[CANCER RESEARCH 48, 5387-5390, October 1. 1988] Benzene Metabolism by Ethanol-, Acetone-, and Benzene-inducible Cytochrome P-450 (IIE1) in Rat and Rabbit Liver Microsomes1

Inger Johansson2 and Magnus Ingelman-Sundberg2

Department of Physiological Chemistry, Karolinska Institute, P. O. Box 60 400, S-104 01 Stockholm, Sweden

ABSTRACT tion of a specific form of liver microsomal cytochrome P-45C active in the microsomal metabolism of, e.g., ethanol, oth; Ethanol is known to exert a synergistic effect on the toxicity of benzene. aliphatic alcohols, carbon tetrachloride, and acetone (15-28). In the present investigation it was found that benzene was metabolized The finding that the ethanol-inducible form of cytochrome P- at a rate 20-65-fold higher in liver microsomes from ethanol- or acetone- 450 oxidizes ethanol partially in a mechanism involving hy- treated rats than in microsomes from control animals. One high affinity site \Ka = 19 ±5 (SD) urn] and one low affinity site [K„=0.3 ±0.1 droxyl radicals (20), that benzene can be oxidized in liver IIIM| for benzene metabolism were present in microsomes of acetone- microsomes and reconstituted cytochrome P-450-containing treated rats, and similar sites were seen in microsomes from control or membranes in a mechanism involving hydroxyl radicals (29), ethanol-treated rats. Treatment of the animals with either ethanol or and that benzene appears to be an inducer of this type of acetone mainly influenced the Vm.»valuesfor benzene metabolism. Also cytochrome P-450 in the rabbit (30) suggested that benzene benzene treatment of rats caused an increased rate of microsomal benzene could provide an efficient substrate for this type of cytochrome metabolism. The hepatic microsomal NADPH-dependent metabolism of P-450. In the present paper we present data indicating that benzene was inhibited by compounds known to interact with the ethanol- benzene is mainly metabolized by ethanol-inducible cytochrome inducible form of P-450 such as ¡midazole,ethanol, aniline, and acetone P-450 in liver microsomes and that the induction of this iso but was unaffected by addition of metyrapone. Anti-IgG against ethanol- inducible cytochrome P-450 from rat (P-450J) or rabbit liver (P-450 zyme by ethanol can provide an explanation for the synergistic LMeb) inhibited the microsomal benzene metabolism effectively in rat action of ethanol on benzene toxicity. or rabbit liver microsomes, respectively, whereas preimmune IgG was without effect. The level of rat ethanol-inducible P-450 (P-450J) was induced to an extent similar to that for the microsomal benzene metabo MATERIALS AND METHODS lism, by either benzene, acetone, or ethanol. The data indicate that Materials. [HC]Benzene (specific activity, 20-60 mCi/mmol) was benzene is metabolized mainly by the ethanol-inducible P-450 form in liver microsomes and that the induction of this isozyme by ethanol can obtained from New England Nuclear. NADPH and mannitol were purchased from Sigma. Imidazole and aniline were from Merck. Aniline provide an explanation for the synergistic action of ethanol on benzene was redistilled and stored at -20°C. Metyrapone was a gift from toxicity. Professor Sten Orrenius, Department of Toxicology, Karolinska Insti tute!. INTRODUCTION Animals. Male Sprague-Dawley rats (175 g) were used in these studies. Some animals were starved and given i.g.3 injections of acetone Exposure to benzene, a known hematotoxic compound, may (5 ml/kg) for 2 days as described previously (16). Other groups of rats cause leukopenia or aplastic anemia in animals and humans ( I- were given s.c. injections (1.05 g/kg) of benzene or treated with ethanol 4). A relationship between chronic benzene exposure and the for 20 days as described by DeCarli and Lieber (31). The alcohol diet occurrence of leukemia in humans has been recognized (3, 5). was purchased from Bioserv, Inc. (Frenchtown, NJ). Control rats were Benzene exposure appears to be mainly connected with the given food and water ad libitum. Male rabbits (2.5 kg) were obtained development of acute myelogenous leukemia (4). It has not yet from a local farm and received acetone (2-4%, v/v) in their drinking been clarified whether benzene-induced leukemia develops sec water for 2-3 weeks. ondary to aplastic anemia or if these symptoms are initiated via Isolation of Liver Microsomes. The livers were homogenized in 2 volumes of 10 mM sodium/potassium phosphate buffer, pH 7.4, con different mechanisms. Benzene has also been described to cause taining 1.14% (w/v) KC1. Microsomes were prepared by ultracentrifu- leukemia in mice (6-9). gation, washed once before suspension in 50 mM potassium phosphate Bioactivation of the compound appears to be a prerequisite buffer (pH 7.4) to a concentration of about 30 mg of protein/ml, and for the hematotoxic effects, although the reactive metabolite stored under nitrogen at —70°C. has not hitherto been identified (2). Partial hepatectomy of rats, Enzymes. The ethanol-inducible cytochrome P-450 was isolated from which decreased the overall metabolism of benzene by 70%, has liver microsomes of acetone-treated rabbits essentially according to the been shown to cause a complete prevention of the benzene- method of Koop et al. (15) as described previously (20) and from liver induced hematotoxicity (10). Accordingly, it appears that the microsomes of acetone-treated rats as described elsewhere (27). IgG hepatic metabolism of the compound is of importance for its from preimmune and immunized goat and rabbit was prepared by adverse effects. This is further substantiated by the fact that ammonium sulfate fractionation as described (18). Incubation Conditions. Benzene metabolism was determined using ethanol, which potentiates the toxicity of benzene (11,12), also the head space technique described by Sato and Nakajima (32). The acts as an inducer of the liver microsomal metabolism of incubations were performed at 37°Cfor 15 min in 5-ml tubes equipped benzene (13). In line with this hypothesis, Nakajima et al. (14) with rubber membrane caps. The incubation mixtures contained liver suggested that ethanol acts synergistically on benzene toxicity microsomes corresponding to 1 mg of protein, benzene (added in water), by induction of the rate of benzene metabolism in liver. and 0.4 mM NADPH in a total volume of 1 ml 50 mM potassium Treatment of rats or rabbits with, e.g., ethanol, acetone, phosphate buffer, pH 7.4. Control incubations contained no NADPH. pyrazole, imidazole, starvation, or isoniazid causes the induc- The incubations were stopped by the addition of 0.1 ml 4.5 M trichlo- roacetic acid and after a further incubation period of 15 min at 37'C, Received 7/23/87; revised 6/15/88; accepted 6/21/88. 1-ml aliquots of the head space volumes were analyzed by gas chro- The costs of publication of this article were defrayed in part by the payment matography at 140'C. A Carbopack C 0.1% SP 1000 column was used of page charges. This article must therefore be hereby marked advertisement in and the gas Chromatograph (Shimadzu GC-8A) was equipped with a accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ' Supported by grants from Magn. Bergvalls Stiftelse and from the Swedish flame ionization detector. Medical Research Council. 1To whom requests for reprints should be addressed. 3The abbreviation used is: i.g., intragastric. 5387 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1988 American Association for Cancer Research. MICROSOMAL BENZENE METABOLISM

Also water-soluble metabolites produced during the benzene incu did not significantly influence the rate of HCHO formation [4.5 bations were quantified. In these cases the incubations were carried out ±0.9 (SD) nmol/mg, min]. The rate of NADPH oxidation was in 7-ml tubes equipped with screw caps in the presence of 0.1 nCi of increased by the Fe-EDTA addition, but the amount of NADPH [MC]benzene. The reactions were stopped by the addition of 50 n\ l M added was still sufficient (not shown). HC1. Sodium chloride (0.1 ml, 9% w/v) was added and the samples Further evidence for the participation of the ethanol-induci were extracted twice with 3.5 ml ethyl acetate. Aliquots of 0.1 ml of ble cytochrome P-450 form in benzene metabolism was ob the water phase were subjected to liquid scintillation counting. tained by incubation of liver microsomes from acetone-treated rats or rabbits in the presence of anti-IgG raised against P- RESULTS 450LMeb (rabbit) or P-450J (rat). Almost 80% inhibition was reached in rabbit liver microsomes by anti-P-450LMeb IgG at Benzene was incubated with liver microsomes from control and acetone-treated rats and the rate of metabolism was quan 4 mg/mg microsomal protein, whereas anti-P-450j IgG inhib tified using head space technique. The rate of metabolism using ited rat liver microsomal benzene metabolism almost com 50 fiM concentration was about 65-fold higher in microsomes pletely, at the same concentration (Fig. 3). The corresponding from acetone-treated compared to control rats and was linear preimmune IgG fractions were without effect (Fig. 3). for 15 and 90 min, respectively (Fig. 1A). Kinetic analysis of the production of water-soluble metabolites from benzene re DISCUSSION vealed the existence of low and high affinity sites in both types of microsomes (Fig. 1Ä;Table 1). The apparent Kmof the high The results presented in this paper indicate that treatment of affinity site in microsomes from acetone-treated rats was about rats with ethanol, acetone, or benzene causes the induction of 20 fiM. Large differences between acetone and control micro the ethanol-inducible form of cytochrome P-450 which is active somes were evident in the apparent Vmaxvalues (Table 1). Liver in the metabolism of benzene. This is supported by the facts microsomes from ethanol-treated rats exhibited a similar type (a) that the increase in the rate of benzene metabolism obtained of kinetics (data not included) for benzene metabolism as did by treatment with the compounds was in the same order as the microsomes from acetone-treated animals. increase in the amount of ethanol-inducible P-450 in the micro Treatment of the rats with ethanol, acetone, or benzene somes, (b) that the oxidation of benzene was inhibited by the caused an induction of the ethanol-inducible form of cyto- compounds known to effectively interact with this form of P- chrome P-450 by 7-9-, 5-9-, and 2-3-fold, respectively, as 450, but unaffected by the presence of metyrapone that interacts evident from quantifications performed using radial immuno- with most other forms of P-450, (c) that benzene induces the diffusion or Western blot (Fig. 2; Table 2). The rate of metab type of P-450 that also metabolizes the compound and (d) that olism of benzene at 20 ¿iMwasincreased in a similar manner the microsomal benzene metabolism in microsomes from rabbit (Table 2), although somewhat more pronounced than the in or rat liver was inhibited by anti-IgG against ethanol-inducible crease in the content of P-450J. Compounds known to interact cytochrome P-450 from either rabbit or rat liver. with the ethanol-inducible form of cytochrome P-450 such as The apparent high affinity Km value for benzene metabolism acetone, aniline, ethanol, and imidazole (cf. Reís.16-18) all in liver microsomes of 15 and 19 ¡J.Minmicrosomes from inhibited the oxidation of benzene in liver microsomes from control and acetone-treated rats, respectively, is in the same acetone-treated rats (Table 3). Carbon monoxide was also in order as observed for several endogenous substrates of P-450, hibitory, whereas neither mannitol, a hydroxyl radical scaven such as steroids (cf. Réf.33).This suggests that the enzyme can ger, nor metyrapone affected the rate of benzene oxidation. Of utilize benzene as a substrate at lexicologically relevant levels special interest was the finding that small amounts of Fe- of benzene. The pharmacokinetics of benzene elimination in EDTA, which is known to increase the rate of ethanol oxidation vivo is altered dramatically in a manner indicative of induced dependent on the ethanol-inducible form of cytochrome P-450 benzene biotransformation, after pretreatment of rats or mice (20), inhibited the microsomal metabolism of benzene (Table with ethanol (34). This further indicates a role of ethanol- 3). In control experiments, where the microsomal NADPH- inducible cytochrome P-450 in benzene metabolism under na dependent TV-demethylation of aminopyrine [2 ITIM]was exam tive conditions. ined, 2.5, 10, or 50 fiM Fe-EDTA in the incubation mixtures The rate of benzene metabolism in liver microsomes from

Fig. 1. Time dependency (A) and effect of substrate concentration (B) of the NADPH- dependent metabolism of benzene measured by head space technique (A) and the formation of water-soluble metabolites (B) in liver micro somes from control (O) and acetone-treated (•)rats. The incubations were carried out as described under "Materials and Methods" us ing a substrate concentration of 50 CM.unless otherwise stated. Inset, Lineweaver-Burk plot of the substrate curve obtained with micro somes from acetone-treated rats.

30 60 90 0 025 050 0.75 minutes m M benzene

5388 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1988 American Association for Cancer Research. MICROSOMAL BENZENE METABOLISM

Table 1 Apparent kinetic constants for the microsomal benzene metabolism in Table 3 Effect of inhibitors, known to interact with the ethanol-inducible form of liver microsomes from control and acetone-treated rats as determined by the cytochrome P450, on the rate of benzene metabolism in liver microsomes from formation of water-soluble metabolites acetone-treated rats The values are from 3 separate experiments performed at 8-10 different The substrate concentration used was 50 I*Mand the metabolism was studied substrate concentrations with each type of liver microsomes and are expressed as both using the head space technique and analysis of water-soluble metabolites. mean ±SD. Rate of benzene metabolism nmol/mg/min (nmol/mg/min) Type of soluble microsomes (mM) space analysis (% ofinhibition)1.00001.000.821.250.550.210.971.120.030.340.970.290100100018457939767371100Watermetabolites Control 15 ±7 0.87 ±0.12 0.015 ±0.003 0.24 ±0.09 AdditionNoneAniline, (% ofinhibition)0.3400.010.370.260.220.050.010.450.320.0500.250.090.021009622358598486100257293 Acetone 19 ±5 0.29 ±0.13 0.40 ±0.10 0.65 ±0.08 1mMEthanol, mMMannitol100200

mM500 mMImidazole0.05

mM0.5 mM5 mMMetyrapone0.1

mM1 mMFerric ¿JMCOEDTA, 10 (30%)Acetone0.5

mM5 mM50 mMHead

BC B Rabbit Fig. 2. Western blot analysis of the level of cytochrome P-450J in rat liver 0.50 microsomes isolated from control rats (C) or rats treated with either ethanol diet (£),control diet (Cc), benzene (B), or acetone (A). Analysis was performed as described elsewhere (27) with microsomes corresponding to 2.5 fig of protein in each lane. <= 025 Table 2 Rate of benzene oxidation, determined by the formation of water-soluble metabolites, and the apparent level of the ethanol-inducible cytochrome P-450 form (P-4SOJ)in liver microsomes from control, ethanol-treated, and acetone- treated rats The substrate concentration was 20 Jphenobarbital-treated rabbits or by phenobarbital-induci- enzyme. These enzymes are subject to distinct regulatory con ble cytochrome P-450 LM2 in reconstituted systems occurred trols. The low Km enzyme is probably the most important for at low affinity sites. Since acetone and ethanol also provide demethylation under in vivo conditions (37, 38) and is likely to inducers of the homologous rat form of cytochrome P-450 (P- be identical with the ethanol-inducible form of cytochrome P- 450b) (27), a corresponding low affinity site in rat liver micro- 5389 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1988 American Association for Cancer Research. MICROSOMAL BENZENE METABOLISM somes could be attributed to this isozyme. In this context it 13. Sato, A., and Nakajima, T. Enhanced metabolism of volatile hydrocarbons in rat liver following food deprivation, restricted carbohydrate intake and should be pointed out that the inhibition experiments of ben administration of ethanol, phénobarbital,polychlorinated biphenyl and 3- zene metabolism in liver microsomes by anti-IgG fractions or methylcholanthrene: a comparative study. Xenobiotica, 75:67-75, 1985. 14. Nakajima, T., Okuyama, S., Yonekura, I., and Sato, A. Effect of ethanol and by various inhibitors presented here were carried out at small phénobarbitaladministration on the metabolism and toxicity of benzene. concentrations of the substrate (20 UM). Chem.-Biol. Interact., 55: 23-38, 1985. We have previously described that benzene might be metab 15. Koop, D. R., Morgan, E. T., Tarr, G. E., and Coon, M. J. Purification and characterization of a unique isozyme of cytochrome P-450 from liver micro olized in reconstituted membranes containing P-450 LM2 in somes of ethanol-treated rabbits. J. Biol. Chem., 257: 8472-8480, 1982. the presence of EDTA, by a mechanism involving the action of 16. Johansson, I., Eliasson, E., Norsten, C., and Ingelman-Sundberg, M. Hy- hydroxyl radicals, generated in a Haber-Weiss reaction from droxylation of acetone by ethanol- and acetone-inducible cytochrome P-450 in liver microsomes and reconstituted systems. FEBS Lett., 196: 59-64, Superoxide anions and hydrogen perioxide (29). Most sub 1986. strates metabolized by the ethanol-inducible form of cyto- 17. Koop, D. R., Crump, B. L., Nordblom, G. D., and Coon, M. 3. Inumino chrome P-450 are effective scavengers of hydroxyl radicals, and chemical evidence for induction of the alcohol-oxidizing cytochrome P-450 of rabbit liver microsomes by diverse agents: ethanol, imidazole, trichloro- evidence has been presented for a mechanism of ethanol oxi ethylene, acetone, pyrazole and isoniazid. Proc. Nati. Acad. Sci. USA, 82: dation by this type of P-450 involving the action of hydroxyl 4065-4069, 1985. 18. Ingelman-Sundberg, M., and Jörnvall,H. Induction of the ethanol-inducible radicals or oxidizing species of equivalent energy (20). It was form of rabbit liver microsomal cytochrome P-450 by inhibitors of alcohol therefore surprising that mannitol, an effective scavenger of dehydrogenase. Biochem. Biophys. Res. Commun., 124: 375-382, 1984. 19. Morgan, E. T., Koop, D. R., and Coon, M. J. Catalytic activity of cytochrome hydroxyl radicals, did not inhibit the microsomal benzene me P-450 isozyme 3a isolated from liver microsomes of ethanol-treated rabbits. tabolism and, furthermore, that Fe(III)-EDTA effectively inhib J. Biol. Chem., 257: I3951-I3957,1982. ited benzene conversion instead of stimulated the metabolism 20. Ingelman-Sundberg, M., and Johansson, I. Mechanisms of hydroxyl radical (Table 3). However, a similar inhibition by Fe-EDTA has formation and ethanol oxidation by ethanol-inducible and other forms of rabbit liver microsomal cytochromes P-450. J. Biol. Chem., 259:6447-6458, previously been registered in systems containing the ethanol- 1984. inducible P-450 when acetone has been used as substrate (16). 21. Johansson, I., and Ingelman-Sundberg, M. Carbon tetrachloride-induced lipid peroxidation dependent on an ethanol-inducible form of rabbit liver It thus appears that the microsomal oxidation of benzene or microsomal cytochrome P-450. FEBS Lett., ¡S3:265-269, 1985. acetone in microsomes from acetone-treated rats does not in 22. Hasumura, Y., Teschke, R., and Lieber, C. S. Increased carbon tetrachloride hepatotoxicity, and its mechanism, after chronic ethanol consumption. Gas- volve the action of free hydroxyl radicals. troenterology, 66:415-422, 1974. In conclusion, evidence has been presented for a major con 23. Ryan, D. E., Ramanathan, L., lida, S., Thomas, P. E., Haniu, M., Shirely, tribution by ethanol-inducible cytochrome P-450, on a quanti J. E., Lieber, C. S., and Levin, W. Characterization of a major form of rat hepatic microsomal cytochrome P-450 induced by isoniazid. J. Biol. Chem., tative basis, to the microsomal metabolism of benzene. This is 260:6385-6393,1985. possibly of toxicological importance under conditions of exten 24. Ryan, D. E., Koop, D. R., Thomas, P. E., Coon, M. J., and Levin, W. sive fasting, ketoacidosis, and alcohol abuse, conditions known Evidence that isoniazid and ethanol induce the same microsomal cytochrome P-450 in rat liver, an isozyme homologous to rabbit liver cytochrome P-450 to cause pronounced induction of this isozyme in liver micro isozyme 3a. Arch. Biochem. Biophys., 246:633-644, 1986. somes. 25. Eliasson, E., Johansson, I., and Ingelman-Sundberg, M. Ligand-dependent maintenance of ethanol-inducible cytochrome P-450 in primary hepatocyte cultures. Biochem. Biophys. Res. Commun., 750:436-443, 1988. 26. Patten, C. J., Ning, S. M., Lu, A. Y. H., and Yang, C. S. Acetone-inducible ACKNOWLEDGMENTS cytochrome P-450: purification, catalytic activity, and interaction with cyto chrome b,. Arch. Biochem. Biophys., 25/: 629-638, 1986. The skillful technical assistance of Elisabeth Wiersma is gratefully 27. Johansson, L, Ekström,G., Schölte,B., Puzycki, D., Jörnvall,and Ingelman- acknowledged. Sundberg, M. 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5390 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1988 American Association for Cancer Research. Benzene Metabolism by Ethanol-, Acetone-, and Benzene-inducible Cytochrome P-450 (IIE1) in Rat and Rabbit Liver Microsomes

Inger Johansson and Magnus Ingelman-Sundberg

Cancer Res 1988;48:5387-5390.

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