In vivo mutagenicity of ethylene oxide at the hprt locus in T-lymphocytes of B6C3F1 lacI transgenic mice following inhalation exposure

Abstract

Ethylene oxide (EO) is a direct-acting alkylating agent with the potential to induce cytogenetic alterations, mutations, and cancer. In the present study, the in vivo mutagenicity of EO at the hypoxanthine guanine phosphoribosyltransferase ( hprt) locus of T-lymphocytes was evaluated following inhalation exposure of male B6C3F1 lacI transgenic mice. For this purpose, groups of male Big Blue ® mice at 6–8 ( n=4/group) and 8–10 ( n=5/group) weeks of age were exposed to 0, 50, 100, or 200 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 concanavalin 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]. The time course for expression of hprt-negative lymphocytes in thymus was determined in mice necropsied 2 h, 2 weeks, and 8 weeks after exposure 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 (M f) in thymus of exposed mice was increased 2 h after exposure and reached a maximum of 7.5±0.9×10 −6 (average M f±SE) at 2 weeks post-exposure, compared with 2.3±0.8×10 −6 in thymus of control mice. Dose-related increases in hprt M fs were found in thymus from mice exposed to 100 and 200 ppm EO. In addition, a nonlinear dose-dependent increase in hprt M fs was observed in splenic T-cells, with greater mutagenic efficiency (mutations per unit dose) found at higher concentrations than at lower concentrations of EO. Average induced M fs (i.e. induced M f=treatment M f−background M f) in splenic T-cells were 1.6, 4.6, and 11.9×10 −6 following exposures to 50, 100, or 200 ppm EO, respectively, while the average control M f value was 2.2±0.3×10 −6. In aliquots of lymphocytes (both B- and T-cells) isolated from spleen for analysis of lacI mutations in the same animals, only two of three EO-exposed mice at the 200 ppm exposure level demonstrated an elevated lacI M f and these elevations were apparently due to the in vivo replication of preexisting mutants and not due to the induction of new mutations associated with EO exposure [Sisk, S., L.J. Pluta, K.G. Meyer and L. Recio (1996) Mutation 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 lacI transgenic mice of B6C3F1 origin.