317

Mutation Research, 40 (1976) 317--324 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

MUTAGENICITY OF INDUSTRIAL COMPOUNDS: STYRENE AND ITS POSSIBLE METABOLITE STYRENE OXIDE *

LOPRIENO, N., A. ABBONDANDOLO, R. BARALE, S. BARONCELLI, S. BONATTI, G. BRONZETTI, A. CAMMELLINI, C. CORSI, G. CORTI, D. FREZZA. C. LEPORINI, A. MAZZACCARO, R. NIERI, D. ROSELLINI and ANNA M. ROSSI

Cattedra di Genetica della UniversitY, and Laboratorio di Mutagenesi e Differenziamento, CNR, Pisa (Italy) (Received May 11th, 1976) (Accepted June 8th, 1976)

Summary Styrene and its presumed metabolite, styrene oxide, were tested for their mutagenic effect on a forward mutation system of yeast and of Chinese hamster cells, and on a gene-conversion system of yeast. Experiments with liver microsomal preparations and host-mediated assay with yeast were also carried out. Styrene oxide was mutagenic in all test systems. Styrene was mutagenic only in the host-mediated assay.

Introduction Styrene (Chem. Abstr. No. 100-42-5), or phenylethylene, has been produced commercially since 1940, and at the present is in the list of the 50 chemicals with the biggest production in 1975. The estimated production in Italy is of 440,000 tons per year and this represents the fifth largest production (after U.S., Japan, Federal Republic of Germany, Netherlands). The main use of styrene is the production of plastics and resins (polystyrene resins, acrylonitrile-butadiene-styrene terpolymers, styrene-acrylonitrile copolymers; styrenebutadiene copolymer resins, etc.), or styrene-butadiene rubber. Miscellaneous applications of styrene include the production of styrene oxide (Chem. Abstr. No. 96-09-3), an intermediate in the preparation of cosmetics and other chemicals of agricultural or biological interest. Styrene vapors occur in the air of manufacturing workshops and in rubber * Publication No. 150 from 56100 Pisa (Italy).

the L a b o r a t o r i o di M u t a g e n e s i e D i f f e r e n z i a m e n t o , C N R , V i a Cisanello

147,

318 vulcanization plants producing butadiene-styrene rubber soles. It has been found as a contaminant of drinking water [2], in cigarette smoke, as a constituent of coal gas, coal tar, and of petrol produced by cracking processes. The International Labour Office reported in 1974 that prolonged exposure to styrene vapors can result in functional disorders of different types; workers employed for over 5 years in polystyrene plants and exposed to an atmospheric concentration of styrene of 0.05 mg/1 (50 mg/m 3) revealed cases of toxic hepatitis [5]. Styrene is known to be biotransformed in animals and in man to hippuric acid, which is excreted in urine. According to Leibman [4], styrene is metabolically converted to styrene oxide, and subsequently to styrene glycol by microsomal mixed oxidase function and microsomal epoxide hydrase from the liver, kidneys, intestine, lungs and skin from several mammals [8]. Since c o m p o u n d s subjected to the formation of epoxides in vivo have been suspected of carcinogenic activity, we decided to undertake an investigation to assess the possible mutagenic activity b y styrene directly or after its biotransformation into biologically active c o m p o u n d s by analyses in vitro (microsomal assay) or in vivo (host-mediated assay). As biological indicators, cells of the yeast Schizosaccharomyces pombe and Saccharomyces cerevisiae, and of V-79 Chinese hamster line were used in several experiments. The mutagenic activity of styrene oxide was also investigated. Preliminary data have been reported in the Fifth Annual Meeting of the European Environmental Mutagen Society [6] and at the Third International Symposium on Detection and Prevention of Cancer (1976). Materials and methods

Chemicals Styrene and styrene oxide were obtained from Aldrich, U.S.A. {kindly supplied by Dr. H. Bartsch, IARC, Lyon), or from Merck-Schuchardt, Darmstadt, FRG. Yeast strains Forward mutations were measured at five adenine loci in strain ade6-60/ rad10-198/h- of Schizosaccharomyces pombe (P~ strain); and gene conversions at the ade2 and trp5 loci of S. cerevisiae in the diploid strain D4 [7]. Mutagenicity assays with yeasts were carried o u t in vitro by incubating 108 cells in a medium (final volume 4 ml) containing purified mouse-liver microsomes, from Swiss albino mice in the presence of NADPH and G-6-P-dehydrogenase for 1 h [ref. 7]. In the host-mediated assay, 6 × 107 cells of the yeast were inoculated into the peritoneum of male Swiss albino mice ( b o d y weight 25 g) and the animals were treated by gavage with 1 ml of styrene or styrene oxide solution. Mutagenic effects were evaluated as described previously [7]. Control animals were analyzed 0 or 12 h after inoculation with yeast cells, depending on the incubation time of the treated animals. Chinese hamster cultures V-79 chinese hamster cells obtained from Dr. C.F. Arlett (MRC Cell Muta-

319

tion Unit, University of Sussex) were cultured as mono-layers in plastic tissue culture flasks or dishes (Falcon) in Dulbecco's modified Eagle's minimal essential medium supplemented with 5% dialyzed fetal calf serum (Eurobio), penicillin G (100 U/ml) and streptomycin sulfate (100/~g/ml). Cultures were incubated at 37°C in a humidified atmosphere containing 10% CO2; for subculturing, a trypsin solution (0.025%) in phosphate-buffered saline (Dulbecco) containing EDTA (0.5 mM) was used. Cells (1.5 × 106) were plated on 25 cm 2 flasks and incubated overnight. They were then washed with Hank's balanced salt solution (HBSS) and incubated at 37°C for 1 h with styrene or styrene oxide solutions in HBSS. Mutagenized cells were washed twice with pre-warmed HBSS; aliquots containing l 0 s cells were seeded into each of 10 plates (90 mm diameter) to which 8-azaguanine (20 pg/ml) was added after cell attachment, and diluted samples were seeded (100 cells in 50-mm plates) for evaluation of cell survival. At the same time, 2--3 × l 0 s viable cells were inoculated into 90-mm plates and incubated till the next sampling (72, 90 and 114 h expression times). Plates were incubated for 6--7 days (survival plates) or 10--12 days (mutation plates) before colonies were stained with methylene blue and counted. Results and discussion Data obtained in forward mutation experiments with yeast (S. pombe) and Chinese hamster cells are reported respectively in Tables I and II (Fig. 2). For both genetic systems styrene oxide was active. It was also able to produce geneconversion in yeast S. cerevisiae (Fig. la,b). Styrene on the contrary, at a 100 mM concentration, was inactive in the production of forward mutations in yeast even in the presence of purified mouse-liver microsomes. The conversion ability of such a metabolizing system is very low, since less than 5% of styrene oxide was produced (styrene oxide at 5 mM concentration was effective in the TABLE I F O R W A R D M U T A T I O N S ( a d e M U T A N T S ) I N D U C E D IN Y E A S T S. P O M B E ( P I S T R A I N )

Compound

mM

Treatment time

Survival % -+ S.E.M.

Number of mutants

a d e - I l O4

± S.E.M

Number of colonies Styrene

Styrene oxide

100 (buffer) 100 (+ p u r i f i e d mierosom.) 5.0 I0.0 15.0 20.0

0 60 t 0 60 t

100.0 93.7 100.0 67.8

0 60 ~ 0 60 t 0 60 t 0 60 t

100.0 71.3 -+ 13.9 I00.0 41.9 + 5.0 100.0 4.6 ± 0 . 7 100.0 3.8

1/23,430 2/45,056 2/17,424 2/22,745

0.43 0.44 1.14 0.88

5/58,878 12/38,627 4/48,325 22/33,590 4/42,052 15/17,974 1/15,096 34/11,739

0.85 3.17 0.82 6.86 1.02 8.31 0.66 28.96

+- 0 . 0 6 ± 1.04 -+ 0 . 1 6 + 3.34 -+ 0 . 2 8 -+ 0 . 0 2

320

T A B L E II F O R W A R D M U T A T I O N S (azg r M U T A N T S ) I N D U C E D IN C H I N E S E H A M S T E R C E L L S ( V 7 9 S T R A I N ) Compound

mM a

Styrene

Styrene oxide

Expression time

P.E. (%)

azg r 6

Viable cells × 10 3

azgr/lO 6 ± S.E.M.

0 8.5 17.0

66 90

100 83.7 77.4

43(5) 11(2) 30(2)

3746 1664 1394

11.37 ± 2.35 13.14 ± 0.32 20.56 ± 11.14

0 4.25 8.50 17.0 25.0

72 72 90 90

89.8 72.4 57.4 14.9 20.0

61(13) 7(2) 53(3) 32 (3) 91(1)

12,177 1385 1721 263 200

4.94± 1.22 5.09 ± 0 . 9 5 29.97 ± 11.32 139.96 ± 28.32 455.00

a T r e a t m e n t , 60 min. b In parentheses the n u m b e r of independent experiments.

production of forward mutation). In the host-mediated assay, on the contrary, both c o m p o u n d s were active for the production of gene conversion (Table III); in the same conditions, they were unable to increase the forward mutation spontaneous frequency in the yeast S. pombe (Table IV). The data are graphiB 100~

"~

50-

18A

16-

30.

-"

20-

14-

~o-

1o-

~-

8-

~

e

o l&. 2

IO

2'o mMolar

o

5

io

2'o mMolar

Fig. 1. (a) F o r w a r d m u t a t i o n i n d u c e d b y s t y r e n e o x i d e on S. p o m b e , PI s t r a i n ( a d e - m u t a t i o n s ) . (b) Survival a n d g e n e - c o n v e r s i o n f r e q u e n c y i n d u c e d b y s t y r e n e o x i d e o n S. cerevisiae, D 4 strain (ade l o c u s ) .

500'

321

? 9

300

=, .m

¢~N 200 < co 100

o

~

,s

I~)

2'0

2's

mMolar

Fig. 2. D o s e - e f f e c t r e l a t i o n s h i p s o b t a i n e d b y t r e a t m e n t o f C h i n e s e h a m s t e r cells w i t h s t y r e n e a n d s t y r e n e o x i d e . (o e) S t y r e n e o x i d e ; (o o) s t y r e n e . 5-

A FORWARD MUTATION (P~)

,...,

)c v U)

i

,I (I-

2

Q

GENE CONVERSION (D 4)

Z

2

Z 0 U uI Z La 2

1 0u.

.cJ

0

treatment ( h o u r s ) 4~

treatment ( h ~ r s )

4-

FORWARD MUTATION

(p,) ¢n

Z

GENE CONVERSION

(D4)

3 Z

_o D

=E Q n,

2~

W I¢ W

2-

Z O U W

Z

W t~

-j,

1

treotment

(hours)

Fig. 3. H o s t - m e d i a t e d assay ( f o r w a r d m u t a t i o n a n d g e n e c o n v e r s i o n ) w i t h s t y r e n e a n d s t y r e n e o x i d e o n y e a s t s . (a) S t y r e n e . (e "-) S t y r e n e ( 1 0 0 0 m g / k g ) ; (o o) c o n t r o l (b) S t y r e n e o x i d e , (e e) S t y r e n e o x i d e ( 1 0 0 m g / k g ) ; (o o) c o n t r o l ,

100 mg/kg

control

trp

ade

trp

ade

trp

1 . 0 0 ± 0 . 0 7 X 10 -5 (3) 1 . 3 0 ± 0 . 2 8 X 1 0 -5 (3)

0.51 ± 0.17 X 10 - s (6)

trp

ade

1.03 ± 0.26 X 10 - s (6) b

0

I n c u b t a i o n t i m e (h)

ade

Locus

a Treatment by gavage: 1 ml of DMSO solution. b Number of mice treated and analyzed.

Styrene oxide

Control

Styrene

1000 mg/kg

Dose

Compound

1 . 4 4 ± 0 . 0 8 x 10 -5 (3) 2 . 1 4 ± 0 . 1 5 x 10 - s (3)

1 . 4 2 + 0 . 1 8 x 10 - s (3) 1 . 4 3 -+ 0 . 1 4 x 10 -S (3)

4.75 ± 0.64 X 10 -s (6) 2 . 5 0 ± 0 . 3 5 x 10 -S (4)

3

(S. C E R E V I S I A E , D 4 S T R A I N )

4 . 2 3 + 0 . 7 2 × 10 -5 (3) 1 . 9 4 ± 0 . 2 5 x 10 - s (3)

1

MUTAGENICITY TEST WITH HOST-MEDIATED ASSAY (MICE) a: GENE CONVERSION

TABLE III

0.92 (3) 1.37 (3) 5.38 (5) 2.64 (4) 1.18 (3) 0.91 (3) 2.29 (3) 1.84 (3)

6

± 0 . 2 7 X 1 0 -5

+ 0 . 5 3 X 1 0 -5

_-!- 0 . 2 4 X 1 0 -5

+ 0 . 1 1 X 10 -S

± 0 . 6 1 X 10 -S

+- 1 . 3 7 X 1 0 -5

± 0 . 7 3 X 10 -5

± 0 . 6 9 X 10 -5

f,o t~

100 mg/kg

Control

1 . 1 6 +- 0 . 1 5 X 1 0 - 4 (7)

1 . 2 0 -+ 0 . 2 0 × 1 0 -4 (6) b

0

I n c u b a t i o n time (h)

a T r e a t m e n t b y g a v a g e : 1 m l o f a q u e o u s solution. b Number of mice and analyzed.

Styrene oxide

Control

Styrene

1000 mg/kg

Dose

Compound

0.96 ± 0.05 X 10-4 (8)

2 . 0 7 +- 1 . 1 8 X 1 0 - 4 (4)

3

(6)

1 . 2 3 -+ 0 . 2 3 X 1 0 - 4

(9)

1.67 +- 0 . 5 4 X 10 - 4 (6)

12 1 . 2 9 -4- 0 . 2 2 (4) 3 . 5 2 -+ 1.21 (8) 1 . 2 1 -+ 0 . 2 2 (3) 2 . 0 9 -+ 0 . 4 9

6

X 10 -4

× 10 -4

X 10-4

× 10 - 4

M U T A G E N I C I T Y T E S T W I T H H O S T - M E D I A T E D A S S A Y ( M I C E ) a: F O R W A R D M U T A T I O N (S. POMBE; ade M U T A N T S ; P1 S T R A I N )

T A B L E IV

¢.0 tO

324

cally reported in Figs. 3a,b: increasing frequencies of gene conversion were time dependent. Styrene and its derivative metabolites have been tested recently for their mutagenic activity in five strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537, TA1538) by Milvy and Garro [9] and only styrene oxide produced base-pair substitutions (TA100); previous recent mutagenic analyses with styrene oxide on strains of Salmonella sensitive to frame-shift-producing agents (TA1537 and TA1538) were negative [3]. Since epoxides may be the metabolite(s) of carcinogenic aromatic or olefinic compounds responsible for their mutagenicity and carcinogenicity, our present findings indicating the mutagenic activity in vitro on yeast and Chinese hamster cells of styrene oxide and the activity in vivo on yeast (host-mediated assay) of both compounds (styrene and styrene oxide) suggest that exposure to styrene vapor may be harmful, owing to its metabolic conversion to the active metabolite styrene oxide. In our experience, this compound can be characterized as a potent mutagen in the production of forward mutations in mammalian somatic cells in culture (Chinese hamster), and is more active than the well-known mutagenic chemical ethylmethane sulfonate [1]. It should be stressed, however, that the mutagenic effect observed by styrene in vivo was obtained with very high doses (1000 mg/kg). References 1 A b b o n d a n d o l o , A . , S. B o n a t t i , C. C o l e l l a , G. C o r t i , F. M a t t e u c c i , A. M a z z a c c a r o a n d G. R a i n a l d i , A comparative study of different experimental protocols for mutagenesis assays using the 8-azaguanine r e s i s t a n c e s y s t e m in C h i n e s e h a m s t e r c u l t u r e d cells, M u t a t i o n R e s . , 3 7 ( 1 9 7 6 ) 2 9 3 - - 2 0 6 . 2 D o w t y , B . J . , D . R . Carlisle a n d J . L . L a s e t e r , N e w O r e l a n s d r i n k i n g w a t e r s o u r c e s tested b y gas c h r o m a t o g x a p h y - m a s s s p e c t r o m e t r y , Envir. Sci. T e c h . , 9 ( 1 9 7 5 ) 7 6 2 - - 7 6 5 . 3 G l a t t , H . R . , F. O e s c h , A. F r i g e r i o a n d S. G a r a t t i n i , E p o x i d e s m e t a b o l i c a l l y p r o d u c e d f r o m s o m e k n o w n c a r c i n o g e n s a n d f r o m s o m e c l i n i c a l l y u s e d d r u g s . I. D i f f e r e n c e in m u t a g e n i c i t y , I n t . J. C a n c e r , 16 (1975) 787--797. 4 L e i b m a n , K . C . , M e t a b o l i s m a n d t o x i c i t y o f s t y r e n e , E n v i r o n m e n t a l H e a l t h P e r s p e c t i v e s , 11 ( 1 9 7 5 ) 115--119. 5 IARC, Monograph on the evaluation of carcinogenic risk of chemicals to man: epoxides and some other c h e m i c a l s o f i n d u s t r i a l i m p o r t a n c e , 1 9 7 6 , in press. 6 L o p r i e n o , N., A. A b b o n d a n d o l o , R . B a r a l e , S. B a r o n c e l l i , S. B o n a t t i , G. B r o n z e t t i , A. C a m m e l l i n i , C. C o r s i , G. C o r t i , D. F r e z z a , C. L e p o r i n i , A. M a z z a c c a r o , R. Nieri, D. R o s e l l i n i a n d A n n a M. R o s s i , M u t a genicity of industrial compounds: vinyl chloride, styrene and their possible metabolites, Mutation Res., 38 (1976) 114--115. 7 L o p r i e n o , N., R . B a r a l e , S. B a r o n c e l l i , C. B a u e r , G. B r o n z e t t i , A. C a m m e l l i n i , G. C e r c i g n a n i , C. Corsi, G. G e r v a s i , C. L e p o r i n i , R. Nieri, A n a M. Rossi, G. S t r e t t i a n d G. T u r e h i , E v a l u a t i o n o f the genetic effects i n d u c e d by c h l o r i d e m o n o m e r ( V C M ) u n d e r m a m m a l i a n m e t a b o l i c a c t i v a t i o n : s t u d i e s in v i t r o a n d in vivo, M u t a t i o n R e s . , 4 0 ( 1 9 7 6 ) 8 5 - - 9 6 . 80esch, F., M a m m a l i a n e p o x i d e h y d r a s e : i n d u c i b l e e n z y m e s c a t a l y z i n g t h e i n a c t i v a t i o n o f c a r c i n o g e n i c and cytotoxic metabolites derived from aromatic and definic compound, Xenobiotica, 3 (1973) 305-340. 9 Milvy, P. a n d A . J . G a r r o , M u t a g e n i c a c t i v i t y o f s t y r e n e o x i d e ( 1 , 2 - e p o x y e t h y l - b e n z e n e ) , a p r e s u m e d styrene m e t a b o l i t e , M u t a t i o n R e s . , 4 0 ( 1 9 7 6 ) 1 5 - - 1 8 .