Studies on mutagenesis by ethylene oxide and styrene oxide in the hprt gene in human cells

Abstract: Studies on Mutagenesis by Ethylene Oxide and Styrene Oxide in the hprt Gene in Human Cells Tatiana Bastlova Department of Biosciences at Novum, Center for Nutrition and Toxicology, The Karolinska Institute, Novum, S-14157 Huddinge, SwedenEnvironmental, occupational and life style-related exposures to mutagenic agentsmay contribute to cancer risk in humans. To prevent the potentially hazardouseffects of such agents it is important to understand their mechanisms of action. In thisthesis, the mutagenic effects of two important industrial epoxides, ethylene oxideand styrene oxide, have been studied. Ethylene oxide is used as a chemical interme-diate in the production of many industrial chemicals and as a disinfectant andsterilising agent. It is a direct acting alkylating agent which has recently beenclassified as carcinogenic to humans. Styrene-7,8-oxide is the primary in vivometabolite of styrene, which is used in the production of polymers and copolymers,and as an important part of unsaturated polyester resins in the production of glass-reinforced plastics products. Styrene and styrene-7,8-oxide have been classified aspossibly and probably carcinogenic to humans.The frequency and nature of ethylene oxide and styrene oxide-induced mutationswere studied in the hypoxanthine guanine phosphoribosyltransferase (hprt) gene inhuman fibroblast and T-lymphocyte cultures, respectively. Polymerase chain reac-tion-based techniques, Southern blotting and direct DNA sequencing were used tostudy the molecular nature of mutations induced by the two compounds. Ethyleneoxide caused a 5-19-fold increase of hprt mutant frequency relative to the back-ground frequency in fibroblasts. Base substitutions and large hprt gene alterationswere the major types of mutation induced by ethylene oxide. Among base substitu-tions, both transitions and transversions were identified, the most frequent being ATto TA transversions. Among the large alterations, both total and partial genedeletions were found. The results suggest that several different target sites might beinvolved in ethylene oxide mutagenesis.Styrene-7,8-oxide caused a 4-fold increase of the hprt mutant frequency in culturedhuman T-lymphocytes. Base substitutions were the major type of mutation and themost frequent were AT to GC transitions. Approximately 60% of the base substitu-tions in the coding region and the splice site sequences were at AT base pairs,suggesting that DNA adducts at these sites may be important in the mutagenesis ofstyrene oxide. The frequency of hprt mutant peripheral blood T-lymphocytes wasstudied in a group of styrene-exposed hand lamination workers. The mutantfrequency was higher in the laminators than in a group of of fice workers from thesame factory at three of four sampling times within a seven-month period. Anothercontrol group, working outside the factory and not exposed to styrene, showed asignificantly lower hprt mutant frequency than the styrene exposed laminators.Taken together, these results have contributed to the knowledge on mutagenesis byethylene oxide and styrene oxide in human cells in vitro and support the possiblemutagenic effect of human styrene exposure in vivo.Key words: ethylene oxide, styrene, styrene-7,8-oxide, human diploid fibroblasts,human T-lymphocytes, hprt mutation ISBN 91-628-1909-7