Reduced synthesis of 5-aminolevulinate dehydratase in styrene-treated rats

Biochimica et Biophysica Acta 867 (1986) 89-96 Elsevier

89

BBA 91586

Reduced synthesis of 5-aminolevulinate dehydratase in styrene-treated rats Hiroyoshi Fujita

Akio Koizumi a,**, Norio Hayashi b and Masayuki Ikeda a

a,.,

a Department of Environmental Health and b Department of Biochemistry, Tohoku University School of Medicine, Sendal 980 (Japan)

(Received December 2nd, 1985)

Key words: 5-Aminolevulinate dehydratase; Porphobilinogen synthase; Styrene; Cell-free translation; (Rat liver)

5-Aminolevulinate dehydratase (porphobilinogen synthase; 5-aminolevulinate hydro-lyase, EC 4.2.1.24) preparations purified from rat liver and erythrocytes are indistinguishable in terms of molecular weight, subunit size, immunoreactivity, amino-acid composition and kinetic properties, suggesting that the enzyme from liver and erythrocytes are identical. Intraperitoneal injection of styrene to rats decreased 5-aminolevulinate dehydratase activity in both erythrocyte (to 8% of the control) and the liver (to 40% of the control). Studies utilizing polysome-directed cell-free translation indicated that hepatic synthesis of the enzyme was inhibited by styrene at the transcriptional level. In vitro addition of styrene 7,8-oxide, a major intermediate of styrene, to purified 5-aminolevulinate dehydratase resulted in a loss of immunoassayable enzyme protein to less than 1% of the untreated control. These findings suggest that the decrease in 5-aminolevulinate dehydratase caused by in vivo treatment of styrene is partially due to a transcription-dependent decrease in the enzyme synthesis, and partially to post-translational alteration of the structure of the enzyme protein. Introduction 5-Aminolevulinate dehydratase (porphobilinogen synthase; 5-aminolevulinate hydro-lyase, EC 4.2.1.24) is the second enzyme of the heme biosynthetic pathway [1]. The enzyme activity is dependent on the essential SH groups in the enzyme protein [2,3], and is inhibited by SH reagents such as silver ions, 1,3-dibromoacetone, 5,5'-dithio[2nitro]benzoic acid and lead ions [2]. Recently, we described that trichloroethylene [4] and bromobenzene [5] also decrease the activity of the en-

* To whom correspondence should be addressed at (present address): The Rockefeller University, 1230 York Avenue, New York, N.Y. 10021, U.S.A. ** Present address: Department of Entomology, College of Natural and Agricultural Sciences, University of California, Riverside, CA 92521, U.S.A.

zyme after metabolic modification by cytochrome P-450-dependent mixed-function oxygenases to form reactive intermediates, which are assumed to bind to the SH groups [4,5]. Styrene, one of the most important industrial materials used in production of polymers, copolymers and reinforced plastics, is also metabolized by cytochrome P450-dependent monooxygenase system, forming styrene 7,8-oxide as a major intermediate [6,7]. In a preliminary study (Fujita, H., Koizumi, A., Furusawa, T. and Ikeda, M., unpublished data), we demonstrated a significant depression of 5-aminolevulinate dehydratase activity with a parallel decrease in the enzyme concentration in men and other animals exposed to styrene vapor. It is possible that styrene decreases the dehydratase concentration and activity after metabolic conversion by cytochrome P-450-dependent monooxygenase system, since styrene 7,8-oxide can covalently bind to proteins and nucleic acids [8,9]; however, little

0167-4781/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)

90 attention has been paid to the mechanism of this decrease in the dehydratase. The present study examines the mechanism of decreased 5-aminolevulinate dehydratase concentration by styrene. We report in this paper that in vivo treatment of rats with styrene decreases the synthesis of the specific m R N A for the enzyme, and that it may also lead to a structural alteration of the enzyme protein with decreased stability.

Experimental Materials 5-Aminolevulinate dehydratase was purified from rat erythrocytes and liver as previously described [2,10]. The purified preparations were homogeneous on gel-filtration chromatography and in analytical polyacrylamide gel electrophoresis both in the absence and presence of sodium dodecyl sulfate. The enzyme preparations were stable for at least 1 month when stored at 4°C after precipitation with a m m o n i u m sulfate (to 55% saturation) in the presence of dithiothreitol and zinc. Prior to the assay, dithiothreitol and zinc were added to ensure that the enzyme was activated as described previously [2,10]. The erythroid enzyme was labeled with ~2Sl (Amersham International, U.K.) as previously reported [10]. The specific radioactivity of the ~25I-labeled enzyme was 6.9/~Ci//~g. Antiserum against the erythroid enzyme was prepared in rabbits and an IgG fractions was purified using a D E A E Affi-Gel blue (Bio-Rad Laboratories, Richmond, CA) column. At a dilution of 1:15000, the IgG preparation bound approx. 50% of the labeled antigen. 5-Aminolevulinic acid hydrochloride was purchased from sigma Chemical Co. (St. Louis, MO), and a goat anti-rabbit lgG serum was obtained from the Medical and Biological Laboratories (Nagoya, Japan). L-[35S]methionine and nucleasetreated rabbit reticulocyte lysates for cell-free protein synthesis were purchased from Amersham International. Other chemicals used were all of analytical grade. Aqueous solutions of styrene 7,8-oxide were prepared as we will report elsewhere. All glassware used was washed with H N O 3 / H C I and rinsed throughly with metal-free

distilled water. Buffer solutions, chemicals, and water were routinely checked for lead and zinc contamination with a flameless and a flame atomic absorption spectrophotometer and, if contaminated, the solutions were discarded. 5-A rninolet, ulinate dehvdratase assay The enzyme activity was assayed both in the absence and presence of 10 mM dithiothreitol and 0.1 mM zinc acetate [2,10]. Unless otherwise stated, the enzyme activity represents the activity after reactivation with dithiothreitol and zinc. One unit of the enzyme activity was defined as 1 p,mol porphobilinogen formed at 37°C per h. To examine the effect of styrene 7,8-oxide on the enzyme in vitro, the activated enzyme (0.9 >M) was incubated with the highest obtainable concentration of styrene 7,8-oxide (17 mM) in 100 mM Tris-acetate buffer (pH 7.1), under N z at 4°C for 16 h. After the incubation, the enzyme was reactivated by treatment with dithiothreitol and zinc at 37°C for 30 min. The mixture was passed through a Sephadex G-50 column (0.75 × 12.0 cm) under N, to remove excess reagents, and the purified enzyme fraction was used to determine the enzyme activity and concentration, and concentration of SH groups. The radioimmunoassay of the enzyme was performed as described previously [4,10]. The specific binding of the antibody with the 1251-labeled enzyme was expressed as the percentage of the radioactivity of the bound fraction to the radioactivity of the same fraction in the absence of unlabeled antigen ( B / B o ). Other assays Amino-acid compositions of 5-aminolevulinate dehydratase preparations from rat erythrocytes and liver, human erythrocytes, and bovine liver were determined with a Hitachi KLA-3B amino acid analyzer after hydrolysis with 6 M HC1 at 110°C for 24 h in evacuated tubes. The concentration of cysteine and tryptophan were determined by the methods of Moore [11] and Bence and Schmidt [12], respectively. Differences in aminoacid composition betwen two enzyme preparations were evaluated using the amino-acid deviation f r o m the rat liver e n z y m e as follows: ]Y~(M

X

)-" / 1 8 ,

where M is the concentration

91 of an amino acid in the rat liver enzyme, and X is the concentration of the same amino acid in another enzyme preparation. The number of sulfhydryl groups in the enzyme was determined by titration with 5,5'-dithio[2-nitro]benzoic acid using the method of Tsukamoto et al. [2]. Protein concentration was determined by the method of Lowry et al. [13].

Chemicals, Uppsala, Sweden). Immunoprecipitable materials were collected, washed and released from protein A-Sepharose to estimate the radioactivity as previously described [15].

Styrene treatment of rats and preparation of polysomes

The purified rat-liver enzyme had a specific

Male Wistar rats, 6 weeks old and weighing 164 + 6.2 g (mean + S.D., n = 8), were divided into the control group (n = 4) and the styrenetreated group (n = 4). The latter group received styrene intraperitoneally in soybean oil, 1400 m g / k g body weight (on day 1), 1500 m g / k g (on day 2), 2000 m g / k g (on day 3), and 2500 m g / k g (on day 4), in a final volume of 2 m l / k g body weight every day, while control animals received the vehicle only. After fasting for 24 h, animals were killed on the fifth day and blood and livers were collected. Total polysomes were prepared from 5 g of the liver as described previously [14]. Polysome pellets were suspended in a solution of 25 mM KC1 and 5 mM MgC12 containing 50 mM Tris-HC1 buffer (pH 7.4) to give a final concentration of 80-150 A260 units/ml.

Results

Comparison of hepatic 5-aminolevulinate dehydratase with the enzyme in erythrocytes

!

a

15 I ,w

7i0

0 i/substrote

I

I

5

i0 (mM-1)

I

b

I

Polysome-directed cell-free protein synthesis Cell-free protein synthesis was carried out with rabbit reticulocyte lysate in the presence of 1 A260 unit of polysome and 19.6 /~Ci L-[35S]methionine (specific radioactivity: 1270 C i / m m o l ) at 30°C for 30 min, using the method of Yamamoto et al. [15]. The reaction was stopped by chilling, followed by the addition of 4 vol. of a solution containing 50 mM Tris-HC1 buffer (pH 7.5), 150 mM NaC1, 5 mM methionine, 5 mM EDTA-Na, 1% Triton X-100, and 4 0 / x g / m l each of antipain, chymostatin, elastatinal, leupeptin and pepstatin (Peptide Institute Inc., Osaka, Japan). A portion (5 /~1) of the mixture was used for the determination of ratioactivity incorporated into total protein. Newly synthesized rat-liver 5-aminolevulinate dehydratase was isolated from an aliquot (320/~1) of the mixture by immunoprecipitation using 5/~1 of specific antibody against the enzyme and 20 mg of protein A-Sepharose CL-4B (Pharmacia Fine

10

E_

i

-5

0 5 1/substrote

!

10 (mM-1)

Fig. 1. Fig. 1. Effect of lead on rat 5-aminolevulinate dehydratase from liver (a) and erythrocytes (b). The activated enzymes were incubated without (©), or with 10 6 M (O), or with 3-10-6 M (11)lead acetate in 100 mM Tris-acetatebuffer (pH 7.1) under N2 for 10 min at 37°C. Each assay contained 19/~g/ml liver enzyme (a) or 22.5 ~tg/ml erythrocyteenzyme (b).

92

~%) i00

O

I

I

I

5O

I

0

10 -2

1

102 (ng)

5-Aminolevul |note dehydratose Fig. 2. Radioimmunoassay of 5-aminolevulinate dehydratase from rat erythrocytes and liver with rabbit IgG versus the rat erythrocyte enzyme. An assay was carried out in the presence of rabbit anti-rat erythrocyte &aminolevulinate dehydratase IgG, 12SI-labeled erythrocyte enzyme and the erythrocyte (O O) or the liver (e . . . . . . O) enzyme in a saline-50 m M phosphate buffer (pH 7.0) containing 0.2% bovine serum albumin.

activity of 23.8 u n i t s / m g protein, which is similar to that of the rat erythrocyte enzyme (25.5.-27.0 units/rag protein) [4,10]. K m values for the liver and the erythrocyte enzyme were 2.44.10 4 M and 2.56.10 4 M , respectively (Fig. 1). Assuming one active site per subunit, maximal turnover numbers were calculated to be 11.7 tool of produ c t s / m i n for the liver enzyme and 12.7 mol of p r o d u c t s / m i n for the erythrocyte enzyme (Fig. 1). Addition of lead, a potent inhibitor of the enzyme, inhibited the activity of both enzyme preparations in a competitive manner with a K~ of 6.02- 10 6 M and 5.94.10 - 6 M , respectively, for the liver and erythrocyte enzyme (Fig. 1). These findings indicate that the kinetic properties of the liver and the erythrocyte enzymes are essentially identical. The apparent molecular weight of both enzymes was 280000 as determined by gel-filtration on Ultrogel AcA 34. The subunit size of both enzymes was 35500 in sodium dodecyl sulfate polyacrylamine slab gel electrophoresis, lmmunochemical determination of the erythrocyte and the liver enzyme showed identical binding for

TABLE I A M I N O - A C I D C O M P O S I T I O N OF T H E LIVER A N D E R Y T H R O C Y T E E N Z Y M E S Data are the means of three analyses. A m i n o acid

5-Aminolevulinate dehydratase liver

Asp Thr Ser Glu Gly Ala Cys/2 Val Met lie Leu Tyr Trp Phe Lys His Arg Pro

erythrocyte

m o l / 3 5 500 g of protein

nearest integer/ 35 500 g of protein

m o l / 3 5 500 g of protein

nearest integer/ 35 500 g of protein

23.6 11.3 15.6 54.4 17.2 36.8 7.8 20.4 8.1 11.6 35.0 5.7 2.9 8.0 18.2 6.3 20.9 16.2

24 11 16 54 17 37 8 20 8 12 35 6 3 8 18 6 21 16

23.6 11.3 15.6 54.3 17.2 36.7 8.1 19.9 8.1 11.8 34.6 5.9 3.1 7.9 18.6 6.3 20.7 15.6

24 11 16 54 17 37 8 20 8 12 35 6 3 8 19 6 21 16

93 TABLE II 5-AMINOLEVULINATE DEHYDRATASE ACTIVITY AFTER IN VIVO TREATMENT WITH STYRENE Animals were received styrene intraperitoneally for 4 days. On the day 5, animals were killed to determine the activity of 5-aminolevulinate dehydratase in the presence and absence of treatment with 10 mM dithiothreitol and 0.1 mM zinc acetate. Values are the mean + S.D. (n = 4). * Difference from the control is significant at P < 0.01.

Erythrocyte enzyme activity ( × 10- 3 unit/ml packed cell) Liver enzyme activity ( x 10 -3 unit/mg protein)

Treatment with dithiothreitol and zinc

Styrene-treated rats

Control rats

+

27.1 +15.8" 16.7 + 12.1 * 5.03+ 0.73* 4.97+ 0.69*

320.5 +67.5 253.4 + 84.6 12.58+ 0.96 11.64+ 1.28

-

+ -

Experimental), 5 - a m i n o l e v u l i n a t e d e h y d r a t a s e activity in e r y t h r o c y t e a n d in the liver was decreased to 8% a n d 40%, respectively, of the c o n t r o l values ( T a b l e II). T a b l e II also indicates that the d i m i n i s h e d enzyme activity was n o t r e s t o r e d b y reactivation with d i t h i o t h r e i t o l a n d zinc. Since it has been shown that there is a high c o r r e l a t i o n b e t w e e n the r e a c t i v a t e d - e n z y m e activity a n d the e n z y m e c o n c e n t r a t i o n in e r y t h r o c y t e s f r o m s t y r e n e - e x p o s e d rats ( r = 0.93, n = 20) (Fujita, H., K o i z u m i , A., F u r u s a w a , T. a n d Ikeda, M., u n p u b lished data), the decrease in the e n z y m e activity o b s e r v e d in erythrocytes a n d in liver in this s t u d y p r o b a b l y also reflects a decrease in the e n z y m e c o n c e n t r a t i o n . T h e b o d y weight of a n i m a l s after t r e a t m e n t with styrene for 4 d a y s was 85 + 6% of the control. N o significant changes were n o t e d for the liver wet weight (7.8 + 0.52 g for c o n t r o l rats vs. 8.3 + 0.50 g for s t y r e n e - t r e a t e d rats), a n d for

b o t h enzymes (Fig. 2). A m i n o acid analysis showed that b o t h the liver a n d the e r y t h r o c y t e enzymes have essentially identical a m i n o acid c o m p o s i t i o n s ( T a b l e I). These studies strongly suggest that the rat-liver a n d the r a t - e r y t h r o c y t e enzymes are identical. C a l c u l a t e d a m i n o acid deviations (see Experim e n t a l ) of the rat-erythrocyte, h u m a n - e r y t h r o c y t e , a n d bovine-live e n z y m e f r o m rat-liver 5-aminolevulinate d e h y d r a t a s e were 0.06, 1.35, a n d 1.67 respectively. This finding indicates that, while the two rat enzymes show little difference in their a m i n o acid c o m p o s i t i o n s , the h u m a n - e r y t h r o c y t e a n d the bovine-liver e n z y m e differ significantly f r o m the rat-liver e n z y m e a n d in this order.

Decrease in 5-aminolevulinate dehydratase activity following styrene administration A f t e r t r e a t m e n t for 4 d a y s with styrene (see TABLE III

EFFECT OF STYRENE TREATMENT IN VIVO ON POLYSOME-DIRECTED CELL-FREE SYNTHESIS OF 5-AMINOLEVULINATE DEHYDRATASE AND TOTAL PROTEIN Total liver polysomes from styrene-treated rats and control rats were incubated with L-[35S]methionine for 30 min at 30°C in a translation system containing nuclease-treated rabbit reticulocyte lysate. * Difference from the control is significant at P < 0.02. Data are the means of two analyses. Styrene-treated rats 5-Aminolevulinate dehydratase (dpm × 103) Total protein (dpm× 10 s ) (A)/(B)

Control rats

No: 1

2

3

4

mean + S.D.

1

2

3

(A)

3.85

3.74

4.08

3.91

-

5.75

5 . 3 2 4 . 7 6 5.61

(B) (%)

1.86 2.07

2.08 1.80

2.13 1.92

2.28 1.72

1.86+0.16 *

2.74 2.10

2 . 2 2 2 . 1 9 2.23 2 . 4 0 2 . 1 7 2.51

4

mean + S.D.

2.29+0.19

94

the total liver protein (0.98 _+ 0.11 g for control vs. 0.96 _+ 0.11 g for the treated animals).

Effect of styrene treatment on the level o[ mRNA for 5-aminolevulinate dehydratase To investigate the mechanism causing the decrease in 5-aminolevulinate dehydratase activity and concentration by styrene administration, polysomes were obtained from livers of both styrene-treated and control rats for polysome-diretted cell-free synthesis studies. The polysome content in 5 g of the liver in the control and in the treated rats were 56.5 _+ 8.8 and 60.8 _+ 13.3 A>0 units, respectively. Polysomes from the styrenetreated rats showed a significant reduction ( P < 0.02) in the synthesis of 5-aminolevulinate dehydratase (Table IlI). This finding indicates that polysomes isolated from the styrene-treated animals had less mRNA for 5 aminolevulinate dehydratase. Effect of styrene 7,8-oxide on 5-aminolevulinate dehydratase in vitro As a previous study suggests that styrene 7,8-oxide rather than styrene reacts with the enzyme in vitro (our unpublished data), 0.9 /~M 5-aminolevulinate dehydratase was incubated with

1

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8

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I

A ~

~_

6

u~

I

4

2

o

0 0

/f i0

20

30

40

Titrotlon time with DTNB (min)

Fig. 3. Titration of SH groups in the enzyme by 5,5'-dithio[2nitro]benzoic acid (DTNB). The enzyme, in 50 mM Tris-acetate buffer (pH 7.1), was incubated with 125/~M DTNB for 30 min, and for another 20 min after the addition of sodium dodecyl sulfate at a final concentration of 0.4% at room temperature. The time of addition of sodium dodecyl sulfate is indicated by the arrow. Calculations of the number of SH groups were made according to Tsukamoto et al. [2]. (a) Activated holo-enzyme; (b) styrene 7,8-oxide-treated enzyme.

17 mM styrene 7,8-oxide. After incubation, the enzyme activity was undetectable and only 0.8C~ of the enzyme protein could be detected by radioimmunoassay. As shown in Fig. 3, tile rat-liver enzyme had 8 tool of SH groups per subunit: however, only 2 mol of SH groups were detected per subunit in the styrene 7,8-oxide-treated enzyme. Styrene 7,8-oxide treatment decreased intraand intersubunit SH groups from 5 and 3 to 0.8 and 1.1, respectively (Fig. 3). However, the styrene 7,8-oxide-treated enzyme showed a homogeneous band in polyacrylamide slab gel electrophoresis in the presence of sodium dodecyl sulfate, whose M,., at 35500, was identical to that of the intact enzyme. Discussion

We have observed a decrease in erythrocyte 5-aminolevulinate dehydratase activity by styrene administration, which reduced the enzyme concentration (Fujita, H., Koizumi, A., Furusawa, T. and Ikeda, M., unpublished data). The reduction in the dehydratase was observed both in rats exposed to 0.21 g / m -~ for 7 days and in men occupationally exposed to the chemical in a fiberreinforced plastic boat plant (our unpublished data), in which type of operation the exposure levels were ranged from 0.14 to 1.09 g / m 3 [16]. The present study indicates that 5-aminolevulinate dehydratase activity was decreased by styrenetreatment not only in rat erythrocytes but also in rat liver, and the decrease in the erythrocyte enzyme (to 8% of the control) was more pronounced than the decrease in the liver enzyme (to 40% of the control) (Table II). This observation is consistent with those of lead poisoning [17] and trichloroethylene poisoning [4], and suggests that the liver can detoxify these hazardous chemicals more rapidly than erythrocytes. According to our previous investigation, a decrease in the reactivated enzyme activity indicates a reduction in the enzyme protein concentration in styrene-treated animals. Liver polysome-directed cell-free synthesis of 5-aminolevulinate dehydratase showed a significant decrease (to 80% of the control, P < 0.02) in the specific mRNA for the enzyme with styrene treatment in comparison to total protein synthesis (Table III). This finding suggests that in

95 vivo styrene treatment leads to an inhibition of enzyme synthesis occurring at the transcriptional level. Identity between the liver and the erythroid enzymes was suggested by molecular weight (280000), subunit size (35 500), immunoreactivity (Fig. 2), amino-acid analysis (Table I), and kinetic properties (Fig. 1). These findings confirm previous reports on the immunochemical identity of 5-aminolevulinate dehydratases in various tissues [17-19], and suggest that this enzyme, while occurring in various tissues, is the product of the same gene. Styrene 7,8-oxide, a major intermediate of styrene metabolism [6,7], may play a significant role in the reduction of enzyme concentration observed after styrene treatment (unpublished data). In vitro studies showed that the addition of styrene 7,8-oxide at a concentration of 17 mM to the purified enzyme preparation (0.9 /~M) decreased both the enzyme activity and immunoassayable protein to less than 1% of the control: this suggests that styrene 7,8-oxide treatment leads to a structural alteration of the enzyme protein which, perhaps, includes the antigenic domain such that the styrene 7,8-oxide-treated enzyme no longer reacts with the specific antibody. The styrene 7,8-oxide-mediated structural alteration probably involves covalent binding of styrene 7,8-oxide to SH groups in the enzyme, since neither enzyme activity or reduced SH groups could be restored by treatment with dithiothreitol and zinc (Fig. 3). Alternatively, the altered enzyme protein might be subject to faster degradation than the unmodified enzyme, leading to a decrease in the enzyme protein. Thus, the present study suggests that the decrease in 5-aminolevulinate dehydratase after styrene treatment in vivo is due both to an inhibition of enzyme synthesis at the transcriptional level and to an alteration in the enzyme conformation. Calculated amino-acid deviations of 5-aminolevulinate dehydratase show a closer similarity between the human enzyme and the rat enzyme than between the bovine enzyme and the rat enzyme. This observation consistent with immunochemical studies of 5-aminolevulinate dehydratase [10,18,20] and the genealogical tree of fl-globin

[211.

Acknowledgements The authors are grateful to Dr. S. Sassa and Dr. R.A. Galbraith (The Rockefeller University, New York) for their interest and fruitful discussions during this study. The amino-acid analysis was kindly performed Ms. A. Kikuchi (Department of Agricultural Chemistry, Tohoku University Faculty of Agriculture, Sendai). Radioisotopic experiments were carried out in the Radioisotopic Research Center, Tohoku University School of Medicine, Sendai, with the kind assistance of Mr. and Mrs. Ohta.

References 1 Shemin, D. (1972) in the Enzymes (Boyer, P.D., ed.), 3rd Edn., Vol. 7, pp. 323-337, Academic Press, New York 2 Tsukamoto, I., Yoshinaga, T. and Sano, S. (1979) Biochim. Biophys. Acta 570, 167-178 3 Gibbs, P.N.B., Gore, M.G. and Jordan, P.M. (1985) Biochem. J. 225, 573-580 4 Fujita, H., Koizumi, A., Yamamoto, M., Kumai, M., Sadamoto, T. and Ikeda, M. (1984) Biochim. Biophys. Acta 800, 1-10 5 Koizumi, A., Fujita, H., Sadamoto, T., Ohmachi, T., Watanabe, M. and Ikeda, M. (1984) Toxicology32, 1-10 6 Sandmeyer, E.E. (1981) in Patty's Industrial Hygiene and Toxicology (Clayton, G.D. and Clayton, F.E., eds.), 3rd Revised Edn., Vol. 2, pp. 3312-3319, John Wiley & Sons, New York 7 World Health Organization (1983) Styrene, pp. 37-40, Geneva 8 Marniemi, J., Suolinna, E.M., Kaatinen, N. and Vainio, H. (1977) in Microsomes and Drug Oxidations (Ullrich, V., Roots, I., Hildebrandt, A., Estabrook, R.W. and Conney, A.H., eds.), pp. 698-702, Pergamon Press, London 9 van Anda, J., Smith, B.R., Fouts, J.R. and Bend, J.R. (1979) J. Pharmacol. Exp. Ther. 211,207-211 10 Fujita, H., Orii, Y. and Sano, S. (1981) Biochim. Biophys. Acta 678, 39-50 11 Moore, S. (1963) J. Biol. Chem. 238, 235-237 12 Bencze, W.L. and Schmid, K. (1957) Anal. Chem. 29, 1193-1196 13 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275 14 Yamauchi, K., Hayashi, N. and Kikuchi, G. (1980) FEBS Lett. 115, 15-18 15 Yamamoto, M., Hayashi, N. and Kikuchi, G. (1982) Biochem. Biophys. Res. Commun. 105, 985-990 16 Ikeda, M., Koizumi, A., Miyasaka, M. and Watanabe, T. (1982) Int. Arch. Occup. Environ. Health 49, 325-339 17 Fujita, H., Yamamoto, R., Sato, K. and Ikeda, M. (1985) Toxicol. Appl. Pharmacol. 77, 66-75 18 Chang, C.S., Sassa, S. and Doyle, D.A. (1984) Biochim. Biophys. Acta 797, 297-301

96 19 Yamamoto, M., Fujita, H., Watanabe, N., Hayashi, N. and Kikuchi, G. (1986) Arch. Biochem. Biophys. 245, 76-83 20 Fujita, H., Sato, K. and Sano, S. (1982) Int. Arch. Occup. Environ. Health 50, 287-297

21 Goodman, M., Moore, G.W. and Barnabas, J. (1974) J. Mol. Evol. 3, 1-48