IN-VIVO MUTAGENICITY OF ETHYLENE-OXIDE AT THE HPRT LOCUS IN T-LYMPHOCYTES OF B6C3F1 LACI TRANSGENIC MICE FOLLOWING INHALATION EXPOSURE

Ethylene oxide (EO) is a direct-acting alkylating agent with the poten tial to induce cytogenetic alterations, mutations, and cancer. In the present study, the in vivo mutagenicity of EO at the hypoxanthine guan ine phosphoribosyltransferase (hprt) locus of T-lymphocytes was evalua ted following inhalation exposure of male B6C3F1 lad transgenic mice. For this purpose, groups of male Big Blue(R) mice at 6-8 (n = 4/group) and 8-10 (n = 5/group) weeks of age were exposed to 0, 50, 100, or 20 0 ppm EO for 4 weeks (6 h/day, 5 days/week). At necropsy, T-cells were isolated from thymus and/or spleen and cultured in the presence of co ncanavalin A, IL-2, and 6-thioguanine [Skopek, T.R,, V.E. Walker, J.E. Cochrane et al. (1992) Proc. Natl. Acad. Sci. USA, 89, 7866-7870]. Th e time course for expression of hprt-negative lymphocytes in thymus wa s determined in mice necropsied 2 h, 2 weeks, and 8 weeks after exposu re to 200 ppm EO. The dose-response for hprt mutant T-cells in thymus and spleen was defined in mice necropsied 2 and 8 weeks post-exposure, respectively. The hprt mutant frequency (Mf) in thymus of exposed mic e was increased 2 h after exposure and reached a maximum of 7.5 +/- 0. 9 x 10(-6) (average Mf +/- SE) at 2 weeks post-exposure, compared with 2.3 +/- 0.8 x 10(-6) in thymus of control mice. Dose-related increase s in hprt Mfs were found in thymus from mice exposed to 100 and 200 pp m EO. In addition, a nonlinear dose-dependent increase in hprt Mfs was observed in splenic T-cells, with greater mutagenic efficiency (mutat ions per unit dose) found at higher concentrations than at lower conce ntrations of EO. Average induced Mfs (i.e. induced Mf = treatment Mf-b ackground MS) in splenic T-cells were 1.6, 4.6, and 11.9 x 10(-6) foll owing exposures to 50, 100, or 200 ppm EO, respectively, while the ave rage control MS value was 2.2 +/- 0.3 x 10(-6). In aliquots of lymphoc ytes (both B-and T-cells) isolated from spleen for analysis of loci mu tations in the same animals, only two of three EO-exposed mice at the 200 ppm exposure level demonstrated an elevated lacl Mf and these elev ations were apparently due to the in vivo replication of preexisting m utants and not due to the induction of new mutations associated with E O exposure [Sisk, S., L.J. Pluta, K.G. Meyer and L. Recio (1996) Mutat ion Res., submitted]. These data demonstrate that repeated inhalation exposures to high concentrations of EO produce dose-related increases in mutations at the hprt locus of T-lymphocytes in male lad transgenic mice of B6C3F1 origin. (C) 1997 Elsevier Science B.V.