Mutation Research, 257 (1991) 107-126 © 1991 Elsevier Science Publishers B.V. 0165-1110/91/$03.50 ADONIS 016511109100056Z

107

MUTREV 07292

The genetic toxicology of styrene and styrene oxide Roberto Barale lstituto di Zoologia, Universitgt di Ferrara, Ferrara (Italy)

(Received 30 January 1990) (Revision received 30 July 1990) (Accepted 30 July 1990)

Keywords: Styrene; Styrene oxide; Genetic toxicology

Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Mutagenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Alkylation activity of styrene oxide in vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Genetic effects of styrene and styrene oxide in prokaryotes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.1. Point mutation: Salmonella typhimurium, Ames assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.2. Other genetic effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Genetic effects of styrene and styrene oxide in eukaryotic non-mammalian systems . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.1. Yeasts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.2. Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.3. Drosophila melanogaster . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Genetic effects of styrene and styrene oxide in mammalian cells in vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.1. DNA binding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.2. DNA breaks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.3. DNA-repair assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.4. Point mutation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.5. Chromosomal effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. Genetic effects of styrene and styrene oxide in mammals in vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.1. Experimental animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.2. Occupationally exposed workers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction S t y r e n e ( C A N : e t h e n y l b e n z e n e ; C A S N o . 10042-5) is a c o l o r l e s s to y e l l o w i s h , v e r y r e f r a c t i v e , o i l y l i q u i d w h i c h o n e x p o s u r e to air o r light u n d e r g o e s p o l y m e r i z a t i o n a n d o x i d a t i o n . S t y r e n e is u s e d in t h e p r o d u c t i o n o f plastics, resins ( p o l y s t y r e n e

Correspondence: Prof. Roberto Barale, Istituto di Zoologia, via Borsari 46, 1-44100 Ferrara (Italy).

107 108 109 109 109 109 113 113 113 113 113 115 115 115 115 115 115 117 117 118 122

resins, a c r y l o n i t r i l e - b u t a d i e n e - s t y r e n e tetrapolymers, styrene-acrylonitrile copolymers; styreneb u t a d i e n e c o p o l y m e r resins) a n d s t y r e n e - b u t a d i e n e r u b b e r ( I A R C , 1979). T h e w o r l d c o n s u m p t i o n in 1990 h a s b e e n e s t i m a t e d to b e 13.6 m i l l i o n s m e t r i c t o n s ( P a r k k i , 1985). O c c u p a t i o n a l e x p o s u r e to s t y r e n e o c c u r s in w o r k p l a c e s w h e r e t h e air c o n c e n t r a t i o n h a s b e e n o b s e r v e d to r a n g e b e t w e e n 0.8 a n d 570 m g / m 3 ( 0 . 2 - 1 3 6 p p m ) d u r i n g t h e f a b r i c a t i o n o f glass-reinforced plastic pipeline joints and 105-605

108

m g / m 3 (25-144 ppm) during the production of plastic boat components (IARC, 1979). In these cases the main route of exposure to styrene is via inhalation. Styrene has also been detected in ambient air at levels of 0.2 ppb (0.84 t~g/m 3) in Nagoia (Japan); 0.1-0.7 ppb (0.4-2.9 /~g/m 3) in Delft (The Netherlands). It has been found in foods packaged in polystyrene containers at levels of 10-70/~g/kg of food (IARC, 1979). Styrene oxide (styrene 7,8-oxide; CAN: phenyloxirane; CAS No. 96-09-3) is a colorless to pale straw-colored liquid. It is considered to be the major primary metabolite of styrene in mammals. It is also industrially produced as a reactive diluent in epoxy resins or as an intermediate in the preparation of agricultural and biological chemicals, cosmetics, surface coatings, and in the processing of textiles and fibers (IARC, 1979). The extensive literature concerning the biological effects of styrene and styrene oxide, including

metabolism and toxicology, has been the object of previous reviews (IARC, 1979; 1987; ICPS, 1983; Parkki, 1985; Bond, 1989). The aim of this paper is to update, review and briefly discuss the current body of information about the genetic toxicology of styrene and styrene oxide. 2. Metabolism

The metabolism of styrene has been widely described and recently summarized; its flow chart is reported in Fig. 1 (ICPS, 1983). In brief, styrene is mainly converted into styrene oxide by the cytochrome P-450-mediated monooxygenase system. Styrene oxide can enzymatically be transformed through several steps into hippuric acid or directly conjugated with glutathione to produce, finally, mercapturic acids. Some intermediates of the main way of styrene oxide detoxification, such as styrene glycol and mandelic acid, can be con-

coniugates

T

?" O CH' H CH2 + ~ / C ~ "cH2 H "(~,,~ H Styrene 3 4-oxide

styrene 1.2-oxide

' ~ CH=CH2 v ~OH 2- vmylphenol

~(~

ethv[mercapturic

l

l

H

r

L ~

O

OH S s t y r e n e 7.8-oxide ,

GSH conlugate 2

phenylacet aldehyde

i ~

~

.C_CH 2

l

I

Hacetate

~CH2-C

~OH

//mandilic acid ~COOH benzoic acid

CH2-COOH

phenylacetic acid

r~------yc-cooH

phenylglyoxylic acid

O

- -

i[JZY 2-phenyl L~. .)J 6H 2-hydroxy ~ J et hylmercapturlc phenylet hylene ~ acid glycol glucuronide

0 II .,-C-COOH

/H

.~ ~ C H 2 - C \

A /L.".H-L.PI _. 2 J__~

OH + rf ~T

GSH conlugate 1 1- p h e n y l 2- hydroxy

CH-CH2

HO" 4-vmylphenol

SG. H-CH2

(C-Y

acid

~

oli

- i -CH2-COOH

phenylaceturic acid

Lo ~,~ C -

IJ

hippur=c acid

Fig. 1. Metabolism of styrene.

N-CH2-COOH

109 jugated with glucuronic acid or dehydrogenated to phenylglyoxylic acid. In man these compounds represent the two major final styrene metabolites. These can be found in urine after occupational exposure. Two other metabolic sideways have been described: (1) the oxidation of styrene to styrene 3,4-oxide which is converted spontaneously into 4-vinylphenol and (2) styrene oxidation to phenylacetaldehyde (Bakke and Scheline, 1970; Pantarotto et al., 1978). In this review, styrene oxide will mean styrene 7,8-oxide. Both styrene and styrene oxide are mainly metabolized in the liver and to a lesser extent in other tissues, such as kidney, intestine, lung and skin (Leibman and Ortiz, 1968; Ryan et al., 1976). Human lymphocytes and, to a larger extent, blood erythrocytes (hemoglobin) have been shown to convert styrene to styrene oxide (Belvedere and Trusi, 1981). For wider information on the pharmacokinetics, metabolism and general toxicity of styrene and styrene oxide some reviews are available (Parrki, 1985; Bond, 1989).

3. Mutagenesis 3.1. Alkylation activity of styrene oxide in vitro Styrene requires metabolic activation to bind covalently to macromolecules and styrene oxide is considered to be the primary mammalian metabolite of styrene. Due to the lability of the oxirane ring, styrene oxide is a direct electrophilic reactant forming covalent adducts with nucleophilic groups in the cells including proteins and nucleic acids (Marniemi et al., 1977). Hemminki and Falck (1979) found a direct correlation between the reactivity of 5 simple epoxides, including styrene oxide, with the nucleophile 4-(p-nitrobenzyl)-pyridine and their mutagenicity in Salmonella typhimurium TA100. However, later, Hemminki et al. (1981) showed that while the rates of 4-(p-nitrobenzyl)pyridine alkylation correlated to some extent with the hydrolysis rates of 4 epoxides, including styrene oxide, the rates of guanosine alkylation were quite different. The styrene oxide rate of DNA alkylation seems to depend on the molecular structure of the nucleophile. In fact Hemminki (1979) showed that deoxyguanosine was alkylated faster than single-

stranded DNA, which in turn was alkylated faster than double-stranded DNA. More recently Vodicka and Hemminki (1988a) have shown that 95% of styrene oxide alkylation in the case of single-stranded DNA is identified as the N-7, N 2 and 0 6 adducts, but a selective suppression of alkylation at N 6 and particularly 0 6 atoms in double-stranded DNA was observed. In an initial study Hemminki (1984) demonstrated that 57%, 28% and 15% of the guanosine alkylations were due to N-7, N 2 and 0 6 adducts respectively. A1kylation by styrene oxide of deoxynucleosides and DNA in aqueous buffer was further investigated by Savela et al. (1986); the relative yields of alkylated deoxinucleosides were dG > dC > dA > dT, the main products being identified as 7-alkyl-, N2-alkyl - and O6-alkyl-deoxyguanosine, 1-alkyl-, and Nr-alkyl-deoxyadenosine, Na-alkyl -, 3-alkyland O2-alkyl-deoxycytydine and 3-alkylthymidine. In the reaction of styrene oxide with DNA the main product was 7-alkylguanine, but N2-alkylguanine was also detected. As a consequence of alkylation, ring-opening and depurination of a and fl isomers of 7-alkylguanine was' also observed (Vodicka and Hernminki, 1988b). Six different adducts were detected with DNA-adduct 32p-postlabehng method and 2 of them have been shown to affect the O6-position of guanosine (Pongracz et al., 1989). These findings are of great interest to determine whether styrene oxide-DNA adducts result from occupational exposure to styrene. Styrene oxide appears to be a highly reactive, DNA-binding epoxide with the properties of a direct-acting mutagen. As a consequence of base alkylations, DNA depurination has been observed. The feasibility of detecting styrene oxide-DNA adducts by 32p-postlabeling opens new possibilities for quantification of DNA damage in workers exposed to styrene or in experimentally treated animals.

3.2. Genetic effects of styrene and styrene oxide in prokaryotes 3.2.1. Point mutation: Salmonella typhimurium, Ames assay On this subject, conflicting results have been reported from many laboratories, possibly due to

110

the great variety of experimental conditions used. For this reason descriptions and comments are reported here in detail. Glatt et al. (1975) obtained negative results with styrene oxide assayed on S. typhimurium strains TA1537 and TA1538, which are known typically to respond to frameshift mutagens. In fact all the following literature reports that styrene oxide and styrene, in the presence of in vitro metabolic activation systems, are active only on the base-substitution-sensitive strains, namely TA1530, TA1535 and TA100. Milvy and Garro (1976) tested a number of styrene metabolites using the spot test on strains TA1535, TA1537, TA1538, TA98 and TA100 showing that, in the absence of in vitro metabolic activation, styrene oxide only was clearly active on TA1535 and TA100. Styrene glycol, D-mandelic acid, L-mandelic acid, phenylglyoxylic acid, benzyl alcohol, benzoic acid, hippuric acid as well as styrene in the absence of metabolic activation were inactive on all strains, demonstrating that styrene oxide was the only direct-acting styrene metabolite known at that time and that it was able to induce mutations of the base-pair substitution type. The role of mammalian metabolism in converting styrene to a mutagenic epoxide was stressed by Vainio et al. (1976), who showed that styrene was mutagenic toward the same strains, TA1535 and TA100, after the addition of liver $9 obtained from Sprague-Dawley rats pretreated with Clophen, a polychlorinated biphenyl compound. The addition of diethylmaleate, known to deplete cytoplasmic glutathione, and of 3,3,3-trichloropropene oxide (TCPO), an inhibitor of epoxide hydratase, slightly enhanced the mutagenicity of styrene in the presence of liver $9, suggesting the possible participation of epoxide hydratase and glutathione S-oxide transferase in the detoxification of styrene oxide. The mutagenicity of styrene to strain TA1535 in the presence of liver $9 prepared from Aroclor-pretreated Wistar rats was confirmed by De Meester et al. (1977) with styrene effective doses ranging between 1 x 10 -5 and 1 x 10 - 6 mole/plate. However, Stoltz and Withey (1977) obtained negative results both with the spot and the plate test using different amounts per plate of $9 from rats and hamsters pretreated with Aro-

clor. Negative results were also obtained by Greim et al. (1977), who exposed the TA1535 strain to styrene for up to 2 h before plating, in a liquid suspension containing $9 mix at 37 ° C. Liver microsomes were obtained from mice pretreated with phenobarbital or Clophen A50. Watabe et al. (1978) found that styrene was positive on the TA100 strain in the presence of liver $9 from 3-methylcholanthrene- or phenobarbital-pretreated Wistar rats, only when the epoxide hydratase inhibitor TCPO was added and the frequency of revertants was corrected taking into account the survival fraction. No net increment of revertants was obtained nor mutagenic activity observed when $9 was obtained from rats pretreated with a polychlorinated biphenyl mixture. Watabe et al. (1978b) concluded that possibly other styrene active metabolites may be involved in styrene S9-mediated mutagenicity. In fact the styrene oxide formed by styrene metabolism can account for only a small fraction of the mutagenic activity of styrene on Salmonella TA100 (Watabe et al., 1978a,b). Finally Watabe et al. (1982) showed that 1-vinylbenzene 3,4-oxide, a putative precursor of 4-vinylphenol, which has been isolated from the urine of rats treated with styrene (Bakke and Scheline, 1970; Pantarotto et al., 1978) was 40-50-fold more potent than styrene oxide to Salmonella TA100. Loprieno et al. (1978) obtained negative results with styrene assayed over a wide range of doses, including toxic ones, in the presence of $9 from phenobarbital-pretreated mice on the TA1535 strain. Busk (1979) confirmed the positivity of styrene oxide both in the absence and in the presence of $9 on strains TA1535 and TA100, but no mutagenic activity was obtained with styrene either in the presence of liver $9 from Aroclor- or Clophen-pretreated Sprague-Dawley rats, or after the addition of T C P O and diethylmaleate. It is noteworthy that Vainio et al. (1976) assayed styrene at doses of 1 x 1 0 - 4 - 1 x l0 -8 mole/plate. Their range thus included toxic (1 x 10 -4, 1 x 10 -5) ineffective (1 X 1 0 - 6 ) , and effective (1 x 10 -7 and 1 x 10 -8) doses on TA1535. Watabe et al. (1978) assayed with the direct plate test 4 styrene doses from 1 x 10 -5 to 1.5 x 10 -6 m o l e / p l a t e (negative results) and, with the pre-incubation test (20 rain) 2 doses, 3 and 6 mM,

111

obtaining positive results only when the net revertant colony numbers were corrected for survival (no net increase of revertants over the control was observed); Busk (1979) assayed 14 styrene doses from 1.5 x 10 -5 to 1 x 10 -8 mole/plate covering those of Vainio (1976) with negative results. Poncelet et al. (1980) reported positive results for styrene with $9 on TA1535 in the plate test, but negative results with other treatment conditions, such as the pre-incubation test, bacterial fluctuation test and incubation of plates in gaseous atmosphere. In this study only one dose was assayed, 1 x 10 -5, which was reported by other authors to be toxic in the plate test (Vainio et al., 1976; Loprieno et al., 1978; De Meester, 1977). Subsequently other papers reported negative mutagenicity results assaying styrene in different treatment conditions such as the Ames plate test, with strains TA1535, TA1527, TA1538, TA97,

TA98 and TA100 (De Flora et al., 1984), the standard Ames plate test or pre-incubation assay using liver $9 prepared from induced and uninduced rats, mice and hamsters (Dunkel et al., 1985). The discrepancies are evident and it is difficult to determine whether the non-reproducibility of positive results with styrene is due to impurities in the compounds used, the metabolic systems employed or other methodological differences (Table 1). The low or negative mutagenic responses obtained with styrene under metabolic activation could also be explained by the rapid detoxification of styrene oxide due to the $9 mix itself. Moreover a rapid loss of monooxygenase activity has been shown when styrene was present in the incubation mixture, whereas the hydratase activity was much more stable (Bauer et al., 1980). The volatility and the poor solubility of styrene in aqueous solution can also explain the conflict-

TABLE 1 SUMMARY O F M U T A G E N I C I T Y ASSAYS W I T H STYRENE IN S. typhimurium Strain TA1535, TA1535, TA100 TA1535, TA1535 TA100 TA1535 TA100 TA100 TA1535, TA1535 TA1535 TA1535 TA1535, TA1535, TA1535, TA1535, TA100

TA100

TA100

TAI00

TA100 TA100 TA100 TA100

Experimental procedure a

Metabolic activation b

Dose range (mole/plate)

Result c

Reference

ST PT PT ST, PT PT PT PT LP LP PT PT LP, FT PT PT PT PT PT, LP PT

No RC RC R.A, HA RA RA MP RM RP RA, RE RA RA RA RA RAt RA R, M, H, RA, MA, HA RA

5 x 10-5 1 X 10 - 4 / - 9 1 X 10 - 4 / - 9 up to 1 x 10-5 1 X 10 - 6 / - 9 1 X 10 - 6 / - 9 1 x 10 - 5 / - 9 1-2 × 10- 6 1-2 x 10 -6 1 x 10 - 6 / - 9 1 × 10-5 1 × 10 - s gaseous gaseous ~ up to 5 × 10-6 up to 5 × 1 0 - 6 1 x 10 - 6 / - 9 1 X 10 - 6 / - 7

_ + in _ + + a + + _ _ -

Milvi et al., 1976 Vainio et al., 1976 Vainio et al., 1976 Stoltz and Withey, 1977 De Meester et al., 1977 De Meester et al., 1977 Loprieno et al., 1978 Watabe et al., 1978 Watabe et al., 1978 Busk, 1979 Poncelet et al., 1980 Poncelet et al., 1980 Poncelet et al., 1980 De Meester et al., 1981 De Flora, 1981 De Flora, 1984 Dunkel et al., 1985 Brains et al., 1987

a ST, spot test; PT, plate incorporation assay; LP, liquid pre-incubation assay (time of pre-incubation at 37 o C); FT, fluctuation test. b RC, Clophen-induced rat liver $9; RA, Aroclor-induced rat liver $9; RM, 3-methycholanthrene-induced rat liver $9; HA, Aroclor-induced hamster liver $9; IL rat uninduced liver $9; M, mouse uninduced liver $9; H, hamster uninduced liver $9; MP, mouse phenobarbital-induced liver $9. c + , positive (dose-related increase in mutant rate/number); ?, doubtful (poorly reproducible a n d / o r not dose-related increase); - , negative; in, inconclusive. Positive only after correction of the obtained revertant number for surviving fraction. Styrene (24% v / v of air) in gaseous atmosphere.

112 TABLE 2 S U M M A R Y OF M U T A G E N I C I T Y ASSAYS W I T H S T Y R E N E O X I D E IN S. typhirnuriurn Strain

Experimental procedure a

Metabolic activation b

Dose range (mole/plate)

Result c

Reference

TA1535, TA100 TA1535, TA100 TA1535, TA100 TA1535, TA100 TA1535 TA100 TA100 TA1535, TA100 TA1535, TA100 TA100 TA100 TA1530, TA1535, TA100 TA1535, TA100 TA100 TA100, TA97 TA100 TA100

ST PT ST, PT PT PT LP LI (1 h) PT PT LP PT PT PT PT PT PT, LP LP

+/+ / + / + + +/+/+/ +/ +/ +/-

4 × 10 -5 1 × 10-5/-8 0.4-8.3 x 1 0 - 6 4 × 10 - 5 / - 7 1 × 10 - 4 / - 6 0.08-4 x 1 0 - 6 0.4-2 x 1 0 - 6 / m l lxl0 -5/-6 1 - 4 × 10 -6 1-6 × 10- 3 11 × 10 -6 gaseous d 2.4 × 1 0 - 5/ - 6 0.5 × 10 -6 6X10 4 1 ×10 -4/-3 23.8 × 1 0 - 3 e

+ + + + + + + + + + + + + + + + +

Milvi et al., 1976 Vainio et al., 1976 Stoltz and Withey, 1977 De Meester et al., 1977 Loprieno et al., 1978 W a t a b e et al., 1978 Sugiura et al., 1978 Busk, 1979 EI-Tantawy and Hammock, 1980 Yoshikawa et al., 1980 Bartsch et al., 1980 De Meester et al., 1981 De Flora, 1981 R o s m a n et al., 1986 Brams et al., 1987 Hughes et al., 1987 W a t t e m b e r g et al., 1987

RA, H A - RA - MP

RA, RC M, R, G - R - RA - RA

RA, HA

a ST, spot test; PT, plate incorporation assay; LP, liquid pre-incubation assay (time of preincubation at 3 7 ° C ) ; LI, liquid

incubation. b R, rat, M, mouse, H, hamster, G, guinea pig uninduced liver $9; RC, Clophen-, RA, Aroclor-, RM, 3-methylcholanthrene-induced rat liver $9; HA, Aroclor-inducedhamster liver $9; MP, mouse phenobarbital-inducedliver $9. c +, positive(dose-relatedincrease in mutant rate/number); ?, doubtful (poorly reproducible and/or not dose-relatedincrease); - , negative; in, inconclusive. a Styrene oxide (24% v/v of air) in gaseous atmosphere. e Concentration in the pre-incubation mix.

ing results obtained as suggested by De Meester et al. (1981), who obtained weak but positive results by incubating Salmonella strains TA1530, TA1535 and TA100 in gaseous atmospheres containing styrene. These findings are in apparent contradiction with those reported in 1980 by the same research group (Poncelet et al., 1980) and this was not discussed further by the authors. The direct mutagenicity of styrene oxide on Salmonella strains TA1535 and TA100 was confirmed by several authors (Loprieno et al., 1978; Sugiura et al., 1978; Wade et al., 1978; Planche et al., 1979; Bartsch et al., 1980; De Flora, 1981; De Flora et al., 1984; Rosman et al., 1986; Brams et al., 1987; Wattenberg et al., 1987) and is summarized in Table 2. The role played by cytosol glutathione S-transferase and microsomal hydratase in styrene oxide detoxification evaluated as a reduction of the mutagenicity on Salmonella TA100 has been in-

vestigated by Yoshikawa et al. (1980) and by E1-Tantawy and Hammock (1980), with conflicting results. The former obtained data suggesting a major role of glutathione while the latter did not reach the same conclusion. However, it should be remembered that Yoshikawa et al. (1980) treated the bacteria in suspension for 20 min and then separated them from the suspension by centrifugation prior to plating, whereas E1-Tantany and Hammock applied the standard plate test according to Ames (1975). It is possible that these treatment differences could partially explain the observed discrepancies. Recently, Hughes et al. (1987) used a Tedlar bag vaporization technique (which increases the contact time between some volatile direct-acting mutagens and bacteria) and obtained negative results with styrene oxide, whereas positive results are obtained with the standard plate test and even more so with the pre-incubation test

113 (strains TA100 and TA102). In these experiments the addition of liver $9 from Aroclor-pretreated rats of hamsters increased styrene oxide mutagenicity possibly by reducing toxicity.

3.2.2. Other genetic effects Styrene was found to be negative in back-mutation systems (gal-, arg- and nad-) and in the MTR forward mutation system in E. coli K-12 (Ellemberger and Mohn, 1975) in the presence of $9 from phenobarbital- or Clophen A 50-pretreated mice (Greim et al., 1977). Negative results in inducing reverse mutation in E. coli WP2uvrA were also obtained by Norppa et al. (1985), even when human erythrocytes were added to the mixture. These blood cells have been suggested to possess some styrene activation system (Norppa et al., 1980a). Using several DNArepair-proficient and -deficient (WP2, WP67, CM 817) E. coli strains in the liquid micromethod procedure including $9 activation, De Flora (1984) did not obtain any evidence of DNA-damaging activity induced by styrene, whereas styrene oxide was effective in the same assay. Styrene gave negative results with the SOS chromotest (in the presence of $9) over a range of doses from 10 ng up to 1 mg/ml. Styrene oxide also gave negative results in the same assay (Brains et al., 1987). Nakamura et al. (1987) obtained positive results with styrene oxide assayed with the SOS umu test (Salmonella typhimurium TA1535/pSK1002).

From the review of the existing literature on the mutagenicity of styrene in bacteria, it emerges that considerable uncertainty still exists on the ability of the bacterial system to assess the mutagenic power of this molecule. This seems a case in which a molecule could probably escape detection as a mutagen, if only one type of assay system were used. Its putative reactive metabolite, styrene oxide, gives clear positive responses. Therefore it appears that the exogenous metabolic activation systems used in these assays play a relevant role in determining the reported difficulties in reproducing positive data with styrene.

3.3. Genetic effects of styrene and styrene oxide in eukaryotic non-mammalian systems 3.3.1. Yeasts Styrene was reported to be negative in inducing both forward gene mutation and mitotic gene conversion in the yeasts Schizosaccharomyces pombe (P1 strain) and Saccharomyces cerevisiae (D4 strain) respectively, in the presence of purified (S100) mouse liver microsomes (Loprieno et al., 1976). Using logarithmically growing yeast cells (S. cerevisiae, D7 strain), which possess enough monooxygenases to actively metabolize indirect mutagens (Callen and Philpot, 1977), Del Carratore et al. (1983) were able to show some convertogenic and recombinogenic activity of styrene (1-10 raM), whereas the addition of external metabolic activation (liver $9 from phenobarbitalpretreated mice) to stationary cells gave completely negative results. These data seem to confirm that external metabolic activation ($9) in most cases cannot form enough active styrene metabolites to reach nuclear DNA. Styrene and styrene oxide tested in the intraperitoneal host-mediated assay with yeasts were weakly active in inducing mitotic gene conversion after 12 h of incubation (Loprieno et al., 1976). Later, however, Loprieno and Abbondandolo (1980) considered these styrene data as negative on the basis of a larger set of control values. Low conversion rates of styrene to active metabohtes and their short half-lives in animals can explain the negative results obtained in the intraperitoneal host-mediated assay. 3.3.2. Plants Cytogenetic studies on Allium meristematic root tips revealed that styrene and styrene oxide were able to induce chromosome breaks, anaphase bridges and micronuclei. The described disordered metaphases and strong C-mitotic effect in treated samples are suggestive that styrene can be also a mitotic poison (Linnainmaa et al., 1978a,b). 3.3.3. Drosophila melanogaster Styrene and styrene oxide were shown to be weakly positive in inducing X-linked recessive lethals when flies were fed 200 ppm of chemical, for 24 h, on tissue paper. Pretreatment of flies

114 TABLE 3 SUMMARY OF M U T A G E N I C I T Y ASSAYS W I T H STYRENE IN LOWER E U K A R Y O T E S Test organism

Strain

S. pombe S. pombe S. cerevisiae S. cereoisiae

P1 P1 D4 D7

Endpoint

Forward mutation Forward mutation Gene conversion Reversion and Gene conversion Allium CA c Allium Micronuclei Allium Anaphase aberration Allium Micronuclei Drosophila melanogaster Recessive lethals Drosophila melanogaster Non-disjunction

Experimental procedure a

Metabolic activation b

Dose range

Result

Reference

LI (1 h) HMA (3-12 h) HMA (3-12 h) LI (2 h)

PMM HMA HMA EMA

100 mM 1 g/kg 1 g/kg 1-5 mM

4-

Loprieno et al., 1976 Loprieno et al., 1976 Loprieno et al., 1976 Del Carratore et ai., 1983

LI (2 h) LI (2 h) LI (2 h) LI (12 h) F (24 h) F

-

4.33 mM 4.33 mM 4.33 mM 1.73 mM 200 p p m 500 p p m

+ + + + -

Linnainmaa, 1978a Linnainmaa, 1978a Linnainmaa, 1978b Linnainmaa, 1978b Donner et al., 1979 Penttila et al., 1980

a LI, liquid incubation; HMA, host-mediated assay; F, feeding flies on solution containing styrene at 200 or 500 p p m in 1% sucrose solution in water. b PMM, purified mouse microsomes; EMA, endogenous metabolic activation. c CA, chromosomal aberrations.

with phenobarbital or TCPO resulted in an increase of styrene or styrene oxide mutagenicity, respectively (Donner et al., 1979). Styrene was ineffective in inducing sex-chromosome non-disjunction in flies fed on 500 ppm for 24 h (Penttila et al., 1980).

In lower eukaryotes, plants and Drosophila, it appears that styrene is either negative or weakly positive in inducing different genetic endpoints (Table 3). Styrene oxide gives much more positive outcomes, but it appears to be only marginally active in vivo in the host-mediated assay suggesting that in complex organisms it can be rapidly

TABLE 4 SUMMARY OF M U T A G E N I C I T Y ASSAYS W I T H STYRENE O X I D E IN LOWER E U K A R Y O T E S Test organism

Strain

S. pombe S. pombe S. cerevisiae

P1 P1

Endpoint

Forward mutation Forward mutation D4 Gene conversion Allium CA Allium Micronuclei Allium Anaphase aberration Allium Micronuclei Drosophila melanogaster Recessive lethals

Experimental procedure a

Metabolic activation b

Dose range

Result

Reference

LI (1 h) HMA (3-12 h) HMA (3-12 h) LI (2 h) LI (2 h) LI (2 h) LI (12 h) F (24 h)

HMA HMA HMA -

5-20 mM 100 m g / k g 100 m g / k g 5 mM 5 mM 5 mM 1 mM 200 p p m

+ + w + w + + + +

Loprieno et al., 1976 Loprieno et al., 1976 Loprieno et al., 1976 Linnainmaa, 1978a Linnainmaa, 1978a Linnainmaa, 1978b Linnainmaa, 1978b Donner et al., 1979

a LI, liquid incubation; HMA, host-mediated assay; F, feeding flies on solution containing styrene at 200 p p m in 1% sucrose solution in water. b PMM, purified mouse microsomes; EMA, endogenous metabolic activation. w, weakly positive.

115 excreted a n d / o r inactivated (Table 4). Tentatively, these results can be attributed to different metabolic conditions in the few organisms tested. 3.4. Genetic effects of styrene and styrene oxide on mammalian ceils in vitro 3. 4.1. DNA binding Belvedere et al. (1984) have shown that isolated rat hepatocytes acted more efficiently than $9 and pure microsomes in converting styrene to styrene oxide, suggesting that hepatocytes could be one of the most suitable systems for the study of indirect mutagens in vitro. However, incubation of hepatocytes from phenobarbital-pretreated rats with 1 mM [~4C]styrene for 3-5 h did not result in styrene oxide DNA binding, suggesting the possibility that D N A alkylation by other reactive intermediates can occur (Legraverend et al., 1984). 3.4.2. DNA breaks When freshly isolated rat hepatocytes were exposed to 3 mM styrene for 3 h, increases of DNA single-strand breaks were measured by alkaline elution. Styrene oxide was positive in the same assay at a dose 10-fold lower (Sina et al., 1983). 3. 4.3. DNA-repair assays EUE human cells in the presence of liver $9 from phenobarbital-pretreated mice were exposed to 14.3, 28.4 and 42.8 mM styrene and the unscheduled DNA synthesis (UDS) assay was performed by measuring hydroxyurea-resistant 3HTdR DNA incorporation evaluated by liquid scintillation counting. Negative results were obtained, while styrene oxide was positive without $9 (Loprieno et al., 1978). These results were confirmed by autoradiographic observation of 3HTdR nuclear incorporation (Bianchi et al., 1982). Styrene oxide turned out to be negative in the UDS assay performed in freshly isolated rat hepatocytes (Brouns et al., 1979). Exposure for 15 rain of human lymphocytes to styrene (10-750 /~M) gave an increased UDS caused by subsequent treatments with the mutagen/carcinogen N-acetoxy-2-acetylaminofluorene (Pero et al., 1982). These authors concluded that styrene does not alter the efficiency of DNA-re-

pair synthesis but only the susceptibility of DNA to damage from other chemical mutagens. Using DNA-repair-proficient and -deficient strains of Chinese hamster ovary cells Hoy et al. (1984) obtained differential cytotoxicity (positive results) with styrene oxide. 3.4.4. Point mutation Styrene was ineffective in inducing mutations at the HGPRT locus in Chinese hamster cells in the presence of mouse $9. In the same assay (without $9) styrene oxide induced a large number of mutants (Loprieno et al., 1978). Bonatti et al. (1978) analyzed styrene oxide-induced HGPRT mutants and found them to be indistinguishable from EMS-induced ones on the basis of some phenotypic properties, such as stability, reversion frequencies and residual enzyme activity. The mutagenicity of styrene oxide was confirmed in this assay by Turchi et al. (1981) and by Nishi et al. (1984). It was also confirmed in the L5178Y/ TK (TK ÷/- TK - / - ) mouse lymphoma assay by Amacher and Turner (1982). When styrene oxide was introduced into perfused rat fiver no mutagenicity on V79 cells was observed by testing either the perfusion medium or the bile, suggesting that styrene oxide is rapidly metabolized. In the same system styrene was effective in inducing HGPRT mutants when added to the perfusion medium, but the low styrene oxide concentration detected suggests that this metabolite is not the cause of the mutagenic effect observed (Beije and Jenssen, 1982). 3. 4. 5. Chromosomal effects Styrene (2.6 mM) added to human whole-blood cultures for 72 h was shown to induce chromosome breaks, aneuploidy and micronuclei in lymphocytes, without exogenous metabolic activation (Linnainmaa et al., 1978a, b). Styrene oxide in the same experiments was active at a lower dose (0.7 mM). De Raat (1978) obtained negative results in the SCE assay by treating Chinese hamster ovary (CHO) cells with styrene in the presence of $9 mix. In th same conditions styrene oxide markedly decreased its activity, but it was restored by the addition of cyclohexene oxide, an inhibitor of epoxide hydratase. The author suggests that

116 TABLE 5 SUMMARY OF IN VITRO M A M M A L I A N CELLS ASSAYS W I T H STYRENE Cell type

Endpoint

Experimental procedure a

Metabolic activation b

Dose range

Result c

Reference

Rat hepatocytes V79 V79 V79 V79 EUE CHL H u m a n lymphocytes Human lymphocytes

D N A binding Forward mutation Forward mutation Forward mutation Forward mutation UDS CA SCE SCE

H u m a n lymphocytes H u m a n lymphocytes

SCE SCE

Human lymphocytes CHO

SCE

H u m a n lymphocytes H u m a n lymphocytes

CA CA

LI LI LI LI LI LI LI LI LI LI LI LI LI LI LI LI LI LI LI LI LI

M M R RLP M MR RBC _ d RBC RBC _ d CR _ d RBC RBC CR RBC RBC _ d RBC

1 mM 8.5-17 mM 51 mM 60-240 mM 2.4-4.8 mM 14.3-42.8 mM 2.4 mM 0.33-4 mM 2 mM 2 mM 0.5-4 mM 1-20 mM 1-20 mM 1-20 mM 1-20 mM 0.5-4 mM 1-20 mM 1.15 mM 0.1-0.5 mM 1--6 mM 0.5-6 mM

+ +w + + + + + -+ + + + +w +

Legraverend et al., 1984 Loprieno et al., 1976 Loprieno et al., 1978 Beije and Jenssen, 1982 Beije and Jenssen, 1982 Loprieno et al., 1978 Matsuoka et al., 1979 Norppa et al., 1980 Norppa et al., 1983 Norppa et al., 1983 Norppa and Vainio, 1983 Norppa and Tursi, 1984 Norppa and Tursi, 1984 Norppa and Tursi, 1984 Norppa and Tursi, 1984 Norppa et al., 1985 Norppa et al., 1985 Norppa et al., 1985 Pohlova et al., 1985b Jantunen et al., 1986 Jantunen et al., 1986

(3-5 h) (1 h) (4 h) (4 h) (1 h) (3 h) (24 h) (48 h) (48 h) (48 h) (4 h) (4 h) (24-34 h) (24-34 h) (48 h) (4 h) (24-34 h) (54 h) (24 h) (24 h)

a LI, liquid incubation. b M, mouse liver $9; R, rat liver $9; MR, 3-methylcholanthrene-induced rat liver $9; CR, Clophen-induced rat liver $9; RLP, rat liver perfusion; RBC, red blood cells. c w, very weakly positive. d Purified lymphocyte cultures.

TABLE 6 SUMMARY OF IN VITRO M A M M A L I A N CELL ASSAYS W I T H STYRENE O X I D E Cell type

Endpoint

Experimental procedure a

Metabolic activation b

Dose range

Result

Reference

V79 V79 V79 V79 V79 EUE HL HL CHO V79 HL HL V79 H u m a n lymphocytes

Forward mutation Forward mutation Forward mutation Forward mutation Forward mutation UDS CA Micronuclei SCE Micronuclei CA and SCE SCE SCE CA

LI LI LI LI LI LI LI LI LI LI LI LI LI LI

RLP WBC WBC WBC WBC WBC

4.25-25 mM 4.2-17 mM 8.5-17 mM 2.1 mM 2-7 mM 4.4-8.7 mM 0.7 mM 0.7 mM 0.1 mM 0.75 mM 0.07-0.34 mM 0.15 mM 2-7 mM 0.05-0.5 mM

+ + + + + + + + + + + + +

Loprieno et al., 1976 Loprieno et al., 1978 Abbondandolo et al., 1978 Beije and Jenssen, 1982 Nishi et al., 1984 Loprieno et al., 1978 Linnainmaa et al., 1978 Linnainmaa et al., 1978 De Raat, 1978 Turchi et al., 1981 Norppa et al., 1981 Norppa et al., 1983 Nishi et al., 1984 Pohlova et al., 1985

(1 h) (1 h) (1 h) (4 h) (3 h) (1 h) (8 h) (8 h) (1 h) (1 h) (48 h) (48 h) (3 h) (54 h)

a LI, liquid incubation. b RLP, rat liver perfusion; WBC, whole blood culture.

117 styrene oxide formed in vitro by styrene metabolism is immediately deactivated by epoxide hydratase present in the $9 mix; in fact by adding cyclohexene oxide to the incubation mixture of styrene an increase in SCEs was observed. Norppa et al. (1980a) confirmed that styrene was active in whole-blood human lymphocyte cultures and explained it by the metabolic capability of blood cells, indicated by a measurable production of styrene oxide in treated cultures. However, styrene oxide inactivation by erythrocytes was also shown in the same incubation system. The role of erythrocytes in styrene activation was further assessed by the induction of SCEs and chromosome aberrations (Jantunen et al., 1986) in human lymphocytes in the presence of different erythrocyte amounts (Norppa et al., 1983a; Norppa and Tursi, 1984). This effect seems to be mediated by the presence of oxyhemoblobin (Tursi et al., 1983). Styrene showed some SCE induction in isolated human lymphocytes, and this is probably due to the capacity of the lymphocytes themselves to metabolize styrene (Belvedere and Tursi, 1981). However, it did not induce SCEs in C H O cells in the presence of erythrocytes or liver $9 from Clophen A 50-pretreated rats, confirming previous data from De Raat (Norppa et al., 1985). Norppa and Vainio (1983b) have shown that styrene and some methyl-substituted derivatives were able to induce SCEs by a 48-h treatment in human whole-blood lymphocyte cultures. According to these authors the negative or weak effects of styrene analogues without a double bond in the side chain suggested that the reactive metabolites are derived from the conversion of the vinyl group and are possibly, in this system, styrene 7,8-oxides. Styrene oxide confirmed its clastogenicity by inducing micronuclei and anaphase chromosome bridges (Turchi et al., 1981), chromosome aberrations (Fabry et al., 1978) and SCEs in cultured human lymphocytes (Norppa et al., 1981). Nishi et al. (1984) found that styrene oxide was much more effective in inducing SCEs than point mutations in Chinese hamster V79 cells, suggesting a stronger clastogenic than mutagenic activity of this styrene metabolite. All these results are summarized in Tables 5 and 6. In mammalian cell systems, styrene shows prevailingly positive results in the induction of geno-

toxicity. Target cells such as lymphocytes seem to possess their own metabolic activation systems, which activate styrene to genotoxic derivatives. Further, it was shown that even liver nuclear membrane activates styrene (Gazzotti et al., 1980). This last form of activation may be of considerable importance, given the proximity of the membrane to the genetic target. 3.5. Genetic effects of styrene and styrene oxide in mammals in vivo 3.5.1. Experimental animals Loprieno et al. (1978) reported negative results of styrene in inducing chromosomal damage in bone marrow cells of mice treated by gavage with a single dose (500, 1000 mg/kg). Styrene oxide was positive in the same assay at 3 doses tested (50, 500, 1000 mg/kg). In another group of experiments styrene oxide did not induce statistically significant increases in micronuclei in bone marrow cells of mice treated by i.p. injection with 250 mg/kg. Higher doses were lethal within a few hours. The dominant lethal test and meiotic chromosome analysis also gave negative results (Fabry et al., 1978). Following exposure of mice for 4 days to 565 ppm styrene by inhalation, 3-4-fold SCE increases in bone marrow, in liver regenerating cells and alveolar macrophages were observed by Conner et al. (1979, 1980). Applying several doses for different treatment times, these authors observed that chronic treatments were more effective than acute ones even if the total dose was lower in the former. Moreover styrene activation in the target cells (bone marrow and macrophages) was suggested by the results obtained in hepatectomized mice. Styrene oxide exposure by inhalation (50 ppm, 5 h) resulted in a very slight increase in SCE frequencies only in alveolar macrophages and regenerating liver cells (Conner et al., 1982). However exposure of Chinese hamsters to styrene by inhalation (300 ppm per 4 days or 3 weeks) or to 25, 50, 75, and 100 ppm styrene oxide for 2 days or longer did not result in any increase of chromosomal aberrations or SCE in the bone marrow cells. This apparent lack of responsiveness in Chinese hamsters could be due to the presence of higher levels of epoxide hy-

118 TABLE 7 S U M M A R Y OF IN VIVO MUTAGENICITY ASSAYS WITH STYRENE Species

Sex

Strain

Endpoint or assay a

Organ or tissue b

Route

Dose range

Result

Mouse Rat

male male

CD1 Wistar

CA CA

BM BM

p.o. inhalation

+

Mouse Mouse Mouse Mouse Mouse Hamster Hamster Hamster Mouse Mouse Mouse Mouse Mouse Mouse

male male male male male male male male male male male male male male

BDF1 BDF1 BDF1 BDF1 BDF1 Chinese Chinese Chinese C57BL/6 CD1 CD1 C57BL/6 C57BL/6 NMRI

SCE SCE SCE SCE SCE MN CA CA MN CA CA SCE CA SSB

inhalation inhalation inhalation inhalation inhalation i.p. inhalation inhalation i.p. p.o. p.o. i.p. i.p. i.p.

+ 250 + (all) 500

Norppa, 1981 Sbrana et al., 1983 Sbrana et al., 1983 Sharief et a1.,1986 Sharief et al., 1986 Solveig and Orsen, 1983

Mouse

male

NMRI

CB

Liver BM Liver BM AM BM BM BM BM BM BM BM BM K, Li, Lu, T,B Li, B, Lu,

500-1000 m g / k g 300 ppm (6 h / d a y , 5 days/week; 11 weeks 565 ppm (6 h / d a y , 4 days) 104-992 ppm (6 h / d a y , 4 days) (6 h / d a y , 4 days) 1 g/kg 300 p p m (6 h / d a y , 4-21 days) 250-1500 m g / k g 200 mg/kg, 70 days 500 mg/kg, 4 days 50-1000 m g / k g 50-1000 m g / k g 177-1000 m g / k g

i.p.

312 m g / k g

+ (all) 105

Nordquist et al., 1985

Mouse

male

S,T SMT

inhalation

Mouse

male

C3H × C57BL C3H× C57BL

SMT

i.p.

150-300 ppm (6h/day, 6 days) 175-700 m g / k g (5 days)

+ + + + + -

LEC c

11 weeks

387 ppm 387 ppm 387 ppm

Reference Loprieno et al., 1978 Meretoja et al., 1978

Conner et al., 1979 Conner et al., 1979 Conner et al., 1980 Conner et al., 1980 Conner et al., 1980 Penttila et al., 1980 Norppa et al., 1980

Salomaa et al., 1985 Salomaa et al., 1985

a CA, chromosomal aberrations; CB, (DNA) covalent binding; MN, micronuclei, SCE, sister-chromatid exchanges; SMT, sperm morphology test; SSB, (DNA) single-strand breaks. b BM, bone marrow; AM, alveolar macrophages; B, brain; K, kidney; Li, liver; Lu, lung; S, spleen; T, testis. c LEC, lowest effective concentration.

dratase than in mice (Norppa et al., 1979, 1980b). No increase of micronuclei in Chinese hamster bone marrow cells was seen, either with a single i.p. injection of 1000 m g / k g styrene or with 250 m g / k g styrene oxide (Penttila et al., 1980). D N A covalent binding and single-strand breaks in DNA were induced in several tissues in mice after a single i.p. injection of styrene oxide comparable to that given in the above-mentioned studies (Walles and Orsen, 1983; Nordquist et al., 1985). The positive effect of styrene on mammalian cells in vivo seems reasonably established particularly when the induction of DNA-covalent binding, D N A single-strand breaks and SCE is in-

vestigated; the administration route, inhalation or i.p., and the duration of exposure and species of animals seem to be the most important variables that determine genotoxic responses (Table 7). Styrene oxide showed relatively week effects possibly due to its inactivation on the way from the administration site to the target cells.

3.5.2. Occupationally exposed workers The first investigation of the possible clastogenic activity of styrene due to occupational exposure of humans was stimulated by the assessed carcinogenic and mutagenic properties of vinyl chloride which, like styrene, contains a vinyl group that can be oxidated to a biologically reactive epoxy form (Meteroja et al., 1977). These authors

119 TABLE 8 SUMMARY OF IN VIVO MUTAGENICITYASSAYSWITH STYRENE OXIDE Species

Sex

Strain

Endpoint Organor or assay a tissue b

R o u t e Dose

Result

LEC

Reference

Mouse Mouse Mouse Hamster

male male male male

CD1 BALB/c BALB/c Chinese

CA MN DL CA

BM BM

p.o. i.p.

50-500mg/kg 250 mg/kg

+ -

50

Loprienoet al., 1978 Fabry et al., 1978

BM

25-100ppm

-

Norppa et al., 1979

SCE

BM

(2, 4, 20 days)

-

Norppa et al., 1979

BM K, Li, Lu, T,B Li, B, Lu, S,T

inhalation inhalation i.p. i.p.

250 mg/kg 216-840mg/kg

+ (all)

Penttila et al., 1980 Solveig and Orsen, 1983

i.p.

120 mg/kg

+ (all)

Nordquist et al., 1985

Hamster Mouse

male Chinese male NMRI

MC SSB

Mouse

male NMRI

CB

a CA, chromosomal aberrations; CB, (DNA) covalent binding; DL, dominant lethal test; MN, micronuclei; SCE, sister-chromatid exchanges; SMT, sperm morphologytest; SSB, (DNA) single-strand breaks. b BM, bone marrow; AM, alveolar macrophages; B, brain; K, kidney; Li, liver; Lu, lung; S, spleen; T, testis.

reported an increased frequency (11-26% vs. 3%) of aberrant chromosomes, mainly of chromatid aberrations, in peripheral lymphocytes of 10 styrene-exposed workers compared to 5 controls. Increased frequencies of micronucleated lymphocytes and cells connected with nuclear bridges were also observed in exposed workers. N o quantitative correlation between the extent of chromosomal damages and the level of urinary mandelic acid or phenylglyoxylic acid was obtained (Camurri et al., 1984). This is not surprising, since these styrene metabolites may be considered reliable markers only for assessing current styrene exposure (Engstrom et al., 1976), while chromosomal aberrations can derive from past exposure, even years before, to genotoxicants. Since then, several human cytogenetic monitoring studies have been carried out on workers exposed to styrene in different industrial processes. In some cases styrene concentrations of ambient air were measured, in others the exposure was estimated by urinary mandelic acid concentrations, while in others both values were reported. Exposure length was also reported, ranging from months to many years, but in some cases exposure to other chemicals was not excluded. Two reviews have been published (IPCS, 1983; Maki-Paak-

kanen, 1987) and are here updated with the integration of more recent papers. A Table (9) summarizing this updating is reported.

(a) Chromosomal aberrations (CA) In general, even if it is difficult to assess the presence of a possible relationship between levels of exposure and the extent of CA detected, it appears that damages to chromosomes were preferentially found in workers exposed to higher levels of styrene. For instance, the negative findings reported by Fleig and Thiess (1978), reviewed with full particulars by N o r p p a et al. (1981), were related to very low styrene exposure levels (0-200 mg/m3). However, Watanabe et al. (1981), Pohlova et al. (1985) and more recently Jablonika et al. (1988) reported negative findings for higher exposure levels (0-1000, average 200 and 118-582, average 253 m g / m 3, respectively), largely overlapping those associated with positive results obtained by other investigators (see Table 9). However, the majority of styrene air concentrations reported, often estimations or averages, were not obtained by continuous and detailed workplace air monitoring and therefore cannot be useful for a reliable assessment of actual exposure. This is also supported by Camurri et al. (1984), who did

-

10 8 _

22/22

21/21 11/11 20/22 46 e

1-25 1-25 _

1-11

1-11

0.6-8.5 1-15 2-16 14-25 3-39 2-24 < 1-10 4-27 1500 225-2100 0-320 90-2640 0-1041 45-1108 9-316 171-1200 < 151-302

Range

1.2/1.5

4.5/4.9 1.3/1.4

-

(5) (50-52) (48)

(54)

6.41

7.6/7,4 -

N

2.3/1.6 h N N N

13.9/9.4

(50) (54)

P N

20.7/15.1 2.8/2.7 29.5/6.3 1.4/1.3 h

SCEs/cell mean Exp/Con

5.3/4.4 8.4/7.5 7.3/7.6 8.9/8.5 13.9/10.8 6.6/6.5 -

Result ( c u l t u r e time) (h)

OF WORKERS

P (64-66) P (66-68) P (66-68) N (70-72) N (70-72) P (70-72) P (70) P ¢ (70-72) P (66-68) N a N (50) P (50) a (48-53) N (64-68)

16.6/1.8 15.1/2.0 16.2/3.8/5.5 5.1/5.5 9.2/5.5 10.2/4.9 5.1/3.8 12.3/6.7 3.4/3.3 6.5/4.7 34.5/7.0

Exp/Con

Cells (%) with CA, g a p s ineluded

AND SCEs IN THE LYMPHOCYTES

Urinary MAf

ABERRATIONS

Concentration of s t y r e n e in a i r ( m g / m 3)

CHROMOSOMAL

Y e a r s of exposure

10/5 16/6 11/3 b 5/20 12/20 14/20 6/6 24/24 36/37 16/13 18/6 24/21 38/20 18/9 15/13

Exp/ Con a

STRUCTURAL

TABLE 9

P

N -

-

1.5,/1.6 0.4/0.4

-

0.6/0.6 0.4/0.1

P N N P N P

-

0.9/0.1

Cells (%) w i t h micronuclei Exp/Con

N

Result

N

N

P

P

P

Resuit

EXPOSED TO STYRENE

M e r e t o j a et al., 1977 M e r e t o j a et al., 1978 M e r e t o j a et al., 1978 F l e i g a n d Thiess, 1978 F l e i g a n d Thiess, 1978 F l e i g a n d Thiess, 1978 H o g s t e d t et al., 1979 T h i e s s et al., 1 9 8 0 A n d e r s s o n et al., 1980 W a t a n a b e et al., 1981 W a t a n a b e et al., 1983 C a m u r r i et al., 1983 H o g s t e d t et al., 1983 H a n s t e e n et al., 1984 Nordenson and Beckman, 1984 C a m u r r i et al., 1984 Pohlova and Sram, 19853 Pohlova and Sram, 1985a M a k i - P a k k a n e n , 1987 J a b l o n l k a et al., 1988 H a g m a r et al., 1 9 8 9 Y a g e r et al., 1989

Reference

7~

121 not find any significant correlation between CA frequencies in exposed workers and styrene air concentrations in each plant when control values were subtracted. More direct exposure evaluation through the measurement of styrene metabolites present in the urine of workers is also a pitfall. In fact this type of monitoring should be performed much more frequently than it has been done in the past in order to decrease the probability that occasional high exposure episodes (Maki-Paakkanen, 1987), occurring at unknown frequency with possible genotoxic effects, pass undetected. However, recently no associations of CA with low styrene exposure levels (34-236, average 98 mg/m3), carefully assessed, were observed by Maki-Pakkanen (1987). Finally, Hagmar et al. (1989) did not find any chromosome aberrations correlated with an average exposure of 56 m g / m 3 (15 ppm).

(b) SCE In all but 2 studies reported here, SCE analyses were performed in parallel with CA assays. The only 2 studies with positive findings were performed on groups characterized by high styrene exposure levels (0-1008 and 3 0 - > 400 mg/m3; Andersson et al., 1980; Camurri et al., 1983, respectively). In the latter study particularly high SCE values were found in workers exposed to concentrations over 250 m g / m 3. So increased SCE frequencies in human lymphocytes showed a weaker association with styrene exposure than CA. A possible explanation is the shorter life of SCE and consequently their induction can reflect only rather recent (months) effective exposures. It is therefore evident that SCE study can be more reliable when performed in combination with accurate recent exposure evaluations. In this regard, Yager et al. (1989) have reported a significant increase of SCE correlated with styrene exposure as low as 64.2 m g / m 3 (15.1 ppm) in the air. In this case, however, full-shift breathing-zone styrene concentrations were measured 7 times over the year on the same 46 individual workers and styrene in the exhaled air was also determined up to 3 times per day. Moreover, to increase the sensitivity of the assay, 80 metaphases were scored per sample in this study.

(c) Micronuclei Increased frequencies of micronuclei in cultured lymphocytes of styrene workers were observed in 3 out of the 5 studies in which they were considered (Meretoja et al., 1977; Hogstedt et al., 1983; Nordenson and Beckman, 1984). These latter authors suggest that exposure to low styrene levels (average 101 m g / m 3, 24 ppm) may be too weak to induce chromosome breaks, but high enough to cause disturbances of the mitotic spindle apparatus. For exposure at levels of 56 m g / m 3 (15 ppm), Hagmar et al. (1989) did not find any increase of micronuclei when the effects of age and smoking were considered.

(d) Other genotoxic effects When lymphocytes of 38 styrene-exposed workers (1-10 ppm) were treated in vitro with N-acetoxy-2-acetylaminofluorene, a significant increase ( p < 0.001) of unscheduled D N A synthesis was observed compared to controls (Pero et al., 1982). It was concluded that styrene exposure predisposes lymphocytes to increased mutagen sensitivity for subsequent genotoxic exposures. Recently Liu et al. (1988), using the 32p post-labeling technique, were able to detect 2 styrene oxideD N A adducts in mononuclear cells of 2 workers exposed to 96 ppm styrene. The 2 adducts corresponded to those obtained by D N A treatments in vitro with styrene oxide. These encouraging results prompted them to extend their investigations to a larger number of workers exposed to styrene to a lesser extent (Bodell et al., 1989). Their preliminary data suggest the presence of a correlation between styrene exposure (1-20 ppm) and levels of styrene oxide-DNA adducts. These data indicate that styrene oxide may be the major active styrene metabolite in human lymphocytes. Moreover Walles et al. (1988) found D N A single-strand breaks in peripheral lymphocytes of styrene-exposed workers (50-100 ppm). A good correlation was also found between the observed damage, the excretion of mandelic acid and the concentration of styrene glycol in the blood, indicating that exposure to styrene was the causative agent. Exposures below 20 ppm were apparently ineffective. There is sufficient evidence that occupational exposure to styrene may cause genotoxic effects in humans. No clear threshold has been identified

122 for the different genetic endpoints observed either because of the lack of reliable data on environmental exposure or because of the increased sensitivity of new studies that show genotoxic effects at exposure levels once considered ineffective. To date, the studies have been confined to blood cells only; however, these cannot be considered the only biomonitoring assays for assessing human exposure because the genotoxic effects detected in lymphocytes cannot exclude damage in other ceils or tissues as suggested by some animal studies. 3.6. Conclusions Styrene has been proven to induce a variety of mutagenic effects in several experimental assays and in peripheral human lymphocytes of professionally exposed workers. It appears well established that styrene requires metabolic activation to produce the observed genotoxic effects. Whatever active metabolite is involved, styrene 1,2-, 3,4- or 7,8-oxide, genotoxic effects are likely to be produced only when a sufficient amount of epoxide is formed and is available for D N A reactions. Conseqtiently a favorable balance between activating enzymes and detoxifying systems appears to be critical for obtaining positive outcomes. This seems particularly important for in vitro assays where microsomal preparations are generally used. In such cases, the above-mentioned balances may differ from one system to another determining apparently conflicting results. This view is supported by the results of studies in which adequate enzyme activity modifiers were added to the incubation mixtures. In in vitro assays, styrene epoxide shows strong genotoxic effects which can be partially modulated by the presence of exogenous metabolic systems. In several in vivo experimental assays, styrene shows some genotoxic activities, even if not unequivocally. What appears more relevant, in these cases, is the route of administration and the duration of treatments, chronic treatments being more effective than acute ones. The rapid inactivation and excretion of styrene oxide can explain its negative results in some studies.

In man, investigations of the effects of occupational exposure to styrene have demonstrated a relationship between exposure and the induction of a variety of genotoxic effects in peripheral lymphocytes. So far, there are insufficient data to establish whether the observed positivity is confined to an activation system peculiar to the red blood cells and lymphocytes or whether it is present in all cell types to a comparable extent. However, data from animal studies reported here indicate that other cell types, such as hepatocytes or macrophages, may show genotoxic effects due to styrene exposure. Current recommended threshold limits range from 215 m g / m 3, 50 ppm (value-time-weighted average) to 425 m g / m 3, 100 ppm for short-term exposure (American Conference of Governmental Industrial Hygienists, 1987); 100 ppm in The Netherlands and the U.K.; 50 ppm in France. In Sweden, the hygienic standard was reduced in 1984 to 106 m g / m 3 (25 ppm), while the F.R.G. and Finland have an even lower limit, 20 ppm. From the data reviewed here we can try to define a threshold limit for chromosomal aberrations of approximately 200 m g / m 3 (50 ppm); for SCE, micronuclei and D N A single-strand breaks attempts to make a reasonable estimate are not possible due to the scanty data. As to D N A adducts, it seems reasonable to forecast that with the improvements in the detecting methodologies actual thresholds might be further reconsidered. For this latter endpoints, however, it remains to clarify what it means in terms of genotoxic risk, even if it remains a strong and significant biochemical marker for genotoxic exposure dosimetry. All these facts lead to the conclusion that, even if carcinogenicity of styrene is far from being demonstrated, current occupational exposure thresholds, approaching the level of no clastogenic effects, should be reconsidered in order to further reduce the possibility of induction of other genotoxic effects in human somatic cells, independently of their possible significance in terms of somatic risks. However, the refinement of these levels needs much more extensive data about 'external' and 'internal' doses, which would allow a more precise correlation between genotoxic events and occupational exposure to styrene.

123

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