Document nk8JgnYoGYLpnQKZrJRnVpZrm

(CANCER RESEARCH 53. I02.VI026. March I. 1993] Benzene and Its Phenolic Metabolites Produce Oxidative DNA Damage in HL60 Cells in Vitro and in the Bone Marrow in Vivo1 Prema Kolachana, Vangala V. Subrahmanyam,2 Kathleen B. Meyer, Luoping Zhang,3 and Martyn T. Smith4 Department of Biomdicaland Environmental Health Sciences, School of Public Health. University of California, Berkeley, California 94720 ABSTRACT Benzene, an important industrial chemical, is myelotoxic and leuke- mogenic in humans. It is metabolized by cytochrome P450 2E1 to various phenolic metabolites which accumulate in the bone marrow. Bone marrow contains high levels of myeloperoxidase which can catalyze the further metabolism of the phenolic metabolites to reactive free radical species. Redox cycling of these free radical species produces active oxygen. This active oxygen may damage cellular DNA (known as oxidalive DNA dam age) and induce genotoxic effects. Here we report the induction of oxida- tive DNA damage by benzene and its phenolic metabolites in HIM) cells in vitro and in the bone marrow of C57BL/6 x C3H F, mice in vivo utilizing 8-hydroxy-2'-deoxyguanosine as a marker. III.Ml cells (a human leukemia cell line) contain high levels of myeloperoxidase and were used as an in vitro model system. Exposure of these cells to phenol, hydroquinone, and 1,2,4-benzenetriol resulted in an increased level of oxidative DNA damage. An increase in oxidative DNA damage was also observed in the mouse bone marrow in vivo l h after benzene administration. A dose of 200 mg/kg benzene produced a 5-fold increase in the 8-hydroxydeoxyguanosine level. Combinations of phenol, catechol, and hydroquinone also resulted in sig nificant increases in steady state levels of oxidative DNA damage in the mouse bone marrow but were not effective when administered individu ally. Administration of 1,2,4-benzenetriol alone did, however, result in a significant increase in oxidative DNA damage. This represents the tirsi direct demonstration of active oxygen production by benzene and its phenolic metabolites in rim. The conversion of benzene to phenolic me tabolites and the subsequent production of oxidative DNA damage may therefore play a role in the benzene-induced genotoxicity, myelotoxicity, and leukemia. convert the phenolic metabolites of benzene to reactive semiquinones and quiones (6-10). These semiquinones, quiones, and, perhaps, iran-.fran.--muconaldehyde are generally believed to be the ultimate toxic species derived from benzene. Benzene and various combinations of its metabolites produce ge netic damage, including numerical and structural chromosomal aber rations in blood and bone marrow of exposed animals and humans (11-16). One mechanism by which benzene induces these genotoxic effects may be by generating one or more active oxygen species such as Superoxide anin radicals (O2 ),hydrogen peroxide (H2O2), hydroxyl radicals (HO'), and singlet oxygen ('O2) in the bone marrow (6). Active oxygen could be generated by peroxidatic metabolism or autoxidation of phenolic metabolites in the bone marrow (6, 8, 17). Previous in vitro studies have shown that semiquinone radicals formed during the peroxidatic metabolism of HQ can reduce dioxygen to O2 (8) and have the potential to undergo cyclic reduction and oxidation reactions (redox cycling) to produce large amounts of active oxygen. However, the ability of benzene or its phenolic metabolites to induce the formation of active oxygen in vivo and cause damage to cellular DNA and proteins has not been studied. The primary objective of the present study was to test this hypothesis. Recently, Kasai and Nishimura (18) and Floyd el al. (19) have shown that active oxygen including HO' and 'O2 can hydroxylate deoxyguanosine residues in DNA resulting in the formation of 8OHd- Gua. This SOHdGua can be easily detected by HPLC with electro chemical detection, following enzymatic hydrolysis of DNA. This method has become a useful tool to measure the formation of active INTRODUCTION oxygen in complex biological systems (such as in vivo situations) where active oxygen cannot be directly detected (20-22). In addition, Chronic exposure to benzene, an ubiquitous pollutant, induces my elotoxicity and leukemia in humans (reviewed in Ref. l). However, the precise mechanisms by which benzene induces these effects are not known. Various studies have, however, shown that benzene me tabolism is required to exert its myelotoxic effects (reviewed in Refs. 2 and 3). The majority of benzene metabolism occurs in liver, where cytochrome P-450 oxidizes benzene to PH,5 CAT, HQ, BT, and a ring-opened product called /ran.y,/ran.?-muconaldehyde, which is ox idized to fran.v./ran.T-muconic acid. These phenolic metabolites and the detection of SOHdGua in DNA provides a direct measure of genotoxic effects of active oxygen (20). Therefore, in this study, we have utilized SOHdGua formation in DNA as a marker for the detec tion of active oxygen formation. Initial studies were performed in vitro in human myeloid leukemia (HL60) cells by exposing them to the phenolic metabolites of benzene, because these cells contain high levels of MPO (23). Further studies were performed in C57BL/6 X C3H F, (hereafter called B6C3F,) mice exposed to benzene or its phenolic metabolites. The data show that benzene or its phenolic- muconic acid accumulate in the bone marrow (4, 5). MPO and other heme-protein peroxidases present in the bone marrow may further Received 9/24/92; accepted 12/22/92. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by Grants P42ES04705 and P30ESOI896 from the National Institute of metabolites produce oxidative DNA damage in vitro in HL60 cells and in vivo in bone marrow (the target organ for benzene) of B6C3F, mice. This study provides the first direct evidence for the production of active oxygen in vivo by benzene. MATERIALS AND METHODS Environmental Health Sciences. P. K. and K. B. M. are trainees of the Health Effects Component of the DC Toxic Substances Program. L. Z. is supported by the William and Ada Isabell Steel Memorial Graduate Scholarship from Simon Fraser University, Bumaby. British Columbia, Canada. 2 Present address: Department of Drug Metabolism and Pharmacokinetics, American Cyanamid Company. Pearl River. NY 10965. 1 Permanent address: Bioenergetic Research Laboratory. Faculty of Applied Science. Simon Fraser University, Burnaby. British Columbia, Canada V5A IS6. 4 To whom requests for reprints should be addressed, at Department of Biomdicaal nd Environmental Health Sciences, School of Public Health, 322-Warren Hall, University of Cali"f"oTrhneia.abbBreevrkiaetlieoyn,s CuAse9d47ar2e0:. PH, phenol; BT, l.2.4-ben/.enetriol; CAT, catechol; dGua. deoxyguanosine; HPLC, high pressure liquid chromatography; HQ, hydroquinone; 8OHdGua. 8-hydroxy-2'-deoxyguanosine; MPO. myeloperoxidase; PBS. phosphate-buffered saline. DMSO. dimethyl sulfoxide. Reagents. Benzene (99.9% pure) was purchased from Aldrich Chemical Co. (Milwaukee. WI). Phenol, hydroquinone, catechol, 1,2,4-benzenetriol. nu- clease P, and alkaline phosphatase (Escherichia coli) were purchased from Sigma Chemical Co. (St. Louis, MO). High purity distilled phenol was pur chased from International Biotechnologies, Inc. (New Haven, CT). All other chemicals and solvents were of the highest grade commercially available. Standard Cell Culture Conditions. HL60 cells obtained from the Amer ican Type Culture Collection (Rockville, MD) were cultured in RPMI 1640 supplemented with fetal bovine serum albumin (107r) and gentamicin sulfate (50 ug/ml). Cells were grown in a humidified atmosphere in 5% CO2 and 37C. Cell viability was determined using trypan blue exclusion in which 200 cells/ culture were analyzed. All initial viabilities were greater than 95%. 1023 OXIDATIVE DNA DAMAGE BY BENZENE AND ITS METABOLITES In Vitro Studies. HL60 cell cultures were preincubated for 0.5 h at 37C before dosing with various metabolites of benzene. Cells (0.5 X I06/ml) were incubated in PBS (20 ml; pH 7.4) with HQ, PH, CAT, or BT (10-100 UM)up to 1 h. The cells were pelleted by centrifugaron (2000 rpm/5 min) and washed twice with PBS prior to DNA extraction. Assay for MPO Activity in HL60 Cells. Myeloperoxidase activity in HL60 cells was determined using 3,3'-tetramethylbenzidine as a substrate as described previously by Bozeman et al. (24). With this method, we found that HL60 cells contain 74 26 pmol of MPO/106 cells. In Vivo Studies. Male B6C3F, mice (25-30 g, about 6 weeks old) were purchased from Simonsen Laboratories (Gilroy, CA) and housed 1-2 weeks prior to use. Mice were fed standard Purina Rat Chow 5012 and water ad libitum and were maintained in a temperature and photoperiod (12 h/day)- controlled room. The animals were housed I/cage. Benzene Studies. Benzene dissolved in corn oil was used for all studies. Time course studies were performed using a single dose (880 mg/kg) of benzene administered i.p. to 4 groups of mice (3 mice/group). Each treatment group had corresponding simultaneous vehicle controls (3 mice/group). All animals were euthanized at 1, 3. 6, or 12 h after treatment by cervical dislo cation. Dose-response studies were performed using 6 groups of mice (3 mice/group) given i.p. injections of single doses of 0, 50, 100, 200, 400, or 800 mg/kg of benzene. All animals were euthanized l h after treatment by cervical dislocation. Phenolic Metabolite Studies. Phenolic metabolites of benzene were dis solved in PBS. Nine groups of mice (3 mice/group) were given i.p. injections of PH (75 mg/kg), HQ (75 mg/kg), CAT (75 mg/kg), BT (25, 50, or 75 mg/kg), or a combination of PH + HQ (75 + 75 mg/kg). HQ + CAT (75 + 75 mg/kg) and PH + CAT (75 + 75 mg/kg). An additional group of 3 mice received an equivalent amount of PBS alone. All the chemicals were preweighed and the solutions were prepared immediately before administration to avoid autoxida- tion. All animals were euthanized l h after treatment by cervical dislocation. Isolation of Bone Marrow Cells. Bone marrow cells were isolated from mouse femurs (9) into 3 ml of ice-cold PBS (pH 7) containing 0.16% (w/v) EDTA. The cells were dispersed by gentle aspiration with a Pasteur pipet, centrifuged at 150 X g for 15 min. and resuspended in the same buffer. Approximately 40-50 x IO6 bone marrow cells (pooled from 2 femurs) were harvested from each mouse by this method. Determination of SOHdGua in DNA. DNA was isolated from mouse bone marrow and HL60 cells by the phenol extraction procedure of Gupta el al. (25). To avoid any additional oxidative damage to the DNA due to peroxide or quinone contaminants in phenol, high purity double distilled phenol was used for extractions. About 200-400 ug DNA were resuspended in 200 ul 20 triMsodium acetate (pH 4.8), and digested to nucleotides with 20 ug nuclease PI at 70Cfor 15 min. To adjust the pH 20 ul of l MTris-HCI (pH 7.4) were added to the nucleoside mixture, which then was treated with 1.3 units of E. coli alkaline phosphatase and incubated at 37Cfor 60 min. These hydrolyzed DNA solutions were then filtered using an Ultrafree Millipore filtration system ( 10,000-dalton cutoff). The HPLC conditions used in the present study have been described previously (26). Briefly, the amount of SOHdGua present in the DNA was analyzed by flow-through electrochemical detection using an ESA model 5100 Coulochem detector equipped with a 5011 or 5100 high sensitivity analytical cell with the oxidation potentials of electrodes 1 and 2 adjusted to 0.10 and 0.35 V, respectively. A Supelco LC-18 HPLC column (15 x 4.6 mm) was utilized for separation of SOHdGua. The mobile phase consisted of 10% methanol and 50 m.MKH2PO4 buffer, pH 5.5, run isocratically at a flow rate of 1 ml/min. Synthesis of SOHdGua Standard. Standard SOHdGua was synthesized by Udenfriend's hydroxylating system as described by Kasai and Nishimura (27). Hydroxylation of dGua at the C-8 position was carried out by sequential addition of 25 ul of 0.1 MdGua, 14 ul of 1 Mascorbic acid, 6.5 ul of l MEDTA, and 13 pi of 0.13 MFeSOj to 0.942 ml of 0.1 Msodium phosphate buffer (pH 6.8) and incubating up to 15 min at 37Cin the dark with vigorous shaking. Following incubation, aliquots of the reaction mixture were analyzed by HPLC. Fractions containing SOHdGua were collected from HPLC eluates, lyophilized, and stored at 4Cfor use as standards. Statistics. All results are expressed as mean SD of at least three exper iments. P values for significance were determined using the two-tailed Stu dent's t test. RESULTS Induction of Oxidative DNA Damage by Phenolic Metabolites in Vitro in HL60 Cells Table 1 shows the effect of different benzene metabolites on the formation of 8OHdGua in the DNA of HL60 cells. HQ (10 UM) induced a 2-fold increase in SOHdGua levels after 30 min of incuba tion, while 100 UMPH produced a 3.5-fold increase. The most effec tive inducer of SOHdGua formation was BT, a minor metabolite of benzene, which produced a maximum of 5-fold increase in SOHdGua after 30 min. No significant increase in SOHdGua level was observed, even after l h incubation, with CAT (100 UM). Little or no cytotoxicity was observed from exposure to these compounds for at least 6 h (assayed by trypan blue exclusion test; data not shown), indicating that SOHdGua formation in these cells does not occur after cell death. In summary, HQ, PH, and BT, but not CAT, induce rapid SOHdGua formation in HL60 cells that returns to normal levels following further incubation presumably due to the rapid repair of DNA damage. Oxidative DNA Damage in Vivo in Mouse Bone Marrow following Exposure to Benzene Time Course. A rapid, 2-fold increase \P < 0.001] in SOHdGua levels in mouse bone marrow DNA was observed l h after benzene administration [880 mg/kg (Fig. 1)]. This increase gradually declined towards background levels after 6 h (P > 0.1) and remained constant through 12 h. As shown in Fig. 1, the vehicle (corn oil) had no observable effect on the steady state levels of SOHdGua throughout treatment. The maximum oxidative damage by benzene was observed after 1 h, and this time point was therefore chosen for additional studies. These results further show that maximum oxidative damage occurs well before cytotoxicity (none prior to 12 h) (28) and may contribute to but is not a consequence of toxicity. Dose Response. The effect of different benzene doses on SOHd Gua levels in mouse bone marrow is shown in Fig. 2. A significant increase (P < 0.05) in oxidative DNA damage was observed even with the lowest dose. 50 mg/kg (Fig. 2), reaching a maximum of 5-fold increase (P < 0.001) in SOHdGua levels at 200 mg/kg. Higher doses of benzene resulted in smaller increases in the SOHdGua level, about 2.5-fold (P < 0.001 ) above control levels at 400 mg/kg and only 2-fold (P < 0.001) at 800 mg/kg. Oxidative DNA Damage in Vivo in Mouse Bone Marrow following Exposure to Phenolic Metabolites of Benzene When administered alone, PH (75 mg/kg), CAT (75 mg/kg), or HQ (75 mg/kg) had no significant effect on the steady state level of SOHdGua (Fig. 3), which ranged from 0.045 to 0.053 pmol. However, when mice were dosed with a combination of HQ (75 mg/kg) + PH (75 mg/kg) or with a combination of HQ (75 mg/kg) + CAT (75 mg/kg), a 2-fold increase (P < 0.01) in the SOHdGua level over the control value was observed (Fig. 3). The combination of PH (75 Table 1 Oxidative DNA damage in HL60 cells b\ benzene metabolites, h\droquinone, phenol, catechol. and 1,2,4-henzenetriol Incubations were performed for 30 min as described in "Malcriis and Methods." Results are expressed as mean SD of 3 experiments. subPshtreanteoNlicone D8NOAH0d.G08u0a/Mg HQ (10 MM) Phenol (100 MM) CAT (100 UM) 1,2,4-BenzenetrioI (10 MM)pmol 0.025 0.1 60 0.030" 0.270 0.032" 0,100*0012*0.3100.018" ' P < 0.05; significantly different from controls (none). ' P > 0.05; not significantly different from controls (none). 1024 OXIDATIVE DNA DAMAGE BY BENZENE AND ITS METABOLITES 0.12-1 0.10- 0) 0.08- en 3 O0a.06- I O oo 0.04- O 0.02- oE. 0.00 Time (h) Fig. 1. Time course of benzene-induced oxidative DNA damage in vivo in thbone marrow of B6C3F| mice. Benzene was administered in com oil at 880 mg/kg. Control mice were given corn oil alone, a, P > 0.05, not significantly different from corresponding corn oil controls; b. P < 0.001. significantly different from 1-h corn oil controls; c, P < 0.05. significantly different from 3-h corn oil controls. (29). This suggests that formation of this hydroxylated base may contribute to the mutagenic and carcinogenic properties of radiation and chemicals that generate active oxygen. Peroxidative metabolism or autoxidation of the phenolic metabolites (PH and HQ) of benzene results in active oxygen generation (6, 8, 17). Here, we report that various phenolic metabolites of benzene increase the steady state level of SOHdGua in the DNA of human leukemia HL60 cells which contains high levels of MPO (23). Further, we have demonstrated that benzene itself and various combinations of its phenolic metabolites produce increases in the steady-state level of SOHdGua in the bone marrow of B6C3F, mice in vivo. These results indicate that activation of the phenolic metabolites of benzene (presumably mediated by MPO) produces active oxygen which is capable of causing oxidative DNA damage. We therefore suggest that oxidative damage to DNA may play a role in benzene-induced genotoxicity, myelotoxicity, and leukemia. Other investigators have previously proposed that oxygen radicals play a important role in benzene toxicity. For example, Anwar et al. (30) showed that DMSO, a hydroxyl radical scavenger, inhibits ben zene-induced genotoxicity in mice. However, it should be noted that DMSO interacts with hepatic cytochrome P-450 enzymes including 0.3 n O> ra 3 Oo I O oo ~5 E o. 1 00 200 400 800 Dose of Benzene (mg/Kg) Fig. 2. Dose response for benzene-induced oxidative DNA damage in the bone marrow of B6C3F, mice. a. P < 0.05; b, P < O.Ol; c. P < 0.001; d, P < 0.001; e, P < 0.001; significantly different from com oil controls. PHENOL D HYDROOUINONE CATECHOL E PHENOL HYDROQUINONE D PHENOL . CATECHOL ID HYDHOOUINONE . CATECHOL PBS Control Treatment Fig. 3. Induction of oxidative DNA damage by the benzene metabolites, phenol, hydroquinone. and catechol in vivo in the bone marrow of B6C3F, mice. Metabolites were administered in PBS at 75 mg/kg. Controls received PBS alone, a. P > 0.05, not significantly different from PBS controls; b. P < 0.01, significantly different from phenol or hydroquinone; c, P < 0.01, significantly different from phenol or catechol; d, P < 0.01, significantly different from hydroquinone or catechol. 0.14 mg/kg) + CAT (75 mg/kg) was less effective, producing only a slight but significant (P < 0.01) increase in the SOHdGua level. No signif icant effect on SOHdGua levels was observed in control mice treated with PBS alone (Fig. 3). 1,2,4-Benzenetriol, a minor but highly reactive phenolic metabolite of benzene, also elicited a significant, 2.5-fold increase (P < 0.001) in SOHdGua levels in vivo in mouse bone marrow when administered at 25 mg/kg (Fig. 4). Thus, BT is the only phenolic benzene metabolite which induced oxidative DNA damage in the mouse bone marrow when administered alone. Treatment with BT at 50 and 75 mg/kg resulted in smaller increases in SOHdGua. This effect is similar to that observed with benzene, where higher doses of benzene resulted in only small increases of SOHdGua in DNA (Fig. 2). 0.12- O) 75 0.10- 3 Oo X 0.08O oo O 0.06- oE. 0.04 2 5 5 O 75 DISCUSSION 1,2,4-benzenetriol (mg/Kg) SOHdGua is the most abundant product of oxidative damage to DNA by active oxygen and induces G-T and A-C base substitutions Fig. 4. Induction of oxidative DNA damage in vivo in bone marrow of B6C3F| mice by l,2.4-benzenetriol, a minor metabolite of benzene, a, P < 0.001, significantly different from PBS controls. 1025 OXIDATIVE DNA DAMAGE BY BENZENE AND ITS METABOLITES benzene and phenol hydroxylase and cytochrome P450 3a (2E1) (31). Altered metabolism rather than radical scavenging may therefore ex plain the inhibitory effect of DMSO on the genotoxic effects of benzene. In addition, recent studies by Khan et al. (32) and Laskin et al. (33) claimed to have shown the production of active oxygen in vivo following benzene administration to rats. However, the detection of active oxygen in these studies was performed following in vitro stim ulation with NADPH/iron (32) and tetradecanoylphorbol acetate (33) (a nonphysiological stimulant) of bone marrow microsomes and mac rophages, respectively, isolated from animals exposed to benzene or its phenolic metabolites. Therefore, the data presented in this paper represent the first direct demonstration of active oxygen formation in bone marrow following benzene administration. Our studies show that the administration of benzene causes a sig nificant increase in SOHdGua levels in the mouse bone marrow after only l h of administration. Interestingly, none of the primary phenolic metabolites of benzene, i.e.. PH, HQ, and CAT, were able to induce any change in SOHdGua levels in the bone marrow when adminis tered alone. However, the administration of BT, a minor phenolic metabolite of benzene, induced a significant increase in SOHdGua formation in the bone marrow. Interestingly, recent studies by Rao et al. (34), have shown that the repeated administration of BT alone to rats for 6 weeks results in myelotoxic effects. BT may, therefore, play some role in benzene toxicity. There is growing evidence that multiple metabolites play a role in benzene toxicity. For example, Eastmond et al. (35) showed that a combination of PH + HQ, each at 75 mg/kg, could reproduce the myelotoxic effects observed following benzene exposure in B6C3F, mice. Barale et al. (16) have recently shown that the combination of PH and HQ is also highly genotoxic to the mouse bone marrow. Results presented here show that the administration of various com binations of primary phenolic metabolites (at 75 mg/kg each) signif icantly increases the SOHdGua level in the mouse bone marrow. PH + HQ was the most effective of the combinations tested, but PH + CAT and CAT + HQ also significantly increased SOHdGua levels. These results add further weight to the hypothesis that benzene tox icity is caused by multiple metabolites (15, 16, 35). Moreover, since SOHdGua has been shown to be mutagenic (20, 29) we propose that the genotoxic, myelotoxic, and leukemogenic effects of benzene may be caused at least in part by active oxygen-induced cell damage. ACKNOWLEDGMENTS We are grateful to Drs. Bruce Ames and Mark Shigenaga for their advice and for allowing us to use their HPLC systems for our preliminary experiments with HL60 cells. REFERENCES 1. Aksoy. M Hemalotoxicity and carcinogenicity of benzene. Environ. Health Perspect.. 82: 193-197. 1989. 2. Snyder, R., and Chatterjee. S. P. Benzene metabolism. In: E. Arinic, J. B. Schenkman. and E. Hodgson (eds.). Proceedings of a NATO Advanced Study Institute on "Mo lecular Aspects of Monooxygenases and Bioactivation of Toxic Compounds," pp. 375-386. New York: Plenum Publishing Corp., 1991. 3. Kalf, G. F. Recent advances in the metabolism and toxicity of benzene. CRC Crit. Rev. Toxicol.. IS: 141-159. 1987. 4. Rickert, D. E., Baker. T. S., Bus. J. S.. Barrow. C. S., and Irons. R. D. Benzene disposition in the rat after exposure by inhalation. Toxicol. Appi. Pharmacol.. 49: 417-423, 1979. 5. Gad-El-Karim, M. M., Ramanujam. V. M., and Legator, M. S. /ranj.rrans-Muconic acid, an open-chain urinary metabolite of benzene in mice. Quantitation by high pressure liquid chromatography. Xenobiotica. 15: 211-220. 1985. 6. Subrahmanyam. V. V., Ross, D., Eastmond. D. A., and Smith. M. T. Potential role of free radicals in benzene-induced myeloloxicity and leukemia. Free Radical Biol. Med., //.-495-5I5. 1991. 7. Eastmond. D. A.. Smith. M. T., Ruzo. L. O.. and Ross, D. Metabolic activation of phenol by human myeloperoxidase and horseradish peroxidase. Mol. Pharmacol., 30: 674-679, 1986. 8. Sadler. A., Subrahmanyam, V. V.. and Ross. D. Oxidation of catechol by horseradish peroxidase and human leukocyte peroxidase: Reactions of o-benzoquinone and obenzosemiquinone. Toxicol. Appi. Pharmacol.. 98: 62-71, 1988. 9. Bhat, R. V.. Subrahmanyam, V. V.. Sadler. A., and Ross. D. Bioactivation of catechol in rat and human bone marrow cells. Toxicol. Appi. Pharmacol.. 94: 297-304. 1988. 10. Subrahmanyam, V. V, Kolachana. P.. and Smith. M. T. Metabolism of hydroquinone by human myeloperoxidase: mechanisms of stimulation by other phenolic com pounds. Arch. Biochem. Biophys., 156: 759-763, 1991. 11. Dean. B. J. Recent findings on the genetic toxicology of benzene, toluene, xylenes and phenols. Mutt.Res., 154: 153-181, 1985. 12. Yardley-Jones, A.. Anderson, D., Lovell, D. P., and Jenkinson, P. C. Analysis of chromosomal aberrations in workers exposed to low level benzene. Br. J. Indust. Med., 47:48-51, 1990. 13. Tice, R., Costa. D.. and Drew, R. Cytogenetic effects of inhaled benzene on murine bone marrow: induction of sister chromatid exchanges, chromosomal aberrations and cellular proliferation inhibition in DBA/2 mice. Proc. Nati. Acad. Sci. USA, 77: 2148-2155. 1980. 14. Yager, J. W., Eastmond. D. A.. Robertson, M. L., Paradisin. W. M.. and Smith, M. T. Characterization of micronuclei induced in human lymphocytes by benzene metab olites. Cancer Res., 50: 393-399, 1990. 15. Robertson, M. L.. Eastmond. D. A., and Smith, M. T. Two benzene metabolites, catechol and hydroquinone. produce a synergistic induction of micronuclei and loxicity in cultured human lymphocytes. Mutt.Res.. 249: 201-209. 1991. 16. Barale, R.. Marrazzini, A.. Beiti, C.. Vangelisti, V.. Loprieno. N.. and Barrai. I. Genotoxicity of two metabolites of benzene: phenol and hydroquinone show strong synergistic effects in vivo. Mutt.Res., 244: 15-20, 1990. 17. Subrahmanyam. V. V., and O'Brien. P. J. Peroxidase-catalyzed oxygen activation by arylamine carcinogens and phenol. Chem.-Biol. Interact., 56: 185-199. 1985. 18. Kasai, H.. and Nishimura, S. Hydroxylation of deoxyguanosine at the C-8 position by polyphenols and aminophenols in the presence of hydrogen peroxide and ferric iron. Jpn. J. Cancer Res., 75: 565-566, 1984. 19. Floyd, R. A.. Walson. J. J., Wong, P. K., Altmiler. D. H., and Rickard. R. C. Hydroxyl free radical adduci of deoxyguanosine: sensitive deteclion and mechanisms of formalion. Free Radical Res. Commun., /: 163-172, 1986. 20. Floyd. R. A. The role of 8-hydroxydeoxyguanosine in carcinogenesis. Carcinogenesis (Lond.), //: 1447-1450, 1990. 21. Kasai. H., Nishimura, S.. Kurokawa, Y, and Hayashi, Y. Oral adminislralion of Ihe renal carcinogen, polassium bramale, specifically produces 8-hydroxydeoxygua nosine in ral large! organ DNA. Carcinogenesis (Lond.). 8: 1959-1961, 1987. 22. Roy, D., Floyd, R. A., and Liehr, J. G. Elevated 8-hydroxydeoxyguanosine levels in DNA of dielhylstilbeslrol-lreated Syrian hamsters: covalent DNA damage by free radicals generaled by redox cycling of dielhylslilbeslrol. Cancer Res., 51: 3882-3885, 1991. 23. Yamada, M., Mori, M., and Sugimura, T. Myeloperoxidase in cultured human pro- myelocylic leukemia cell line HL-60. Biochem. Biophys. Res. Commun., 98: 219- 226, 1981. 24. Bozeman, P. L., Learn, D. B., and Thomas, E. L. Assay of human leukocyte enzymes myeloperoxidase and eosinophil peroxidase. J. Immunol. Melhods. 726: 125-133, 1990. 25. Gupla, R. C. Nonrandom binding of the carcinogen /V-hydroxy-2-acetylaminofluorene to repelilive sequences of ral liver DNA in vivo. Proc. Nail. Acad. Sci. USA, 81: 6943-6947, 1984. 26. Park, E. M., Shigenaga, M. K., Degan, P., Korn. T. S., Kilzler. J. W.. Wehr, C. M., Kolachana. P., and Ames, B. N. Assay of excised oxidalive DNA lesions: isolation of 8-oxoguanine and its nucleoside derivatives from biological fluids with a monoclonal antibody column. Proc. Nati. Acad. Sci. USA, 89: 3375-3379, 1992. 27. Kasai, H., and Nishimura, S. Hydroxylalion of deoxyguanosine al Ihe C-8 posilion by ascorbic acid and other reducing agents. Nucleic Acids Res., 12: 2137-2145, 1984. 28. Lee, E. W.. Kocsis, J. J.. and Snyder, R. Acute effect of 50Fe incorporation inlo circulating erylhrocyles. Toxicol. Appi. Pharmacol.. 27: 431"436. 1974. 29. Cheng, K. C.. Cahill. D. S., Kasai, H.. Nishimura. S., and Loeb, L. A. 8-Hydrox- yguanine. an abundant form of oxidative DNA damage, causes G-T and A-C substilulions. J. Biol. Chem., 267: 167-172, 1992. 30. Anwar, W. A., Au, W. W., Legator, M. S., and Ramanujam, V. M. S. Effect of dimelhylsulfoxide on the genotoxicily and metabolism of benzene in vivo. Carcino genesis (Lond.). 10: 441-445, 1989. 31. Morgan, E. T., Koop. D. R., and Coon, M. J. Catalylic aclivily of cylochrome P-450 isozyme 3a isolated from liver microsomes of elhanol-treated rabbils: oxidalion of alcohols. J. Biol. Chem., 257: 13951-13957, 1982. 32. Khan, S.. Krishnamurlhy, R., and Pandya, K. P. Generalion of hydroxyl radicals during benzene loxicity. Biochem. Pharmacol., 33: 2009-2112, 1990. 33. Laskin. D. L.. MacEashem. L.. and Snyder, R. Activation of phagocytes following benzene treatment of mice. Environ. Health Perspect.. 82: 75-79. 1989. 34. Rao. G. S., Siddiqui, S. M., Pandya, K. P., and Shanker, R. Relative toxicily of metabolites of benzene in mice. Vet. Human Toxicol.. 30: 517-520, 1988. 35. Eastmond, D. A.. Smith. M. T., and Irons, R. D. An interaclion of benzene melaboliles reproduces the myelotoxicily associated with benzene exposure. Toxicol. Appi. Phar macol.. 91: 85-95, 1987. 1026