performed were conducted in the presence of DMSO to maintain the solubility of the PAH trans-dihydrodiols. The DMPO-CH3 secondary adducts that are formed during the oxidation of Npdiol and Bpdiol were abolished by the presence of SOD and catalase, suggesting that the O2-• and H2O2 are obligatory for the formation of DMPOCH3 spin adducts. It is proposed that O2-• is the radical that propagates autoxidation and that the resultant H2O2 undergoes Fenton chemistry in the presence of trace metal ions to yield OH•. Since no metal ions were added to the experimental system, an iron-catalyzed HaberWeiss reaction is favored over a formal Fenton reaction (see eqs 4-7; MSO ) methanesulfinic acid). In these reactions Fe2+ could be regenerated from Fe3+ by a variety of reductants involving O2-• and NADPH.2

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Penning et al.

Figure 2. Superoxide anion formation during the enzymatic oxidation of PAH-trans-dihydrodiols. In parallel reactions the enzymatic oxidation of Npdiol was followed at 340 nm (O), and the rate of cyt c reduction was followed at 550 nm in the presence and absence of SOD. Total cyt c reduction (4) and superoxide anion formation (b) are shown. Initial rates of superoxide anion formation during Npdiol oxidation (panel A). Total cyt c reduction during Npdiol oxidation (panel B). In parallel reactions the enzymatic oxidation of Bpdiol was followed at 340 nm (O), and the rate of cyt c reduction was followed at 550 nm in the presence and absence of SOD. Total cyt c reduction (4) and superoxide anion formation (b) are shown. Initial rates of superoxide anion formation during Bpdiol oxidation (panel C). Total cyt c reduction during Bpdiol oxidation (panel D). Reactions were performed as described in the Experimental Procedures section. Npdiol oxidation was monitored in triplicate, and the mean ( SD is shown. All other measurements were performed in duplicate, and the mean is shown.

O2-• + O2-• + 2H+ ) H2O2 + O2

(4)

H2O2 + Fe2+ ) OH- + OH• + Fe3+

(5)

DMSO + OH• ) CH3• + MSO•

(6)

CH3• + DMPO ) DMPO-CH3

(7)

In support of our spectral assignment the hypoxanthine/xanthine oxidase system was used to generate superoxide anion in the presence of DMSO and DMPO. The resultant six-line hyperfine spectrum was indistinguishable from that observed during diol oxidation (Figure 3C). The DMPO-CH3 spin adduct observed in the hypoxanthine/xanthine oxidase system was not abolished by superoxide dismutase but was attenuated by catalase. It is important to make some distinctions between the spin-trapping experiments performed during diol oxida2 Equation 4 results from the reactions in eqs 8-10, and eq 5 results from the reaction in eqs 11 and 12:

H+ + O2-• h HO2•

(8)

HO2• + O2-• + H+ ) H2O2 + O2

(9)

HO2• + HO2• ) H2O2 + O2

(10)

3+

(11)

Fe (trace) + O2

-•

) Fe

2+

+ O2

Fe2+ + H2O2 ) OH• + OH- + Fe3+

(12)

tion and those performed in the presence of the hypoxanthine/xanthine oxidase system. In the former instance, superoxide anion is hypothesized as being the propagating radical. Therefore, superoxide dismutase would remove this radical, autoxidation of the intermediate catechol would cease, and no H2O2 would be generated for DMPO-CH3 formation. In contrast, in the hypoxanthine/xanthine oxidase system superoxide anion is produced and SOD would catalyze its dismutation to H2O2, which would ultimately contribute to the DMPOCH3 signal. In this instance, the spin adduct would not be abolished by SOD. The formation of the DMPO-CH3 spin adducts during trans-dihydrodiol oxidation was followed kinetically (Figure 4). Measurement of the signal amplitudes of the spin adducts showed that their formation followed the same time courses as that observed for oxygen metabolism during the enzymatic oxidation of Npdiol and Bpdiol (compare Figures 1, 2, and 4). Further, these kinetic measurements indicate that the level of spin adduct formation observed with Npdiol and Bpdiol is similar to that observed with the hypoxanthine/xanthine oxidase system. Thus under the conditions used, the amounts of O2-• produced by the enzymatic oxidation of PAH trans-dihydrodiols and the hypoxanthine/xanthine oxidase system are of the same magnitude.

Discussion This paper provides evidence that ROS are produced during the oxidation of PAH-trans-dihydrodiol (proximate carcinogens) catalyzed by homogeneous DD. This evi-


Figure 3. EPR spectra obtained during the enzymatic oxidation of PAH-trans-dihydrodiols using DMPO as the spintrapping agent. Panel A: Complete system for the enzymatic oxidation of Npdiol plus 50 mM DMPO at 24 min (upper spectrum); complete system for the enzymatic oxidation of Npdiol plus 50 mM DMPO and SOD at 20 min (middle spectrum); complete system for the enzymatic oxidation of Npdiol plus 50 mM DMPO and catalase at 24 min (lower spectrum). Panel B: Complete system for the enzymatic oxidation of Bpdiol plus 50 mM DMPO at 78 min (upper spectrum); complete system for the enzymatic oxidation of Bpdiol plus 50 mM DMPO and SOD at 60 min (middle spectrum); complete system for the enzymatic oxidation of Bpdiol plus 50 mM DMPO and catalase at 71 min (lower spectrum). Panel C: Superoxide generating system (hypoxanthine/xanthine oxidase) plus DMSO as cosolvent and DMPO as spin-trapping agent, complete system at 57 min (upper spectrum); complete system plus SOD at 38 min (middle spectrum); complete system plus catalase at 56 min (lower spectrum). Elapsed times indicate time after addition of the enzyme (DD or xanthine oxidase) to initiate the reaction. Modulation amplitude was 0.1 MT, and the microwave power was 200 mW.

dence includes measurements of oxygen uptake and measurements of O2-• and H2O2 formation. Additional evidence for the production of ROS was obtained by detecting the formation of DMPO-CH3 secondary spin adducts. Both O2-• and H2O2 were obligatory for the formation of the these spin adducts, which speaks to the mechanism of autoxidation of the intermediate catechol. The detection of the DMPO-CH3 adducts also provides evidence that the highly reactive OH‚ radical can be produced as a side product of diol oxidation. The production of superoxide anion during the enzymatic oxidation of Npdiol and Bpdiol was initially measured indirectly by monitoring the rate of cyt c reduction that was inhibited by SOD. With both trans-


Figure 4. Kinetics of DMPO-CH3 adduct formation during the oxidation of PAH-trans-dihydrodiols. The six-line hyperfine splitting pattern observed during the enzymatic oxidation of Npdiol was recorded, and the accumulation kinetics of the EPR spin adduct DMPO-CH3 was measured from the intensity of the sextet, midfield component (panel A). The six-line hyperfine splitting pattern observed during the enzymatic oxidation of Bpdiol was recorded and the accumulation kinetics of the EPR spin adduct DMPO-CH3 was measured from the intensity of the sextet, midfield component (panel B). The six-line hyperfine splitting pattern observed during the generation of O2-• (Hypoxanthine/xanthine oxidase) was recorded, and the accummulation kinetics of the EPR spin adduct DMPO-CH3 was measured from the intensity of the sextet, midfield component (panel C).

dihydrodiols, the amount of cyt c reduction exceeded O2-• formation, suggesting that other one-electron reductions take place. For example, for every mole of diol oxidized, 1 mol of NADPH is produced, and during the autoxidation of the catechol, 1 mol of superoxide anion and 1 mol of o-semiquinone radical are formed as transient species. These reactions would provide a net of 4 electrons which could be accepted by cyt c as terminal acceptor. Because the rate of cyt c reduction that is inhibited by SOD is an indirect measure of O2-• production, it was prudent to spin trap O2-•, and attempts were made to do this in these studies. The ability to observe identical spin adducts either when PAH-trans-dihydrodiols were oxidized by DD or when a hypoxanthine/xanthine oxidase system was substituted provides evidence that O2-• is formed during turnover of these proximate carcinogens.

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The spin adduct observed during either diol oxidation or the generation of superoxide anion by xanthine oxidase was assigned to DMPO-CH3. The ability of superoxide dismutase to prevent the formation of the DMPO-CH3 spin adduct during diol oxidation is taken as evidence that superoxide anion is the radical that propagates the autoxidation. The inability of superoxide dismutase to block the formation of DMPO-CH3 spin adducts observed with xanthine oxidase is attributed to the enzymatic production of hydrogen peroxide. Although the identity of the initiating radical that generates the first molecule of o-semiquinone anion radical is unknown, either trace superoxide anion or contaminating metal ions may be responsible. In preliminary experiments, we have shown that, under anaerobic conditions, NPQ, NADPH, and Zn2+ will produce an EPR signal assigned to an osemiquinone anion radical. This implies that contaminating metal ions will oxidize the catechol to produce the o-semiquinone radical which is stabilized by Zn2+.3 In comparing the autoxidation events that occur following the enzymatic oxidation of Npdiol and Bpdiol, it is apparent that some differences exist. In the case of Bpdiol, oxygen consumption and superoxide anion production lagged behind diol oxidation and H2O2 formation. This would support the mechanism of autoxidation proposed in which oxygen is consumed only in the second one-electron reduction step. It also suggests that intermediate o-semiquinone anion radicals may accumulate. One reason for this accumulation is that the o-semiquinone anion radical may be stabilized by the larger polycyclic aromatic ring system present in benzo[a]pyrene. A similar phenomenon may be observed when other PAH trans-dihydrodiols are oxidized by DD. The accumulation of the o-semiquinone anion radical suggests that it should be possible to spin trap this intermediate. As already described, we have sucessfully spin trapped the o-semiquinone anion radical of NPQ using Zn2+ as a spin-stabilization reagent. However, this approach is not an option in this enzyme study since millimolar concentrations of Zn2+ inhibit DD activity. It has been suggested previously that DD initiates a novel pathway of proximate carcinogen detoxication in which trans-dihydrodiols are oxidized to o-quinones which could be conjugated with cellular thiols for elimination (10, 25). The present study indicates that this is not an innocuous transformation since the autoxidation process generates ROS that may have deleterious cytoand geno-toxic consequences. Furthermore, the production of ROS may be amplified when the enzymatically generated o-quinones enter futile 1e- or 2e- redox cycles. Within the cell, PAH o-quinones are not substrates for DT-diaphorase and prefer to enter 1e- redox cycles catalyzed by microsomal NADPH:cytochrome P450 reductase and NADH:cytochrome b5 reductase as well as mitochondrial NADH:ubiquinone oxidoreductase. In vitro they will redox cycle with NAD(P)H nonenzymatically (14). In terms of cytotoxicity, one consequence of ROS production is that H2O2 accumulates. The glutathione peroxidase/reductase system then attempts to eliminate the peroxide, and as a result there is a concomittant change in redox state, which can ultimately lead to cell death. Certain PAH o-quinones produced by DD have been shown to be cytotoxic by this mechanism in rat and 3

T. Ohnishi, and T. M. Penning, unpublished data.

Penning et al.

human hepatoma cells, and a similar mechanism may operate in rat hepatocytes (22, 26). In terms of genotoxicity, one consequence of ROS production is that OH• radicals arise from Fenton chemistry. Within the cell, OH• radicals are known to cause oxidative damage of DNA, which results in the formation of 8-hydroxy-2′deoxyguanosine (8-OH-dG) and thymine glycol. 8-OHdG causes mutation of DNA in vitro and in vivo (4042), and in animal models of human cancer, the levels of 8-OH-dG in target tissues correlates well with tumor incidence (43, 44). Oxidative damage of DNA by ROS can also result in strand scission (45-47). Studies from this laboratory have shown that isolated rat hepatocytes will convert Bpdiol into BPQ (12, 13). When hepatocytes are incubated with Bpdiol, there is a robust production of O2-• and concomitant strand scission of genomic DNA which are attenuated by inhibitors of DD. Further, extensive O2-• production and DNA fragmentation are observed in hepatocytes treated with BPQ (13, 48). In model studies, BPQ will cause strand scission of plasmid DNA in the presence of NADPH and either Fe3+ or Cu2+, implying that the reactive species responsible is OH• (13, 48). Together, these data support the concept that in hepatocytes the end result of converting Bpdiol into BPQ is to generate OH• which will promote DNA strand scission. The repair of DNA strand breaks can result in recombination events that may result in mutation and tumor initiation. The generation of ROS may also influence tumor promotion. The production of O2-• by the hypoxanthine/ xanthine oxidase system is known to influence the transcription of the c-fos cellular oncogene which is a component of the transcription factor complex, activating protein 1 (AP-1) (49, 50). This effect is seen in mouse fibroblasts (20) and mouse skin, known targets for PAHinduced carcinogenesis. Members of the AP-1 transcription family form homo- and hetero-dimers via the formation of leucine zipppers (49, 50). Heterodimers generally have a synergistic effect on target gene transcription. ROS can regulate the formation of the productive heterodimers via transcriptional and post-translational mechanisms. First, it is known that the promoters of the c-fos and c-jun genes contain phorbol ester (12-Otetradecanoylphorbol 13-acetate) response elements which are trans-activated via the stimulation of protein kinase C (51, 52) and that ROS will activate protein kinase C (20). Second, ROS (produced by UV light and H2O2) activate Src-tyrosine kinase, which leads to increased phosphorylation of c-jun and enhanced AP-1 binding activity (53). Third, a critical cysteine on c-fos may be oxidized to sulfinic and sulfenic acid, which in turn prevents the binding of heterodimers of c-fos:c-jun to target gene sequences (54). It has been suggested that AP-1 factors are activated (maintained in a reduced state) during oxidative stress by the action of redox factor 1 (55). From this discussion, it can be seen that the generation of ROS during the enzymatic oxidation of PAH transdihydrodiols may have a profound influence on PAH induced chemical carcinogenesis. This influence may include changes in redox state, oxidative DNA damage, and alterations in oncogene transcription that mimic activation of protein kinase C by the tumor promotors. Together, these events may contribute to the complete carcinogenic potential of PAH.

Acknowledgment. This study was supported by Public Health Service Grants CA39504 (to T.M.P.),


GM30736 (to T.O.), and ES04266 (to R.G.H.) and by Grant NS30180 (to S.T.O).

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