Effects of Pregnancy, Age and Sex in the Metabolism of Styrene in Rat Liver in Relation to the Regulation of Cytochrome P450 Enzymes

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1 J Occup Health 2005; 47: Journal of Occupational Health Effects of Pregnancy, Age and Sex in the Metabolism of Styrene in Rat Liver in Relation to the Regulation of Cytochrome P450 Enzymes Reiko KISHI 1, Fumihiro SATA 1, Yoko KATAKURA 1, Rui-Sheng WANG 2 and Tamie NAKAJIMA 3 1 Department of Public Health, Hokkaido University Graduate School of Medicine, 2 Department of Hazard Assessment, National Institute of Industrial Health and 3 Department of Occupational and Environmental Health, Nagoya University Graduate School of Medicine, Japan Abatract: Effects of Pregnancy, Age and Sex in the Metabolism of Styrene in Rat Liver in Relation to the Regulation of Cytochrome P450 Enzymes: Reiko KISHI, et al. Department of Public Health, Hokkaido University Graduate School of Medicine To elucidate the effect of maternal styrene exposure, which is due to various postnatal changes in the development and behavior of offspring, we investigated pregnancy-induced changes in the metabolism of styrene in rat liver in relation to the regulation of cytochrome P450 enzymes. We also examined age and sex-induced changes in the metabolism of styrene. Pregnancy appeared to exert a negative effect on cytochrome P450 content at the late stage, whereas microsomal protein content showed little change during pregnancy. Pregnancy significantly decreased the rate of formation of styrene glycol at the late stage. The percentage of remaining activity in microsomes exposed to anti-cyp2e1 was lower than that exposed to anti-cyp2c11/6 in pregnant and nonpregnant female rats and immature male rats, indicating that CYP2E1 contributes to the metabolism of styrene more than CYP2C11/6 in these rats. Although pregnancy seemed to decrease styrene metabolism, the contribution of CYP2E1 seemed to be slightly increasing. In conclusion, pregnancy clearly influences the metabolism of styrene as well as other characteristic factors such as age and sex. It is very important to elucidate the changes in specific P450 isozyme composition related to their characteristic modification and in their affinity for chemicals. (J Occup Health 2005; 47: 49 55) Received May 17, 2004; Accepted Oct 18, 2004 Correspondence to: R. Kishi, Department of Public Health, Hokkaido University Graduate School of Medicine, Kita-15, Nishi- 7, Kita-ku, Sapporo , Japan ( rkishi@med.hokudai.ac.jp) Key words: Styrene, Metabolism, Cytochrome P450, Liver, Pregnancy, Rats Styrene is an important industrial chemical widely used for the production of reinforced plastics and various polymers such as polystyrene, resins, paints, and synthetic rubbers 1, 2). Exposure to styrene can also occur owing to its presence in automobile exhaust, cigarette smoke and packed foods and water 3). Styrene is activated metabolically by cytochrome P450 (CYP) monooxygenases to styrene 7,8-oxide (SO), which is a toxic, mutagenic and potentially carcinogenic form 1). Adducts of styrene to proteins and DNA are formed via this metabolic intermediate SO in hemoglobin 4, 5). In mice and rats, it induces tumors dose dependently after oral administration 6 8). This reactive intermediate is, in humans, mainly detoxified by microsomal epoxide hydrolase (EH) to styrene glycol (SG) 9). In rats, in vivo exposure to styrene results in high SO levels in liver, lung, kidney and brain; so that most of adverse effects of styrene have been attributed to accumulation of SO in tissues 1). 4-Vinylphenol (4-VP) is a minor metabolite of styrene and metabolized primarily by CYP2E1 and CYP2F2 10, 11). 4-VP is a more potent toxicant in mice than either styrene or SO. Approximately 95% of the styrene retained upon inhalation was excreted as urinary metabolites by rats 12). Cytochrome P450 consists of multiple isozymes, which were inducible and are expressed differently in tissues 13). The cytochrome P450 isozymes involved in styrene metabolism and their distribution, including their induction and destruction by chronic treatment, in target tissues must therefore be addressed. The unique ability of monoclonal antibodies (MAbs) to define the P450 responsible for the metabolism of styrene was previously shown both in human and rat tissues 14 16). In humans,

2 50 J Occup Health, Vol. 47, 2005 CYP2E1 is the main isoform responsible for styrene metabolism 15, 17, 18), whereas CYP2B6 is the most active isoform at the high substrate concentrations 15). At least four CYPs, CYP2C11/6, CYP2E1, CYP2B1/2 and CYP1A1/2 had been reported to contribute to the metabolism of styrene in rats 18). CYP2C11/6 is the major form of P450 responsible for the metabolism in untreated rat liver microsomes, whereas Anti-CYP2E1 inhibited styrene oxidation more prominently in microsomes from styrene-treated rats 14). We have reported various postnatal effects of prenatal styrene exposure on development, behavior and neurochemical levels in offspring rats 19 22), but pregnancy-induced changes in styrene exposure remain largely unknown. In the present study, we investigated pregnancyinduced changes in the metabolism of styrene in rat liver to elucidate the effect of maternal styrene exposure, which may cause various postnatal changes in the development and behavior of offspring. Materials and Methods Animals The experiment was performed in accordance with Guidelines for Animal Experiments of the Sapporo Medical College and Hokkaido University. Male and female Wistar rats were obtained from Nippon Clea (Tokyo, Japan) and were maintained three to a cage on a 12-h light/dark cycle in an environment with controlled temperature and humidity, provided with rodent chow (MF and NMF, Oriental Yeast Co.,Tokyo, Japan) and tap water ad libitum. Sixteen female rats were mated by placing them each with one male in a cage overnight; sperm and vaginal plugs were checked the following morning. The day on which the sperm or vaginal plugs were found was recorded as the 1st day of gestation. On the 10th and 2lst days of gestation, five and seven dams, respectively, were sacrificed by decapitation and the liver was removed. The offspring of the other four rats were sacrificed and their livers were removed on the first day (female) and 3 wk after birth (male, female); the livers of rats in the same litters were pooled for each sex. Six intact male and female rats were sacrificed at 18 wk of age. To prepare the microsomal suspension, 25% liver homogenate in 1.15% KCl solution was centrifuged at l0,000 g for 10 min, the supernatant was centrifuged further at 105,000 g for 60 min and the microsomal pellets obtained were suspended in 50 mm potassiumphosphate buffer (ph 7.4) containing 10 % (v/v) glycerin and frozen at 85 C. Microsomal protein was determined by the method of Lowry et al. 23), and P450 content was measured by the method of Omura and Sato 24). Metabolism Assays Styrene metabolism was determined by measuring the rate of formation of styrene glycol, according to the method of Nakajima et al 25). The reaction mixture contained an NADPH-generating system (1 mm NADP, 20 mm glucose 6-phosphate, 2 IU glucose phosphate dyhydrogenase and 50 mm magnesium chloride), 200 µg of liver microsomal protein, 50 mm potassium phosphate buffer (ph 7.4) and mm styrene, to a final volume of 0.5 ml. The reaction was started by adding the substrate, and the reaction vials were placed in a thermoregulated, shaking water bath at 37 C. After 10 min incubation, 0.1 ml of 15% (w/v) zinc sulfate and saturated barium hydroxide was added to stop the reaction for measurement of styrene glycol formation. The mixture was centrifuged at 1,800 g for 15 min, and 20 µl of supernatant was injected into a high-performance liquid chromatograph with an ultraviolet-vis detector and a chromatointegrator. The analytical conditions were as follows: 4.0 mm diameter 250 mm stainless steel column packed with Unisil C18; mobile phase, 10% (v/ v) acetonitrile solution containing 0.25% (v/v) phosphoric acid at a flow rate of 1.1 ml/min; wavelength 200 nm. The retention time of styrene glycol was 10.2 min; the amount of styrene glycol formed increased linearly with respect to incubation time for at least 30 min and up to 1.0 mg microsomal protein. Inhibition by Monoclonal Antibodies In order to explore the relationship between changes in styrene metabolism by sex, age and pregnancy and changes in P450 isoenzymes, MAbs were raised at the U.S. National Cancer Institute, Laboratory of Metabolism, by a modification of the method of Koehler and Milstein 26). In the present study, two MAbs that had been shown to be specific for different cytochrome P450s were used: anti-cyp2c11 MAb (clone ), which is steroid hormone-inducible male-specific and cross-reacts with CYP2C6, and anti-cyp2e1 (clone ), an ethanol-inducible P450 isozyme 27 29) As a control MAb, Hy-Hel against chicken lysozyme was used to determine any nonspecific reaction. MAbs were added to pooled microsomes and buffer at room temperature 30 min before the start of the metabolism assay at 37 C by adding an NADPH-generating system and styrene. In preliminary experiments, maximal inhibition by each MAb was obtained at MAb protein: microsomal protein ratios below 1.0. Statistics Analysis of variance was performed. When there was significant difference among groups, means were tested by Student s t-test or paired t-test. The 0.05 level of probability was the criterion of significance. Results Mocrosomal Protein and Cytochrome P450 Pregnancy, age and sex-induced changes in microsomal

3 Reiko KISHI, et al.: Effects of Pregnancy, Age and Sex in Rat Liver Metabolism 51 Fig. 1. Effect of pregnancy, sex and age on (a) microsomal protein and (b) cytochrome P450 level. The number of rats in each group is shown in parentheses. a Significantly different (p<.01) from male rats at 3 wk of age; b Significantly different (p<.01) from female rats at 3 wk of age; c Significantly different (p<.05) from female rats at 18 wk of age; d Significantly different (p<.05) from rats at the 10th day of pregnancy. Square pillars indicate means of rats in each group; thin bars indicate standard deviations. ND: not determined. protein and cytochrome P450 content are showed in Fig. 1. Pregnancy appeared to exert a negative effect on cytochrome P450 content at the late stage (day 21), whereas microsomal protein content showed little change during pregnancy. The cytochrome P450 content in newborn female rats was lower than that at the age of 3 wk, whereas the microsomal protein contents were similar. At the age of 3 wk, the cytochrome P450 content was the same in males and females, but the microsomal protein content was significantly higher in males than in females; at 18 wk, the cytochrome P450 content in females was significantly lower than that in males. The contents of both microsomal protein and cytochrome P450 increased significantly in male rats between 3 and 18 wk, but in female rats only microsomal protein content increased between these ages. Thus, there was sex difference in microsomal protein content at 3 wk of age and in cytochrome P450 content at 18 wk of age. Formation of Styrene Glycol The metabolism of styrene was evaluated by the formation of styrene glycol (Table 1). Pregnancy

4 52 J Occup Health, Vol. 47, 2005 Table 1. Effects of pregnancy, age and sex on styrene metabolism (mean ± S.D.) in rat liver sex age n styrene glycol nmol/g liver/min Male 3 wk ± 6.6 a Male 18 wk ± 7.1 b Female 3 wk ± 3.4 Female 18 wk ± 3.8 Female pregnancy (day 10) ± 8.2 Female pregnancy (day 21) ± 4.9 c a Significantly different (p<.05) from female rats at 3 wk of age. b Significantly different (p<.01) from female rats at 18 wk of age and male rats at 3 wk of age. c Significantly different (p<.05) from non-pregnant female rats and those 10 days pregnant. Fig. 2. Inhibition of styrene glycol formation from styrene by two MAbs (anti-cyp2e1 and anti-cyp2c11/6) in rat liver. As a control MAb, Hy-Hel against chicken lysozyme was used to determine any nonspecific reaction. MAbs were added to pooled microsomes of rats in each group. significantly decreased the rate of formation of styrene glycol at the late stage (day 21). Age had an effect only on male rats, with a higher rate of formation of styrene glycol at 18 wk of age than at 3 wk. A sex differences was found at 3 and 18 wk of age, with a higher rate of formation in male rats than in female. Inhibition of Styrene Metabolism by MAbs The inhibition of styrene metabolism by MAbs (anti- CYP2E1 and anti-cyp2c11/6) was analyzed, and the remaining activity and the control (Hy-Hel) are showed in Fig. 2. The percentage of remaining activity in microsomes exposed to anti-cyp2e1 was lower than that exposed to anti-cyp2c11/6 in pregnant and non-pregnant female rats and immature male rats, indicating that CYP2E1 contributes more to the metabolism of styrene than CYP2C11/6 among these rats. In adult male rats, on the other hand, CYP2C11/6 seemed to contribute to styrene metabolism no less than CYP2E1. Although styrene metabolism seemed to be gradually decreased during pregnancy, the contribution of CYP2E1 seemed to be relatively increasing styrene glycol formation. Discussion Pregnancy is known to decrease the overall content of the constitutive P450 isozymes in rats and mice 25, 30) The

5 Reiko KISHI, et al.: Effects of Pregnancy, Age and Sex in Rat Liver Metabolism 53 present study shows that pregnancy appeared to exert a negative effect on cytochrome P450 content at a late stage, whereas microsomal protein content showed little change during pregnancy. Pregnancy also decreased the rate of formation of styrene glycol, to which CYP2E1 contributed more than CYP2C11/6. In our previous study, pregnancy slightly decreased the levels of two constitutive isozymes, CYP2E1 and CYP2C11, and correspondingly suppressed the metabolite rates of toluene and trichloroethylene 25). It was also reported that pregnancy decreased CYP2E1 in the metabolism of acetone 31). CYP2E1 expression was suppressed in females particularly in late pregnancy in both acetone-treated and untreated rats, although CYP2E1 induction by acetone was still observed during pregnancy. The decrease in hepatic CYP2E1 in pregnancy and its rapid reversal could result from changes in CYP2E1 apoenzyme accompanied by alteration in CYP2E1 gene transcription or its mrna turnover rate 31). On the other hand, Symons et al. (1982) suggested that the reduction might be due to microsomal phospholipids contents or its composition 32). CYP2E1 has multiple mechanisms of regulation such as transcriptional activation during normal development, pretranslational activation during starvation or diabetes, and various post-translational activation of CYP2E1 by its substrates 31). In the present study, CYP2E1 was found to be one of the major P450 forms involved in styrene oxidation in female rats. An additional pathway has been identified in which the paraposition on the styrene is hydroxylated to yield 4-VP with evidence of the formation of this metabolite 33, 34). 4-VP is metabolized to as yet unidentified metabolites although it has been suggested that these may be ring-opened products 11). Carlson et al. (2001) have shown that the 4-VP is metabolized rapidly by mouse and rat hepatic and pulmonary microsomes 10). This process requires NADPH, and the use of chemical inhibitors has identified CYP2E1 and CYP2F2 as being the most important cytochrome P450s involved 11). Further studies should be needed to clarify changes in CYP2E1 during pregnancy in the metabolism of styrene including minor pathways. Levels of hepatic microsomal monooxygenase activity are very low during fetal development in most mammals but increase rapidly soon after birth. The analysis of immunochemically detectable proteins showed that some P450 isozymes are regulated developmentally in sexspecific fashion. The present study shows that styrene metabolism was dependent on cytochrome P450 content more than microsomal protein content but that was differed according to age and sex. In liver microsomes from male rats, styrene metabolism was regulated developmentally: the rate of styrene glycol formation is much higher in older than in younger rats. In liver microsomes from female rats, on the other hand, styrene metabolism was regulated in a different way from that of male rats: the styrene glycol formation is almost constant in non-pregnant female rats. In addition, little styrene oxidation was found in liver microsomes from fetal rats (data not shown). The study with MAbs indicates that CYP2E1 and CYP2C11/6 contribute to the formation of styrene glycol from styrene but, the contribution of each enzyme differed according to sex. This sex difference can be attributed mainly to a difference in the distribution of male specific CYP2C11 and in the affinity of hydrocarbons for this isozyme: hydrocarbons with a high affinity for CYP2C11 may exhibit a sex difference in their metabolism. There is also a sex difference in the CYP2E1 level, but this is very small compared to the difference in CYP2C11 and would not be reflected as a sex difference in metabolism 25). The major form involved in this metabolism in adult male rat liver microsomes was CYP2C11, because in adult male rats, although CYP2C6 is expressed equally to CYP2C11, the specific activity of the latter for styrene metabolism is 5.4-fold that of the former 35, 36). The male specific CYP2C11 is at significant levels in immature male rats 37, 38) and reflects a more than 30-fold induction of isoenzymes at puberty in male but not in female rat liver; these results are in good agreement with our findings that the CYP2C11/6 level increased at puberty compared with that in immature male rats. In addition, it is of great interest that CYP2C11 was also the major form responsible for the formation of styrene glycol even in microsomes treated with ethanol, which greatly induces CYP2E1 39). On the other hand, the expression of CYP2C6 in liver from adult male rats was found to be equal to that from adult females 37). Cytochrome P450 isoenzymes are characterized by the specificity of their substrates. CYP2E1 was one of the major P450 forms in benzene and trichloroethylene metabolism, whereas CYP2C11/6 was contributed mainly to toluene metabolism 25, 36). Styrene seemed to have relatively high specific activity with one or a few cytochrome P450 isoenzymes and little or no activity with other isozymes. The previous study indicated that CYP2C11/6, CYP2E1, CYP2B1/2 and CYP1A1/2 contributed to the formation of styrene glycol of styrene 18). Apart from CYP2E1 or CYP2C11/6, the female-specific CYP2C12 is developmentally induced in female but not in male rat liver, reaching its adult level by 8 wk of age 37). The major form in the metabolism of styrene in rat lung was CYP2B1/2, which probably corresponds to CYP2B1 40). Rat CYP2B1 was one of the most active of the P450s in catalyzing formation of styrene glycol: the activity was almost doubled that of human CYP2B6 41). Further studies are required to determine the precise mechanisms of styrene oxidation including minor pathways by cytochrome P450 isozymes such as CYP2E1, CYP2C11/6/12, CYP2B1/2 and CYP2F2 during pregnancy according to age, and to evaluate the reproductive toxicity of each metabolite generated by

6 54 J Occup Health, Vol. 47, 2005 P450 isozymes, in order to elucidate how maternal changes in styrene metabolism influence neurochemical levels in newborn rat brain and the neurobehavioral development and activity of offspring. In conclusion, pregnancy clearly influences the metabolism of styrene as well as other characteristic factors such as age and sex. It is very important to elucidate the changes in specific P450 isozyme composition, and not just in the total content of P450, related to these characteristic modifications and in their affinity for chemicals. Acknowledgments: This study was supported in part by Grants-in-aid from the Japan Ministry of Health, Labour and Welfare. References 1) JA Bond: Review of the toxicology of styrene. CRC Crit Rev Toxicol 19, (1989) 2) RR Miller, R Newhook and A Poole: Styrene production, use, and human exposure. Crit Rev Toxicol 24 (Suppl.), S1 S10 (1994) 3) PG Murphy, DA Macdonard and TD Lickly: Styrene migration from general-purpose and high-impact polystyrene into food-stimulating solvents. Food Chem Toxicol 30, (1992) 4) DH Phillips and PB Farmer: Evidence for DNA and protein binding by styrene and styrene oxide. Crit Rev Toxicol 24 (Suppl.), S35 S46 (1994) 5) K Yeowell-O Connell, W Pauwels, M Severi, Z Jin, MR Walker, SM Rappaport and H Veulemans: Comparison of styrene-7,8-oxide adducts formed via reaction with cysteine, N-terminal valine and carboxylic acid residues in human, mouse and rat hemoglobin. Chem Biol Interact 106, (1997) 6) W Lijinski: Rat and mouse forestomach tumors induced by chronic oral administration of styrene oxide. J Natl Cancer Inst 77, (1986) 7) B Conti, C Maltoni, G Perino and A Ciliberti: Longterm carcinogenicity bioassays on styrene administered by inhalation, ingestion and injection and styrene oxide administered by ingestion in Sprague-Dawley rats, and para-methylstyrene administered by ingestion in Sprague-Dawley rats and mice. Ann New York Acad Sci 534, (1988) 8) V Ponomarkov, J Cabral, J Wahrendorf and D Galendo: A carcinogenicity study of styrene-7,8-oxide in rats. Cancer Lett 24, (1984) 9) SJ Sumner and TR Fennell: Review of the metabolic fate of styrene. Crit Rev Toxicol 24 (Suppl.), S11 S33 (1994) 10) GP Carlson, AA Perez Rivera and NA Mantick. Metabolism of the styrene metabolite 4-vinylphenol by rat and mouse liver and lung. J Toxicol Environ Health 63, (2001) 11) GP Carlson: Effect of the inhibition of the metabolism of 4-vinylphenol on its hepatotoxicity and pneumotoxicity in rats and mice. Toxicology 179, (2002) 12) PJ Boogaard, KP de Kloe, SCJ Sumner, PA van Elburg and BA Wong: Disposition of [ring-u- 14 C]styrene in rats and mice exposed by recirculating nose-only inhalation. Toxicol Sci 58, (2000) 13) M Adesnic and M Atchison: Genes for cytochrome P- 450 and their reguration. CRC Crit Rev Biochem 19, (1986). 14) T Nakajima, RS Wang, E Elovaara, FJ Gonzalez, HV Gelboin, H Vainio and T Aoyama: CYP2C11 and CYP2B1 are major cytochrome P450 forms involved in styrene oxidation in liver and lung microsomes from untreated rats, respectively. Biochem Pharmacol 48, (1994) 15) H Kim, RS Wang, E Elovaara, H Raunio, O Pelkonen, T Aoyama, H Vainio and T Nakajima: Cytochrome P450 isozymes responsible for the metabolism of toluene and styrene in human liver microsomes. Xenobiotica 27, (1997) 16) MAM Wenker, S Kezic, AC Monster and FA De Wolff: Metabolism of styrene in the human liver in vitro: interindividual variation and enantioselectivity. Xenobiotica 31, (2001) 17) FP Guengerich, DH Kim and M Iwasaki: Role of human cytochrome P-450 IIE1 in the oxidation of many low molecular weight cancer suspects. Chem Res Toxicol 4, (1991) 18) T Nakajima, E Elovaara, FJ Gonzalez, HV Gelboin, H Raunio, O Pelkonen, H Vainio and T Aoyama: Styrene metabolism by cdna-expressed human hepatic and pulmonary cytochromes P450. Chem Res Toxicol 7, (1994) 19) R Kishi, Y Katakura, T Ikeda, BQ Chen and H Miyake: Neurochemical effects in rats following gestational exposure to styrene. Toxicol Lett 63, (1992) 20) R Kishi, BQ Chen, Y Katakura, T Ikeda and H Miyake: Effect of prenatal exposure to styrene on the neurobehavioral development, activity, motor coordination, and learning behavior of rats. Neurotoxicol Teratol 17, (1995) 21) Y Katakura, R Kishi, T Ikeda and H Miyake: Effects of prenatal exposure to styrene on neurochemical levels in rat brain. Toxicol Lett 105, (1999) 22) Y Katakura, R Kishi, T Ikeda and H Miyake: Effects of prenatal styrene exposure on postnatal development and brain serotonin and catecholamine levels in rats. Environ Res 85, (2001) 23) OH Lowry, NJ Rosebrough, AL Farr and RJ Randall: Protein measurement with the folin phenol reagent. J Biol Chem 193, (1951) 24) T Omura and R Sato: The carbon monoxide-binding pigment of liver microsomes. 1. Evidence for its hemoprotein nature. J Biol Chem 239, (1964) 25) T Nakajima, RS Wang, Y Katakura, R Kishi, E Elovaara, SS Park, HV Gelboin and H Vainio: Sex, age-pregnancy-induced changes in the metabolism of toluene and trichloroethylene in rat liver in relation to the regulation of cytochrome P450IIE1 and P450IIC11 content. J Pharmacol Exp Ther 261, (1992)

7 Reiko KISHI, et al.: Effects of Pregnancy, Age and Sex in Rat Liver Metabolism 55 26) D Koehler and C Milstein: Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 265, (1975) 27) SS Park, DJ Waxman, DP Lapenson, JB Schenkman and HV Gelboin: Monoclonal antibodies to rat liver cytochrome P-450 2c/RLM5 that regiospecifically inhibit steroid metabolism. Biochem Pharmacol 38, (1989) 28) DJ Waxman, DP Lapenson, SS Park, C Attisand and HV Gelboin: Monoclonal antibodies inhibitory to rat hepatic cytochromes P-450, P-450 from specificities and use as probes for cytochrome P-450-dependent steroid hydroxylation. Mol Pharmacol 32, (1978) 29) IY Ko, SS Park, BJ Song, C Patten, Y Tan, YC Hah, CS Yang and HV Gelboin: Monoclonal antibodies to ethanol-induced rat liver cytochrome P-450 that metabolized aniline and nitrosamines. Cancer Res 47, (1987) 30) GH Lambert, HW Lietz and AN Kotake: Effects of pregnancy on the cytochrome P-450 system in mice. Biochem Pharmacol 36, (1987) 31) JP Casazza, DH Sohn, KS Park and Song BJ: Serum acetone and liver acetone monooxygenase activity in pregnant rats, fetuses, and neonates: reversible pretranslational reduction of cytochrome P450IIE1 (P450IIE1) during pregnancy. Arch Biochem Biophys 309, (1994) 32) AM Symons, RG Turcan and DV Parke: Hepatic microsomal drug metabolism in the pregnant rat. Xenobiotica 12, (1982) 33) OM Bakke and RR Scheline: Hydroxylation of aromatic hydrocarbons in the rat. Toxicol Appl Pharmacol 16, (1970) 34) P Pfaffli, A Hesso, H Vainio and M Hyvonen: 4- Vinylphenol excretion suggestive of arene oxide formation in workers occupationally exposed to styrene. Toxicol Appl Pharmacol, 60, (1981) 35) GL Foureman, C Harris, FP Guengerich and JR Bend: Stereoselectivity of styrene oxidation in microsomes and in purified cytochrome P-450 enzymes from rat liver. J Pharmacol Exp Ther 248, (1989) 36) T Nakajima, RS Wang, E Elovaara, SS Park, HV Gelboin and H Vainio: Cytochrome P450-related differences between rats and mice in the metabolism of benzene, toluene and trichloroethylene in liver microsomes. Biochem Pharmacol 45, (1993) 37) DJ Waxman, GA Dannan and FP Guengerich: Regulation of rat hepatic cytochrome P-450, agedependent expression, hormonal imprinting, and xenobiotic inducibility of sex-specific isoenzymes. Biochemistry 24, (1985) 38) T Kamataki, M Shimada, K Maeda and R Kato: Pituitary regulation of sex-specific forms of cytochrome P-450 in liver microsomes of rats. Biochem Biophys Res Commun, 130, (1985) 39) T Nakajima, RS Wang, E Elovaara, SS Park, HV Gelboin, E Hietanen and H Vainio: Monoclonal antibody-directed characterization of cytochrome P450 isozymes responsible for toluene metabolism in rat liver. Biochem Pharmacol, 41, (1991) 40) CJ Omiecinski: Tissue-specific expression of rat mrnas homologous to cytochromes P-450b and P- 450e. Nucleic Acids Res, 14, (1986) 41) T Nakajima, E Elovaara, FJ Gonzalez, HV Gelboin, H Vainio and T Aoyama: Characterization of the human cytochrome P450 isozymes responsible for styrene metabolism. IARC Sci Publ, 127, (1993)