Phase 11 Metabolism of Benzene Have to Be Identified
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Phase 11 Metabolism of Benzene have to be identified. Application of single metabolites of benzene such as phenol Dieter Schrenk,1 Achim Orzechowski,1 (PH), catechol (CT), hydroquinone (HQ), and 1,2,4-trihydroxybenzene (THB) to Leslie R. Schwarz,2 Robert Snyder,3 Brian Burchell,4 rodents failed to reproduce the characteristic Magnus lngelman-Sundberg,5 and Karl Walter Bock1 toxic effects of benzene in the bone marrow (4,5). In several studies the possible syner- 1'nstitute of Toxicology, University of Tubingen, Tubingen, Germany; gistic action of certain metabolites of ben- 2GSF-lnstitute of Toxicology, Neuherberg/Munchen, Germany; zene on the bone marrow was investigated. 3Department of Pharmacology and Toxicology, EOHSI, Piscataway, It was shown that PH enhanced the conver- New Jersey; 4Department of Biochemical Medicine, University of sion of HQ into 1,4-benzoquinone cat- Dundee, Ninewells Hospital and Medical School, Dundee, United alyzed in vitro by myeloperoxidase (6,7), an Kingdom; 5Department of Physiological Chemistry, Karolinska Institute, enzyme present in abundance in the bone marrow (8). The electrophilic 1,4-benzo- Stockholm, Sweden quinone thus formed is able to bind to cel- The hepatic metabolism of benzene is thought to be a prerequisite for its bone marrow toxicity. lular proteins and DNA. Binding to critical However, the complete pattern of benzene metabolites formed in the liver and their role in bone proteins such as tubulin (9) or DNA poly- marrow toxicity are not fully understood. Therefore, benzene metabolism was studied in isolated merase-a (10) may play an important role rodent hepatocytes. Rat hepatocytes released benzene-1,2-dihydrodiol, hydroquinone (HQ), in benzene toxicity. catechol (CT), phenol (PH), trans-trans-muconic acid, and a number of phase 11 metabolites such In vivo experiments by Eastmond et al. as PH sulfate and PH glucuronide. Pretreatment of animals with 3-methylcholanthrene (3-MC) (11) demonstrated that combined but not markedly increased PH glucuronide formation while PH sulfate formation was decreased. Likewise, single treatment of rats with HQ and PH V79 cells transfected with the 3-MC-inducible rat UGT1.6 cDNA showed a considerable rate of led to a decrease in bone marrow cellularity, PH and HQ glucuronidation. In addition to inducing glucuronidation of phenols, 3-MC treatment while CT was ineffective when combined (reported to protect rats from the myelotoxicity of benzene) resulted in a decrease of hepatic with either PH or HQ. In a report by Guy CYP2E1. In contrast, pretreatment of rats with the CYP2E1-inducer isopropanol strongly enhanced et al. (12) the combination of muconal- benzene metabolism and the formation of phenolic metabolites. Mouse hepatocytes formed much dehyde and HQ was reported to inhibit higher amounts of HQ than rat hepatocytes and considerable amounts of 1,2,4-trihydroxybenzene erythroid iron utilization in mice in a syn- (THB) sulfate and HQ sulfate. In conclusion, the protective effect of 3-MC in rats is probably due ergistic manner. These studies demonstrate to a shift from the labile PH sulfate to the more stable PH glucuronide, and to a decrease in the central role of phenolic metabolites in hepatic CYP2E1. The higher susceptibility of mice toward benzene may be related to the high the myelotoxicity of benzene. rate of formation of the myelotoxic metabolite HQ and the semistable phase 11 metabolites HQ In this report we show that pretreat- sulfate and THB sulfate. Environ Health Perspect 104(Suppl 6):1 183-1188 (1996) ment of rats with 3-methylcholanthrene Key words: benzene metabolism, cytochrome P4502E1, drug-metabolizing enzymes, (3-MC) leads to a shift from sulfation to glucuronidation, hepatocytes, hydroquinone formation, inducers of drug metabolism, sulfate glucuronidation of phenol, the major conjugates, UDP-glucuronosyltransferase phase I metabolite of benzene. Induction of a phenol glucuronosyltransferase (UGT 1.6) may thus protect the organism by forming Introduction stable glucuronides instead ofsemistable sul- It is widely accepted that the bone marrow releasing myelotoxic or pro-myelotoxic fates. Since inhalation experiments had toxicity and carcinogenicity of benzene benzene metabolites into the systemic circu- revealed significantly higher levels of vari- results from the action of reactive metabo- lation (1,2). Principal pathways of hepatic ous benzene metabolites such as HQ, HQ lites that damage essential cellular macro- benzene metabolism include the formation glucuronide, and trans-trans-muconic acid molecules in the target organ, thus leading of phenolic metabolites and their conju- in mice compared to rats (13), we investi- to decreased proliferation, cell death, and gates, and of the ring-opened trans-trans- gated the pattern of benzene metabolites genotoxicity. A number of findings indicate muconaldehyde and its oxidation products released from isolated hepatocytes of NMRI that the metabolism of benzene in the liver (3). However, the metabolites ultimately mice to elucidate whether differences in plays an important role in this scenario by responsible for bone marrow toxicity still benzene metabolism might contribute to the observed differences in metabolite levels and myelotoxicity (14). Major differences This paper was presented at Benzene '95: An International Conference on the Toxicity, Carcinogenesis, and were found in the formation of sulfate con- Epidemiology of Benzene held 17-20 June 1995 in Piscataway, New Jersey. Manuscript received 16 January jugates of phenolic benzene metabolites 1996; manuscript accepted 14 June 1996. between mouse and rat hepatocytes. The authors thank F. Oesch (Institute of Toxicology, University of Mainz) for a sample of benzene-1,2-dihy- drodiol, H. Bartholoma and R. Muller (Institute of Organic Chemistry, University of TObingen) for FAB mass spectrometric analysis of metabolites, and H. Maser and S. Vetter for expert technical assistance. Materials and Methods Address correspondence to Dr. D. Schrenk, Institute of Toxicology, University of TObingen, Wilhelmstrasse 56, D-72074 TObingen, Germany. Telephone: 49 7071 297 4945. Fax: 49 7071 292 273. E-mail: Chemicals [email protected] Abbreviations used: CT, catechol; CYP, cytochrome P450; HQ, hydroquinone; 3-MC, 3-methylcholanthrene; 14C-Benzene was obtained from Amersham PB, phenobarbital; PH, phenol; THB, 1,2,4-trihydroxybenzene; UGT, UDP-glucuronosyltransferase. (Braunschweig, Germany) at a radiochemical Environmental Health Perspectives - Vol 104, Supplement 6 * December 1996 1 183 SCHRENK ETAL. and chemical purity of > 99.5%. It was exclusion of 0.4% Trypan Blue, was a Finnigan 4021 mass spectrometer diluted with unlabeled HPLC-grade greater than 85%. (Finnigan, San Jose, CA) using electron benzene (Baker, Gross-Gerau, Germany) 14C-Benzene (0.5 mM; final specific impact (EI) mass spectrometry, after deriva- to a specific activity of 37 MBq/mmol. activity 37 MBq/mmol) was incubated tization with N-methyl-N-(trimethyl- Collagenase type IV was from Sigma with 20 x 106 freshly isolated hepatocytes silyl)trifluoroacetamide (MSTFA). (Taufkirchen, Germany) or from Boehringer for 1 hr in 20 ml Krebs-Ringer buffer (Mannheim, Germany). HQ sulfate was (NaCl 7.0 g/liter, KCI 0.36 g/liter, Immnunoblotting ofCYP2E1 obtained from TCI Chemicals (Tokyo, MgSO4x7 H20 0.295 g/liter, KH2PO4 Microsomes were prepared from isolated Japan). Benzene-1,2-dihydrodiol was a 0.163 g/liter, NaHCO3 2.016 g/liter) hepatocytes, and microsomal proteins were generous gift of Prof. F. Oesch (Institute supplemented with 5 mM HEPES, 10 separated by SDS-PAGE, transferred to of Toxicology, University of Mainz, mM D-glucose, 5 mM Na2SO4, and 20 g Immobilon P sheets (Millipore, Dreieich, Mainz, Germany). bovine serum albumin per liter as previ- Germany), and incubated with rabbit ously described (15). Incubations were anti-CYP2E1 IgG as described by Schrenk Animal performed in airtight Erlenmeyer flasks and Bock (15). Immunoreactive bands Male Wistar rats weighing 220 to 240 g, (250 ml) which were gently shaken. were visualized by incubation with peroxi- and male NMRI mice weighing 20 to 24 g, dase-conjugated swine anti-rabbit IgG were obtained from Savo (Kisslegg, Separation and Quantification and subsequent reaction with 4-chloro-1- Germany) and the breeding station of the of Metabolites naphthol/H202- GSF (Neuherberg, Germany), respectively. After addition of 10 mM ascorbic acid to The animals were kept under standard avoid oxidation of metabolites, the incuba- Glucuronidation Experiments conditions, and had free access to labora- tions were stopped by adding 2 vol of ice- Glucuronidation of phenols was studied in tory chow (Altromin, Lage, Germany) and cold methanol, flushed with nitrogen, and rat liver microsomes or in V79 Chinese drinking water. Isopropanol (2.5 ml/kg) kept on ice for 30 min. Then, precipitated hamster lung fibroblasts. Liver microsomes was applied by gavage as dilution in saline protein was removed by centrifugation. were prepared as described by Bock and 24 hr before sacrifice. 3-MC (40 mg/kg, ip, Supernatants were evaporated to dryness, White (16) and were incubated with 200 dissolved in corn oil) was adminstered redissolved in 1 ml methanol, and aliquots pM HQ under the same conditions as 3 days before sacrifice, and phenobarbital were analyzed on a high performance liq- described for PH (15). After addition of was given by a single ip injection of 100 uid chromatography (HPLC) system as 10 mM ascorbic acid to avoid oxidation, mg/kg