Detection of Nitrated Benzene Metabolites in Bone Marrow of B6C3F1

Benzene, a constituent of cigarette smoke, is a human leukemogen and induces bone marrow toxicity. The mechanism of benzene-induced toxicity is not ...
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Chem. Res. Toxicol. 2004, 17, 370-377

Detection of Nitrated Benzene Metabolites in Bone Marrow of B6C3F1 Mice Treated with Benzene K.-M. Chen, K. El-Bayoumy, J. Cunningham, C. Aliaga, H. Li, and A. A. Melikian* Institute for Cancer Prevention, American Health Foundation Cancer Center, 1 Dana Road, Valhalla, New York, 10595 Received August 25, 2003

Benzene, a constituent of cigarette smoke, is a human leukemogen and induces bone marrow toxicity. The mechanism of benzene-induced toxicity is not well-established. We hypothesized that relatively high levels of nitric oxide formed in bone marrow can react with oxygen and/or superoxide anion that is generated during redox cycling of ring-hydroxylated benzene metabolites to yield peroxynitrite as well as other NO-derived intermediates. Peroxynitrite can either directly damage cellular macromolecules or form nitrated toxic metabolites. Toward this end, we investigated whether nitro derivatives of benzene are formed in bone marrow of mice treated with benzene. First, we have characterized products formed during activation of benzene in Fenton’s system in the absence or presence of NO-releasing compound in vitro by GC/MS. The result of above experiment prompted us to determine whether similar products can be formed in vivo. Groups of B6C3F1 male mice, eight weeks of age, were given a single intraperitoneal dose of [14C]benzene (400 mg/kg body wt, 9.7 mCi/mmol) or an equal dose of unlabeled benzene in corn oil, and the mice were killed 0.5 or 1 h posttreatment. The control group received only vehicle injections. Organic solvent extractable metabolites from bone marrow, liver, lungs, and blood of mice treated with [14C]benzene were identified by comparison of their respective retention times under two different HPLC conditions with authentic standard samples. These metabolites were further characterized by comparison of their GC/MS properties to those of reference standards. Nitro metabolites, namely, nitrobenzene, nitrobiphenyl, and nitrophenol isomers, were detected in the bone marrow of the mice 1 h after benzene treatment. Formation of nitro derivatives in other tissues was either not observed or was significantly less than that formed in bone marrow. This study clearly demonstrates that nitric oxide is a contributor to benzene metabolism and can form nitrated derivatives that may, in part, account for bone marrow toxicity.

Introduction Benzene is a major environmental and occupational pollutant. A major source of benzene exposure for the general population is the inhalation of mainstream and sidestream cigarette smoke, gasoline fumes, automotive engine emissions, and various consumer products (1, 2). Benzene is a multisite carcinogen in rodents (3, 4), and in humans, chronic exposure is associated with immunosuppression and hematological disorders, including pancytopenia (decreased number of all blood cell lineages), aplastic anemia, myelodysplasia, and acute myeloid leukemia (5-7). Despite extensive research, the mechanism of benzene-induced bone marrow toxicity and leukemia is still not fully elucidated (8). It is recognized that benzene requires hepatic metabolism to produce reactive metabolites that mediate benzene-induced toxicity (5, 9, 10). The major metabolism of benzene in the liver is catalyzed by cytochrome P450 2E1 (CYP2E1) isoenzyme (5, 9-13); benzene can also be activated nonenzymatically by interaction with a hydroxyl radical to yield a hydroxycyclohexadienyl intermediate (14, 15). Gorsky and Coon have shown that the latter pathway is important at low concentrations of benzene (16). Both * To whom correspondence should be addressed. Tel: 914-789-7117. Fax: 914-592-6317. E-mail: [email protected].

pathways eventually yield a series of ring-hydroxylated metabolites, including PH,1 hydroquinone (HQ), benzoquinone (BQ), catechol (CAT), BT (benzene triol), and ring-opened products, such as trans,trans-muconaldehyde that is oxidized to t,t-MA, as well as dimeric products, such as biphenyl (Figure 1) (5, 17-23). Benzene and its hepatic metabolites can be transported via the bloodstream to the bone marrow and to other organs, where further activation can generate hematotoxic and genotoxic metabolites (24-26). It has also been postulated that ROS are formed during benzene metabolism (27-30). Benzene and its metabolites enhance the expression of inducible nitric oxide synthase (iNOS) gene, which can lead to the formation of excessive •NO (31, 32). Administration of the inflammatory agent lipopolysaccharide increases the production of •NO in bone marrow of mice and enhances benzene-induced genotoxicity (31-34). To test the hypothesis that •NO directly or indirectly via formation of peroxynitrite or other NO-derived in1 Abbreviations: BT, 1,2,4-trihydroxybenzene; BQ, 1,4-benzoquinone; BQ-epoxide, 2,3-epoxy-1,4-benzoquinone; CAT, 1,2-dihydroxybenzene; DEA-NO, sodium 1-(N,N-diethylamino)diazen-1-ium-1,2-diolate; HQ, 1,4-dihydroxybenzene; t,t-MA, trans,trans-muconic acid; •NO, nitric oxide; PH, phenol; ROS, reactive oxygen species; SIM, selected ion monitoring.

10.1021/tx030039s CCC: $27.50 © 2004 American Chemical Society Published on Web 01/30/2004

Nitro Derivatives as Benzene Metabolites in Mice

Figure 1. Metabolic activation pathways of benzene.

Figure 2. Hypothetical pathways for the formation of nitro derivatives of benzene.

termediates (Figure 2) contributes to benzene metabolism, we have investigated whether nitro-containing benzene metabolites are formed during metabolic activation of benzene in the bone marrow of mice. First, we have characterized the products formed in vitro during oxidation of benzene in Fenton’s system in the absence or presence of •NO by GC/MS. Then, we examined whether nitro products similar to those formed in vitro are also produced in bone marrow cells of mice treated either with [14C]-labeled or unlabeled benzene. We employed both radioactive tracer analysis and GC/MS for characterization of in vivo metabolites.

Experimental Procedures Caution: Benzene and its metabolites should be regarded as potential carcinogens and should be handled with extreme caution using the guidelines for handling carcinogenic materials developed by the National Cancer Institute. Animals. Male B6C3F1 mice were obtained from Charles River Laboratories (Wilmington, MA) at 7 weeks of age. They were then maintained, four to a cage, in a pathogen-free area of the Research Animal Facility for the next week while their health titers were confirmed; 1 week later, they were transferred to the bioassay laboratory. All laboratories in the Research Animal Facility are maintained at 55 ( 15% relative humidity and 22 ( 2 °C with a 12 h light/dark cycle. The mice received NIH-07 (Harlon Teklad 7022C) diet and water ad libitum. The bioassay was carried out under established federal regulation for the care and use of laboratory animals and was approved by our Institutional Animal Care and Use Committee. Chemicals. Freshly double distilled [14C]benzene (52 mCi/ mmol) that HPLC analysis indicated was free of biphenyl was obtained from ChemSyn Laboratories (Lenexa, KS). PH, HQ, BQ, 2,5-dihydroxybenzoquinone, BT, CAT, t,t-MA, biphenyl, 2and 3-hydroxybiphenyl, p-terphenyl, 2,2′- and 4,4′-biphenols, nitrobenzene, 2-, 3-, and 4-nitrophenol, 2-, 3-, and 4-nitrobiphen-

Chem. Res. Toxicol., Vol. 17, No. 3, 2004 371 yl, sodium periodate, and cis-3,5-cyclohexadien-1,2-diol were obtained from Aldrich Chemical Co. (Milwaukee, WI). Thiobarbituric acid (TBA) was purchased from Sigma Chemical Co. (St. Louis, MO). DEA-NO was bought from the Cayman Chemical Co. (Ann Arbor, MI), and unlabeled benzene (>99% pure) was from J. T. Baker (Phillipsburg, NJ). Synthesis of Muconaldehydes. trans,trans- and cis,cisMuconaldehydes were synthesized by a method described previously (35) and further characterized by UV and MS. The UV spectrum of muconaldehyde in MeOH had a λmax at 269 nm. Both cis,cis- and trans,trans-muconaldehydes were analyzed by HPLC using two serially connected 150 mm × 3.9 mm, 10 µm Bondaclone columns (Phenomenex, Torrance, CA). The columns were eluted with a linear gradient from 100% H2O to 25:75 MeOH:H2O for 35 min at a flow rate of 1 mL/min to elute cis,cisand trans,trans-muconaldehydes at 22.6 and 24.2 min, respectively. These analytes were collected from HPLC and further analyzed by GC/MS (Table 1). A characteristic feature of the MS is the molecular ion at m/z 110 and a major fragment at m/z 81 (M - CHO). Synthesis of BQ-Epoxide. BQ-Epoxide was synthesized by a method previously described (26). A mixture of BQ (2 mM) and H2O2 (88 mM) in 100 mM phosphate buffer (pH 7.6) was incubated at 37 °C for 20 min. An aliquot of the mixture was analyzed by HPLC on a 250 mm × 4.6 mm, 5 µm ODS column (Beckman, Fullerton, CA). The column was eluted with 100% H2O for 30 min, followed by a gradient from 100% H2O to 100% MeOH over 5 min at a flow rate of 0.6 mL/min for 20 min then maintained at 1.5 mL/min; BQ eluted at 34.1 min and HQ eluted at 19.3 while a new product eluted at 21.3 min. The UV spectrum of the product eluting at 21.3 min corresponded to that of BQ-epoxide, λmax ) 212 nm, and its EI-MS showed a molecular ion at m/z 124 (relative intensity 100) and a main fragment at m/z 68 [58%, (M - 2CO)]. Derivatization of Muconaldehyde with TBA. About 10 µM muconaldehyde solutions in H2O (0.9 mL) or samples collected from the HPLC were added to 0.1 mL of 0.67% TBA in 10% trichloroacetic acid. The flask containing this mixture was immersed in boiling water for 30 min. An aliquot of the mixture was used to take the UV/vis spectrum, which was identical to that reported in the literature, showing a λmax at 490 nm (36). Another aliquot of the muconaldehyde-TBA mixture was analyzed by reverse phase HPLC with monitoring at 490 nm. Nonenzymatic Oxidation of Benzene in the Absence or Presence of •NO-Releasing Compound in Vitro. A mixture of Fe2+ (FeSO4‚7H2O, 1 mM), H2O2 (10 mM), and benzene (22 mM) was incubated for 15 min in the absence or presence of DEA-NO (4 mM). An aliquot was analyzed by HPLC, and another aliquot was extracted with 1 vol of CH2Cl2 and analyzed by GC/MS. Control incubations contained all chemicals except benzene. Metabolism of [14C6]Benzene in Bone Marrow and Other Tissues of Mice. At the age of 8 weeks, the mice were set up as four groups of six each. In two of the groups, mice were injected i.p. with a single dose of 400 mg/kg body weight of [14C6]-labeled freshly distilled benzene (9.7 mCi/mmol) in 0.2 mL of corn oil. One-half hour and 1 h postadministration, blood samples were collected by cardiac puncture and mice were killed by CO2 asphyxiation. The mice in a third group received the same dose of unlabeled benzene in corn oil and were killed 1 h posttreatment. The control mice received only corn oil. Livers, lungs, and femurs were removed and kept at -80 °C. Isolation of Organic Soluble [14C6]-Labeled or Unlabeled Benzene Metabolites from Various Tissues. Tissues were thawed, cut into small pieces, and homogenized; similarly, bone marrow was flushed out with 1-2 mL of distilled water and was then homogenized. A mixture of internal standard samples consisting of unlabeled benzene metabolites (PH, HQ, CAT, BT, BQ, t,t-MA, S-phenylmercapturic acid, biphenyl, nitrobenzene, 2-, 3-, and 4-nitrophenols, and 3-nitrobiphenyl, terphenyl) with known concentration was added to each tissue homogenate. Each homogenate was extracted with 4 × 2 vol of

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Table 1. GC-EI-MS Spectral Data of Products Formed from Activation of Benzene in Fenton’s System in the Presence of •NO-Releasing Compound in Vitroa peak no. (Figure 3B)

GC retention time (min)

compound

MS spectra m/z (relative intensity)

1 2 3

6.09 6.91 7.47 7.67 7.85 8.05 8.1 8.55 9.01 9.19 9.58 10.1 4 10.45 11.03 11.32 11.4 12.48 12.59 12.50 13.20 13.20

BQ PH BQ-epoxide 2-hydroxybenzo-quinone cis,cis-muconaldehyde trans,trans-muconaldehyde nitrobenzene 2-nitrophenol CAT 2-aminophenol HQ 4-nitrosophenol biphenyl 3-nitrophenol 4-nitrophenol 2-hydroxybiphenyl 2-nitrobiphenyl S-phenylmercapturic acid 4-hydroxybiphenyl 3-nitrobiphenyl 4-nitrobiphenyl

108 (100), 95 (5), 82 (39), 54 (75) 94 (100), 66 (39), 55 (4) 124 (100), 123 (17), 95 (17), 69 (43), 68 (58), 54 (47) 124 (5), 94 (23), 92 (23), 66 (100), 55 (34) 110 (47), 94 (2), 81 (100), 53 (77) 110 (100), 94 (2), 81 (98), 53 (87) 123 (65), 107 (3), 93 (13), 77 (100), 65 (12), 51 (27) 139 (100), 122 (6), 109 (20), 93 (8), 81 (23), 65 (30) 110 (100), 92 (10), 81 (15), 64 (29) 109 (100), 80 (40), 53 (13), 52 (10), 51 (5) 110 (100), 81 (24), 53 (15) 123 (100), 95 (15), 80 (19), 65 (36), 52 (29) 154 (100), 153 (42), 152 (28), 151 (8), 76 (10) 139 (100), 123 (6), 109 (6), 93 (59), 81 (20), 65 (69) 139 (100), 109 (15), 93 (50), 81 (20), 65 (88) 170 (100), 169 (81), 141 (51), 139 (100), 115 (51) 199 (31), 152 (100), 151 (50) 242 (100), 227 (100) 170 (100), 141 (18), 115 (13) 199 (90), 152 (100), 153 (85) 199 (100), 153 (50), 152 (100), 151 (50)

4 5 6 7 8 9 10 11 12 13 14 15 15 a

From Figure 3B.

ethyl acetate, and one aliquot of the extract was set aside for measurement of total radioactivity; then, the solvent was removed under a stream of N2. The residue was dissolved in 0.2 mL of methanol or methylene chloride and analyzed by HPLC or by GC/MS, respectively. Weights for bone marrow samples were estimated by multiplying dry weight content of the aqueous layer by 4.5, a value derived from previously published studies measuring bone marrow weight (37, 38). HPLC Analysis of [14C6]Benzene Metabolites. The organic solvent extractable metabolites from various tissues were analyzed by two HPLC elution systems as described below, using a 250 mm × 4.6 mm, 5 µm Luna C18 column (Phenomenex); 0.5 mL fractions were collected, and the radioactivity of each fraction was measured. System 1: Isocratic elution with 100% H2O for 10 min was followed by a linear gradient from 100% H2O to 100% MeOH over 60 min at a flow rate of 1 mL/ min. System 2: This HPLC elution program was identical to that in system 1, except that instead of H2O, a 10 mM phosphate buffer (pH 3.5) was used. GC/MS Analysis of Benzene Metabolites. A HewlettPackard (HP) 5973 Mass Spectrometer coupled to HP 6890 GC (Wilmington, DE) was used to analyze organic soluble benzene metabolites. An EC-5 (30 m × 0.25 mm i.d. × 0.25 µm film thickness) capillary GC column (Altech Associates, Deerfield, IL) was interfaced directly with the MS source. The GC oven temperature was held at 50 °C for 4 min, followed by programming to 310 °C at 20 °C/min. The temperature was then kept at 310 °C for 2 min. MS conditions were as follows: ion source, 230 °C; emission current, 34.6 µA; and electron energy, 70 eV.

Results Characterization of unknown benzene metabolites in bone marrow samples of mice that contain only a very small number of cells will be difficult, even by GC/MS. Therefore, we first conducted model studies to characterize products formed during nonenzymatic activation of benzene in the absence or presence of •NO in vitro. The outcome of these model studies prompted us to determine whether similar products can be formed in in vivo using GC-SIM-MS. Characterization of Products Formed in Nonenzymatic Oxidation of Benzene (Fenton’s System) in the Absence or Presence of NO-Releasing Compound in Vitro. Figure 3A,B shows the GC profile of

organic solvent extractable products formed in vitro by activation of benzene in Fenton’s reagent (Fe2+, H2O2) in the absence and presence of the NO-releasing compound, DEA-NO, respectively. Each GC peak was initially characterized by an electron impact mass spectrum (EI-MS) and use of the NIST 98/mass spectral library database. Structural assignment of each product was confirmed by analyzing authentic reference samples that either were synthesized in our laboratory or were obtained commercially. The mass spectral data of characterized products in vitro are summarized in Table 1. Figure 3A (insert) indicates that the major products derived from benzene in the absence of •NO are PH (peak 2) and biphenyl (peak 11), and the minor ones are BQ (peak 1), BQ-epoxide (peak 3), muconaldehyde (peaks 4 and 5), CAT (peak 8), HQ (peak 9), hydroxylated biphenyl (peaks 13 and 14), and other unknown products. The formation of muconaldehyde was further confirmed by its UV spectrum and the UV/vis absorption of adducts formed between muconaldehyde and TBA (36). In the presence of NO-releasing compound, a major GC peak eluted at 5.9 min (insert of Figure 3B), and the MS spectrum of the peak showed a molecular ion at m/z 102 and a cluster of fragmentation ions at m/z 56 and 57. The GC retention time and the MS fragmentation pattern of this peak correspond to the dimethyl-N-nitrosamine that is derived from the decomposition of NO-releasing compound, DEA-NO. The products derived from benzene in the presence of NO-releasing compound are nitrobenzene (peak 6), p-nitrosophenol (peak 10), 3-nitrophenol (peak12), 3- and/or 4-nitrobiphenyl (peak 15), and all metabolites that were present in the activation of benzene in the absence of NO-releasing compound including BQ-epoxide (peak 3) and muconaldehydes (peaks 4 and 5). BQepoxide was identified based on its GC retention time that is distinct from that of 2-hydroxybenzoquinone and by its MS spectrum (Table 1). Determination of Organic Soluble [14C]Benzene Metabolites in Target and Nontarget Tissues of B6C3F1 Male Mice. Figure 4A-D shows HPLC radiochromatogram elution profiles of organic soluble metabolites of benzene in liver, bone marrow, lungs, and blood,

Nitro Derivatives as Benzene Metabolites in Mice

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unknown nonpolar metabolites (possibly polymeric products) were elevated in liver, bone marrow, and lungs 1 h after treatment by comparison to levels in tissues evaluated 0.5 h posttreatment (Table 2). One of the unknown nonpolar eluting products cochromatographed with synthetic p-terphenyl. One hour after treatment, levels of benzene metabolites were higher in bone marrow than in any other tissue (Figure 5), and levels of both nitro derivatives and polymeric products were significantly higher than after 0.5 h of exposure. Among nitro derivatives, 3- and/or 4-nitrobiphenyl were relatively higher in the bone marrow of the mice. Characterization of Benzene Metabolites in Bone Marrow of Mice by GC-SIM-MS. To ensure that [14C]benzene metabolites detected by radioactive tracer method in the bone marrow of mice (Figure 4B) are unequivocally identified, these compounds were further characterized by GC-SIM-MS. Organic solvent extracts of the bone marrow of mice treated with the same dose of benzene over the same exposure periods as the mice that received [14C]benzene were analyzed by GC-SIMMS either directly (Figure 6A-H) or after HPLC fractionation. In the latter case, fractions corresponding to characterized benzene metabolites were collected, extracted with organic solvent, and then analyzed by GC-SIM-MS. The left panels of Figure 6A-H are GC-EI-SIM-MS of molecular ions of selected analytes monitored from bone marrow extract; the other ions that also were monitored for each analyte are not shown in Figure 6 but are reported later on; the middle panels represent GC retention times of authentic reference standards; and the right panels depict the mass spectra of reference standards. The selected ions monitored for each analyte are as follows: nitrobenzene at m/z 123 (M) and 77 (M - NO2); 2-nitrophenol at m/z 139 (M) and 81 (M - CNO2); 3- and 4-nitrophenol at m/z 139 (M) and 93 (M - NO2); 2-, 3-, and 4-nitrobiphenyl at m/z 199 (M), 153 (M - NO2), and 152 (M - HNO2); biphenyl at m/z 154 (M), 153 (M - H), and 152 (M - 2H); S-PMA at m/z 242 (M) and 227 (M - CH3) (spectra not shown); PH at m/z 94; HQ, CAT at m/z 110 (M) and 81 (M - COH) (spectra not shown); BQ-epoxide at m/z 110 (M) (spectrum not shown); and cis,cis-MA and trans,trans-MA at m/z 110 (M) and 81 (M - COH). None of the nitro derivatives of benzene were detected in organic solvent extracts of bone marrow from corn oil-treated control mice, except for a trace of 2-nitrophenol. The level of 2-nitrophenol in this control sample was estimated to be 20 times below that present in the bone marrow of benzene-treated mice.

Figure 3. GC chromatograms of products formed from benzene in Fenton’s system in vitro: (A) in the absence of NO-releasing compound; (B) in the presence of NO-releasing compound and DEA-NO (inserts are complete GC chromatograms).

respectively, 1 h after [14C]benzene administration. The arrows designate where unlabeled internal standards elute. The levels of each metabolite are estimated on the basis of total radioactivity associated with each peak after subtraction of background values and recoveries estimated from unlabeled internal standards. Table 2 compares levels of several key benzene metabolites, including nitrated derivatives, in different tissues 0.5 and 1 h after [14C]benzene administration. At both time points, PH and HQ were the major metabolites in circulating blood. PH was also the most abundant metabolite in liver, lung, and bone marrow of mice 0.5 h after [14C]benzene administration. Some unknown nonpolar products were found in tissue samples collected 1 h posttreatment and especially in bone marrow (Figure 4B). Collectively, levels of

Table 2. Levels of Organic Soluble Benzene Metabolites in Target and Nontarget Tissues of B6C3F1 Micea,b total metabolites (pmol/mg tissue) bone marrowc

liver metabolites unknown early eluting polar metabolite HQ PH nitro derivatives (nitrobenzene, nitrophenols, nitrobiphenyls) biphenyl unknown nonpolar metabolites c

lung

blood

0.5 h

1h

0.5 h

1h

0.5 h

1h

0.5 h

1h

0.01 0.5 4.5 0.1

3.4 9.7 0.3

1.2 6.7 0.1

4.7 5.6 13.3 9.1

0.8 21.2 0.3

0.5 4.9 0.2

0.4 2.1

0.01 0.6 3.8 0.01

0.6 0.9

1.1 11.1

0.3 0.4

10.9 27.4

0.5 0.3

0.1 8.4

0.05 0.04

0.1 0.1

a Mice are treated with a single dose of [14C]benzene (400 mg/kg body wt) in corn oil. b Means estimated from two or more experiments. The bone marrow weights were estimated by multiplying their dry weight by 4.5 (37, 38).

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Figure 4. (A-D) HPLC elution profiles of organic phase extractable [14C]benzene metabolites in male B6C3F1 mice from (A) liver, (B) bone marrow, (C) lungs, and (D) blood.

Figure 5. Levels of estimated unconjugated PH, HQ, biphenyl, and total identified nitrated [14C]benzene metabolites in various tissues of mice 1 h after i.p. injection of [14C]benzene to B6C3F1 male mice.

Discussion On the basis of two experimental approaches (radio tracer analysis and GC/MS), the results of this study clearly demonstrate that various nitro derivatives of benzene occur in the bone marrow of mice treated with benzene. These metabolites are identified by comparison of their retention times with those of authentic reference standards in two different HPLC analyses systems and

also by comparison of GC-SIM-MS characteristics of metabolites collected from the HPLC with those of synthetic standards. The major nitro metabolites of benzene in the bone marrow of mice, estimated from HPLC radiograms, are nitrobiphenyls, followed by nitrophenols and nitrobenzene (Figure 4B). As for nitrobenzene, as well as biphenyl, the higher volatility may encumber accurate assessment of levels in mouse tissues.

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Figure 6. (A-H) Left panels are the GC-EI-SIM-MS of organic phase extractable selected benzene metabolites in bone marrow of B6C3F1 mice 1 h after benzene i.p. injections (only molecular ions are shown in the figure); the middle panels are GC retention times of reference standards; and the right panels are mass spectra of reference standards.

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The levels of total nitro derivatives of benzene in the bone marrow of the mice 1 h posttreatment were greater than at the early time point (0.5 h) and were higher than in any of the other tissues examined. This observation suggests that nitro metabolites are formed primarily in the bone marrow rather than being transported there from the liver. Nitro derivatives of benzene can be formed from direct interaction of •NO with metabolically activated benzene intermediates (e.g., cyclohexadienyl radical), as well as from indirect interaction of •NO. For example, interaction of •NO with superoxide ion that is generated by redox cycling of ring-hydroxylated benzene metabolites can form peroxynitrite, and/or reaction of • NO with oxygen may lead to the formation of other reactive nitrogen oxide intermediates (Figure 2) that can nitrate aromatic compounds and cellular macromolecules (39-46). •NO can also facilitate oxidation of HQ to BQ and BT to 2-hydrox-BQ. Indeed, we have noticed that activation of benzene in the presence of •NO in the Fenton’s system forms relatively more BQ than is generated during the activation of benzene in the absence of • NO (Figure 3A,B). Using supercoiled DNA as the target, Yoshi and Ohshima have shown that HQ in the presence of •NO induces DNA strand breaks, while in the absence of •NO no significant DNA damage is observed (47). We found that DNA scission, mediated by HQ in the presence of •NO, is comparable with DNA strand breaks induced by peroxynitrite in a dose responsive manner, whereas other benzene metabolites, such as CAT and BQ, induce DNA damage to a lesser degree than HQ or peroxynitrite (48). We have also found that exposure of mice to benzene enhances the formation of protein-bound 3-nitrotyrosine in the bone marrow when analyzed by GC/MS (49). These observations suggest that •NO may play a role in benzene-induced toxicity. Some of the benzene-derived nitro products can be toxic. For example, nitrobenzene has been shown to induce male reproductive toxicity in laboratory animals (50) and 4-nitrobiphenyl leads to the formation of 4-aminobiphenyl DNA adducts in humans (51). Our findings complement those reported previously by Laskin et al., showing that benzene enhances the activation and/or maturation of bone marrow macrophages in mice and increases the production of TNF-R, IL-1, and hydrogen peroxide (52). Laskin et al. also have demonstrated that TNF-R and IL-1 increase the level of iNOS, which, in turn, stimulates the production of •NO (53). The result of the current study is also in agreement with findings by Tuo et al. who have demonstrated the formation of p-nitrophenol following the incubation of benzene with human neutrophils pretreated with the tumor promoter phorbol-12-myristate 13-acetate (54). Characterization of biphenyl in vivo clearly demonstrates that benzene can be metabolically activated via a cyclohexadienyl radical. We have also identified biphenyl in bone marrow and other organs of rats treated with benzene by gavage or intraperitoneally (55). [14C]Benzene metabolism in the bone marrow of mice leads to the formation of some products that remain unidentified. One of the minor radioactive products cochromatographed with synthetic BQ-epoxide (Figure 4B). A similar product was formed in the activation of benzene in Fenton’s system in vitro, especially in the presence of • NO (peak 3 of Figure 3) when analyzed by HPLC or GC/MS within 24 h after formation. Brunmark and Cadenas have demonstrated that BQ-epoxide reacts with

Chen et al.

nucleophiles such as glutathione to yield glutathionyl2,3,5-trihydroxybenzene, which readily undergoes autoxidation and can participate in redox cycling that generates significant quantities of superoxide (26). It is also likely that BQ-epoxide may react and modify cellular DNA. In conclusion, we have for the first time demonstrated that in vivo benzene metabolism in the bone marrow of mice leads to the formation of nitro derivatives. Further studies are required to investigate the role of •NO and nitrated benzene metabolites in benzene-induced bone marrow toxicity.

Acknowledgment. This work was supported by National Cancer Institute PO1 Grant CA70972 and the Cancer Center Support Grant CA17613.

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