Intercorrelations and sources of variability in three mutagenicity assays: a population-based study

The purpose of this study was to evaluate the intercorrelation between three genetic assays in 112 subjects. The group was pooled from two originally separate but homogeneous subgroups of 56 persons each. Procedures included assays for hprt mutant frequencies, micronuclei in human lymphocytes, and mutations at the glycophorin A (gpa) loci. We found no statistically significant or biologically important intercorrelations among the three biomarkers. We did, however, observe significant correlations between log_e hprt mutant frequency and cloning efficiency (inverse correlation for these 2 variables), age and log_e hprt mutant frequency, an inverse relationship between cloning efficiency and age, and an important differential sex effect favoring a greater micronuclei frequency in females than males. No significant correlations between the covariates of interest and glycophorin A variant frequencies NN or NO were observed. Using multivariable linear regression, age was found to account for the majority of the variability in hprt mutant frequency (greater than sex and/or smoking); for micronuclei data, only sex contributed a statistically significant and biologically important proportion to the total variation. We conclude that despite observing no significant intercorrelations between the three assays performed simultaneously from the same individuals in a large population database, a significant correlation between age and hprt mutant frequency and an inverse association between cloning efficiency and hprt do exist; furthermore, we verified the strong differential sex-specific effect on micronucleus frequencies.     

Age-related accumulation of autosomal mutations in solid tissues of the mouse is gender and cell type specific

Most cancers in solid tissues increase with age and invariably contain causal mutations eliminating expression of one or more autosomal tumor suppressor genes. However, very little is known about the effect of age on autosomal mutations, often large in size, in cells of solid tissues. In this study, the frequency and spectrum of autosomal mutations were examined as a function of age for kidney epithelial cells and ear mesenchymal cells in B6D2F1 mice heterozygous for the selectable Aprt locus. Aprt mutant frequencies were found to increase with age in the kidneys of both male and female mice, but at all ages the mutant frequencies were approximately twice as high in the females, which in this strain have shorter lifespans than the males. An age-related increase in Aprt mutant frequencies was also observed for ear cells from female mice, but no significant increases in mutant frequencies were observed for the ear cells of male mice. A molecular analysis showed that the kidney and ear mutational spectra were distinct and that the age-related increases in mutant frequencies did not involve significant shifts in the mutational spectra. In total, the results demonstrate both gender and cell-type-specific patterns of autosomal mutational accumulation as a function of age in two solid tissues of the mouse.     

Influence of sex, smoking and age on human hprt mutation frequencies and spectra.

Examination of the literature for hprt mutant frequencies from peripheral T cells yielded data from 1194 human subjects. Relationships between mutant frequency, age, sex, and smoking were examined, and the kinetics were described. Mutant frequency increases rapidly with age until about age 15. Afterward, the rate of increase falls such that after age 53, the hprt mutant frequency is largely stabilized. Sex had no effect on mutant frequency. Cigarette smoking increased mean mutant frequency compared to nonsmokers, but did not alter age vs. mutant frequency relationships. An hprt in vivo mutant database containing 795 human hprt mutants from 342 individuals was prepared. No difference in mutational spectra was observed comparing smokers to nonsmokers, confirming previous reports. Sex affected the frequency of deletions (>1 bp) that are recovered more than twice as frequently in females (P = 0. 008) compared to males. There is no indication of a significant shift in mutational spectra with age for individuals older than 19 yr, with the exception of A:T --> C:G transversions. These events are recovered more frequently in older individuals.     

Suppression of intestinal polyposis in Apcmin/+ mice by targeting the nitric oxide or poly(ADP-ribose) pathways

Mutant frequency at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene in the peripheral blood lymphocytes obtained from 44 healthy individuals (23 non-smokers and 21 smokers) of an Indian male population was studied using T-lymphocyte cloning assay. It was found that ln MF increased with age at a rate of 2.5% per year (P < 0.001). Blood samples from smokers showed a significant (P < 0.037) increase in HPRT mutant frequency (MF) (10.43 ± 4.74 × 10⁻⁶) as compared to that obtained from non-smokers (7.69 ± 3.69 × 10⁻⁶). This study also showed a significant (P < 0.027) inverse correlation between ln MF and non-selected cloning efficiency (CE). However, with respect to age no variation was observed in cloning efficiency. The results obtained in this study showed a good comparison with those reported in different populations of the world.     

Frequency of mutant T lymphocytes defective in the expression of the T-cell antigen receptor gene among radiation-exposed people.

The frequency of mutant T lymphocytes defective in T-cell receptor gene (alpha or beta) expression was measured using the 2-color flow cytometric technique. Results for a total of 203 atomic bomb survivors, 78 of whom were proximally exposed (DS86 doses of greater than or equal to 1.5 Gy) and 125 of whom were distally exposed (DS86 dose of less than 0.005 Gy), showed that the mutant frequency was significantly higher in males than in females. No significant dose effects were observed. In contrast, a significant increase of mutant frequency was observed for 6 patients treated with Thorotrast, a contrast medium containing thorium-232 formerly used for radioligands. In addition, thyroid disease patients treated with 131I showed a dose-related increase of mutant frequency. It was suggested that the present T-cell receptor mutation assay has a unique characteristic as a biological dosimeter for measurement of recent exposures to genotoxic agents.     

Mutagenesis of a single AT basepair in mice transgenic for PhiX174 am3, cs70. II. Brain.

Most cell divisions in the mouse brain have ceased by 14 days after birth. Therefore, spontaneous mutations that occur in brain cells can be assumed to be fixed by replication during brain development. Spontaneous and ethylnitrosourea (ENU)-induced reverse mutations at a single AT base pair were measured in brain tissue by using mice transgenic for PhiX174 am3, cs70. The line (am54) has 50 PhiX genomes per haploid genome integrated in a tandem array and is maintained by random breeding on a C57BL/6 background. For mutagenesis studies, homozygous am54 males were mated to non-transgenic C57BL6/J females. Four-day old offspring from this cross were treated with 50 mg/kg ENU and were euthanized at 68-80 days of age. The ENU-treated animals had a significantly higher frequency of am3 revertants in brain than did concurrent controls. In a second experiment, hemizygous male offspring (85 to 94 days old) were treated with 150 mg/kg ENU and euthanized at various post-injection intervals (3, 10 and 110 days). The revertant frequencies 3 and 10 days after treatment were not significantly different from control values. At the 110 days post-injection interval, however, the average revertant frequency in the treated group was significantly lower than controls. In a second study animals were euthanized 3, 10 and 74 days after treatment. Two groups (3 and 74 days post-injection) also showed a significant decrease in the revertant frequency as compared to controls. Additional sets of adult animals were treated with 50 and 150 mg/kg ENU and were euthanized 195 to 201 days after treatment. The average revertant frequency of the animals that were treated with 50 or 150 mg/kg ENU was not significantly different from the control value. Thus, although an increase in mutant frequency is detected in the PhiX174 system when neonatal mice are treated with ENU, treatment of mature mice with ENU did not result in an increase in the mutant frequency. Moreover, under certain conditions, ENU-produced a significantly lower mutant frequency than was observed in the control animals. This decrease in the revertant frequency among the treated animals was likely due to selective killing of cells with a higher spontaneous revertant frequency than cells with lower frequency.     

Elevated frequencies of hypoxanthine phosphoribosyltransferase lymphocyte mutants are detected in Russian liquidators 6 to 10 years after exposure to radiation from the Chernobyl nuclear power plant accident

This study was conducted to determine whether the frequency of hypoxanthine phosphoribosyltransferase (HPRT) deficient lymphocyte mutants would detect an effect of radiation exposure in a population of Russians who were exposed to low levels of radiation while working in 1986 and 1987 as liquidators cleaning up after the Chernobyl nuclear power reactor accident. The HPRT lymphocyte cloning assay was performed on peripheral blood lymphocytes collected between 1992 and 1996 from 142 liquidators and 66 Russian controls, and between 1989 and 1993 from 231 American controls. Russian and American controls were not significantly different for either cloning efficiency or mutant frequency (MF); inclusion of both sets of controls in the analysis increased the ability to detect a Chernobyl exposure effect in the liquidators. After adjusting for age and smoking, the results revealed no significant difference in cloning efficiency of Chernobyl liquidators relative to Russian controls but a significant, 24% increase in liquidator HPRT mutant frequency over Russian controls (90% confidence interval was 7% to 45% increase). The analytical method also accounted for differences in precision of the individual estimates of log CE and log MF and accommodated for outliers. The increase in HPRT mutant frequency of liquidators is an attribute of the exposed population as a whole rather than of individuals. These results demonstrate that, under appropriate circumstances, the HPRT specific locus mutation assay of peripheral blood lymphocytes can be used to detect a semi-acute, low dose radiation exposure of a population, even 6 to 10 years after the exposure.     

A further assessment of factors influencing measurements of thioguanine-resistant mutant frequency in circulating T-lymphocytes

We have used the T-Lymphocyte cloning technique as a method of monitoring the human population for somatic cell mutant frequency. We present a statistical analysis of the experimental factors which may influence the observed mutant frequency. We have obtained consistently high plating efficiencies of T-cells from the mononuclear cell fraction from donor blood samples (mean of 56%, based on 123 observations from 70 individuals). Nevertheless, an inverse correlation of mutant frequency with plating efficiency was observed, and some experimental factors (serum and interleukin-2 batch, and worker) may have a significant effect on the observed mutant frequency. We discuss the difficulties that these possible effects present in establishment of a reference database and design of long-term studies. No significant effect of donor sex on mutant frequency was observed, but age (1.3% increase per year for normal adults) and smoking (56% increase over normal non-smokers) both significantly increased the mutant frequency. We discuss the utility of the assay for the monitoring of populations for heritable DNA damage, and we compare the results to those obtained with lymphocytes using other endpoints, e.g. chromosome aberrations, micronuclei and sister-chromatid exchange.     

Genotoxicity of 3'-azido-3'-deoxythymidine in the human lymphoblastoid cell line, TK6: relationships between DNA incorporation, mutant frequency, and spectrum of deletion mutations in HPRT.

Perinatal treatment with 3'-azido-3'-deoxythymidine (AZT) has been found to reduce the rate of maternal-infant transmission of HIV; however, AZT is genotoxic in mammalian cells in vitro and induces tumors in the offspring of mice treated in utero. The purpose of the present study was to investigate the relationships between incorporation of AZT into DNA, and the frequency and spectrum of mutations at the HPRT locus of the human lymphoblastoid cell line, TK6, following in vitro exposures to AZT. Cells were cultured in medium containing 0 or 300 microM AZT for 1, 3, or 6 day(s) (n = 5/group). The effects of exposure duration on incorporation of AZT into DNA and HPRT mutant frequency were determined using an AZT radioimmunoassay and a cell cloning assay, respectively. AZT accumulated in DNA in a supralinear manner, approaching a plateau at 6 days of treatment (101.9 +/- 14.7 molecules AZT/10(6) nucleotides). After 3 days of AZT exposure, HPRT mutant frequency was significantly increased (1.8-fold, p = 0.016) compared to background (mutant frequency = 3.78 x 10(-6)). Multiplex PCR amplification of genomic DNA was used to determine the frequency of exon deletions in HPRT mutant clones from untreated cells versus AZT-treated cells. Molecular analyses of AZT-induced mutations revealed a significant difference in the frequency of total gene deletions (44/120 vs. 18/114 in controls, p = 0.004 by the Mann-Whitney U-statistic). In fact, the Chi-square test of homogeneity demonstrate that the differences between the control and AZT-treatment groups is attributed mainly to this increase in total gene deletion mutations (p = 0.00001). These data indicate that the primary mechanism of AZT mutagenicity in human TK6 cells is through the production of large deletions which occur as a result of AZT incorporation into DNA and subsequent chain termination. The data imply that perinatal chemoprophylaxis with AZT may put children of HIV-infected women at potential risk for genetic damage.     

Mutation induction by different dose rates of gamma rays in radiation-sensitive mutants of mouse leukemia cells

Induction of cell killing and mutation to 6-thioguanine resistance was examined in a radiation-sensitive mutant strain LX830 of mouse leukemia cells following gamma irradiation at dose rates of 30 Gy/h (acute), 20 cGy/h (low dose rate), and 6.2 mGy/h (very low dose rate). LX830 cells were hypersensitive to killing by acute gamma rays. A slight but significant increase was observed in cell survival with decreasing dose rate down to 6.2 mGy/h, where the survival leveled off above certain total doses. The cells were also hypersensitive to mutation induction compared to the wild type. The mutation frequency increased linearly with increasing dose for all dose rates. No significant difference was observed in the frequency of induced mutations versus total dose at the three different dose rates so that the mutation frequency in LX830 cells at 6.2 mGy/h was not significantly different from that for moderate or acute irradiation.     

The frequency of illegitimate TCRbeta/gamma gene recombination in human lymphocytes: influence of age, environmental exposure and cytostatic treatment, and correlation with frequencies of t(14;18) and hprt mutation.

Chromosome translocations in lymphoid malignancies often involve V(D)J recombinase mediated events giving rise to aberrant T-cell receptor (TCR) and immunoglobulin genes, which have been suggested to be useful as markers of genomic instability, genotoxic exposure and cancer risk. Illegitimate rearrangements involving the TCRbeta/gamma loci on chromosome 7 create TCRbeta/gamma hybrid genes which occur at low frequency in peripheral blood lymphocytes (PBLs) of normal healthy individuals. To evaluate the utility of this marker, we studied the possible effects of age and genotoxic exposures on the TCRbeta/gamma gene variant frequency (VF), and compared the frequencies of hypoxanthine guanine phosphoribosyl transferase (hprt) mutation, hprt exon 2/3 deletion, t(14;18) and TCRbeta/gamma gene rearrangements in cells from the same donors. The TCRbeta/gamma VF ranged five-fold among 16 middle aged blood donors with a mean of 0.74+/-0.29/10(5) PBLs, which is consistent with our previous estimate in healthy subjects. The TCRbeta/gamma VF was found to increase from birth until early adult life, and then to decrease with increasing age. Four testis cancer patients, who 6 years earlier had been treated with etoposide and other cytostatic drugs, showed TCRbeta/gamma VF similar to that in healthy controls. No increase of the TCRbeta/gamma VF was found among non-smoking PAH-exposed aluminum smelter workers compared to non-smoking controls. Smoking smelter workers showed decreased TCRbeta/gamma VF compared to non-smoking workers and controls, but in a follow-up study 2 years later the difference was no longer statistically significant, although the smoking smelter workers still showed a lower TCRbeta/gamma VF than the controls. No correlation was obtained between the TCRbeta/gamma VF and the t(14;18) or hprt mutant frequency (MF) in a group of healthy individuals. However, there was a statistically significant correlation between the TCRbeta/gamma VF and the hprt exon 2/3 deletion frequency in PBL DNA from the same donors. These results show that the TCRbeta/gamma VF in healthy individuals changes with age and correlates with the frequency of hprt exon 2/3 deletion, another marker of aberrant V(D)J recombination in T-cells. However, no effect of smoking or present or previous exposure to genotoxic agents on TCRbeta/gamma VF was observed in this study. Thus, further studies are needed to prove the utility of TCRbeta/gamma gene rearrangement as a marker of genotoxic exposure.     

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 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.     

Mutation frequency and specificity with age in liver, bladder and brain of lacI transgenic mice

Mutation frequency and specificity were determined as a function of age in nuclear DNA from liver, bladder, and brain of Big Blue lacI transgenic mice aged 1.5-25 months. Mutations accumulated with age in liver and accumulated more rapidly in bladder. In the brain a small initial increase in mutation frequency was observed in young animals; however, no further increase was observed in adult mice. To investigate the origin of mutations, the mutational spectra for each tissue and age were determined. DNA sequence analysis of mutant lacI transgenes revealed no significant changes in mutational specificity in any tissue at any age. The spectra of mutations found in aging animals were identical to those in younger animals, suggesting that they originated from a common set of DNA lesions manifested during DNA replication. The data also indicated that there were no significant age-related mutational changes due to oxidative damage, or errors resulting from either changes in the fidelity of DNA polymerase or the efficiency of DNA repair. Hence, no evidence was found to support hypotheses that predict that oxidative damage or accumulation of errors in nuclear DNA contributes significantly to the aging process, at least in these three somatic tissues.     

Damage to lymphocytes by X-ray and bleomycin measured with the cytokinesis-block micronucleus technique.

Chromosome damage induced by X-irradiation or bleomycin was measured using the cytokinesis-block micronucleus assay in the peripheral blood lymphocytes of 6 newborn, 8 young and 10 elderly individuals. An increase in the frequency of spontaneous micronuclei with age was observed. There was no difference in the X-irradiation-induced micronucleus frequency between the 3 groups. There was a significant increase with age in the number of micronuclei induced by bleomycin. Kinetochore-labelling studies revealed that the percentage of kinetochore-positive induced micronuclei was higher for bleomycin (36.2-43.3%) than for X-irradiation (17.1-19.7%). The age-related increase in frequency of spontaneous or bleomycin-induced micronuclei was due to increases in both kinetochore-positive and kinetochore-negative micronuclei. The frequency of kinetochore-positive or -negative micronuclei induced by X-irradiation was not different between the 3 age groups. These results suggest that bleomycin is more potent in inducing whole-chromosome loss than X-rays, and that lymphocytes from aged individuals are more sensitive to bleomycin in terms of both chromosome breakage and whole chromosome loss.     

Role of the mismatch repair gene, Msh6, in suppressing genome instability and radiation-induced mutations

Mismatch repair (MMR) is critical for preserving genomic integrity. Failure of this system can accelerate somatic mutation and increase the risk of developing cancer. MSH6, in complex with MSH2, is the MMR protein that mediates DNA repair through the recognition of 1- and 2-bp mismatches. To evaluate the effects of MSH6 deficiency on genomic stability we compared the frequency of in vivo loss of heterozygosity (LOH) between MSH6-proficient and deficient, 129S2xC57BL/6 F1 hybrid mice that were heterozygous for our reporter gene Aprt. We recovered mutant cells that had functionally lost APRT protein activity and categorized the spectrum of mutations responsible for the LOH events. We also measured the mutant frequency at the X-linked gene, Hprt, as a second reporter for point mutation. In Msh6-/-Aprt+/- mice, mutation frequency at Aprt was elevated in both T cells and fibroblasts by 2.5-fold and 5.7-fold, respectively, over Msh6+/+Aprt+/- littermate controls. While a modest increase in mitotic recombination (MR) was observed in MSH6-deficient fibroblasts compared to wild type controls, point mutation was the predominant mechanism leading to APRT deficiency in both cell types. Base substitution, consisting of multiple types of transitions, accounted for all of the point mutations identified within the Aprt coding region. We also assessed the role of MSH6 in preventing mutations caused by a common environmental mutagen, ionizing radiation (IR). In Msh6-/-Aprt+/- mice, 4Gy of X-irradiation induced a significant increase in point mutations at both Aprt and Hprt in T cells, but not in fibroblasts. These findings indicate that MutS alpha reduces spontaneous and IR-induced mutation in a cell type-dependant manner.     

Involvement of eddy currents in the mutagenicity of ELF magnetic fields

Possible carcinogenic and/or mutagenic activity of extremely low frequency magnetic fields was examined using somatic mutation and recombination test system of Drosophila melanogaster. An X-linked semi-dominant DNA repair defective mutation mei-41(D5) was introduced into the conventional mwh/flr test system to enhance mutant spot frequency. Virgin females of w mei-41(D5)/FM6; flr/TM6 were crossed with w mei-41(D5)/Y; mwh jv; spa(pol) males. The F(1) third instar larvae were exposed to a 50Hz, 20mT sinusoidal AC magnetic field for 24h. After moulting from pupal cases, their wings were examined under a bright field microscope to detect hair spots with mwh or flr mutant morphology. The exposure caused a statistically significant enhancement in somatic recombination spot frequency. Mutant spots arising due to chromosomal non-disjunction or terminal deletion also increased but the frequency of spots resulting from point mutation was not altered. The enhancement in the recombination spot frequency was suppressed to the control level when a culture medium without electrolytes was used during exposure. When larvae were exposed to a magnetic field in an annular dish, flies from the outer ring showed more mutant spots compared to those from the inner ring. These results suggest that the detected mutagenic activity was that of the induced eddy current, rather than that of the magnetic field itself.     

The paternal-age effect in Apert syndrome is due,in part,to the increased frequency of mutations in sperm

A paternal-age effect and the exclusive paternal origin of mutations have been reported in Apert syndrome (AS). As the incidence of sporadic AS births increases exponentially with paternal age, we hypothesized that the frequency of AS mutations in sperm would also increase. To determine the frequency of two common FGFR2 mutations in AS, we developed allele-specific peptide nucleic acid-PCR assays. Analyzing sperm DNA from 148 men, age 21-80 years, we showed that the number of sperm with mutations increased in the oldest age groups among men who did not have a child with AS. These older men were also more likely to have both mutations in their sperm. However, this age-related increase in mutation frequency was not sufficient to explain the AS-birth frequency. In contrast, the mutation frequency observed in men who were younger and had children with AS was significantly greater. In addition, our data suggest selection for sperm with specific mutations. Therefore, contributing factors to the paternal-age effect may include selection and a higher number of mutant sperm in a subset of men ascertained because they had a child with AS. No age-related increase in the frequency of these mutations was observed in leukocytes. Selection and/or quality-control mechanisms, including DNA repair and apoptosis, may contribute to the cell-type differences in mutation frequency.     

Characteristics of genomic instability in clones of TK6 human lymphoblasts surviving exposure to 56Fe ions

Genomic instability in the human lymphoblast cell line TK6 was studied in clones surviving 36 generations after exposure to accelerated 56Fe ions. Clones were assayed for 20 characteristics, including chromosome aberrations, plating efficiency, apoptosis, cell cycle distribution, response to a second irradiation, and mutant frequency at two loci. The primary effect of the 56Fe-ion exposure on the surviving clones was a significant increase in the frequency of unstable chromosome aberrations compared to the very low spontaneous frequency, along with an increase in the phenotypic complexity of the unstable clones. The radiation-induced increase in the frequency of unstable chromosome aberrations was much greater than that observed previously in clones of the related cell line, WTK1, which in comparison to the TK6 cell line expresses an increased radiation resistance, a mutant TP53 protein, and an increased frequency of spontaneous unstable chromosome aberrations. The characteristics of the unstable clones of the two cell lines also differed. Most of the TK6 clones surviving exposure to 56Fe ions showed unstable cytogenetic abnormalities, while the phenotype of the WTK1 clones was more diverse. The results underscore the importance of genotype in the characteristics of instability after radiation exposure.     

Effect of pH shifts on the mutant frequency at the thymidine kinase locus in mouse lymphoma L5178Y TK+/- cells

Evidence has been accumulating that conditions of nonphysiological pH may affect the results of in vitro genetic tests by mechanisms unrelated to the chemical being tested. Medium was pH-adjusted with HCl, NaOH or with organic buffers (Good's zwitterions). In the absence of S9 mix, no changes in mutant frequency were observed over a pH range of 6.4-9.2; a small, 1.9-fold increase was observed for a moderately toxic treatment (24% relative growth) at pH 6.3. However, in the presence of S9 mix, the mutant frequency increased sharply for pH values below 6.8. At pH 6.4, a 4-fold increase was induced, and pH 6.0 resulted in a 10-fold increase in mutant frequency. Basic pH shifts in the presence of S9 mix caused no changes in mutant frequency up to pH 8.0; treatment with pH 8.8 was highly toxic (5.3% relative growth) and caused a 3-fold increase in mutant frequency. Thirteen mutant clones induced at pH 6.0 with S9 mix were challenged with trifluorothymidine after their expansion in nonselective medium and all retained their resistance; another 14 clones were tested for thymidine utilization and all incorporated only 0.1-5.5% of the 14C-labeled thymidine used by the parental line. The induced mutants were primarily of the small-colony phenotype, which indicated clastogenic activity. This was confirmed with chromosome studies which showed a large increase in cells with aberrations consisting of chromatid breaks and complex rearrangements. The results show that the combination of weak acidity (pH 6-6.8) and S9 mix is mutagenic and clastogenic to L5178Y TK+/- cells.     

Elevated hprt mutant frequency in circulating T-lymphocytes of xeroderma pigmentosum patients.

The mutant frequency to 6-thioguanine resistance in circulating T-lymphocytes from 10 xeroderma pigmentosum patients (including complementation groups D and G and XP variants) has been determined. A highly significantly elevated frequency was observed, compared to age-matched, non-smoking control donors (x 2.1-fold higher than the mutant frequency in normal control donors, adjusted for age and cloning efficiency, p less than 0.001). The mutant frequency of 5 XP heterozygotes was in the normal range, when age, smoking habit and log cloning efficiency were taken into account. A number of possible factors which may account for the elevated mutant frequency seen in the XP donors (including an elevated spontaneous mutation rate, UV mutagenesis of the T-cells as they pass through the skin, an effect of environmental mutagens such as tobacco smoke, or as a consequence of immune deficiency) are discussed.