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Rheumatoid arthritis (RA) is an inflammatory disease in which high levels of reactive nitrogen oxygen species (RNOS) may be present in the affected joints. RNOS are known to produce small-scale mutational events (transitions, transversions, small insertions, and small deletions) but the ability of these compounds to cause deletion of large segments of genomic DNA has not been previously determined. To address this question, a human lymphoblastoid cell line (WIL2-NS) was exposed to nitric oxide (NO)-donating drugs and hypoxanthine phosphoribosyltransferase (hprt)-negative clones were selected and analyzed by multiplex-PCR. Large-scale deletions accounted for 60-80% of hprt mutations arising in drug-treated cultures compared to 12% in untreated cultures (P-values of 0.006 and 0.0001, respectively, in two experiments). Deletion mutations in untreated cultures affected exon 9, whereas 75% of drug-induced deletion mutations affected exons 2, 3, and 9, and the remainder were very large, ranging from 26 to 1200 kbp. To compare this spectrum of NO-induced mutations in a lymphoblastoid line to that arising in vivo in arthritis patients, T-cells from RA patients, osteoarthritis (OA) patients, and controls were cloned and similarly analyzed. We previously showed that the overall frequency of Hprt mutant clones from patients is appreciably elevated compared to that of control subjects. Large-scale hprt deletions (0.5 to >26 kb) were detected in mutant T-cell clones from both RA and OA patients and also from control subjects. A total of 54 mutant clones from 16 RA patients and 19 mutant clones from 6 OA patients were studied. Of these, 6 clones (from 3 RA and 1 OA patient) had suffered large-scale deletions. A total of 9 control subjects were studied and 62 mutant clones were obtained. Of these, 19 had suffered large-scale deletions, arising in 7 of 9 control subjects. In conclusion, (1) RNOS are capable of inducing large-scale deletion mutations in a human lymphoblastoid cell line and (2) large-scale deletion mutations were found in 10-30% of T-cell clones from RA and OA patients and controls, which we hypothesize may be induced by RNOS., (Copyright 2001 Wiley-Liss, Inc.)

In a previous study, we found that treating transgenic Big Blue rats with the hepatocarcinogen N-hydroxy-2-acetylaminofluorene (N-OH-AAF) produced the same major DNA adduct in the target liver and the nontarget spleen lymphocytes and bone marrow cells, induced lacI mutants in the liver, and induced much lower frequencies of lacI and hprt mutants in spleen lymphocytes. In the present study, sequence analysis was conducted on lacI DNA and hprt cDNA from the mutants, to determine the mutational specificity of N-OH-AAF in the rat. All the mutation spectra from N-OH-AAF-treated rats differed significantly from corresponding mutation profiles from untreated animals (P = 0.02 to P < 0.0001). Although there were similarities among the mutational patterns derived from N-OH-AAF-treated rats (e.g., G:C --> T:A transversion was the most common mutation in all mutation sets), there were significant differences in the patterns of basepair substitution and frameshift mutation between the liver and spleen lymphocyte lacI mutants (P = 0.02) and between the spleen lymphocyte lacI and hprt mutants (P = 0.04). Also, multiplex PCR analysis of genomic DNA from the hprt mutants indicated that 12% of mutants from treated rats had major deletions in the hprt gene; no corresponding incidence of large deletions was evident among lacI mutations. All the mutation profiles reflect the general mutational specificity of the major DNA adduct formed by N-OH-AAF. The differences between N-OH-AAF mutation in the endogenous gene and transgene can be partially explained by the structures of the two genes. The tissue-specificity of the mutation spectra may contribute to targeting tumor formation to the liver. Environ. Mol. Mutagen. 37:203-214, 2001. Published 2001 Wiley-Liss, Inc.

N-Hydroxy-2-acetylaminofluorene (N-OH-AAF) is the proximate carcinogenic metabolite of the powerful rat liver carcinogen 2-acetylaminofluorene. In this study, transgenic Big Blue(R) rats were used to examine the relationship between in vivo mutagenicity and DNA adduct formation by N-OH-AAF in the target liver compared with that in nontarget tissues. Male rats were given one, two, or four doses of 25 mg N-OH-AAF/kg body weight by i.p. injection at 4-day intervals, and groups of treated and control rats were euthanized up to 10 weeks after beginning the dosing. Mutant frequencies were measured in the spleen lymphocyte hprt gene, and lacI mutant frequencies were determined in the liver and spleen lymphocytes. At 6 weeks after beginning the dosing, the hprt mutant frequency in spleen lymphocytes from the four-dose group was 16.5 x 10(-6) compared with 3.2 x 10(-6) in control animals. Also at 6 weeks, rats given one, two, or four doses of N-OH-AAF had lacI mutant frequencies in the liver of 97.6, 155.6, and 406.8 x 10(-6), respectively, compared with a control frequency of 25.7 x 10(-6); rats given four doses had lacI mutant frequencies in spleen lymphocytes of 55.8 x 10(-6) compared with a control frequency of 20.4 x 10(-6). Additional rats were evaluated for DNA adduct formation in the liver, spleen lymphocytes, and bone marrow by (32)P-postlabeling. Adduct analysis was conducted 1 day after one, two, and four treatments with N-OH-AAF, 5 days after one treatment, and 9 days after two treatments. N-(Deoxyguanosin-8-yl)-2-aminofluorene was the major DNA adduct identified in all the tissues examined. Adduct concentrations increased with total dose to maximum values in samples taken 1 day after two doses, and remained essentially the same after four doses. In samples taken after four doses, adduct levels were 103, 28, and 7 fmol/microg of DNA in liver, spleen lymphocytes, and bone marrow, respectively. The results indicate that the extent of both DNA adduct formation and mutant induction correlates with the organ specificity for N-OH-AAF carcinogenesis in the rat. Environ. Mol. Mutagen. 37:195-202, 2001. Published 2001 Wiley-Liss, Inc.

1,3-Butadiene (BD) has been shown to be a potent animal carcinogen and a probable human carcinogen, yet the molecular mechanisms of BD genotoxicity and carcinogenicity still are not fully understood. Our hypothesis is that metabolites of BD induce specific structural changes in the human hprt gene like those observed in vitro in TK6 cells and in vivo in the mouse. Characteristic mutations in BD-exposed subjects can be identified and used as biomarkers for monitoring genotoxic effects associated with BD exposure. Molecular analysis of hprt mutant lymphocytes from BD-exposed workers and unexposed control subjects was carried out to identify changes in the structure of the hprt gene. A multiplex polymerase chain reaction (PCR) assay was used to detect exon deletions in 360 hprt mutant clones. We determined that exon deletions were significantly more frequent (P < 0.05) in BD-exposed workers (17.5%) than in control subjects (9.7%). Sequence analysis of hprt cDNA from 175 independent mutants indicated that the distribution of the types of mutations was different between the workers and the unexposed control subjects. There was a significant increase in -1 frameshift mutations in BD-exposed workers, predominantly in repeated DNA sequences, and single-base substitutions were decreased to 66% in the workers compared to 83% in the control subjects (P < 0.05). In addition to the spectral changes, hprt clonal assays revealed an elevation in mutant frequency in the lymphocytes of workers (N = 10) when compared with that in unexposed control subjects (N = 11; P < 0. 05). There also was a twofold increase of A:T --> T:A transversions in BD-exposed workers (16% in BD-exposed workers compared to 8% in controls, P = 0.25). Some of the BD-associated changes in mutational spectra observed in our study have the potential for application in monitoring genotoxic effects related to butadiene exposure., (Copyright 2000 Wiley-Liss, Inc.)

An important question regarding the use of transgenic reporter genes to detect mutation in rodents is how the types of mutations recovered in transgenes compare with the types of mutations found in endogenous genes. In this study, we examined mutations induced by 7,12-dimethylbenz[a]anthracene in the lacI transgene and the endogenous hprt gene of lymphocytes from Big Blue rats and in the hprt gene of lymphocytes from nontransgenic Fischer 344 rats. The overall mutation profiles found in these genes were remarkably similar: the majority of mutations were base pair substitutions, with the most common mutation being A:T-->T:A transversion. Differences were found for the mutational profiles in the endogenous gene and transgene with respect to the location of the mutations and the orientation of basepair substitutions in the DNA strands. In most cases, these differences could be explained by the nature of the target genes. The results support the use of the lacI transgene for detecting in vivo mutation.

Phenolphthalein is a cathartic agent widely used in non-prescription laxatives. For the simultaneous assessment of in vitro carcinogenicity and mutagenicity of phenolphthalein, the ability of this chemical to induce cell transformation and genetic effects was examined using the Syrian hamster embryo (SHE) cell model. Cell growth was reduced by treatment with phenolphthalein at 10-40 microM in a dose-related manner. Treatment with phenolphthalein for 48 hr induced a dose-dependent increase in morphological transformation of SHE cells. Over the dose range that resulted in cell transformation ( 10-40 microM), treatment of SHE cells with phenolphthalein induced gene mutations at the hprt locus but not at the Na+/K+ ATPase locus. A statistically significant level of chromosomal aberrations was elicited in SHE cells treated with phenolphthalein at the highest dose (40 microM). Meanwhile, neither numerical chromosomal changes nor DNA adduct formation, analyzed by the nuclease P1 enhancement version of 32P-post-labeling, were induced by treatment with phenolphthalein at any concentrations examined. We thus report cell-transforming activity and mutagenicity of phenolphthalein assessed with the same mammalian cells in culture. Our results provide evidence that phenolphthalein has cell-transforming and genotoxic activity in cultured mammalian cells. The mutagenic and clastogenic activities of phenolphthalein could be a causal mechanism for carcinogenicity in rodents.

In January 1993 the oil tanker Braer ran aground in the Shetland Islands, Scotland. Approximately 80,000 tons of crude oil were released. Exceptionally high winds caused extensive pollution and exposure of the local population to crude oil. We describe the study which was immediately set in place to examine the exposed population for evidence of genotoxic exposure. Blood samples were taken and primary DNA damage was measured in the mononuclear cell fraction by the butanol modification of the 32P-postlabelling method. Mutation was measured at the hprt locus in T lymphocytes. No evidence of genotoxicity was obtained for either end point, but nevertheless, we believe that useful lessons were learnt, which should be incorporated into the design of future studies: (1) A rapid response is essential, and even if sufficient funds are not immediately available, it is still worth attempting to obtain samples quickly and use cryopreservation, also to attempt to estimate exposure. (2) Adequate numbers of volunteers must be sought, together with enough controls, not just to allow meaningful analysis but to overcome loss of samples and failure of things to go according to plan. (3) Points concerning laboratory practice include: (i) samples should be coded, (ii) clearly defined and proven protocols should be used, (iii) irreplaceable samples should not be used for method development, (iv) should a problem become apparent during the study, work on such samples should cease immediately until the problem is solved, (v) all critical experimental components should be pretested against a laboratory standard. (4) The study design should include replicate experiments to monitor experimental variability and reproducibility, as well as internal standards and cryopreserved "in house" samples. Care must be taken that samples from any one exposure group are spread between a number of independent experiments and that each experiment includes samples from a number of exposure groups. (5) A computerised data base should be maintained with full details of experimental variables, donor attributes, and raw data so that any contribution of experimental artefacts to "outlier" results can be monitored. (6) Because of the nature of the statistical variation for many environmental genotoxicity end points, only a large-scale study is likely to be capable of yielding useful information.

Previous work with the autoradiographic mutant lymphocyte assay has provided information about the time-course of development of hprt mutations and the persistence of detectable mutant cells in human subjects following therapeutic exposures to genotoxic agents. These early studies also revealed elevations in frequencies of mutant cells in pretreatment blood samples from patients who were current tobacco smokers, but no information was available on former smokers. In the present study, blood samples were obtained from 21 healthy former tobacco smokers who had quit smoking at least 1 year before sampling, 42 subjects who had never smoked, and 23 tobacco smokers. Plasma from all samples was tested for cotinine, a metabolite of nicotine. Current smokers were categorized as heavy smokers (> or = 10 cigarettes per day, cotinine > or = 90 ng/ml plasma) and light smokers (< 10/day, cotinine < 90 ng/ml). Lymphocytes from the blood samples were isolated, cryopreserved, and later thawed and assayed with the autoradiographic hprt assay. The 21 former tobacco smokers had a mean variant (mutant) frequency (Vf +/- standard error) of 1.97 (+/-0.13) per million evaluatable cells. The Vf of 42 subjects who had never smoked was 1.74 (+/-0.13) x 10(-6), not significantly different from the former smokers. The smokers had Vfs of 8.09 (+/-0.78) x 10(-6) for 18 heavy smokers and 5.22 (+/-1.02) x 10(-6) for five light smokers. The two categories of smokers had frequencies of mutant cells significantly different from each other, and each was significantly higher than non-smokers and former smokers (P < 0.05). Vfs were significantly correlated with both cotinine concentrations and the number of cigarettes smoked per day, P < 0.001. This study demonstrates the sensitivity of the autoradiographic hprt assay for detecting mutagenic effects related to chronic low-level exposures to genotoxins, and indicates that this assay is more likely to detect the effects of recent rather than past exposures.

Big Blue (BB) and generic B6C3F1 mice were given one to three i.p. injections of 50 mg/kg benzo[a]pyrene (B[a]P) in DMSO every other day to achieve cumulative doses of 50 to 150 mg/kg. Three weeks after treatment, the mutation frequency at the endogenous hprt gene and lacI transgene was measured in splenic T cells. Generic mice given 50, 100, and 150 mg/kg B[a]P displayed induced hprt frequencies (observed hprt frequency minus control frequency) of 5.5 +/- 1.0, 11 +/- 2.0, and 19 +/- 2.6 x 10(-6), respectively (average +/- SEM). In contrast, BB mice given 50 and 150 mg/kg B[a]P displayed induced hprt frequencies of 0.9 +/- 0.6 and 9.1 +/- 1.5 x 10(-6). 32P postlabelling revealed that the lower hprt response in BB mice correlated with lower amounts of BP-DNA adducts in spleen, liver, and lung 24 hours after B[a]P exposure. Western blot analysis of liver samples from B[a]P-treated mice suggests that the reduced adduct load in turn may be due to lower P450 1A1 levels in BB mice. The frequency of induced, nonsectored blue plaques (observed blue plaque frequency minus control frequency) in BB mice receiving 50 and 150 mg/kg B[a]P was 41 +/- 9 and 134 +/- 10 x 10(-6) (15- to 40-fold higher than the induced hprt frequency in the same treated animals). Sectored plaques were observed in both control and B[a]P groups but their frequency showed no relationship to dose (sectored frequency in all groups was approximately 20 x 10(-6)). To test whether persistent DNA adducts in the packaged lambda vector were contributing to the observed blue plaque frequency, purified lambda-LIZ DNA was treated in vitro with B[a]P diol epoxide (BPDE), packaged, and plated on E. coli lawn cells. Treatment with BPDE did not produce significant increases in homogeneous blue plaques, suggesting that the majority of mutants obtained from B[a]P-treated BB mice occurred in vivo. These results indicate that B[a]P exposure produces many more mutations at the lacI transgene than at the endogenous hprt locus.

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.

1-Nitrosopyrene, a metabolite of the tumorigenic environmental pollutant 1-nitropyrene, is a potent mutagen at the hprt locus in Chinese hamster ovary (CHO) cells. A single DNA adduct, N-(deoxyguanosin-8-yl)-1-aminopyrene, is produced in CHO cells treated with 1-nitrosopyrene, and this adduct is found in rats and mice exposed to 1-nitropyrene. In this study, the structure of the hprt gene and the structure and amount of hprt mRNA were analyzed in 43 CHO cell mutants (16 isolated from solvent control cultures and 27 isolated from 1-nitrosopyrene-treated cultures). Pstl- and BamHl-digested DNA from the mutants were subjected to Southern blot analysis using a hamster hprt cDNA probe. None of the 1-nitrosopyrene-induced mutants and only one of the control mutants displayed hybridization patterns that were different from the parent CHO cells. Northern blot analysis revealed that two control mutants had truncated hprt mRNAs, while 56% of the control mutants and 78% of the induced mutants had reduced levels of hprt mRNA. Using polymerase chain reaction amplification of cDNA synthesized from RNA, the hprt protein-coding region could be amplified from 23 of the 1-nitrosopyrene-induced mutants and 11 of the control mutants. The amplification products from 3 of the control mutants and 5 of the induced mutants were smaller than that found with RNA from parental CHO cells. These results indicate that the mutagenic DNA damage produced by 1-nitrosopyrene in CHO cells does not cause major structural alterations in the hprt gene and suggest that 1-nitrosopyrene acts as a point mutagen. A large number of both control and 1-nitrosopyrene-induced mutants exhibited a marked reduction in hprt mRNA concentration or possessed truncated mRNA hprt protein coding sequence. These alterations may contribute to the 6-thioguanine-resistant phenotype.