Phase 11 Metabolism of have to be identified. Application of single metabolites of benzene such as Dieter Schrenk,1 Achim Orzechowski,1 (PH), catechol (CT), (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 , 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 (dissolved in saline) followed by described by Schrenk and Bock (15). the incubations were stopped by adding application in drinking water (0.1%, w/v) Metabolites were detected using a Lambda- 2 vol of ice-cold methanol, flushed with over 3 days. Control rats received saline or Max 491 UV-detector (Millipore, Dreieich, nitrogen, and kept on ice for 30 min. Then, corn oil injections, respectively. Germany) at 280 nm and a Beckmann 171 precipitated protein was removed by cen- radioisoptope detector (Beckman Instru- trifugation. Supernatants were evaporated Hepatocyte Preparation ments, Fullerton, CA). The radiodetector to dryness, redissolved in 1 ml methanol, and Incubation was set for liquid scintillation using Ultima and aliquots were analyzed on the HPLC For hepatocyte preparation animals were Gold scintillation cocktail (Packard system used for benzene metabolites. A anesthetized with pentobarbital sodium Instruments, Meriden, CT) at a flow rate distinct peak of HQ monoglucuronide (100 mg/kg bw), and the liver was perfused of 3 ml/min. Alternatively, fractions of eluting slightly earlier than HQ was in situ via the portal vein using the sequen- 0.3 min were collected and quantified by detected by UV absorbance (280 nm) and tial ethyleneoxyethylenenitrilotetraacetic liquid scintillation counting. was quantified by peak area integration. acid (EGTA)/calcium-collagenase method. Both wild-type V79 cells and a clone Rat hepatocytes were prepared as described Identification ofMetabolites stably transfected with the rat UGT1.6 by Schrenk and Bock (15). Only cell The metabolites PH sulfate, PH glucuro- cDNA were cultured as described by Bock preparations exceeding a viability of 90% nide, S-phenylglutathione, CT, HQ, PH, et al. (17). HQ or PH was freshly dis- were used for incubations. benzene-1,2-dihydrodiol, and metabolite solved in sterile phosphate-buffered saline For preparation of mouse hepatocytes, G (presumably trans-trans-muconic acid) and was added to the culture dishes. After livers were perfused sequentially with were identified after preparative HPLC as various time points, aliquots of the super- 50 ml preperfusion buffer 1 (Ca2+-free described by Schrenk and Bock (15). Two natants were removed and HQ glucuro- modified Hank's medium containing novel benzene metabolites separated by nide or phenylglucuronide were separated 100 pM [EGTA]), with 50 ml pre-perfu- HPLC analysis of supernatants from mouse and quantified by HPLC/UV detection. sion buffer 2 (Ca2+-free modified Hank's hepatocyte incubations were isolated by Both glucuronides could be identified medium, without EGTA), and with 100 ml preparative HPLC. Fractions eluting at the by FAB-MS. of Dulbecco's minimal essential medium respective retention intervals were col- (DMEM) containing 1.8 mM CaCl2 and lected. After adjustment to pH 7.8, frac- Results 12 U collagenase/100 ml at a flow rate of tions were evaporated to dryness, kept In addition to a number of highly polar 10 ml/min. Then cells were dispersed in under nitrogen, and were analyzed by Fast metabolites designated as fraction A, seven DMEM containing 1% albumin. After atom bombardment (FAB) mass spectrom- radioactive peaks could be separated from filtration through 80-pm and subsequently etry using a Varian MAT 71 IA mass spec- supernatants of rat hepatocytes incubated through 40-pm nylon mesh filters, the cells trometer (Varian, Bremen, Germany) after with 14C-labeled benzene (Figure 1). were washed 3 times in DMEM at 50g dissolving the sample in a glycerol matrix. Further analysis of peak 3 revealed the pres- for 45 sec. Viability, as determined by Alternatively, the samples were analyzed in ence of two metabolites that were identified

1 184 Environmental Health Perspectives - Vol 104, Supplement 6 * December 1996 PHASE 11 METABOLISM OF BENZENE

by mass spectrometry (not shown) as acid, could not be identified because the number of benzene metabolites. The most phenylsulfate and S-phenylglutathione amount formed was insufficient for mass dramatic effects were obtained for HQ/HQ (15). Based on the amount of radioactivity, spectrometric identification. glucuronide (-8-fold) and for benzene-1,2- a quantitative pattern of metabolites was While total metabolism (at a substrate dihydrodiol (-5-fold). Enhanced glu- obtained (Table 1). In hepatocytes from concentration of 0.1 mM) was not affected curonidation of PH in liver microsomes untreated rats, PH sulfate, PH glucuro- by pretreatment with 3-MC or phenobar- from 3-MC-pretreated rats has been shown nide, and S-phenylglutathione represented bital (PB), 3-MC caused a significant (15). In a similar experiment, glucuronida- the major metabolites, while benzene-1,2- increase in PH glucuronide formation at tion of HQ was also found to be markedly dihydrodiol, HQ, HQ glucuronide, CT, the expense of PH sulfate and unconju- inducible by 3-MC and, to a lower extent, and unconjugated phenol (PH) were gated PH. In contrast, isopropanol pretreat- by PB (Figure 2). From Lineweaver-Burk formed in smaller amounts. Another ment resulted in a pronounced increase in diagrams an apparent Km value for the metabolite, possibly trans-trans-muconic total metabolism and in the release of a high-affinity UGT activity toward HQ of approximately 0.1 mM was calculated (not shown). Figure 3 shows the time course of 3 glucuronidation of PH and HQ in V79 cells stably expressing rat UGT 1.6 cDNA, an isozyme that is induced in rat liver by 3-MC treatment. While almost no

0.0 3 E E cL 0 5 r_ E 2

c0 I ccco cc

A 50 100 200 1 2 7 6 Hydroquinone, jM lure 2. Glucuronidation of hydroquinone in liver 0 IL micFigi,rosomes isolated from rats treated with saline or 0 5 10 15 corrn oil (o), phenobarbital (*), or 3-methylcholan- Retention time, min th.,,Ircene (v). Microsomes supplemented with UDP- glu curonic acid were incubated with various conicentrations of hydroquinone. The reaction was lin- Figure 1. HPLC radiochromatogram of 14C-benzene metabolites formed in isolated rat hepatocytes. Hepatocytes ear *over 30 min. Data represent mean ± SD from three (20 x 106/20 ml) were incubated for 1 hr with 0.5 mM 14C-benzene (specific activity, 37 MBq/mmol). Fractions were expieriments. collected at 0.3-min intervals and radioactivity was determined by liquid scintillation counting. Numbers represent polar metabolites (A), benzene-1,2-dihydrodiol (1), hydroquinone/hydroquinone glucuronide (2), phenylsulfate/S- phenylglutathione (3), catechol (4), phenylglucuronide (5), unidentified metabolite coeluting with trans-trans- 0.20 muconic acid (6), phenol (7). 0.15 Table 1. Pattern of benzene metabolites formed in isolated rat hepatocytesa: effects of inducers. nmol/hrx 106 CellSb 0.10 f Metabolite Control Phenobarbital 3-MC Isopropanol 0.05 Polar metabolitesc 0.18 ± 0.04 0.22 ± 0.05 0.30 ± 0.06 0.37 ± 0.09* E Benzen:i,2-dih drJod0 0.07 0.0 03 ...... 3 ...3.± 8* Hydroquinone/HQ glucuronide 0.20 ± 0.06 0.20 ± 0.07 0.13 ± 0.06 1.76 ± 0.31* 0.00- I IF Phenlsfate ::94±0:15 0.71 aa0.11::: :.: 1.51 0 1 2 3 Phenylglutathione 0.32 ± 0.09 0.27 ± 0.10 0.25 ± 0.09 1.60 + 0.24* Incubation time, hr Cateehol: :::- :--: ----: 0.27±005 0.32: 0 --:6 01. Phenylglucuronide 0.41 0.07 0.60 0.07 0.96 0.09* 0.67 0.12 Fi lure 3. Glucuronidation of hydroquinone and phenol (Muco.ic eddY1 0.03±0.02 0.06 ±0.0 .:0,05±0.02 0.. in\V79 cells expressing rat UGT1.6. V79 wild Phenol 0.16 0.05 0.09 0.04 0.08 0.02* 0.24 ±0.08 type Others.;,,,:::,:' , ,:: 0.14± 0.08 0.1 :0....0: :; ..... 0...... '. A) and stably UGT1.6-transfected (,A) V79 cells Total 2.67 2.62 2.79 7.51 werre incubated with 200 pM phenol (o,e) or hydro- ~~~--~~~ ------~~quir- none (A,A). Glucuronides were analyzed by HPLC "Rat hepatocytes were incubated with 0.1 mM benzene. bMean ± SD from four experiments. clncluding conjugates ascdescribed in "Materials and Methods." Data repre- of catechol. dCharacterized on the basis of retention. *Significantly different from controls at p< 0.05. senit mean ± SD from three experiments.

Environmental Health Perspectives * Vol 104, Supplement 6 a December 1996 1 185 SCHRENK ET AL.

4000 IsoC3MC 2

Iso C 3MC PB 3000 = 0 Figure 4. Western blotting of microsomal fractions of C,4- 0. I hepatocytes isolated from untreated controls (C), and from rats treated with isopropanol (Iso), 3-methyl- cholanthrene (3-MC) or phenobarbital (PB). Each lane E2C;, 4 contained 4.2 microsomal protein. ~2000 pg ._ 1 6 co glucuronides were formed in untransfected 0 3 8 .a_ A cells, UGT1.6-expressing cells showed a co ' considerable rate of glucuronidation of 1000 - 5 both PH and HQ. I The most important phase I enzyme 71 involved in benzene metabolism is CYP2E1 Western blot analysis of 1 (18,19). microsomes prepared from freshly isolated 0-i hepatocytes showed that both 3-MC and 3 6 12 15 PB treatment decreased hepatic CYP2E1, Retention time, min whereas the well-known inducing action of short-chained aliphatic alcohols could be Figure 5. HPLC radiochromatogram of 14C-benzene metabolites formed in isolated mouse hepatocytes. confirmed in parallel experiments with Hepatocytes (20x 106/20 ml) were incubated for 1 hr with 0.5 mM 14C-benzene (specific activity 37 MBq/mmol). isopropanol (Figure 4). Fractions were collected at 0.3-min intervals and radioactivity was determined by liquid scintillation counting. Numbers represent polar metabolites (A), 1,2,4-trihydroxybenzene sulfate (1), hydroquinone/hydroquinone glu- Comparatively, the metabolism of curonide (2), hydroquinone sulfate (3), phenylsulfate/S-phenylglutathione (4), catechol (5), phenylglucuronide (6), benzene in mouse hepatocytes was inves- unidentified metabolite coeluting with trans-trans-muconic acid (7), phenol (8). tigated. At least nine peaks detectable by liquid scintillation counting could be sepa- rated by HPLC (Figure 5). Most of the Table 2. Comparative analysis of metabolites formed in isolated rat and mouse hepatocytes. metabolites were identical to those found nmol/hrx 106 cellsa % of total metabolites in rat hepatocytes, as revealed by mass Metabolite Wistar rat NMRI mouse Wistar rat NMRI mouse spectrometric analysis. However, two addi- Trihydroxybenzene sulfate - 0.72 ± 0.41* - 5.3 tional peaks were observed at a retention Be no.dihyd:iol 0:- :!009 .04 -:.;:i.. 2.1 time of 7.0 min (peak 1) and around 10.0 Hydroquinone/HQ glucuronide 0.54 ± 0.04 6.14 ± 1.10* 12.4 45.0 min (peak 3). The compound eluting at Hydrvquina;Isulfate:1.65...0.2..-2. . 7.0 min could be isolated and identified by Phenyl sulfate/phenylglutathione 1.76 ± 0.51 2.09 ± 0.62 40.3 15.3 FAB-MS as THB sulfate (not shown). The ...... 0.65±0.40 031...0.12...49..2.3 compound eluting at around 10.0 min Phenylglucuronide 0.97 0.61 1.45± 0.23 22.2 10.6 IuM-foiccdbW0.2 .X. id 0.2012090.9 coeluted with synthetic HQ sulfate but did Phenol 0.20 ± 0.06 0.81 ± 0.32* 4.6 5.9 not reveal a concisive FAB signal. However, ~~~~~~~~~~~~~~~~~~~~~~ ;!...... : after treatment with diluted hydrochloric Total 4.37 13.64 100.0 100.0 acid overnight, HQ was detected in the El -, not detectable. aMean + SD from four experiments. bCoeluting with trans-trans-muconic acid. *Significantly mass spectrum (not shown). different from rat hepatocytes (p< 0.05). Mouse hepatocytes metabolized 0.5 mM benzene at a 3-fold higher rate than rat hepatocytes (Table 2). Furthermore, distinguishable by radiodetection. Further- conjugates. The overall pattern of metabo- considerable differences were found when more, PH and metabolite 7, presumably lites was similar to that found in urine of the quantitative patterns of metabolites trans-trans-muconic acid, were found in laboratory animals treated with benzene were compared. Rat hepatocytes did not mouse hepatocytes at a 4-fold and 3-fold (20,21), a finding that emphasizes the role show formation ofTHB sulfate or HQ sul- higher level, respectively. of the liver in benzene metabolism. fate, whereas in mouse hepatocytes ben- Treatment of rats with 3-MC, leading zene-1,2-dihydrodiol was not found and Discussion to the induction of drug-metabolizing catechol formation was significantly lower. This study's major aim was to explore the enzymes that belong to the Ah gene bat- The most pronounced differences were seen pattern of benzene metabolites in hepato- tery, did not result in dramatic changes in with HQ (including HQ glucuronide), cytes, including phase II metabolism. The total benzene metabolism. However, which was found at a 10-fold higher combined radiodetection/direct mass spec- increased glucuronidation of PH at the amount in mouse hepatocytes. The HQ trometry methods allowed the sensitive expense of PH sulfate formation suggest fraction probably comprises both HQ and detection, identification and quantification involvement of UGT1.6 (a member of the HQ glucuronide, which were not clearly of phase I metabolites and of intact Ah receptor gene battery that catalyzes the

1 186 Environmental Health Perspectives - Vol 104, Supplement 6 * December 1996 PHASE 11 METABOLISM OF BENZENE

glucuronidation of planar phenols with CYP2B1 by these inducers at 0.1 mM ben- suggestion that benzene-1,2-dihydrodiol, high affinity). Both 3-MC induction of zene counterbalances the decrease in which was detected only in rat hepatocytes, PH and HQ glucuronidation in liver CYP2E1. It can be speculated that modifi- may be a precursor of catechol (3). The microsomes and the high glucuronidation cations of the hepatic CYP2E1 level may fact that some catechol was also found in rates of both substrates in V79 cells express- have considerable consequences for the mouse hepatocytes, however, suggests ing rat UGT1.6 suggest that UGT1.6 toxicity of benzene at lower concentrations either that it may be formed by an alterna- induction is the cause of the metabolic shift in the range ofhuman exposure. tive pathway or that benzene-1,2-dihydro- toward PH glucuronidation. Apparent Km Since mice have been reported to be diol is converted more effectively to values of 0.14 mM for PH (15) and of 0.1 more sensitive toward the carcinogenic catechol in mouse hepatocytes. mM for HQ characterize both compounds action of benzene (14), the patterns of The most prominent differences found as intermediate-affinity substrates of the benzene metabolism in rat and mouse by Henderson et al. (13) in an in vivo enzyme when compared to other planar hepatocytes were compared. A major study are basically in agreement with the phenols (17,22). No glucuronide was finding was that hepatocytes isolated from data presented here. The authors report detectable in incubations of liver micro- NMRI mice metabolize benzene at a 3-fold much higher levels of the putative myelo- somes with THB, possibly as a result of the higher rate than hepatocytes from Wistar toxic metabolites HQ and HQ glu- high polarity ofTHB. rats (Table 2). This agrees with data pub- curonide in mouse tissues. However, the Another major effect of inducing agents lished by Henderson et al. (13) showing reported considerable formation of trans- was the pronounced enhancement of ben- that mice had considerably higher levels trans-muconic acid was not confirmed in zene metabolism by isopropanol. Similar of various benzene metabolites in liver, the present study. This discrepancy may be observations were made by measuring ben- lung, and blood after a 6-hr inhalation of due to limitations in the comparison zene metabolism in laboratory animals and 50 ppm benzene. between whole animals and cell cultures. liver microsomes (23,24). In fact, the iso- The comparison of metabolite patterns The pronounced differences in benzene propanol-inducible isozyme CYP2E1 acts formed in isolated hepatocytes from both metabolism between rat and mouse hepa- as a high-affinity benzene monooxygenase species also revealed qualitative differences. tocytes may have an important impact on (18,19), and its induction thus led to an A benzene metabolite detectable in super- benzene toxicity. Since HQ is suspected to increased formation of a broad spectrum of natants from mouse hepatocytes only was mediate the myelotoxicity of benzene in benzene metabolites, which are derived identified as THB sulfate. Marked quanti- concert with other benzene metabolites directly or indirectly from benzene oxide. tative differences were also found for HQ such as phenol or trans-trans-muconic acid, A suprising result was the clear suppression and its conjugates. HQ (including HQ glu- a higher risk of myelotoxicity in mice of CYP2E1 expression in hepatocytes curonide) and HQ sulfate taken together could be concluded from our experiments. freshly isolated from 3-MC- (and PB-) accounted for more than 50% of total Furthermore, the higher rate of formation treated rats. Although the molecular basis metabolites in mouse hepatocyte incuba- of sulfate conjugates may represent an of this suppression is not known, it may tions, whereas HQ was only a minor additional pro-myelotoxic factor. Experi- contribute to the protection toward the metabolite in rat hepatocytes. In rats, evi- mental evidence in our laboratory suggests, myelotoxicity of benzene provided by 3- dence for the excretion of HQ sulfate in at least for HQ sulfate, a tendency for MC-type inducers (25,26). At a substrate bile was obtained in a previous study (27). hydrolysis and oxidation in an aqueous concentration of 0.1 mM, this suppression Nevertheless, the rate of formation in rat environment. Recently, the instability of was not reflected by a decreased rate of hepatocyte incubations was probably too the sulfate conjugate of 1,4-dihydroxy- total benzene metabolism in rat hepato- low to allow a clear separation and identifi- naphthalene in mice was demonstrated by cytes. However, incubations at lower cation of this compound. No evidence was Tsuruda et al. (29). Similarly, THB sulfate substrate concentrations showed a decrease obtained for the formation of HQ disulfate may provide a semistable transport form in total benzene metabolism after 3-MC in accordance with a report by Divincenzo for THB. THB was shown to be genotoxic or PB treatment (27). The low yield of et al. (28). in Chinese hamster V79 cells (30); it may metabolites, however, prevented the Benzene-1,2-dihydrodiol, usually formed also play a role in benzene myelotoxicity quantification and identification of indi- in small amounts in rat hepatocytes, was (31). Thus, the role of HQ sulfate and vidual metabolites. Apparently, the con- completely absent in mouse hepatocytes. THB sulfate as putative pro-myelotoxic comitant induction of low-affinity benzene Similarly, rat hepatocytes produced more metabolites and their activation in bone monooxygenases such as CYP1A2 or catechol, which is in agreement with the marrow cells warrants further investigation.

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