Relationships between exposure, cell loss and proliferation, and manifestation of Hprt mutant T cells following treatment of preweanling, weanling, and adult male mice with N-ethyl-N-nitrosourea

Experiments were performed to characterize the age-related patterns of appe arance and frequency of hypoxanthine-guanine phosphoribosyl transferase (Hp rt) mutant T lymphocytes in thymus and spleen following exposure of prewean ling (12-day-old), weanling (22-day-old), and young adult (8-week-old) male B6C3F1 mice to ethylnitrosourea (ENU). Mice were given single i.p. injecti ons of 0 or 40 mg ENU/kg and then groups of animals were necropsied from 2 h to 116 days after treatment to examine the relationships between exposure , cell loss and proliferation, and the frequency of Hprt mutant T cells in thymus and spleen. Hprt mutant frequency (Mf) data for thymus of ENU-expose d (0, 11.7, 35, 58, or 72 mg/kg, or five weekly doses of 1.7 mg/kg i.p.) ma le C57BL/6 mice (12- or 62-week-old), obtained during an earlier study of s pleen cells [I.M. Jones, K. Burkhart-Schultz, C.L. Strout, T.L. Crippen, Fa ctors that affect the frequency of thioguanine-resistant lymphocytes in mic e following exposure to ethylnitrosourea, Environ. Mutagen. 9 (1987) 317-32 9.], were compared to results in B6C3F1 mice. Isolated T cells were culture d in the presence of mitogen, growth factor, and 6-thioguanine to detect Hp rt mutants. The time required to achieve maximum Mfs in thymus was uniforml y found at 2 weeks after ENU treatment, while the times needed to reach pea k values in spleen were proportional to animal age at treatment. These data indicate that age-related differences in the appearance of Hprt mutant cel ls in spleen are largely defined by the physiologically based, age-dependen t trafficking of mutant cells from or through the thymus. Three modes of ha ndling the resulting Hprt MS data were evaluated: (i) comparing the Mfs at a single time point, (ii) comparing the maximum Mfs observed. and (iii) com paring the change in Mfs over time (or the mutant T cell 'manifestation' cu rves in treated vs. control mice) in each age group post-exposure. Measurin g the Mfs in spleen at multiple time points after cessation of exposure and integrating the frequency of mutants as a function of time appeared to be the superior method for comparing mutagenic responses in different age grou ps. Some of the underlying assumptions of this approach, as well as its str engths and weaknesses, are discussed. (C) 1999 Elsevier Science B.V. All ri ghts reserved.