Mean Mutant Frequency Research Articles

Effect of plating medium and phage storage on mutant frequency and titer in the lambdacII transgenic mutation assay

We examined several experimental parameters of the lambda cI/cII transgenic mutation assay. In the assay, clear plaque lambda phage mutants are identified in a positive selection scheme following rescue of the lambda/LIZ shuttle vector from frozen tissues of Big Blue" transgenic mice. Mutant frequency and titer of phage from various tissues of control and ENU-treated animals was essentially the same on LB or TB1 plating medium, and storage of isolated DNA at 4 degrees C for up to 4 months did not affect either mutant frequency or titer. Storage of packaged phage for 28 days at 4 degrees C did not affect titer. The mean mutant frequency of packaged phage stored 28 days at 4 degrees C was consistently higher than phage plated the same day as packaging (day 0), though the difference was statistically significant in only two of the four samples tested. Reconstruction experiments in which numerically defined titers of known cII mutants were plated on both G1217 and G1225 E. coli strains and incubated at 37 degrees C or 24 degrees C showed highest titers on G1217 at 37 degrees C. The fraction of the G1217, 37 degrees C titer seen in the other strains and conditions varied widely with the cII mutation.

Effect of plating medium and phage storage on mutant frequency and titer in the lambda cII transgenic mutation assay

We examined several experimental parameters of the lambda cI/cII transgenic mutation assay. In the assay, clear plaque lambda phage mutants are identified in a positive selection scheme following rescue of the lambda/LIZ shuttle vector from frozen tissues of Big Blue® transgenic mice. Mutant frequency and titer of phage from various tissues of control and ENU-treated animals was essentially the same on LB or TB1 plating medium, and storage of isolated DNA at 4°C for up to 4 months did not affect either mutant frequency or titer. Storage of packaged phage for 28 days at 4°C did not affect titer. The mean mutant frequency of packaged phage stored 28 days at 4°C was consistently higher than phage plated the same day as packaging (day 0), though the difference was statistically significant in only two of the four samples tested. Reconstruction experiments in which numerically defined titers of known cII mutants were plated on both G1217 and G1225 E. coli strains and incubated at 37°C or 24°C showed highest titers on G1217 at 37°C. The fraction of the G1217, 37°C titer seen in the other strains and conditions varied widely with the cII mutation. Environ. Mol. Mutagen. 32: 325–330, 1998 © 1998 Wiley-Liss, Inc.

Mutation frequencies at codon 248 of the p53 tumour suppressor gene are not increased in colon cancer cell lines with the RER+ phenotype.

The replication-error positive (RER+) phenotype characterizes tumour cells with microsatellite instability. This 'mutator phenotype' is thought to induce spread mutations throughout the genome, thus increasing the risk of tumour development. Here we analyse spontaneously arising mutations at the tetranucleotide CCGG ( Msp I recognition site), at positions 14 067-14 070 of the p53 gene sequence, in three colon cancer cell lines, two with microsatellite instability and one without this characteristic. This restriction site covers hot-spot codon 248, which is often mutated in colon carcinomas. Using the Msp I RFLP-PCR assay we found that the mean mutation frequency at this site was not different among the cell lines considered. Taking the substitutions separately, none of the mutations involving codon 248 arose with significantly higher frequency in each of the RER+ cell lines (HCT116 and DLD1) compared with the RER-one (SW480). Only the CG transversion at nt 14 067 (codon 247) occurred with a slightly higher, but biologically insignificant, frequency in one of the RER+ cell lines (HCT116). Our in vitro data support the previously reported lack of correlation between microsatellite instability and p53 mutations in RER+ tumour specimens.

In vivo antioxidant status, DNA damage, mutation and DNA repair capacity in cultured lymphocytes from healthy 75- to 80-year-old humans

The accumulation of damage to cellular biomolecules, including DNA, over time may play a significant role in the aetiology of the ageing process. We have previously quantified DNA damage and mutation within cultured lymphocytes from healthy human male subjects in three different age groups (35–39, 50–54 and 65–69 years). The results of that study showed an age-related increase in DNA damage and mutations in lymphocytes. In addition, an age-related decrease in the capacity of the lymphocytes to repair H 2O 2-induced DNA damage was found. In this article, we report the findings of an extension to the earlier study. Thirty-one generally healthy male and female subjects between the ages of 75 and 80 years were recruited. Using a number of bioassays, we were able to determine; basal levels of DNA damage (for 18 subjects) and mutant frequency at the hypoxanthine phosphoribosyltransferase ( hprt) gene locus (for 16 subjects) within cultured lymphocytes. In addition, in vivo antioxidant status (for all study subjects) and the capacity of lymphocytes to repair H 2O 2-induced DNA damage (for 18 subjects) were also assessed. The results obtained showed: that the mean basal level of DNA damage in lymphocytes from subjects in the 75- to 80-year age group (12.6±4.7%) was similar to that of the 35- to 39-year age group (13.3±3.3%), p=0.42 (Mann–Whitney); there was no significant difference between log mean mutant frequency at the hprt gene locus in lymphocytes from the 75- to 80-year age group (0.31±0.33) compared to that observed in the 35- to 39-year age group (0.24±0.21; Student's t-test, t=0.68, p>0.05). Levels of the antioxidants glutathione peroxidase (GPx EC 1.11.1.9), catalase (CAT; EC 1.11.1.6) and caeruloplasmin (CPL; EC 1.16.3.1) were significantly elevated in the 75- to 80-year age group, compared to the 35- to 39-, 50- to 54- and 65- to 69-year age groups. Levels of bilirubin (BR) were reduced in the 75- to 80-year age group, the decrease being contributed by the female subjects. No differences in levels of superoxide dismutase (SOD; EC 1.15.1.1) or uric acid (UA) were found between the 4 age groups. Following treatment of lymphocytes with H 2O 2, we did not find any difference in the susceptibility of lymphocytes to DNA damage in the 75- to 80-year age group, compared to the other age groups. The DNA repair capacity in lymphocytes from individuals in the 75- to 80-year age group was similar to that of the 35- to 39-year age group, for all time points assessed. These results highlight the importance of DNA repair processes and antioxidant defence systems for maintaining genomic stability in vivo.

The detection of gene mutation in the tubular sperm of Muta™Mice following a single intraperitoneal treatment with methyl methanesulphonate or ethylnitrosourea

Transgenic mouse assays, such as Muta™Mouse, provide a method to predict the potential target organ carcinogenicity of chemical compounds. As part of a collaborative study, the effects of the direct-acting mutagens, methyl methanesulphonate (MMS) and ethylnitrosourea (ENU), were investigated for gene mutation in the tubular sperm of Muta™Mice testes after a single intraperitoneal exposure. Groups of male Muta™Mice were dosed intraperitoneally with either 1/15 M phosphate buffer, pH 6.0 (vehicle control), 40 mg/kg methyl methanesulphonate (MMS) or 150 mg/kg ethylnitrosourea (ENU). The animals were sacrificed 14 days after the single dose. Mutation frequencies were determined in tubular sperm DNA. The results showed a mean mutation frequency (MF) of 2.1×10 5 (64 mutants per 3.05×10 6 PFU) for the 10 vehicle-treated mice, a mean MF of 2.8×10 5 (78 mutants per 2.75×10 6 PFU) for the 10 MMS-treated mice and a mean MF of 9.1×10 5 (194 mutants per 2.14×10 6 PFU) for the 8 ENU-treated mice; this latter value representing a 4.5-fold increase over the vehicle control values.

A new T-lymphocyte cloning assay for detection of in vivo mutations in the human hypoxanthine-guanine phosphoribosyltransferase gene

The X-linked hypoxanthine-guanine phosphoribosyl transferase (hprt) gene is a target of analyses of in vivo mutation frequencies in circulating T-lymphocytes. We established a novel, accessory cell-free cloning method of T-lymphocytes with a hprt mutation by a combined use of recombinant interleukin-2, conditioned medium from activating T-lymphocytes and culture plates coated with anti-CD3 monoclonal antibody. Using the method, we examined mutation frequencies of the hprt gene in T-lymphocytes from six healthy individuals, nine patients with colon cancer including two patients from different families with hereditary nonpolyposis colon cancer and six cancer-free relatives of the patients. In six healthy individuals, the mean cloning efficiency and mutation frequency (MF) of the hprt gene in T-lymphocytes were 0.51 +/- 0.28 and 9.4 +/- 7.5 x 10(-6), respectively. These data were similar to the reported values. The mean MFs in the nine colon cancer patients (10.6 +/- 7.3 x 10(-6)) were not significantly different from those of the 12 cancer-free individuals (11.6 +/- 9.4 x 10(6)). The correlation between mutation frequencies and age of the individuals was significant regardless of the presence or absence of cancers. The single-strand conformation polymorphism analyses of nested RT-PCR products of hprt mRNA were done in 33 mutant clones from five members of a family of which MF values were high. All the analyzed mutant clones show a genetic aberration in the coding region of the hprt gene. At least 28 of 33 mutants were independent. Our method provides a new versatile tool for in vivo analysis for mutations of the hprt gene.

A new T‐lymphocyte cloning assay for detection of in vivo mutations in the human hypoxanthine‐guanine phosphoribosyltransferase gene

The X-linked hypoxonthine-guanine phosphoribosyltransferase (hprt) gene is a target of analyses of in vivo mutation frequencies in circulating T-lymphocytes. We established a novel, accessory cell-free cloning method of T-lymphocytes with a hprt mutation by a combined use of recombinant interleukin-2, conditioned medium from activating T-lymphocytes and culture plates coated with anti-CD3 monoclonal antibody. Using the method, we examined mutation frequencies of the hprt gene in T-lymphocytes from six healthy individuals, nine patients with colon cancer including two patients from different families with hereditary nonpolyposis colon cancer and six cancer-free relatives of the patients. In six healthy individuals, the mean cloning efficiency and mutation frequency (MF) of the hprt gene in T-lymphocytes were 0.51 ± 0.28 and 9.4 ± 7.5 × 10−6, respectively. These data were similar to the reported values. The mean MFs in the nine colon cancer patients (10.6 ± 7.3 × 10−6) were not significantly different from those of the 12 cancer-free individuals (11.6 ± 9.4 × 10−6). The correlation between mutation frequencies and age of the individuals was significant regardless of the presence or absence of cancers. The single-strand conformation polymorphism analyses of nested RT-PCR products of hprt mRNA were done in 33 mutant clones from five members of a family of which MF values were high. All the analyzed mutant clones show a genetic aberration in the coding region of the hprt gene. At least 28 of 33 mutants were independent. Our method provides a new versatile tool for in vivo analysis for mutations of the hprt gene. Environ. Mol. Mutagen. 30:31–39, 1997 © 1997 Wiley-Liss, Inc.

Biological effect monitoring in industrial workers from the Czech Republic exposed to low levels of butadiene

Blood samples were collected twice (in 1993 and 1994) from 19 workers exposed to 1,3-butadiene and 19 matched controls. Three exposed and three control subjects were the same in 1993 and 1994. Personal passive dosimetry was performed in 1993 and twice in 1994 on the day preceding blood sampling. Mean exposure level in 1994 was 1.76 ± 4.20 ppm (S.D.) and individual exposure levels ranged between 0.012 ppm (detection limit) and 19.77 ppm. Using the clonal assay, geometric mean of hprt mutant frequencies adjusted for cloning efficiency, age and smoking were, respectively, 7.85 (± 7.09) × 10 −6 and 10.14 (± 9.16) × 10 −6 in pooled (1993 plus 1994) exposed and control subjects. The difference was not statistically significant indicating that 1,3-butadiene did not induce a detectable increase in mutations at the hprt locus. A similar result was obtained for the 1994 subjects alone. There was no difference between adjusted geometric mean mutant frequencies of exposed and unexposed non-smokers or between exposed and unexposed smokers. Analysis of chromosomal aberrations in lymphocytes from 1994 subjects indicated that the percentage of aberrant cells was significantly enhanced in exposed subjects. In 1993 (data not shown), it was impossible to demonstrate a significant increase of aberrant cells in subjects exposed to 1,3-butadiene. Frequencies of micronuclei in cytochalasin-B blocked binucleate lymphocytes in exposed and unexposed 1994 subjects were not significantly different. This was also the case for earlier samples analyzed in the same plant. Using the comet assay for 1994 subjects, no statistically significant difference was found between the whole group of exposed and unexposed subjects. This was true for both the comet tail length and the percentage of DNA in the tail. In exposed smokers, however, the comet tail length was significantly longer than in unexposed smokers. Unexpectedly, in unexposed smokers the tail length was significantly shorter than in unexposed non-smokers. It was also unexpected that the percentage of DNA in the comet tail was significantly lower in exposed non-smokers than in unexposed non-smokers.

Reduced proliferation rate of hypoxanthine-phosphoribosyl transferase mutant human T-lymphocytes in vitro.

The clonal hprt mutation assay in human T-cell is based on the assumption that wild-type cells and hypoxanthine-phosphoribosyl transferase (hprt) mutant cells survive and proliferate at the same rate during the expression phase which is required for the expression of in vitro-induced mutants. We have tested this assumption in a study of mutant frequency (MF) and proliferation rate at different time points during in vitro expansion of human T-cells in non-selective medium. Peripheral blood lymphocytes from 11 individuals were studied using standard cloning procedures to determine the cloning efficiency (CE) and the hprt MF by 6-thioguanine (TG) selection. Another cell portion from each individual was allowed to proliferate in bulk culture for 8 days in vitro, before measuring CE and MF as above. In the directly plated cell population the CE was 45% and the MF 18.7 +/- 15.3 x 10(-6) (mean +/- S.D.), whereas the in vitro expanded cell population showed a CE of 38% and a significantly reduced MF of 8.3 +/- 6.9 x 10(-6) (P = 0.0033). Thus, the mean MF was 56% lower in the in vitro expanded than in the directly plated cell population. The experiment was repeated in another group of ten individuals with essentially the same result. In a third experiment, freshly prepared cells from two donors were allowed to grow for up to 15 days in bulk culture in vitro. Cell growth, CE, and MF were determined every third day. The MF decreased gradually, and at day 12-15 it was only 25% of the initial value. The total number of clonable cells increased 13-fold during the 15 days of in vitro expansion, while the mutant, TG-resistant cell population increased only 3-fold. These results suggest that human hprt mutant T-lymphocytes have a reduced proliferation rate compared to wild-type cells during in vitro proliferation. Thus, measurements of chemical and radiation induced MF with the T-cell clonal assay may underestimate the true MF by a factor of 2 or more.

Frequency of HPRT mutant lymphocytes in a human control population as determined by the T-cell cloning procedure

The T-cell-cloning assay wa established to determine the frequency of hypoxanthine-guanine phosphoribosyl-transferase (HPRT) mutant lymphocytes in the presence of the selective agent 6-thioguanine in peripheral blood from a human control population. We investigated 44 healthy adults (blood donors) and found a mean mutant frequency of 7.2 × 10 −6 (geometric mean 5.6 × 10 −6). An elevted mean mutant frequency occurred in smokers as compared to non-smokers. However, a statistically significant increase was only observed between female smokers and female non-smokes while there was only a slight difference in the male group. A significant difference in mutant frequency could be found between individuals younger than 35 years and those above 35. But the difference of the mutant frequency with age showed up only among smokers. No significant effect of the gender was observed. Mutant frequency was inversely related to the cloning efficiency.

Inhalation of benzene leads to an increase in the mutant frequencies of a lacI transgene in lung and spleen tissues of mice

The goal of this study was to determine if inhalation of benzene leads to an increase in the mutant frequencies in the tissues of male C57 BL 6 mice. Mutant frequencies were measured using a previously described assay in which bacteriophage lambda lacI transgenes are rescued from mouse genomic DNA as infectious phage and scored for their LacI phenotype. Eight experimental mice were exposed to a target concentration of 300 ppm of benzene for 6 h/day × 5 days/week × 12 weeks, and eight control mice were treated similarly except that they were not exposed to benzene. Mutant frequencies were calculated as the ratio of LacI −/total phage recovered from organs of interest. The mean mutant frequency measured in lung tissues of mice exposed to benzene was (10.6 ± 1.4) × 10 −5, which is about 1.7-fold higher than that of the unexposed controls. In spleen tissues from benzene-exposed mice the mean mutation frequency was (12.6 ± 4.1) × 10 −5, which is about 1.5-fold higher than that of spleen tissues from unexposed controls. The differences in mean mutant frequencies between benzene-exposed and unexposed lung and spleen tissues are statistically significant. In liver tissues, however, the mean mutant frequencies of benzene-exposed mice and unexposed mice are not significantly different. These results demonstrate that inhaled benzene results in a statistically significant increase in the mutant frequencies in lung and spleen, but not in liver tissues of mice.

Interlaboratory comparison: liver spontaneous mutant frequency from lambda/ lacI transgenic mice (Big Blue ®) (II)

Spontaneous mutant frequency in livers of two transgenic mouse strains, each carrying identical lambda shuttle vectors with a lacI target gene, was evaluated by two laboratories. These studies investigated variability in spontaneous mutant frequency between animals and as a function of the number of phage screened. Liver DNA was independently isolated from 7–11 week old C57 BL 6 and B6C3F1 Big Blue ® transgenic mice. At least 500 000 phage were screened for mutation at lacI for each animal using standardized assay procedures. In the two labs, the C57 BL 6 liver spontaneous mutant frequency was 45 ± 9 × 10 −6 and 41 ± 7 × 10 −6. The B6C3F1 liver spontaneous mutant frequency was 42 ± 10 × 10 −6 at one lab and 43 ± 12 × 10 −6 and 41 ± 8 × 10 −6 in two trials at the second lab. Mean mutant frequency data from both labs, calculated in increments of 100 000 plaque forming units (pfu) scored for each mouse strain, show stabilized mean mutant frequency and standard deviation after approximately 200 000–300 000 pfu screened. The frequency of spontaneous lacI mutants was reproducible both within and between labs and was comparable between the two transgenic mouse strains.

Southern blot analysis of T-cell receptor gene rearrangements in cynomolgus monkeys, and identification of a progenitor cell HPRT mutation.

Increases in peripheral blood T-lymphocyte HPRT mutant frequency may reflect either a number of independent HPRT gene mutational events or clonal proliferation of a single HPRT mutant. Sequence analysis of HPRT mutations in conjunction with T-cell receptor (TCR) gene rearrangement pattern analysis can distinguish these possibilities. Our laboratory previously characterized a nonhuman primate model for in vivo mutation studies using the clonal HPRT mutation assay. In the present study we report the use of probes for human TCR beta and gamma genes to characterize TCR rearrangements in cynomolgus monkeys. Together, these methods were used to examine a monkey which exhibited a mean spontaneous HPRT mutant frequency (MF) of 16.4 x 10(-6), compared to the normal mean MF of 3.03 x 10(-6). The elevated MF resulted from the occurrence of a single HPRT mutation in a lymphocyte progenitor cell or stem cell, since T-cell clones isolated from the monkey exhibited a G to T transversion at base pair 539 in the HPRT coding region, and had unique rearrangements of TCR gamma along with an apparent germline TCR beta configuration. In a preliminary in vivo mutation study, the animal was treated with the investigational potent mutagen and antitumor agent adozelesin (U-73975). No increase in HPRT mutant frequency was observed. The HPRT mutant clones isolated after treatment showed rearrangement of both TCR gamma and beta genes. Possible explanations for these findings are discussed.

Hprt mutant frequency and GSTM1 genotype in non-smoking healthy individuals.

The T-cell cloning assay which combines mitogen- and growth factor-dependent expansion of lymphocyte clones with thioguanine selection of hypoxanthine-guanine phosphoribosyl transferase (hprt)-negative cells has been extensively used for studying human somatic gene mutation in vivo. However, large interindividual variations in the hprt mutant frequency (MF), much of which is not explained by donor attributes such as age and smoking habit, and interlaboratory variations in the experimental methodology, including cloning efficiency (CE), call for further developments of the cloning protocol and additional population studies. Using an improved T-cell cloning method, we have studied in vivo hprt MF of 76 non-smoking healthy males aged 23-77 years. The addition of 5% human serum to the growth medium was found to produce a consistently high CE of 61% in average. The MF, ranging from 1.4 to 22.6 x 10(-6) with a mean of 8.6 x 10(-6), increased significantly (P < 0.0001) with age, by 2% per year. A significant (P = 0.002) inverse relationship between MF and CE was observed. Using a PCR-based technique for GSTM1-genotyping, we also studied the relationship between MF and GSTM1 polymorphism. The 38 (50%) GSTM1-negative individuals showed a 20% higher mean MF than the 38 (50%) GSTM1-positive individuals. The difference was however not significant, neither before (P = 0.1) nor after (P = 0.5) correction for CE and the significantly (P = 0.04) higher mean age in the GSTM1-negative group. This study shows that age contributes more than GSTM1 polymorphism to the large interindividual variation in the hprt MF of non-smokers. The relationship between GSTM1 polymorphism and hprt MF in smokers remains to be investigated.

An investigation of mutation as a function of age in humans.

An accumulation of mutations on their own or together with other age-related changes may contribute to aging and the development of age-related pathologies. The aim of this investigation was to assess the extent of DNA mutations as a function of age in humans. The mutant frequency (MF) at the hypoxanthine-guanine phosphoribosyl-transferase (hgprt) locus was assessed in lymphocytes isolated from male volunteers in each of three age groups (35-39, 50-54 and 65-69 years). Results show that the mean MF in the 65-69 years group was approximately twice that in the 35-39 and 50-54 years groups (4.1/10(6) cells, 1.9/10(6) cells and 1.79/10(6) cells, respectively) increasing by about 1.33% per year, after 54 years. In addition, there was an increased frequency of chromosomal aberrations in the 65-69 years group compared to the other two age groups. The results of this investigation show an increase in DNA mutations in cultured human lymphocytes with age. Factors which may influence the extent of DNA damage in human lymphocytes are discussed.

Analysis of hprt gene mutation following anti-cancer treatment in pediatric patients with acute leukemia

We evaluated the genotoxic effect of cancer therapy on somatic cell mutation by isolating 6-thioguanine-resistant mutants in peripheral lymphocytes. The study population comprised 45 children with acute lymphoblastic leukemia (ALL), 13 children with acute myclogenous leukemia (AML) and 28 age-matched healthy controls. The geometric mean mutant frequency for ALL patients was 7.8 × 10 −6, which was significantly higher than that for AML patients (1.7 × 10 −6) or for healthy controls (1.1 × 10 −6). Fifteen patients with ALL showed a high mutant frequency above 10 × 10 −6, although 10 of them had completed their treatment at least 24 months earlier. Moreover, repeated measurements of mutant frequency at intervals of 12 or more months revealed that the values were very stable. Structural hypoxanthine-guanine phosphoribosyl transferase ( hprt) gene alterations, as determined by Southern blot analysis, were seen in 23% (12/52) of mutant clones derived from ALL patients, but not in those from the controls. These results suggest that intensive anti-cancer therapy of children may produce persistent somatic mutations, which could be related to the appearance of second neoplasms.

In vivo mutations in human blood cells: biomarkers for molecular epidemiology.

Mutations arising in vivo in recorder genes of human blood cells provide biomarkers for molecular epidemiology by serving as surrogates for cancer-causing genetic changes. Current markers include mutations of the glycophorin-A (GPA) or hemoglobin (Hb) genes, measured in red blood cells, or mutations of the hypoxanthine-guanine phosphoribosyltransferase (hprt) or HLA genes, measured in T-lymphocytes. Mean mutant frequencies (variant frequencies) for normal young adults are approximately: Hb (4 x 10(-8)) < hprt (5 x 10(-6)) = GPA (10 x 10(-6)) < HLA (30 x 10(-6)). Mutagen-exposed individuals show decided elevations. Molecular mutational spectra are also being defined. For the hprt marker system, about 15% of background mutations are gross structural alterations of the hprt gene (e.g., deletions); the remainder are point mutations (e.g., base substitutions or frameshifts). Ionizing radiations result in dose-related increases in total gene deletions. Large deletions may encompass several megabases as shown by co-deletions of linked markers. Possible hprt spectra for defining radiation and chemical exposures are being sought. In addition to their responsiveness to environmental mutagens/carcinogens, three additional findings suggest that the in vivo recorder mutations are relevant in vivo surrogates for cancer mutations. First, a large fraction of GPA and HLA mutations show exchanges due to homologous recombination, an important mutational event in cancer. Second, hprt mutations arise preferentially in dividing T-cells, which can accumulate additional mutations in the same clone, reminiscent of the multiple hits required in the evolution of malignancy.(ABSTRACT TRUNCATED AT 250 WORDS)

In Vivo Mutations in Human Blood Cells: Biomarkers for Molecular Epidemiology

Mutations arising in vivo in recorder genes of human blood cells provide biomarkers for molecular epidemiology by serving as surrogates for cancer-causing genetic changes. Current markers include mutations of the glycophorin-A (GPA) or hemoglobin (Hb) genes, measured in red blood cells, or mutations of the hypoxanthine-guanine phosphoribosyltransferase (hprt) or HLA genes, measured in T-lymphocytes. Mean mutant frequencies (variant frequencies) for normal young adults are approximately: Hb (4 x 10(-8)) &lt; hprt (5 x 10(-6)) = GPA (10 x 10(-6)) &lt; HLA (30 x 10(-6)). Mutagen-exposed individuals show decided elevations. Molecular mutational spectra are also being defined. For the hprt marker system, about 15% of background mutations are gross structural alterations of the hprt gene (e.g., deletions); the remainder are point mutations (e.g., base substitutions or frameshifts). Ionizing radiations result in dose-related increases in total gene deletions. Large deletions may encompass several megabases as shown by co-deletions of linked markers. Possible hprt spectra for defining radiation and chemical exposures are being sought. In addition to their responsiveness to environmental mutagens/carcinogens, three additional findings suggest that the in vivo recorder mutations are relevant in vivo surrogates for cancer mutations. First, a large fraction of GPA and HLA mutations show exchanges due to homologous recombination, an important mutational event in cancer. Second, hprt mutations arise preferentially in dividing T-cells, which can accumulate additional mutations in the same clone, reminiscent of the multiple hits required in the evolution of malignancy.(ABSTRACT TRUNCATED AT 250 WORDS)

In vivo mutations at the H-2 locus in mouse lymphocytes

An experimental system has been developed to study in vivo autosomal mutations in murine splenic lymphocytes. Mutant lymphocytes were isolated by immunocytotoxicity using monoclonal antibodies directed against the k and d alleles of the K and D H-2 histocompatibility loci and were enumerated using limiting-dilution cloning. Genomic allele loss in mutant clones was detected using allele-specific primers in a polymerase chain reaction. Mutant clones were classified on the basis of phenotypic and genotypic criteria into “no change”, deletion or recombination mutants. The geometric mean mutation frequency in 102 mice was 2.42 × 10 −4. Detailed phenotypic and genotypic study of 87 mutant clones from 4 mice revealed “no change” mutants in 83%, mutants due to deletion in 7% and mutants due to recombination in 7%. Anomalous results were obtained in 3% of mutant clones. The development of an animal model for study of in vivo mutations at an autosomal locus will further advance study of mutations, particularly those involving chromosomal changes such as mitotic recombination.

Tradescantia stamen-hair mutation bioassay on the mutagenicity of radioisotope-contaminated air following the Chernobyl nuclear accident and one year later

This paper presents results of the research on the mutagenic effect of ambient air in the Cracow area. Initial studies were conducted in May 1986, following the Chernobyl accident. Other studies were performed at various sites within the Cracow area in the Spring of 1987. Counts were made of stunted hair and pink cells in the stamen hairs of Tradescantia clone 4430. Mutations scored from the 11th day after the beginning of exposure were used as a measure of the mutagenic effect. The mean mutation frequencies measured in 1986 and 1987 were 0.43 and 0.21 per 100 hairs respectively. The time-dependent development of mutation frequencies observed after the Chernobyl accident showed a correlation with the time-dependent development of total radioactivities measured in the air at that time. The results obtained in 1987 showed on average a significant decrease of ambient air mutagenicity. Still, the variation of mutation rates observed during the investigated period at different sites in the Cracow area was rather high (0.09–0.38 mut/100 hairs). Only the highest frequencies observed in the Spring of 1987 were comparable to the level detected after the Chernobyl accident.