Factors that affect the frequency of thioguanine‐resistant lymphocytes in mice following exposure to ethylnitrosourea

Abstract

The frequency of thioguanine(TG)-resistant lymphocytes in mice treated with ethylnitrosourea (ENU) was followed for a period of 51 wk using our clonogenic assay [Jones et al, 1985a,b]. The effects of dose (0-58 mg/kg), time since treatment (2-51 wk), dose rate (5 weekly X 11.7 mg/kg versus 1 X 58 mg/kg), and age at time of treatment (3 vs 15 mo) on the frequency of TG-resistant, concanavalin A-responsive spleen cells were evaluated. The frequencies of TG-resistant spleen cells were generally dose responsive for 51 wk after exposure to ENU. They also were dependent upon the time that had elapsed since treatment with ENU, increasing to maximal values at 10 wk as previously reported [Jones et al, 1985a], and holding essentially stable at values of approximately 20% of the maximum frequency from week 15 until at least week 40 for the 3-month-old mice. Fractionation of 58 mg ENU/kg into 5 weekly doses did not affect the frequency of ENU-induced TG-resistant cells detected in the spleen but did increase the rate of appearance in the spleen, and the efficiency of induction by the unit dose, of TG-resistant cells. The mice exposed to ENU at 15 mo of age appeared to have a 4-fold reduction in the rate of increase in frequency of ENU-induced TG-resistant spleen cells. One set of control mice was found to have a 10-fold elevated frequency of TG-resistant cells in both the spleen and thymus, indicating that mutations can occur in stem cells of untreated animals.

Mutational spectrum at the Hprt locus in splenic T cells of B6C3F1 mice exposed to N-ethyl-N-nitrosourea.

“…The Hprt-mutant fraction in control animals was =2 x 10-6; this is similar to the values reported previously for the BALB/c (1) and C57BL/6 (2, 3) mouse strains. Mutant frequency in treated animals was =35-fold higher (7.3 x 10-5), similar to previously reported values obtained with ENU (2). Reconstruction experiments showed that the PE of mutants on plates containing 6TG was indistinguishable from the PE on the PE plates (data not shown), thus demonstrating that the observed mutation frequencies measured in our experiments are good approximations of true mutation frequency existing in the T-cell populations.…”

“…Nevertheless, at the present time we cannot exclude the possibility that some of the mutations observed could have resulted from in vitro replication of DNA containing extremely persistent DNA adducts. However, this seems unlikely given the stable mutation frequency observed in mice up to a year after ENU exposure (2). If replication of promutagenic lesions in vitro were contributing a substantial fraction to the total number of observed mutations, then one would anticipate that the mutation frequency would decrease with time as adducts were slowly repaired.…”

“…The complexity of this pathway suggests that in vivo mutagenesis assays may be more relevant than in vitro systems for modeling possible mutagenic consequences in humans. In vivo mutation assays based on the cloning of hypoxanthine (guanine) phosphoribosyltransferase (HPRT)-negative T cells have been developed in the mouse (1)(2)(3), rat (4), monkey (5), and human (6,7). Recently, transgenic mouse systems have also been described that contain bacterial transgenes as mutational targets (8,9).…”

DNA sequence analysis of hprt mutations in lymphocytes from Sprague-Dawley rats treated with 7,12-dimethylbenz[a]anthracene

“…For instance, there is trafficking of cells between different lymphoid tissues, uneven expansion and turnover of individual lymphocyte clones, and a moderate selection pressure against the mutant phenotype [Freitas and Rocha, 1993;Deubel et al, 19961. In rodents, it is also unclear in which organs and tissues lymphocyte mutations are induced [Jones et al, 1987;Aidoo et al, 1991, 19931. The types of mutations produced in rodent lymphocytes by simple alkylating agents appear to reflect the damage produced by these compounds Mittelstaedt and Heflich, 1994;Mittelstaedt et al, 1995;Jansen et al, 19951.…”

Quantification of thioguanine-resistant lymphocytes from mice irradiated in vivo.

“…Jones and co-workers (1)(2)(3) have demonstrated that cloning mouse lymphocytes permits analysis of mutations in the hprt gene that have occurred in vivo in T-cell progenitors. Possibilities and limitations of this method were investigated, and preliminary results on the mutant frequency in gammairradiated animals will be given.…”