Ethylbenzene Induces Microsomal Oxygen Free Radical Generation: Antibody-Directed Characterization of the Responsible Cytochrome P450 Enzymes

Toxicology and Applied Pharmacology 164, 305–311 (2000) doi:10.1006/taap.2000.8910, available online at http://www.idealibrary.com on

Ethylbenzene Induces Microsomal Oxygen Free Radical Generation: Antibody-Directed Characterization of the Responsible Cytochrome P450 Enzymes Sonia C. Serron, Neelam Dwivedi, and Wayne L. Backes 1 Department of Pharmacology and Experimental Therapeutics and The Stanley S. Scott Cancer Center, Louisiana State University Health Sciences Center, 1901 Perdido Street, New Orleans, Louisiana 70112 Received October 19, 1999; accepted January 31, 2000

Ethylbenzene Induces Microsomal Oxygen Free Radical Generation: Antibody-Directed Characterization of the Responsible Cytochrome P450 Enzymes. Serron, S. C., Dwivedi, N., and Backes, W. L. (2000). Toxicol. Appl. Pharmacol. 164, 305–311. Small aromatic hydrocarbons cause changes in oxidative metabolism by modulating the levels of cytochrome P450 enzymes, with the changes in these enzymes being responsible for qualitative changes in aromatic hydrocarbon metabolism. The goal of this study was to determine if exposure to the small alkylbenzene ethylbenzene (EB) leads to an increase in hepatic free radical production. Male F344 rats were treated with ip injections of EB (10 mmol/kg) and compared to corn oil controls. Hepatic free radical production was examined by measuring the conversion of 2ⴕ,7ⴕ-dichlorofluorescin diacetate (DCFH–DA) to its fluorescent product 2ⴕ,7ⴕ-dichlorofluorescein (DCF). A significant elevation of fluorescent DCF production was observed after treatment with EB, despite the lack of effect on overall cytochrome P450 levels. This process was shown to be inhibitable by metyrapone, an inhibitor of P450. DCF production was also inhibited by catalase, suggesting that hydrogen peroxide (H 2O 2) is one of the reactive oxygen intermediates involved in EB-mediated reactive oxygen species (ROS) formation. Interestingly, superoxide dismutase (SOD) did not inhibit DCF production in corn oil-treated rats but was an effective inhibitor in the EB-treated groups. In an effort to determine if the increase in ROS production was related to changes in specific P450 enzymes, DCF production was measured in the presence of anti-CYP2B, anti-CYP2C11, anti-CYP2E1, and anti-CYP3A2 inhibitory antibodies. Anti-CYP2B antibodies inhibited DCF production in EB-treated, but not corn oil groups, which is consistent with the low constitutive levels of this enzyme and its induction by EB. The data also demonstrate that CYP2B contributes to ROS production. Anti-CYP2C11 did not influence DCF production in either group. ROS formation in corn oil-treated rats as well as in ethylbenzene-treated rats was also inhibited with antibodies to anti-CYP2E1 and anti-CYP3A2. These results suggest that CYP2C11 does not appear to influence free radical production and that the increase in free radical production in EB treated rats is consistent with the EB-mediated elevation of 1

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CYP2B, CYP 2E1, and CYP3A2. Such alterations in free radical generation in response to hydrocarbon treatment may contribute to the toxicity of these compounds. © 2000 Academic Press

Aromatic hydrocarbons are extensively used throughout the world. Simple aromatic hydrocarbons like benzene, toluene, and ethylbenzene are constituents of numerous industrial and commercial products such as solvents, adhesives, paints, household aerosols, and unleaded gasoline (Fishbein, 1984; Fishbein, 1985a,b,c). Widespread use of these compounds provides opportunities for industrial and environmental exposure (Von Burg, 1992; Ashley et al., 1994). Their high lipid solubility and toxic effects make these compounds potential health hazards. It is well documented that benzene can promote blood abnormalities, genotoxicity, and certain types of leukemia (Dean, 1985; Aksoy, 1985). Exposure to low levels of aromatic hydrocarbons can produce CNS depression, nausea, vomiting and fatigue, while high level exposure can produce neurotoxicity and damage to the liver, heart, and kidney (Fishbein, 1985a,b,c; Tegeris and Balster, 1994). Aromatic hydrocarbons are primarily metabolized by the cytochrome P450 (P450) 2 superfamily of enzymes. In general, the P450 enzymes catalyze the hydroxylation of the aromatic ring and/or the aliphatic side chain, making the substrate molecule more polar. Hydroxylation is followed by conjugation usually to glycine or glucuronic acid (Fishbein, 1985a,b,c; Engstro¨m et al., 1987). Several basic cellular processes lead to the production of reactive oxygen species (ROS) within a cell. The P450 enzymes, which utilize molecular oxygen to oxidize their substrates, comprise an important source of ROS formation in the liver cell. Superoxide anion (O 2•⫺), and hydrogen peroxide (H 2O 2) can be formed during P450 turnover. The active oxygen species can be generated due to uncoupling of 2 Abbreviations used: BCIP, 5-bromo,4-chloro,3-indolyl phosphate; DCF, 2⬘,7⬘-dichlorofluorescein; DCFH, 2⬘,7⬘-dichlorofluorescin; DCFH–DA, 2⬘,7⬘dichlorofluorescin diacetate; EB, ethylbenzene; NBT, 4-nitroblue tetrazolium chloride; P450 (or CYP), cytochrome P450; ROS, reactive oxygen species; O 2•⫺, superoxide radical.

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microsomal monooxygenase reactions. For example, superoxide can be produced during the autooxidation of the oxycytochrome P450 complex (Sligar et al., 1974; Kuthan and Ullrich, 1982). Although the superoxide radical shows intermediate reactivity, it can undergo enzymatic or spontaneous dismutation in the presence of superoxide dismutase (SOD) to produce H 2O 2 and molecular oxygen (Estabrook et al., 1979). H 2O 2 can also be formed by the protonation and decay of the peroxy complex (Fe 3⫹O 22⫺RH) (Nordblom and Coon, 1977; Oprian and Coon, 1980; Karuzina and Archakov, 1994). Alternatively, O 2•⫺ and H 2O 2, in the presence of transition metals, can further be converted to the highly reactive hydroxyl radicals via the Haber–Weiss or Fenton reactions (Halliwell, 1984). In recent years free radicals have been linked in the pathogenesis of several diseases, including atherosclerosis, cancer, cardiovascular disease, reperfusion injury, and brain diseases (Cerutti, 1985; Kolata, 1986; Sparrow et al., 1992; Caraceni et al., 1994). The free radicals produced during the catalytic cycle of cytochrome P450 could contribute to organ damage. Under basal conditions, the body can protect itself from free radicalinduced damage with the help of the vast array of antioxidant defenses including SOD, glutathione peroxidase, and catalase. However, a large increase in free radical production (oxidative stress) can override these protective mechanisms, leading to a variety of diseases. The production of ROS via the cytochrome P450 enzymes is of great interest as these enzymes are extensively involved in the metabolism of xenobiotics. The fact that P450 enzymes can be induced by exogenous compounds is well established (Conney, 1967; Koop and Tierney, 1990; Gonzalez, 1990) and increases the importance of P450-related ROS generation. Studies in this laboratory have shown ethylbenzene (EB), an alkylbenzene present in gasoline, motor vehicle exhaust, and cigarette smoke (Von Burg, 1992), to be an effective inducer of several P450 enzymes (Imaoka and Funae, 1991; Backes et al., 1993; Gut et al., 1993; Yuan et al., 1997b). EB treatment in male Holtzman rats altered the expression of CYP1A1, CYP2B1/2, CYP2C11, CYP2E1, and CYP3A1/2 with different isozymes exhibiting multiphasic induction patterns (Yuan et al., 1997b). Literature reports indicate that the P450 system can contribute to generation of significant quantities of ROS, even under basal conditions (Bondy and Naderi, 1994). Consequently, induction of specific P450 enzymes, because of exposure to EB, could potentially alter the levels of ROS compared to normal physiological conditions. ROS-induced tissue injury may thus be involved in the toxicity produced by aromatic hydrocarbons. The aim of the present study was to investigate the effect of ethylbenzene on the generation of ROS and to identify a possible role for cytochrome P450 in this effect. ROS generation was estimated by fluorometric techniques using 2⬘,7⬘dichlorofluorescin diacetate (DCFH–DA), a probe that has been utilized extensively for the measurement of free radical generation (Bass et al., 1983; Bondy and LeBel, 1992; Punta-

rulo and Cederbaum, 1996; Hoare et al., 1999; Liu et al., 1999). The thiobarbiturate/malondialdehyde complex, the characteristic chromogenic adduct utilized traditionally to measure lipid peroxidation, has been demonstrated to be unstable in the presence of H 2O 2 (Kostka and Kwan, 1989). Additionally, thiobarbituric acid and malondialdehyde, cross-react with several endogenous substances such as amino acids and deoxyribose (Halliwell and Gutteridge, 1981; Gutteridge, 1981). The use of DCFH–DA to quantify ROS formation has been adapted to subcellular systems and has been shown to be rapid and sensitive (LeBel and Bondy, 1990). In cellular systems, the nonfluorescent probe DCFH–DA readily crosses the cell membrane and undergoes hydrolysis by intracellular esterases to nonfluorescent DCFH (Bass et al., 1983). DCFH is then rapidly oxidized in the presence of reactive oxygen species to highly fluorescent 2⬘,7⬘-dichlorofluorescein (DCF) (Szejda et al., 1984). The present experiments were designed to determine if EBmediated induction of P450 caused an increase in the potential for free radical production. Inhibitory antibodies to specific P450 enzymes were used in an attempt to identify those enzymes most responsible for the changes in free radical production. Finally, ROS formation was measured in the presence of the free radical scavengers, SOD, and catalase to investigate the nature of the reactive oxygen species involved in EBmediated ROS formation. MATERIALS AND METHODS Chemicals. Ethylbenzene was obtained from Aldrich Chemical Co (Milwaukee, WI), DCFH–DA was purchased from Molecular Probes, Inc. (Eugene, OR) and DCF was obtained from Polysciences, Inc. (Warrington, PA). AntiCYP2B, anti-CYP2C11, anti-CYP2E1, and anti-CYP3A2 inhibitory antibodies were obtained from Gentest Corporation (Woburn, MA). Anti-CYP2B, -CYP2E1, -CYP2C11, and -CYP3A2 used for immune blotting were obtained from Oxygene (Dallas, TX). Other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). Animals and treatments. Male Fischer 344 rats (200 –300 g, Harlan Sprague Dawley, Inc., Madison, WI) were placed on a 12-h day/night cycle with each group housed separately in hanging wire-bottom cages. The rats were maintained on an unrestricted diet of rodent chow and deionized water passed through activated charcoal to avoid organic contamination. Rats were treated with a single ip injection of a 2 M suspension of ethylbenzene in corn oil (10 mmol/kg body wt). The cytochrome P450 system has been well characterized in the rat model. Previous studies in our laboratory investigating the dose–response effect of EB in male rats have shown the dose of 10 mmol/kg to be an effective inducer of several P450s without causing toxicity. An inbred strain of rats (F344) was used in this study in an effort to decrease the variability between groups. The response to EB was the same between F344, Sprague Dawley and Holtzman rats; however, we did not observe a significant reduction in the intragroup variability using the inbred strain (not shown). Control animals received ip injections of corn oil (vehicle). The animals were killed by decapitation 24 h after the final injection and liver microsomes were prepared by differential centrifugation (Sequeira et al., 1992). Protein concentrations were determined by the method of Lowry et al. (1951). Microsome aliquots were frozen at ⫺80°C for subsequent use. Estimation of ROS generation. In cellular systems, the nonfluorescent probe DCFH–DA readily crosses the cell membrane and undergoes hydrolysis

EB-MEDIATED ROS GENERATION by intracellular esterases to nonfluorescent DCFH (Bass et al., 1983). DCFH is then rapidly oxidized in the presence of reactive oxygen species to highly fluorescent DCF (Szejda et al., 1984; LeBel and Bondy, 1990). In our study, hepatic microsomes (0.2 mg) were incubated at 37°C in Tris buffer (40 mM, pH 7.4 at 37°C) and DCFH–DA (5 ␮M) for 25 min. At the end of the incubation period, NADPH (0.6 mM) was added and the rate of formation of fluorescent product was measured using a Shimadzu spectrofluorometer (excitation wavelength ⫽ 488 nm, emission wavelength ⫽ 525 nm). In studies measuring ROS in the presence of free radical scavengers, superoxide dismutase (SOD) (final concentration 400 U/ml) and catalase (final concentration 3400 U/ml) were added after the initial incubation period and samples were further incubated for 5 min, before initiating the reaction with NADPH. The P450 inhibitor, metyrapone (1 mM) was added during the preincubation with buffer and DCFH–DA. Inhibitory antibodies to CYP2B, CYP2C11, CYP2E1, and CYP3A2 were added to microsomes and preincubated at room temperature for 30 min. A control reaction was also set up using normal serum in place of antiserum. A standard curve using 0.0 –1.0 ␮M DCF was prepared and the extinction coefficient was determined from the slope of this curve. To ensure that DCFH was oxidized by microsomes alone, controls measuring fluorescence development in the absence of tissue and in the buffer alone were used. Antibodies to CYP2B, CYP2E1, and CYP3A2 do not cross-react with other than the specified enzymes. Antibody to CYP2C11 weakly cross-reacts with CYP2B1 and CYP2B2 (Gentest, Woburn, MA). In the inhibition experiments we used 20 ␮l of antibody as recommended by the vendor. Separate experiments showed that anti-CYP2B (20 ␮l) caused a 50% inhibition of DCF production, whereas a 70% inhibition was observed after addition of 30 ␮l of antibody (not shown). The other anti-CYP antibodies were also used according to the recommendations of the vendor (20 ␮l per assay). Western immunoblotting of P450 enzymes. Changes in specific cytochrome P450 isozyme levels were estimated by immunoblotting techniques (Towbin et al., 1979; Sequeira et al., 1992; Backes et al., 1993). Liver microsomes (6 ␮g protein) were subjected to SDS–polyacrylamide gel electrophoresis. Proteins were transferred electrophoretically from polyacrylamide gels to nitrocellulose membranes (Costar, Cambridge, MA). The washed membranes were then incubated with antibodies to P450 2B, P450 2C11, P450 2E1, or P450 3A (primary antibodies). The membranes were then washed and incubated with an alkaline-phosphatase-linked goat anti-rabbit IgG (secondary antibody). 4-Nitroblue tetrazolium chloride (4-NBT) and 5-bromo,4-chloro,3indolyl phosphate (5-BCIP) reagents were used for color development of the immunoreactive proteins. Intensities of the blots were quantified on a Hewlett Packard Scan Jet 4C scanner and using the image scanning software Scion Image, which is based on NIH Image modified for windows by Scion Corporation. Statistical analyses. Rats were separated into different groups of at least five animals. All data are presented as mean ⫾ SEM. Significance was estimated using ANOVA followed by Tukey’s Multiple Comparison Procedure with p ⬍ 0.05 being considered significant.

RESULTS

The ability of EB to induce ROS generation was studied in liver microsomes from rats treated with EB using DCFH–DA to quantify ROS production (Bass et al., 1983; Bondy and LeBel, 1992). Figure 1 demonstrates the effect of EB on DCF formation. EB (10 mmol/kg body wt) produced a significant elevation in DCF formation (⬃40%) compared to the vehicle (corn oil)-treated group. Although DCF levels in the control group were lower than the EB-treated group, it is worth noting that there was a high level of DCF production in the control group. In order to determine the contribution of the cytochrome P450 enzymes to EB-mediated free radical production, DCF

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FIG. 1. Effect of ethylbenzene administration on hepatic microsomal free radical production. Rats were treated with either EB (10 mmol/kg ip) or with corn oil as a control. Microsomes were prepared and assayed for the formation of 2⬘,7⬘-dichlorofluorescein as an index of free radical production. Where indicated, the microsomal preparations were treated with the P450 inhibitor metyrapone (1 mM). *Values are significantly different from the corn oil control. }Values are significantly different from the corresponding group without metyrapone. Values represent the mean ⫾ SEM with p ⬍ 0.05 being considered significant. Number of determinations in each group: corn oil (n ⫽ 11), EB (n ⫽ 8), corn oil plus metyrapone (n ⫽ 5), and EB plus metyrapone (n ⫽ 5).

formation was measured in the presence of the P450 inhibitor metyrapone (Fig. 1). Metyrapone (1 mM) suppressed the formation of DCF in the EB treatment groups as well as in the vehicle-treated group (62 and 60%, respectively). These results support an important role for cytochrome P450 in EB-mediated ROS formation. Since metyrapone inhibited ROS production in the control group, it is likely that P450 enzymes are contributing to the basal levels of active oxygen species measured. However, metyrapone did not produce a complete inhibition of basal DCF formation, suggesting that there may also be other sources of ROS formation in the hepatic microsomes. In an attempt to identify the nature of the prooxidant species produced in our studies, ROS formation was measured in the presence of catalase and superoxide dismutase, inhibitors of oxygen free radical production. Catalase, an enzyme that degrades hydrogen peroxide, significantly inhibited ROS generation in control as well as the EB-treated group (Fig. 2). As a control experiment, DCF production was not altered by the addition of 100 ␮M sodium azide, which would inhibit endogenous catalase activity. Furthermore, in separate experiments inhibition of DCF production was obtained with dialyzed catalase (The catalase preparation was dialyzed to remove the contaminating antioxidant thymol.). These results indicate that H 2O 2 was one of the reactive intermediates in free radical production. Interestingly, superoxide dismutase inhibited DCF formation in EB-treated rats but had no effect on ROS generation in the vehicle-treated group (Fig. 2), suggesting association of superoxide anion radical with the EB-induced free radical generation. Previous reports using Holtzman and Sprague Dawley rats have demonstrated that EB modulates the expression of several P450 enzymes (Imaoka and Funae, 1991; Sequeira et al., 1994;

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FIG. 2. Effect of superoxide dismutase and catalase on free radical production. Hepatic microsomal ROS generation was measured in the absence and presence of SOD (400 U/ml) and catalase (3,400 U/ml) in corn oil-treated and EB-treated rats. Rats were treated as described under Materials and Methods and in Fig. 1. *Values are significantly different from the corn oil control. }Values are significantly different from the corresponding group without the inhibiting enzyme. Values represent the mean ⫾ SEM with p ⬍ 0.05 being considered to be significant. Number of determinations in each group: corn oil (n ⫽ 11), EB (n ⫽ 9), and all other groups (n ⫽ 5).

Yuan et al., 1997a,b). EB treatment causes an induction of CYP2B, CYP2E1, and CYP3A and a decrease in CYP2C11 levels (Yuan et al., 1997a,b). Similar results were found with the F344 rats (Fig. 3). EB treatment caused a 20-fold increase in CYP2B protein levels. Ethylbenzene treatment significantly induced the expression of CYP3A2 (67%) and CYP2E1 (80%), whereas CYP2C11 levels were decreased. The results shown in Fig. 4A illustrate the effect of inhibitory antibodies to CYP2B1/2 and CYP2C11 on ROS formation. Anti-CYP2B antibody failed to significantly inhibit ROS in corn oil-treated rats, an expected result due to the extremely low constitutive levels of CYP2B in uninduced rats. However, this antibody produced about a 45% inhibition in free radical production after EB treatment. The data suggest that CYP2B1/2 contributes to the increase in active oxygen species

FIG. 3. Effect of EB administration on CYP2B, CYP2C11, CYP2E1, and CYP3A protein levels. Rats were treated with either EB or corn oil as described in Fig. 1. Hepatic microsomes were examined for changes in immunoreactive CYP protein levels by immune blotting techniques. Values represent the mean ⫽ SEM (n ⫽ 5 in each group), with p ⬍ 0.05 being considered significant.

FIG. 4. Effect of inhibitory anti-CYP antibodies on DCF production. Microsomal preparations were assayed for DCF formation in the absence and presence of inhibitory antibodies to various CYP enzymes. Rats were treated as described under Materials and Methods and in Fig. 1. Antibodies were added as indicated and preincubated for 30 min at RT. A control reaction was also set up using normal serum. }Values were significantly different from the corresponding group without the inhibiting antibody ( p ⬍ 0.05) using a two-way analysis of variance. Values represent the mean ⫾ SEM. (A) Effects of anti-CYP2B and anti-CYP2C11 inhibitory antibodies. The rates of DCF production in each uninhibited group were 0.75 and 0.92 nmol (min) ⫺1(mg protein) ⫺1 for the corn oil and EB-treated groups, respectively, and were assigned values of 100%. Number of determinations in each group: corn oil (n ⫽ 10), EB (n ⫽ 8), corn oil plus anti-CYP2B (n ⫽ 4), and all other groups (n ⫽ 5). (B) Effects of anti-CYP2E1 and anti-CYP3A2 inhibitory antibodies. The rates of DCF production in the uninhibited groups were 0.43 and 0.61 nmol (min) ⫺1(mg protein) ⫺1 for the corn oil and EB-treated groups, respectively, and were assigned values of 100%. Each group represents the mean ⫾ SEM for six determinations.

generation in EB-treated rats. Anti-CYP2E1 and anti-CYP3A2 antibodies showed a significant inhibition in both corn oil- and EB-treated rats. Anti-CYP2E1 showed an inhibition of about 20% in both groups (Fig. 4B). Anti-CYP3A2 caused a significant inhibition of ROS formation to the extent of 22% in control rats and 25% in EB treated rats. Interestingly, addition of antibody to CYP2C11, the other major P450 enzyme modulated by EB, did not inhibit DCF production in either corn oilor EB-treated rats, suggesting that this isozyme does not play an important role in the production of ROS. Taken together, these results indicate that EB influences hepatic microsomal ROS generation by modulating the levels of different P450

EB-MEDIATED ROS GENERATION

enzymes and that the increase in ROS production in EB-treated rats could be explained by the increase in CYP2B (and to a lesser extent CYP3A) expression. DISCUSSION

In addition to being oxidatively metabolized by the P450 enzyme system, alkylbenzenes can also modulate the levels of particular P450 enzymes. These newly induced enzymes metabolize small alkylbenzenes such as toluene in a qualitatively different manner, dramatically increasing the amount of aromatic hydroxylation without significantly affecting hydroxylation of the aliphatic side chain (Sequeira et al., 1994; Yuan et al., 1997a). An increased production of phenolic metabolites could potentially increase the risk of toxicity for individuals exposed to these chemicals. The data reported in this manuscript extend these observations by demonstrating that the P450 enzymes induced by aromatic hydrocarbon exposure can also cause an increase in the potential for ROS production. These reactive oxidants produced could contribute to the toxic effects seen with aromatic hydrocarbon exposure. This is especially true in the liver (where the majority of the P450 enzymes are located) and in the CNS. Due to its high lipid content, the CNS is a potential site for free radical damage of cellular membranes and lipoproteins by lipid peroxidation. P450 enzymes can produce superoxide anion and hydrogen peroxide through the breakdown of oxygenated intermediates of the enzyme. Superoxide and hydrogen peroxide, in the presence of transition metals, can produce the highly reactive hydroxyl radical via the Fenton and Haber–Weiss reactions. Preliminary studies indicated an inhibition of DCF production by mannitol (500 mM) or catechol (0.3 mM), further supporting the view that DCF is produced by a free radical-mediated mechanism (not shown). We measured ROS formation in the presence of the antioxidants catalase and SOD in an attempt to determine the nature of the prooxidant species. The inhibition of both basal and EB-induced ROS generation by catalase while SOD inhibited only EB-induced ROS formation suggests that there may be some qualitative differences in the prooxidant species in EB and corn oil groups. This is in contrast to literature reports, which indicated a complete inhibition of DCF formation by SOD with microsomes from previously untreated rats. (Bondy and Naderi, 1994). This effect could be due to differences in the rat strains used or the specific assay conditions. Further studies will be required to address this discrepancy. It is important to determine if aromatic hydrocarbon exposure does in fact increase the production of ROS and if the cytochrome P450 enzymes are involved in this effect. Previous studies in this lab have shown that both acute and continued exposure to EB altered the expression of several P450 isozymes including P450 2B1/2, 2E1, 2C11 and 3A1/2 (Backes et al., 1993; Yuan et al., 1997b; Bergeron et al., 1999). The results from the present study demonstrate that exposure to EB

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induces ROS generation. The role for the P450 system in ROS production is strengthened by the increase in ROS generation after induction of P450 enzymes, as well as ROS inhibition by the P450 inhibitor metyrapone. As pointed out, a high level of ROS was observed not only after induction of P450 by EB treatment, but also in corn oil-treated rats. ROS generation in both groups could be inhibited with metyrapone. This result is consistent with other reports, which demonstrated that basal levels of P450 could make significant contributions to ROS formation (Ingelman-Sundberg and Hagbjork, 1982; Bondy and Naderi, 1994). These results are also consistent with others demonstrating that ROS production is increased by induction of P450 enzymes after treatment with ethanol, benzene, or naphthalene (Ingelman-Sundberg and Hagbjork, 1982; Vuchetich et al., 1996; Bagchi et al., 1998). An extremely interesting aspect of this study is related to the antibody-directed inhibition of ROS formation. In corn oiltreated rats, antibodies to CYP2E1 and CYP3A caused a 20 and 22% inhibition of ROS generation, respectively, whereas CYP2B and CYP2C11 were without a significant effect. Interestingly, despite the extremely high levels of CYP2C11 enzyme in corn oil groups, the antibody to CYP2C11 shows no inhibitory effect. This effect on ROS generation is much greater than expected based on the relative amount of the P450 enzymes present in these microsomal preparations. CYP2E1, CYP3A, and CYP2B comprise about 6, 10, and 0.4% of the total P450 present in corn oil-treated rats (Imaoka and Funae, 1991). Whereas CYP2E1 only constitutes about 6% of the total P450, inhibition of this enzyme caused a 22% inhibition of ROS production. A similar effect was observed with CYP3A, which constitutes about 10% of the total P450, yet inhibition of this enzyme produced a 22% inhibition of ROS generation. This inhibitory effect on ROS generation was also observed in EB-treated rats. CYP2E1, CYP3A, and CYP2B constitute about 9, 12.2, and 6% of the total P450 (Imaoka and Funae, 1991). Inhibition of these enzymes with selective CYP antibodies caused 17, 25, and 45% inhibition of ROS production. One possible explanation for this effect is that there is an interaction among P450 enzymes in the microsomal membrane, with individual enzymes combining to form multisubunit complexes. Inhibition of a single P450 (e.g., CYP3A) could not only inhibit CYP3A monooxygenase activities but could cause a greater inhibition of ROS generation by tying up the reductase. Interactions among different P450 enzymes have been reported previously and have been shown to have a significant influence on their catalytic activities (Cawley et al., 1995; Tan et al., 1997; Yamazaki et al., 1997; Lovern et al., 1997; Backes et al., 1998). The potential for such interactions to influence ROS production is currently being examined. In conclusion, exposure to the aromatic hydrocarbon ethylbenzene elevates ROS formation in rat hepatic microsomes and appears to be mediated by the cytochrome P450 enzymes. An increase in ROS generation was produced without a significant alteration in overall P450 content. This effect appears to be

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related to alterations in the relative levels of CYP2B, CYP3A, and CYP2E1. There has been evidence that the toxicity of the aromatic hydrocarbon toluene may be related to induction of excess ROS formation (Mattia et al., 1993a,b). Consequently, the alteration of specific CYP protein levels by treatment with hydrocarbons or other inducers could lead to an increase in hydrocarbon toxicity by an alteration in ROS production. In summary, we have shown that exposure to ethylbenzene induces various P450 enzymes that are responsible for the generation of ROS. The importance of this P450-mediated induction is supported by the antibody-directed inhibition of ROS generation. The magnitude of the antibody-directed inhibition of ROS appears to be greater than that predicted by the relative quantity of the particular P450 present in the microsomal preparation. These results suggest a possible interaction among the P450s leading to this inhibitory response. Future studies correlating the induction of P450 enzymes capable of stimulating free radical production with the toxic effects produced by EB and the other alkylbenzenes are required. The antioxidant defenses present in the body such as glutathione peroxidase, SOD, and catalase help protect against free radicalinduced damage. Additionally, free radical scavengers such as vitamins A, E, and C, diets rich in fruit and vegetables (Vuchetich et al., 1996; Lampe, 1999), and pharmacological agents such as selegiline (Rosler et al., 1998; Mitsuyoshi et al., 1999) may provide us with protection against free radical-induced damage. Further characterization of the antioxidants that inhibit EB-mediated ROS production could provide new information that could be used in developing protective measures against exposure to aromatic hydrocarbons. ACKNOWLEDGMENT These studies were supported in part by a U.S. Public Health Services research grant from the National Institute of Environmental Health Sciences, R01-ES04344.

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