Xenobiotica the fate of foreign compounds in biological systems
ISSN: 0049-8254 (Print) 1366-5928 (Online) Journal homepage: http://www.tandfonline.com/loi/ixen20
Metabolism of inhaled styrene in acetone-, phenobarbital- and 3-methylcholanthrenepretreated rats: Stimulation and stereochemical effects by induction of cytochromes P450IIE1, P450IIB and P450IA E. Elovaaraf, K. Engström, T. Nakajima, S. S. Parkii, H. V. Gelboin & H. Vainioj To cite this article: E. Elovaaraf, K. Engström, T. Nakajima, S. S. Parkii, H. V. Gelboin & H. Vainioj (1991) Metabolism of inhaled styrene in acetone-, phenobarbital- and 3-methylcholanthrenepretreated rats: Stimulation and stereochemical effects by induction of cytochromes P450IIE1, P450IIB and P450IA, Xenobiotica, 21:5, 651-661 To link to this article: http://dx.doi.org/10.3109/00498259109039505
Published online: 27 Aug 2009.
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Date: 05 November 2015, At: 14:10
XENOBIOTICA,
1991, VOL. 21,
NO.
5 , 651-661
Downloaded by [Australian National University] at 14:10 05 November 2015
Metabolism of inhaled styrene in acetone-, phenobarbital- and 3-methylcholanthrene-pretreated rats: stimulation and stereochemical effects by induction of cytochromes P450IIE1, P450IIB and P450IA E. ELOVAARAtj, K. ENGSTROMj, T. NAKAJIMAg, S. S. PARKII, H. V. GELBOINll and H. VAINIOjT $ Institute of Occupational Health, Topeliuksenkatu 41 A a, SF-00250 Helsinki, Finland
0 Department of Hygiene, Shinshu University, School of Medicine,
Matsumoto, 390, Japan 11 Laboratory of Molecular Carcinogenesis, National Cancer Institute, Bethesda, Maryland 20892, USA
Received 15 July 1990; accepted 8 December 1990 1. The effect of various cytochrome P-450 inducers, namely acetone, phenobarbital (PB) and 3-methylcholanthrene (MC), on the pharmacokinetics of styrene metabolism was studied. 2. Styrene metabolism in vivo was studied measuring phenylglyoxylic acid (PGA), the enantiomers of mandelic acid (MA), and total thioethers excreted in the urine during a 24 h period of airborne exposure to styrene at 500 cm3/m3(2100mglm’). In acetone-pretreated rats, PGA and MA and thioether formation were elevated 3040%. The R/Sratio of MA enantiomers was about two in all styrene-exposed groups except PB-pretreated rats, which showed a ratio of four. 3. Styrene metabolism in liver microsomes measured in oitro was increased by styrene 140%, acetone plus styrene by 190%, methylcholanthrene plus styrene by 180% and phenobarbital plus styrene by 250%. 4. N-Nitrosodimethylamine demethylation (NDMAD) and 7-pentoxyresorufin dealkylation (PROD) in liver microsomes were enhanced 100-1 50% by styrene inhalation. The metabolism of 7-ethoxyresorufin was not significantly enhanced. 5. Monoclonal antibodies to P-450 IAl, IA2, IIBl and I I E l were utilized to identify cytochrome P-450s by Western blot analysis. These studies showed clearly that styrene inhalation induced principally cytochrome P450IE1, whereas styrene given by gavage at a high narcotic dosage induced both P450IIE1 (NDMAD, 60%)and P450IIB (PROD, 3000%). 6. Our conclusions are that styrene metabolism in Vim is both autoinduced and induced by other foreign compounds, that cytochrome P450IIE1 induction has a major impact on styrene metabolism and that P450IIB1 induction yields an altered MA metabolite enantiomer ratio.
Introduction Styrene (ethenylbenzene) is widely used in the production of plastics, resins and synthetic rubber. Although the pharmacokinetics and health effects of styrene have been extensively studied (IPCS 1983, Vainio et al. 1984, Bond 1989), the biological effects related to stereoselective biotransformation are still poorly understood Author to whom correspondence should be addressed.
7 Present address: International Agency for Research on Cancer, 150 Cours Albert-Thomas, F-69372 Lyon Cedex 08, France. 0049-8254/91 t 3 9 0
0 1991 Taylor & Francis Ltd.
Downloaded by [Australian National University] at 14:10 05 November 2015
652
E. Elovaara et al.
(Delbressine 1981, Korn et al. 1987, Vermeulen 1987, Drummond et al. 1989, Foureman et al. 1989). A cytochrome P-450 enzyme system is required for the initial oxidation of styrene into styrene-7,8-oxide, the first chiral intermediate of styrene metabolism (Foureman et al. 1989). Styrene-7,8-oxide is also a potentially toxic intermediate and has been classified by IARC (1987) as an animal carcinogen. It is hydrated to phenylethylene glycol by epoxide hydrolase or conjugated by glutathione S-transferases. Upon conjugation, tissue glutathione is consumed, and thioethers (mercapturic acids) are formed and excreted in the urine (Steele et al. 1981, Delbressine et al. 1981). The biotransformation of phenylethylene glycol into mandelic acid or phenylglyoxylic acid is of occupational health interest since they are the metabolites monitored in the urine of styrene-exposed workers (Engstrom 1984, Bartolucci et al. 1985, Korn et af. 1987). It is apparent that multiple forms of the cytochrome P-450 enzyme family are catalytically involved (Foureman et al. 1989) and the specificity of each cytochrome P-450 may contribute to interindividual variation in the formation of specific metabolites and their toxicity. We have studied the effects of different types of cytochrome P-450 isozyme inducers on the metabolic handling of inhaled styrene. We report that acetone, phenobarbital and 3-methylcholanthrene, model inducers of distinct P-450 isozymes (Nebert et a1 1989), affect differentially the metabolism and urinary elimination of styrene. We also immunochemically characterized with monoclonal antibodies the hepatic profile of cytochrome P-450s induced by styrene. Materials and methods Chemicals Styrene (>99% purity by g.1.c.) was purchased from Fluka AG (Buchs, Switzerland), S(+)-and R( -)-mandelic acids from Merck Suchardt, (Darmstadt, Germany). All other chemicals were of the highest quality commercially available. Animal treatment Male Han/Wistar rats (from the National Laboratory Animal Centre, Kuopio, Finland), housed in cages with aspen wood shavings as bedding, were divided by pretreatment into four subgroups: untreated, acetone- and phenobarbital- (PB)-induced rats (given 1% acetone of 0.1% PB in the drinking water for 1 week) and 3-methylcholanthrene (MC)-induced rats (20mg/2 ml olive oil per kg by gavage on the 7th. 5th and 3rd days before the onset afstyrene exposure). Acetone and PB treatments were discontinued 18 h and 24 h prior to exposure, respectively. Exposure to styrene took place in a dynamic exposure chamber (size 1 0 8 m3) equipped with stainless steel metabolic cages with access to 2% sucrose water, as described earlier (Elovaara et al. 1990). Styrene vapour (volatilized by air streaming through the liquid solvent in a gas-wash bottle placed in a water-bath, 45°C) was mixed into the main stream of ambient air flowing into the chamber. T h e pumps, which were feeding air through the liquid styrene, were regulated by a Power Integrate Derivative controller (type 070, Eurotherm Ltd, Sussex, UK) based on the feedback circuit signal from a infrared analyser (Miran IA, Wilks Scientific Corp., USA), which was used for continuous monitoring of styrene concentrations in chamber air at the wavelength 11.1 p n . Chamber air exchange rate was 6/h, and short-term deviations from nominal concentrations were less than f5%. Six animals (2945 15g) were exposed to ambient air (sham-treated) and 18(299 f 13g) to 500cm3/m3 (2100mg/m3)styrene vapour for a single period of 24 h. The animals received tap water and rat chow (R3, Ewos AB, Sweden) ad libitum,except during the period of styrene exposure. The light-dark cycle was 12 h/12 h, the temperature 23°C and humidity about 58%. Urine samples were collected over 24 h (during the exposure) and stored frozen (-20") until analysis of styrene metabolites. The rats were anaesthetized by CO, asphyxiation and killed within 1 h after the end of the 24 h styrene exposure, autopsied, injected with heparin ( 5 0 0 i.u.) in vena cava and perfused with cold phosphatebuffered saline (PBS, Dulbecco, Orion Diagnostica, Espoo, Finland) to clear the liver, lungs and kidneys from blood before they were removed, weighed and stored at -70°C for biochemical analyses. Twenty-five male Han/Wistar rats (281k78g) were divided into five subgroups of which four (untreated, acetone, PB and MC) received the above-described pretreatments and the fifth subgroup was
Acetone-stimulated metabolism of styrene
653
given liquid styrene in olive oil (1 : 1, v/v) by gavage (l.Oml/kg per day 1,1.5 ml/kg per day 2 , 2 0 mljkg per day 3) and killed 24 h after the last treatment. Note that 2 ml/kgstyrene was narcotic, and that the rats were killed at a time point corresponding to the onset of the 24 h styrene inhalation exposure to take the livers for isolation of reference microsomes for enzyme activity and Western blot analyses.
Downloaded by [Australian National University] at 14:10 05 November 2015
Urine analyses The enantiomeric composition of mandelic acid in urine was analysed by the g.1.c. method of Korn et al. (1984) which allowed, from the same chromatographic runs, simultaneous quantification of phenylglyoxylic acid. Thioethers were analysed from 0 1 4 5 ml aliquots of urine according to the procedure of van Doorn et al. (1980). The assay is based on the ethyl acetate extraction of acidified samples, alkaline hydrolysis, and spectrophotometric quantification of the -SH equivalents by the Ellman reaction. N-Acetylcysteine was used as a standard (molar absorption coefficient was 14mM-' x cm-') and no correction was made for recovery. Biochemical assays The liver, lungs and kidneys were homogenized in cold 0.15 M KC1-0.05 M K-phosphate buffer (pH 7.4) to measure non-protein-SH groups in tissue homogenates by the method of Saville (1958). Liver microsomal and cytosolic fractions were isolated by differential centrifugation (12,000 g for 20 min, 105,OOOg for 1 h). Microsomes were washed with the isolation buffer (105,OOOg, 1 h) and then resuspended by homogenization in buffer containing 20% glycerol, and stored at -70°C. The concentrations of protein (Lowry et al. 1951), and cytochrome P-450 (Omura and Sato 1964), were assayed. The microsomal activities of 7-ethoxyresorufin 0-deethylase (EROD) (Prough et al. 1978) and 7-pentoxyresorufin 0-dealkylase (PROD) (Lubet et al. 1985) were kinetically recorded at 1 PM final concentrations with fluorometric assay methods. The activity of N-nitrosodimethylamine N-demethylase (NDMAD) was assayed at 5 m M final concentration according to the method of Imai et al. (1966). The microsomal rate of styrene metabolism in witro (styrene concentration 8 7 p ~ 37°C) , was measured with head-space g.1.c. as described for the substrate disappearance method of Sato and Nakajima (1979).
Western blot analysis Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and immunoblotting conditions of liver microsomes were as described earlier (Nakajima et al. 1989). We used three monoclonal antibodies (MAb) which have been generated (at the US National Cancer Institute Laboratory of Molecular Carcinogenesis, Bethesda, MD, USA) against purified rat liver cytochromes P-450: MAb 1-7-1pl2 against MC-inducible cytochrome P450IA (Park et al. 1982), MAb 2-66-3-1-56 against PB-inducible P450IIB (Park et al. 1984) and MAb 1-98-1-1-2 against ethanol-inducible P450IIE1 (KOet al. 1987). Statistics The statistical significance between experimental groups was calculated with Student's t-test.
Results Changes in cytochrome P-450-dependent drug-metabolizing enzyme activities caused by styrene and other inducers are shown in table 1. Styrene metabolism in witro by liver microsomes was greatly increased after a 24 h period of styrene inhalation both in untreated rats (140%) and in rats pretreated with acetone (190%), phenobarbital (PB) (250%) or 3-methylcholanthrene (MC) (180%). T h e activity of N-nitrosodimethylamine N-demethylase (NDMAD) was elevated about 100% after styrene inhalation. A similar increase was observed in rats also receiving acetone or MC. Pretreatment of rats with PB prior to styrene prevented induction of monooxygenase responsible for N-nitrosodimethylamine metabolism despite inhalation of styrene. T h e activity of 7-pentoxyresorufin 0-dealkylase (PROD) was enhanced about 150% by styrene inhalation in rats also receiving acetone, MC, or no other treatment whereas after treatment with PB plus styrene the increase was 700%. 7-Ethoxyresorufin 0-deethylase (EROD) was not significantly increased (20%) by styrene alone. Acetone, PB and M C pretreatment increased the EROD activity significantly over control (50-2500/,) and also over the styrene-pretreated rats. A
+ +
+ +
1.42f0.44 3.42 +071*+* 493 +0.94***tt 4.09+0.31*** 4.00 f0.06***
Styrene metabolism in liver microsomes (nmol/min per mg protein) 586f 77 S62f 78 961 fES***ttt 616+76 555+84
Cytochrome P-450 (pmol/mg protein)
PROD
+
EROD
43Sf78 23+10 195+81 890 f83*** 57 f 18 232+57 500+39ttt 183&46***ttt 707+81***ttt 895+149++* 44+14* 301 29*t 1038+138*** 39+21 394+81*+*tt
NDMAD
Monooxygenases (pmollmin per mg protein)
Styrene vapour was S00cm’/m3 (2100mg/m3) for 24h. Values are the means & S D of 4-6 rats. PB, phenobarbital; MC, 3-methylcholanthrene; NDMAD, N-nitrosodimethylamine demethylase; PROD, 7-pentoxyresorufin 0-depentylase; EROD, 7-ethoxyresorufin 0-deethylase. Difference from (*, *+,***) shamexposed controls and (t, t t , t t t ) styrene alone exposed rats: P
ISSN: 0049-8254 (Print) 1366-5928 (Online) Journal homepage: http://www.tandfonline.com/loi/ixen20
Metabolism of inhaled styrene in acetone-, phenobarbital- and 3-methylcholanthrenepretreated rats: Stimulation and stereochemical effects by induction of cytochromes P450IIE1, P450IIB and P450IA E. Elovaaraf, K. Engström, T. Nakajima, S. S. Parkii, H. V. Gelboin & H. Vainioj To cite this article: E. Elovaaraf, K. Engström, T. Nakajima, S. S. Parkii, H. V. Gelboin & H. Vainioj (1991) Metabolism of inhaled styrene in acetone-, phenobarbital- and 3-methylcholanthrenepretreated rats: Stimulation and stereochemical effects by induction of cytochromes P450IIE1, P450IIB and P450IA, Xenobiotica, 21:5, 651-661 To link to this article: http://dx.doi.org/10.3109/00498259109039505
Published online: 27 Aug 2009.
Submit your article to this journal
Article views: 8
View related articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ixen20 Download by: [Australian National University]
Date: 05 November 2015, At: 14:10
XENOBIOTICA,
1991, VOL. 21,
NO.
5 , 651-661
Downloaded by [Australian National University] at 14:10 05 November 2015
Metabolism of inhaled styrene in acetone-, phenobarbital- and 3-methylcholanthrene-pretreated rats: stimulation and stereochemical effects by induction of cytochromes P450IIE1, P450IIB and P450IA E. ELOVAARAtj, K. ENGSTROMj, T. NAKAJIMAg, S. S. PARKII, H. V. GELBOINll and H. VAINIOjT $ Institute of Occupational Health, Topeliuksenkatu 41 A a, SF-00250 Helsinki, Finland
0 Department of Hygiene, Shinshu University, School of Medicine,
Matsumoto, 390, Japan 11 Laboratory of Molecular Carcinogenesis, National Cancer Institute, Bethesda, Maryland 20892, USA
Received 15 July 1990; accepted 8 December 1990 1. The effect of various cytochrome P-450 inducers, namely acetone, phenobarbital (PB) and 3-methylcholanthrene (MC), on the pharmacokinetics of styrene metabolism was studied. 2. Styrene metabolism in vivo was studied measuring phenylglyoxylic acid (PGA), the enantiomers of mandelic acid (MA), and total thioethers excreted in the urine during a 24 h period of airborne exposure to styrene at 500 cm3/m3(2100mglm’). In acetone-pretreated rats, PGA and MA and thioether formation were elevated 3040%. The R/Sratio of MA enantiomers was about two in all styrene-exposed groups except PB-pretreated rats, which showed a ratio of four. 3. Styrene metabolism in liver microsomes measured in oitro was increased by styrene 140%, acetone plus styrene by 190%, methylcholanthrene plus styrene by 180% and phenobarbital plus styrene by 250%. 4. N-Nitrosodimethylamine demethylation (NDMAD) and 7-pentoxyresorufin dealkylation (PROD) in liver microsomes were enhanced 100-1 50% by styrene inhalation. The metabolism of 7-ethoxyresorufin was not significantly enhanced. 5. Monoclonal antibodies to P-450 IAl, IA2, IIBl and I I E l were utilized to identify cytochrome P-450s by Western blot analysis. These studies showed clearly that styrene inhalation induced principally cytochrome P450IE1, whereas styrene given by gavage at a high narcotic dosage induced both P450IIE1 (NDMAD, 60%)and P450IIB (PROD, 3000%). 6. Our conclusions are that styrene metabolism in Vim is both autoinduced and induced by other foreign compounds, that cytochrome P450IIE1 induction has a major impact on styrene metabolism and that P450IIB1 induction yields an altered MA metabolite enantiomer ratio.
Introduction Styrene (ethenylbenzene) is widely used in the production of plastics, resins and synthetic rubber. Although the pharmacokinetics and health effects of styrene have been extensively studied (IPCS 1983, Vainio et al. 1984, Bond 1989), the biological effects related to stereoselective biotransformation are still poorly understood Author to whom correspondence should be addressed.
7 Present address: International Agency for Research on Cancer, 150 Cours Albert-Thomas, F-69372 Lyon Cedex 08, France. 0049-8254/91 t 3 9 0
0 1991 Taylor & Francis Ltd.
Downloaded by [Australian National University] at 14:10 05 November 2015
652
E. Elovaara et al.
(Delbressine 1981, Korn et al. 1987, Vermeulen 1987, Drummond et al. 1989, Foureman et al. 1989). A cytochrome P-450 enzyme system is required for the initial oxidation of styrene into styrene-7,8-oxide, the first chiral intermediate of styrene metabolism (Foureman et al. 1989). Styrene-7,8-oxide is also a potentially toxic intermediate and has been classified by IARC (1987) as an animal carcinogen. It is hydrated to phenylethylene glycol by epoxide hydrolase or conjugated by glutathione S-transferases. Upon conjugation, tissue glutathione is consumed, and thioethers (mercapturic acids) are formed and excreted in the urine (Steele et al. 1981, Delbressine et al. 1981). The biotransformation of phenylethylene glycol into mandelic acid or phenylglyoxylic acid is of occupational health interest since they are the metabolites monitored in the urine of styrene-exposed workers (Engstrom 1984, Bartolucci et al. 1985, Korn et af. 1987). It is apparent that multiple forms of the cytochrome P-450 enzyme family are catalytically involved (Foureman et al. 1989) and the specificity of each cytochrome P-450 may contribute to interindividual variation in the formation of specific metabolites and their toxicity. We have studied the effects of different types of cytochrome P-450 isozyme inducers on the metabolic handling of inhaled styrene. We report that acetone, phenobarbital and 3-methylcholanthrene, model inducers of distinct P-450 isozymes (Nebert et a1 1989), affect differentially the metabolism and urinary elimination of styrene. We also immunochemically characterized with monoclonal antibodies the hepatic profile of cytochrome P-450s induced by styrene. Materials and methods Chemicals Styrene (>99% purity by g.1.c.) was purchased from Fluka AG (Buchs, Switzerland), S(+)-and R( -)-mandelic acids from Merck Suchardt, (Darmstadt, Germany). All other chemicals were of the highest quality commercially available. Animal treatment Male Han/Wistar rats (from the National Laboratory Animal Centre, Kuopio, Finland), housed in cages with aspen wood shavings as bedding, were divided by pretreatment into four subgroups: untreated, acetone- and phenobarbital- (PB)-induced rats (given 1% acetone of 0.1% PB in the drinking water for 1 week) and 3-methylcholanthrene (MC)-induced rats (20mg/2 ml olive oil per kg by gavage on the 7th. 5th and 3rd days before the onset afstyrene exposure). Acetone and PB treatments were discontinued 18 h and 24 h prior to exposure, respectively. Exposure to styrene took place in a dynamic exposure chamber (size 1 0 8 m3) equipped with stainless steel metabolic cages with access to 2% sucrose water, as described earlier (Elovaara et al. 1990). Styrene vapour (volatilized by air streaming through the liquid solvent in a gas-wash bottle placed in a water-bath, 45°C) was mixed into the main stream of ambient air flowing into the chamber. T h e pumps, which were feeding air through the liquid styrene, were regulated by a Power Integrate Derivative controller (type 070, Eurotherm Ltd, Sussex, UK) based on the feedback circuit signal from a infrared analyser (Miran IA, Wilks Scientific Corp., USA), which was used for continuous monitoring of styrene concentrations in chamber air at the wavelength 11.1 p n . Chamber air exchange rate was 6/h, and short-term deviations from nominal concentrations were less than f5%. Six animals (2945 15g) were exposed to ambient air (sham-treated) and 18(299 f 13g) to 500cm3/m3 (2100mg/m3)styrene vapour for a single period of 24 h. The animals received tap water and rat chow (R3, Ewos AB, Sweden) ad libitum,except during the period of styrene exposure. The light-dark cycle was 12 h/12 h, the temperature 23°C and humidity about 58%. Urine samples were collected over 24 h (during the exposure) and stored frozen (-20") until analysis of styrene metabolites. The rats were anaesthetized by CO, asphyxiation and killed within 1 h after the end of the 24 h styrene exposure, autopsied, injected with heparin ( 5 0 0 i.u.) in vena cava and perfused with cold phosphatebuffered saline (PBS, Dulbecco, Orion Diagnostica, Espoo, Finland) to clear the liver, lungs and kidneys from blood before they were removed, weighed and stored at -70°C for biochemical analyses. Twenty-five male Han/Wistar rats (281k78g) were divided into five subgroups of which four (untreated, acetone, PB and MC) received the above-described pretreatments and the fifth subgroup was
Acetone-stimulated metabolism of styrene
653
given liquid styrene in olive oil (1 : 1, v/v) by gavage (l.Oml/kg per day 1,1.5 ml/kg per day 2 , 2 0 mljkg per day 3) and killed 24 h after the last treatment. Note that 2 ml/kgstyrene was narcotic, and that the rats were killed at a time point corresponding to the onset of the 24 h styrene inhalation exposure to take the livers for isolation of reference microsomes for enzyme activity and Western blot analyses.
Downloaded by [Australian National University] at 14:10 05 November 2015
Urine analyses The enantiomeric composition of mandelic acid in urine was analysed by the g.1.c. method of Korn et al. (1984) which allowed, from the same chromatographic runs, simultaneous quantification of phenylglyoxylic acid. Thioethers were analysed from 0 1 4 5 ml aliquots of urine according to the procedure of van Doorn et al. (1980). The assay is based on the ethyl acetate extraction of acidified samples, alkaline hydrolysis, and spectrophotometric quantification of the -SH equivalents by the Ellman reaction. N-Acetylcysteine was used as a standard (molar absorption coefficient was 14mM-' x cm-') and no correction was made for recovery. Biochemical assays The liver, lungs and kidneys were homogenized in cold 0.15 M KC1-0.05 M K-phosphate buffer (pH 7.4) to measure non-protein-SH groups in tissue homogenates by the method of Saville (1958). Liver microsomal and cytosolic fractions were isolated by differential centrifugation (12,000 g for 20 min, 105,OOOg for 1 h). Microsomes were washed with the isolation buffer (105,OOOg, 1 h) and then resuspended by homogenization in buffer containing 20% glycerol, and stored at -70°C. The concentrations of protein (Lowry et al. 1951), and cytochrome P-450 (Omura and Sato 1964), were assayed. The microsomal activities of 7-ethoxyresorufin 0-deethylase (EROD) (Prough et al. 1978) and 7-pentoxyresorufin 0-dealkylase (PROD) (Lubet et al. 1985) were kinetically recorded at 1 PM final concentrations with fluorometric assay methods. The activity of N-nitrosodimethylamine N-demethylase (NDMAD) was assayed at 5 m M final concentration according to the method of Imai et al. (1966). The microsomal rate of styrene metabolism in witro (styrene concentration 8 7 p ~ 37°C) , was measured with head-space g.1.c. as described for the substrate disappearance method of Sato and Nakajima (1979).
Western blot analysis Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and immunoblotting conditions of liver microsomes were as described earlier (Nakajima et al. 1989). We used three monoclonal antibodies (MAb) which have been generated (at the US National Cancer Institute Laboratory of Molecular Carcinogenesis, Bethesda, MD, USA) against purified rat liver cytochromes P-450: MAb 1-7-1pl2 against MC-inducible cytochrome P450IA (Park et al. 1982), MAb 2-66-3-1-56 against PB-inducible P450IIB (Park et al. 1984) and MAb 1-98-1-1-2 against ethanol-inducible P450IIE1 (KOet al. 1987). Statistics The statistical significance between experimental groups was calculated with Student's t-test.
Results Changes in cytochrome P-450-dependent drug-metabolizing enzyme activities caused by styrene and other inducers are shown in table 1. Styrene metabolism in witro by liver microsomes was greatly increased after a 24 h period of styrene inhalation both in untreated rats (140%) and in rats pretreated with acetone (190%), phenobarbital (PB) (250%) or 3-methylcholanthrene (MC) (180%). T h e activity of N-nitrosodimethylamine N-demethylase (NDMAD) was elevated about 100% after styrene inhalation. A similar increase was observed in rats also receiving acetone or MC. Pretreatment of rats with PB prior to styrene prevented induction of monooxygenase responsible for N-nitrosodimethylamine metabolism despite inhalation of styrene. T h e activity of 7-pentoxyresorufin 0-dealkylase (PROD) was enhanced about 150% by styrene inhalation in rats also receiving acetone, MC, or no other treatment whereas after treatment with PB plus styrene the increase was 700%. 7-Ethoxyresorufin 0-deethylase (EROD) was not significantly increased (20%) by styrene alone. Acetone, PB and M C pretreatment increased the EROD activity significantly over control (50-2500/,) and also over the styrene-pretreated rats. A
+ +
+ +
1.42f0.44 3.42 +071*+* 493 +0.94***tt 4.09+0.31*** 4.00 f0.06***
Styrene metabolism in liver microsomes (nmol/min per mg protein) 586f 77 S62f 78 961 fES***ttt 616+76 555+84
Cytochrome P-450 (pmol/mg protein)
PROD
+
EROD
43Sf78 23+10 195+81 890 f83*** 57 f 18 232+57 500+39ttt 183&46***ttt 707+81***ttt 895+149++* 44+14* 301 29*t 1038+138*** 39+21 394+81*+*tt
NDMAD
Monooxygenases (pmollmin per mg protein)
Styrene vapour was S00cm’/m3 (2100mg/m3) for 24h. Values are the means & S D of 4-6 rats. PB, phenobarbital; MC, 3-methylcholanthrene; NDMAD, N-nitrosodimethylamine demethylase; PROD, 7-pentoxyresorufin 0-depentylase; EROD, 7-ethoxyresorufin 0-deethylase. Difference from (*, *+,***) shamexposed controls and (t, t t , t t t ) styrene alone exposed rats: P
