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Mutations in T lymphocytes (T-cells) are informative quantitative markers for environmental mutagen exposures, but risk extrapolations from rodent models to humans also require an understanding of how T-cell development and proliferation kinetics impact mutagenic outcomes. Rodent studies have shown that patterns in chemical-induced mutations in the hypoxanthine-guanine phosphoribosyltransferase (Hprt) gene of T-cells differ between lymphoid organs. The current work was performed to obtain knowledge of the relationships between maturation events during T-cell development and changes in chemical-induced mutant frequencies over time in differing immune compartments of a mouse model. A novel reverse transcriptase-polymerase chain reaction based method was developed to determine the specific T-cell receptor beta (Tcrb) gene mRNA expressed in mouse T-cell isolates, enabling sequence analysis of the PCR product that then identifies the specific hypervariable CDR3 junctional region of the expressed Tcrb gene for individual isolates. Characterization of spontaneous Hprt mutant isolates from the thymus, spleen, and lymph nodes of control mice for their Tcrb gene expression found evidence of in vivo clonal amplifications of Hprt mutants and their trafficking between tissues in the same animal. Concurrent analyses of Hprt mutations and Tcrb gene rearrangements in different lymphoid tissues of control versus N-ethyl-N-nitrosourea-exposed mice permitted elucidation of the localization and timing of mutational events in T-cells, establishing that mutagenesis occurs primarily in the pre-rearrangement replicative period in pre-thymic/thymic populations. These findings demonstrate that chemical-induced mutagenic burden is determined by the combination of mutagenesis and T-cell clonal expansion, processes with roles in immune function and in the pathogenesis of autoimmune disease and cancer., (© 2023 The Authors. Environmental and Molecular Mutagenesis published by Wiley Periodicals LLC on behalf of Environmental Mutagenesis and Genomics Society.)

Experiments were performed: (i) to investigate potential age- and gender-dependent differences in mutagenic responses in T cells following exposures of B6C3F1 mice and F344 rats by inhalation for 2 weeks to 0 or 1250 ppm butadiene (BD), and (ii) to determine if exposures for 2 weeks to 62.5 ppm BD produce a mutagenic effect in female rats. To evaluate the effect of age on mutagenic response, mutant manifestation curves for splenic T cells of female mice exposed at 8-9 weeks of age were defined by measuring Hprt mutant frequencies (MFs) at multiple time points after BD exposure using a T cell cloning assay and comparing the resulting mutagenic potency estimate (calculated as the difference of areas under the mutant manifestation curves of treated versus control animals) to that reported for female mice exposed to BD in the same fashion beginning at 4-5 weeks of age. The shapes of the mutant T cell manifestation curves for spleens were different [e.g., the maximum BD-induced MFs in older mice (8.0+/-1.0 [S.D.]x10(-6)) and younger mice (17.8+/-6.1 x 10(-6)) were observed at 8 and 5 weeks post-exposure, respectively], but the mutagenic burden was the same for both age groups. To assess the effect of gender on mutagenic response, female and male rodents were exposed to BD at 4-5 weeks of age and Hprt MFs were measured when maximum MFs are expected to occur post-exposure. The resulting data demonstrated that the pattern for mutagenic susceptibility from high-level BD exposure is female mice>male mice>female rats>male rats. Exposures of female rats to 62.5 ppm BD caused a minor but significant mutagenic response compared with controls (n=16/group; P=0.03). These results help explain part of the differing outcomes/interpretations of data in earlier Hprt mutation studies in BD-exposed rodents.

A multiplex PCR procedure for analysis of genomic DNA mutations in the mouse hypoxanthine-guanine phosphoribosyltransferase (Hprt) gene was developed and then used with other established methods for the coincident identification of large- and small-scale genetic alterations in the Hprt gene of mutant T-cell isolates propagated from sham- and 1,3-butadiene (BD)-exposed mice and rats. The spectra data for RT-PCR/cDNA analysis and multiplex PCR of genomic DNA from Hprt mutants were combined, and statistical analyses of the mutant fractions for the classes of mutations identified in control versus exposed animals were conducted. Under the assumption that the mutant fractions are distributed as Poisson variates, BD exposure of mice significantly increased the frequencies of (1) nearly all types of base substitutions; (2) single-base deletions and insertions; and (3) all subcategories of deletions. Significantly elevated fractions of G:C-->C:G and A:T-->T:A transversions in the Hprt gene of BD-exposed mice were consistent with the occurrence of these substitutions as the predominant ras gene mutations in multiple tumor types increased in incidence in carcinogenicity studies of BD in mice. BD exposure of rats produced significant increases in (1) base substitutions only at A:T base pairs; (2) single-base insertions; (3) complex mutations; and (4) deletions (mainly 5' partial and complete gene deletions). Future coincident analyses of large- and small-scale mutations in rodents exposed to specific BD metabolites should help identify species differences in the sources of deletion mutations and other types of mutations induced by BD exposures in mice versus rats., (Copyright 2004 Wiley-Liss, Inc.)

Propylene is a major industrial intermediate and atmospheric pollutant to which humans are exposed by inhalation. In this study, 6-week-old male F344 rates were exposed to 0, 200, 2000, or 10,000 ppm propylene by inhalation for 4 weeks (6 h/day, 5 days/week), and mutant frequencies were determined in the Hprt gene of splenic T-lymphocytes. Twenty milligrams of cyclophosphamide monohydrate (CPP)/kg bw, given on the penultimate day of propylene exposure, was used as a positive control mutagen. Rats (n = 8/group) were necropsied for isolation of T-cells 8 weeks after the last dose, a sampling time that produced peak spleen Hprt mutant frequencies (Mfs) in a preliminary mutant manifestation study using CCP treatment. Hprt Mfs were measured via the T-cell cloning assay, which was performed without knowledge of the animal treatment groups. Mean Hprt Mfs were significantly increased over control values (mean Mf = 5.24 +/- 1.55 (SD) x 10(-6)) in CPP-treated rats (10.37 +/- 4.30 x 10(-6), P = 0.007). However, Hprt Mfs in propylene-exposed rats were not significantly increased over background, with mean Mfs of 4.90 +/- 1.84 x 10(-6) (P = 0.152), 5.05 +/- 3.70 x 10(-6) (P = 0.895), and 5.95 +/- 2.49 x10(-6) (P = 0.500) for animals exposed to 200, 2000, or 10,000 ppm propylene, respectively. No significant increase in F344 rat or B6C3F1 mouse cancer incidence was reported in the National Toxicology Program carcinogenicity studies of propylene across this same exposure range. Taken together, these findings support the conclusion that inhalation exposure of rats to propylene does not cause mutations or cancer., (Copyright 2004 Wiley-Liss, Inc.)

A study was conducted to test the hypothesis that repeated low level exposures to 1,3-butadiene (BD), approaching the OSHA occupational threshold for this chemical, produce a significant mutagenic response in mice. Female B6C3F1 mice (4-5 weeks of age) were exposed by inhalation for 2 weeks (6 h/day, 5 days/week) to 0 or 3 ppm BD, and then necropsied at 4 weeks after the cessation of exposures to measure the frequency of mutations (MF) at the Hprt locus using the T-lymphocyte clonal assay. At necropsy, T cells were isolated from spleen and cultured in the presence of mitogen, growth factors, and a selection agent. Cells were scored for growth on days 8-9 after plating to determine cloning efficiencies (CEs) and Hprt MFs. There was a marginal but significant reduction in the growth of splenic T cells from mice exposed to 3 ppm (n=27) compared with control mice (n=24) (P=0.004), suggesting the occurrence of BD-induced cytotoxicity at this low exposure concentration. In addition, the average Hprt MF in mice exposed to 3 ppm BD [1.54+/-0.82 (S.D.)x10(-6)] was significantly increased by 1.6-fold over the average control value of 0.96+/-0.51 (S.D.)x10(-6) (P=0.004). Comparisons of these data to earlier Hprt mutagenicity studies of mice exposed to high concentrations of BD (where significant mutagenic but not cytotoxic effects were observed) indicate that the ability to detect the cytotoxic and mutagenic responses of T cells to low levels of BD was enhanced by using a much larger sample size than usual for both the control and treatment groups. Additional analyses of the quantitative relationships between CE and MF demonstrated that CE had no significant effect upon MF values in sham-exposed control mice or mice exposed to low-level BD. Furthermore, the approaches for assessing the impact of CE and clonality on Hprt MFs in these control and BD-exposed mice were applied with the same rigor as in in vivo Hprt mutagenicity studies in human children. The overall study results support the conclusion that short-term low-level BD exposure is mutagenic in the mouse.

Experiments were performed to characterize the age-related patterns of appearance and frequency of hypoxanthine-guanine phosphoribosyl transferase (Hprt) mutant T lymphocytes in thymus and spleen following exposure of preweanling (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. injections 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-exposed (0, 11.7, 35, 58, or 72 mg/kg, or five weekly doses of 1.7 mg/kg i.p.) male C57BL/6 mice (12- or 62-week-old), obtained during an earlier study of spleen cells [I.M. Jones, K. Burkhart-Schultz, C.L. Strout, T.L. Crippen, Factors that affect the frequency of thioguanine-resistant lymphocytes in mice following exposure to ethylnitrosourea, Environ. Mutagen, 9 (1987) 317-329.], were compared to results in B6C3F1 mice. Isolated T cells were cultured in the presence of mitogen, growth factor, and 6-thioguanine to detect Hprt mutants. The time required to achieve maximum Mfs in thymus was uniformly found at 2 weeks after ENU treatment, while the times needed to reach peak values in spleen were proportional to animal age at treatment. These data indicate that age-related differences in the appearance of Hprt mutant cells in spleen are largely defined by the physiologically based, age-dependent trafficking of mutant cells from or through the thymus. Three modes of handling the resulting Hprt Mf data were evaluated: (i) comparing the Mfs at a single time point, (ii) comparing the maximum Mfs observed, and (iii) comparing the change in Mfs over time (or the mutant T cell 'manifestation' curves in treated vs. control mice) in each age group post-exposure. Measuring 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 groups. Some of the underlying assumptions of this approach, as well as its strengths and weaknesses, are discussed.

The species specific response to 1,3-butadiene (BD), an important industrial chemical, was investigated by determining the influence of exposure duration and exposure concentration on the mutagenicity of BD in mice and rats and by defining the spectra of mutations in the Hprt gene T-cell mutants from control and BD-exposed mice. Female B6C3F1 mice and F344 rats (4-5 weeks old) were exposed by inhalation to 0, 20, 62.5, or 625 ppm of BD for up to 4 weeks (6 h/day, 5 days/week). Groups of control and exposed animals (n=4-12/group) were necropsied at multiple time points after exposure and the T-cell cloning assay was used to measure Hprt mutant frequencies in lymphocytes isolated from spleen. Mutant clones collected from control and BD-exposed mice were propagated and analyzed by RT-PCR to produce Hprt cDNA for sequencing. In animals necropsied 4 weeks after 2 or 4 weeks of BD exposure (0 or 625 ppm), the rate of accumulation of mutations was greater in mice than in rats. Supra-linear dose-response curves were observed in BD-exposed mice, indicating a higher efficiency of mutant induction at lower concentrations of BD. The mutagenic potency estimates (represented by the differences in the areas under the mutant T-cell 'manifestation' curves of treated vs. control animals) in mice were 11 and 61 following 4 weeks of exposures to 62.5 and 625 ppm of BD, respectively, while mutant frequencies (Mfs) in rats were significantly increased only at 625 ppm BD (mutagenic potency of 7). Molecular analysis of Hprt cDNA from expanded T-cell clones from control and BD-exposed mice demonstrated an increased frequency of mutants in exposed animals that likely contain large deletions in the Hprt gene (P=0.016). These data indicate that both exposure duration and exposure concentration are important in determining the magnitude of mutagenic response to BD, and that mutagenic and carcinogenic properties of BD in mice may be related more to the ability of its metabolites to cause chromosomal deletions than to produce point mutations., (Copyright 1999 Elsevier Science B.V.)

The purpose of this study was to determine if Hprt mutant frequency (Mf) data from rodents exposed directly to individual epoxy metabolites of 1,3-butadiene (BD) can be used to identify the relative significance of each intermediate in the mutagenicity of BD in mice vs. rats. To this end, the relative contributions of the racemic mixtures of BD monoepoxide (BDO) and BD diepoxide (BDO(2)) to BD-induced mutagenicity was investigated by exposing mice and rats to selected concentrations of BDO and BDO(2) (i.e., 2.5 and 4.0 ppm, respectively) and comparing the mutagenic potency of each intermediate to that of BD (at 62.5 ppm) when comparable blood levels of metabolites are achieved (in the mouse). Female B6C3F1 mice and F344 rats (4-5 weeks old) were exposed to rac-BDO (0, 2.5, or 25 ppm) or (+/-)-BDO(2) (0, 2, 4 ppm) by inhalation for 4 weeks (6 h/day, 5 days/week), and then groups of control and exposed animals (n=3-12/group) were necropsied at multiple time points post-exposure for measuring Hprt Mfs in splenic lymphocytes (via the T-cell cloning assay) and estimating mutagenic potencies (represented by the difference in the areas under the mutant T-cell 'manifestation' curves of treated vs. control animals). The resulting Mf data, along with the extant metabolism data, suggest that at lower BD exposures (

The relative sensitivities and specificities of the endogenous Hprt gene and the lacI transgene as mutational targets were evaluated in splenic lymphocytes from male standard B6C3F1 mice (only Hprt assayed) and from lacI transgenic B6C3F1 mice treated at 6-7 weeks- of-age with the indirect-acting agent, cyclophosphamide (CP). To define the effects of the time elapsed since CP treatment on Hprt mutant frequencies (Mfs), nontransgenic mice were given single i.p. injections of 25 mg CP/kg or vehicle (PBS) alone and then necropsied 2, 4, 6, 8, or 10 weeks after treatment. Peak Mfs were found at 6 weeks postexposure, with mean Mf values ranging from 2.27 to 3.27 x 10(-5) using two different lots of CP in standard packaging (compared with mean control Mf values of 0.14 to 0.26 x 10(-5) in various experiments). To determine the dose response for Hprt Mfs, nontransgenic mice were given single doses of 0, 12.5, 25, 50, or 100 mg CP/kg and necropsied 4 weeks postexposure. These treatments produced a supralinear dose response curve for CP-induced Hprt Mfs. Based on these experiments, CP mutagenicities at Hprt and lacI were compared in transgenic mice treated with 0, 25, or 100 mg CP/kg (using another lot of CP in ISOPAC((R)) bottles; Sigma) and necropsied 6 weeks later. There was a significant increase in Hprt Mfs in treated transgenic mice (100 mg CP/kg: 0.75 +/- 0.09 x 10(-5); 25 mg CP/kg: 0.39 +/- 0.05 x 10(-5)) versus controls (0.10 +/- 0.01 x 10(-5)); however, the Mfs in lacI of lymphocytes from the same CP-treated animals were not significantly different from controls (100 mg CP/kg: 9.4 +/- 1.1 x 10(-5); 25 mg CP/kg: 6.7 +/- 0. 8 x 10(-5); control: 7.7 +/- 0.7 x 10(-5)). Hprt mutational spectra data in CP-treated transgenic and nontransgenic mice were different from those of control mice, whereas the spectra of mutations in lacI of lymphocytes from Big Blue((R)) transgenic mice were not significantly changed after CP treatment. These data indicate that, under these treatment conditions, CP-induced mutations in splenic lymphocytes were detectable in the Hprt gene but not the lacI transgene of this nontarget tissue for CP-induced cancer., (Copyright 1999 Wiley-Liss, Inc.)

The optimization of the mouse lymphocyte Hprt mutation assay has been impeded by the relatively poor growth potential of mouse T-cells in vitro, which leads to low cloning efficiencies (CEs) and limited expansion of Hprt mutant clones for molecular analysis of mutations occurring in control and treated mice. In this study, the addition and manipulation of concanavalin A (Con A), mouse interleukin-2 (IL-2), and a commercially available culture supplement, rat T-STIM with Con A, were used to identify growth conditions producing relatively high CEs for mouse T-cells. Supplementation of medium with 10% rat T-STIM, along with appropriate amounts of Con A for priming and exogenous IL-2 for cloning, resulted in average CEs of 15-16% in lymphocytes isolated from spleens of control mice (n = 32) or mice exposed to 1,3-butadiene (n = 27). In addition, several reagents were assessed for their potential to stimulate long-term growth of Hprt mutant clones; these T-cell stimulatory agents included Con A, phytohemagglutinin, and a calcium ionophore ionomycin combined with a tumor promoter phorbol 12-myristate 13-acetate. In a pilot study, stimulation with Con A proved to be the most effective means for propagating mouse T-cell clones under the various conditions tested. In follow-up experiments, transfer of mutant clones to 24-well plates and repeated stimulation with Con A in IL-2 and rat T-STIM supplemented medium was found to expand 76% of 536 mutant clones to about 400,000 to several million cells per clone. These data indicate that rat T-STIM-supplemented medium enhances the initial outgrowth of mouse T-cells, and that repeated mitogenic stimulation with Con A in the presence of IL-2 and rat T-STIM provides a means for propagating mouse T-cell clones for mutation analyses by a variety of methods.

The labeling index of the infiltrate of oral lichen planus lesions was significantly greater than that of normal mucosa, and its labeling activity redistributed, as determined by in vitro autoradiography. An electron microscopic examination of the infiltrate of lichen planus lesions of oral mucosa and skin supported the concept that the lichen planus infiltrate represents a cell mediated immune response in which T lymphocytes and macrophages are predominant.