A study on the mutagenic activity of styrene and styrene oxide.

A study on the mutagenic activity of styrene and styrene oxide. Scand. j. work environ. & health 3 (1976) 147-151. Styrene oxide is mutagenic, without metabolic activation, to S. typhimurium strains TA 1535 and TA 100, which have been devised to detect mutagens causing base-pair substitutions. Styrene seems to be mutagenic toward the same straims, but only after metabolic activation. The toxi cHy of both styrene and styrene oxide make the construction of reliable dose response curves rather difficult. Diethylmaleate and 3,3,3-trichloropropene oxide enhanced the mutagenicity of styrene oxide in the presence of homogenate; this result indicates the participaUon of ,epoxide hydratase and g,lutathione S-oxide transferase in the metabolism of styrene oxide. These two chemioaJls did not influence the mutagenic activity of ,styrene. Styrene glycol and 4-tert.-butyl brenzcatechin were not mutagenic to any of the strains studied. Results show that further, more detailed experimental and, possibly, epidemiologic studies are warranted.

and animals (4). In in vitro experiments vinyl chloride is metabolized by liver microsomes to ,a compound which i:s mutagenic to Salmonella typhlimudum ba,meria (histidine auX!otmp'hs) strains (9) developed by Ames and coworkers for the detectton of mutagenk compounds (1,2).
Because of the wide use of styrene and i'bs resemblanoe to vinyl ohloride we decided to study the mutageni'c potential of styrene and its metahoUtes in the Salmonella ,sy,stem, both in the presence and in the ,absence of liver microsomal enzymes.

Enzyme preparation
Male rats (200 to 300 g) of the Sprague-Dawley strain were injected with a polychlorinated hiphenyl compound, Clophen C (200 mg/kg, obtained from Bayer AG, Leverkusen) in sesame oil 5 days before they werelcilled. Li'vers were removed, homogenized in four volumes of 0.15M KCl, and centrifuged ,at 10,000 X g for 10 min. The superna'tant fraction ("S-9") was divided into 2-ml fractions and stored at -80 D C until used. Tables 1 and 2 show the preliminary screening of the mutagenic potential of styrene oxide and styrene over a wide rang.e of concentrations. As can be seen, both styrene oxide and styrene were toxic to all strains at high concentrations, styrene oxide at about .10-4 moles/pLate and above and styrene at ahout 10-6 moles/ plate and above. This high toxicity made the mutagenicity determinations at this range of concentration difhcult. However, as table 1 shows, styrene oxide was clearly mutagenk to two of the five strains, namely, to TA 1535 and TA 100. It was not mutagenic to TA 1537, 1538, or 98. The enzyme preparati'on did not remarkably affect the mutation rate, and this finding suggests tha,t we were dealing with a direct mutagen. On the other hand, the preliminary screening with styrene showed (table 2) that it may be mutagenic to TA 1535 and TA 100. However, a more detailed dose-response study with TA 1535 revealed that styrene may be both an indirect and direct mutagen, although results suggest tha t the considerable toxicity of styrene disturbs the interpretation of results obtained from concentrations as low as about 10-7 mole's/plate ( fig. 1) A simiIar study wi1!h styrene and TA 100 DiethyImaleate, a compound which is known to deplete cytoplasmic glutathione, and 3,3,3-trkhloropropene oxide, an inhibitor of epoxide hydratase (8), slightly increased the mutation rate caused by styrene (table 3). At the concentration used and in the presence of the S-9 fvachon, diethylmaleate and trichloropropene oxide had no effect on the muta,tion rate of the strains studied.

RESULTS
The ratto styrene oxide/homogenate, when added, affects grea1tly the data obtained. Larger amounts of the liver homogenate seem to protect the bacteria, perhaps due to the presence of the enzymes glutathione S-transferase and epoxide hydratase, which metabolize styrene oxide. The protective effect could be abolished by diethylmaleate and trichloropropene oxide (table 3).
Styrene glycol did not e~hi!bit mutagenicity towards TA 1535 and TA 100 in the concentration range from 10-4 to 10-10 moles/plate. In addition the preservation agent, 4-tert.-butyl-bnmzcatechin, was not mutagenic to any of the five strains used.

DISCUSSION
Earlier studies have demonstrated that styrene is convertoo by microsoma:l monooxygenase systems t.o styrene oxide, which is metabolized further by epoxide hydratase to styrene glycol (5). Because styrene is structurally sim5.:lar to vinyl chloride, a known mutagen and carcinogen, one can suspeot that styr,ene is potentially mutagenic and possibly carcinogenic. This study demonstrates clearly that styrene oxide is mutagenic to S. typhimurium ,strnins that ha,ve been especially constructed to detect mutagens, namely, to TA 153'5 and TA 100. These two strains can be used to detect mutagens causing base-pair suhstituhons, and oonsequently styrene oxide seems to be a base-pair substitution mutagen. ks small an amount as about 10-8 moles of styrene oxide per plate causes a significiant increase in the mutJation rate. Styrene ox,ide seems to be a directly acting mutagen, because the presence of an enzyme preparation does nOit enhance its mutagenicity.
Styrene seems ,to be an indirectly acting mutagen tQo TA 1535 and TA 100, the same strains which are also sensitive tJo styrene oxide. One can speculate that styrene oxide, formed by the monooxygenase, is the adive principle in the enhanced mutag,enesis caused by styrene. However, in tests with TA 100, styrene a'lso seemed to be a direct mutagen, but, because styrene is r,a'1:her toxic to Salmonella, the results of cer,tain doses are dHficult to interpret.
In the presence of liver homogenate, styrene appears to be ev,en more mutagenic to strain TA 1535 than styrene oxide. This phenomenon is not necessarily in nonagreement with the metabolic activation theory of styrene to corresponding epoxide, but it may be due to i'he complex fadors of chemicallbomogenate association. The difference at very low concentrations may be also eX!plained by the presence of an impurity in the styrene preparation. The main impurity, 4-tert.butyl-brenzoateohin, is not, however, mutagenic at all. There is then a possibi:lity that vinylphenol forms perhaps via an ,epoxide, and it might be the toxic and mutagenic metabollite in bacteria.

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While this work was in progress, Milvy and Garro (7) reported the mutagenicity of styrene oxide in S. typhimurium. They found that styrene was una/bie to increase the mutation rate in the Salmonella strains. They have not, however, tested the mutagenic activity ,in the presence of liver homogenate, which transforms styrene to its active metabolites. In addition they reported a high spontaneous mutahon rate, especiaUy with stmins TA 1535 and TA 100 (7). This high rate may perhaps have been a result of eX!posure to styl'ene monomers in the petri dilshes used, since we had similar problems with a certain set of petri dishes. After we changed to another type, the spontaneous mutation rate diminished to the normal level.
In condusion, this study suggests that furtlher, more detailed investigati<ms on the mutagenic and carcinogenic potential of styrene and styrene oxide are warranted and it points to the possible need of an epidemiologic study on workers in industry.