Molecular analysis of styrene oxide-induced hprt mutation in human T-lymphocytes.

Styrene-7,8-oxide (SO), the mutagenic in vivo metabolite of the widely used chemical monomer styrene, has been classified as a probable human carcinogen (IARC, 1994). We examined mutations in the hypoxanthine-guanine phosphoribosyl transferase (hprt) gene of primary human T-lymphocytes exposed to 0.2 mM SO for 6 days in vitro. PCR amplification and direct DNA sequencing were used to identify 55 SO-induced mutations from two experiments in which the mutation frequencies increased 3.6 and 4.8 times respectively, and 44 control mutations from untreated T-cell cultures. Base substitutions were the dominating type of mutation in both groups, with 35 and 23 independent changes, of which nine and six respectively, have not previously been described in human T-cells. Frameshift mutations (+/-1 bp) and small deletions (2-200 bp) were less frequent and splicing mutations more frequent among the SO-induced than among the control mutations. In SO-treated mutants, base substitutions in the coding region occurred at 15 sites, nine of which were AT bp, and in the splice donor and acceptor regions six of 10 mutated sites were AT bp. Altogether six independent mutations were found at site 539 in cells from the two SO experiments (four GC > AT and two GC > TA). In the control cultures, base substitutions in the coding and splicing regions were identified at 20 sites, eight of which were AT bp. In published data on hprt mutation in untreated T-cells in vivo and in vitro, 31 of 88 base substitutions have been reported to occur at AT bp. These results indicate that SO-induced mutations at the hprt locus in human T-lymphocytes are predominantly base substitutions, and suggest that in addition to DNA adducts at guanine bases, adducts at A and/or T bases also deserve attention with regard to the mutagenesis of SO.

Sequence analysis of deletion mutations at the HPRT locus of human T-lymphocytes: association of a palindromic structure with a breakpoint cluster in exon 2.

To study the structure and mechanism of deletion mutation in human somatic cells in vivo, we have identified and sequenced the breakpoints of 16 independent deletions at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in human T-lymphocytes. Seven deletions were found in exon 2, three in each of exon 3 and 6, and one in each of intron 3, exon 8 and exon 9. Most of the deletions seemed to result from non-homologous recombination, possibly by a slippage-misalignment mechanism between short repeat sequences. Putative secondary DNA structures, possibly acting as intermediates in the deletion formation, were identified in several mutants. Six of the seven exon 2 deletions had a breakpoint within a 12 bp region (in the 5' end of exon 2) which contains a 9 nucleotide palindrome (AACCAGGTT) and is preceded by a TGA direct repeat tract. One of the mutants had two deletions in tandem, separated by the palindrome. Another mutant, in which 23 bp containing the palindromic sequence was deleted, had an additional base (C) inserted between the breakpoints forming a direct repeat (gACGAC) in the deletion junction. Taken together with previously reported deletion mutations at the HPRT locus, these results suggest that the deletion cluster in the 5' part of HPRT exon 2 in T-cells in vivo is promoted by the 9 nucleotide palindrome sequence and the TGA repeat tract. The former may act as a stabilizer in a putative intermediate structure, and the latter may induce slippage and misalignment during replication.

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.

Analysis of mutation at the hprt locus in human T lymphocytes.

Studies of mutation at the hypoxanthine phosphoribosyl transferase (hrpt) locus in human T-cells have the potential to elucidate the molecular basis of in vivo mutagenesis, reveal exposure dependent changes in ther background frequency of mutation, and provide knowledge on individual sensitivity. Styrene exposed lamination workers in Bohemia showed a significantly higher frequency of hprt mutant cells than Swedish control populations studied simultaneously. In a study of 47 healthy, non-smoking male bus maintenance workers exposed to diesel exhausts, soot and oil, and 22 unexposed controls, a significant correlation (P = 0.008) was obtained between the levels of aromatic DNA adducts and frequencies of hprt-mutant T-cells. In the group of workers with the highest exposure, subjects with glutathione S-transferase (GSTM1) deficiency showed significantly higher (P < 0.05) frequency of hprt mutant T-cells than GSTM1-positive subjects. The highest adduct levels were found in subjects with the combined genotype of GSTM1 and NAT2 deficiency (GSTM1-negative slow acetylators). These results indicate that GSTM1 and NAT2 genotypes may play a role in determining the individual levels of hprt mutation and DNA adducts. Using PCR-based screening methods, hprt mutations have been classified in 462 T-cell clones from 43 subjects in this study population. Deletions were found in 3% of the mutants, coding errors in 81% and splice mutations in 17%. Transitions and transversions were equally common, and all types of base substitutions were detected.

Styrene oxide-induced HPRT mutations, DNA adducts and DNA strand breaks in cultured human lymphocytes.

Styrene-7,8-oxide (SO) is the major in vivo metabolite of styrene, a widely used plastic monomer. SO has been classified as probably carcinogenic to humans. We studied the genotoxic effects of SO in human peripheral blood lymphocytes (PBL) in vitro. SO-treatment in the range of 0.05-0.6 mM for 24 h resulted in a dose-dependent decrease of cell survival and increase of HPRT mutation, O6-guanine DNA adducts and DNA strand breaks, whereas higher concentrations caused pronounced cell death. SO was a weak mutagen, inducing at most 10-20 mutants per 10(6) clonable cells (approximately 4-fold over the background) after treatment with 0.2-0.4 mM for 24 h or 6 days. The levels of DNA adducts in treated cells correlated with SO-concentrations, but only four adducts per 10(8) nucleotides were detected at the highest treatment concentrations. Yet, adducts were still detectable in cells that had been cultured for 6-8 days after treatment. SO-induced DNA strand breaks, measured with the Comet assay, were detectable after 1 h exposure to 0.05-0.1 mM. Post-treatment incubation for 24 h decreased the level of DNA strand breaks to the control level. There was no correlation between the levels of DNA adducts and frequency of HPRT mutation. The present results indicate that SO is relatively inefficient in inducing HPRT mutation and O6-guanine DNA adducts in human lymphocytes in vitro, which may be related to its pronounced cytotoxicity at concentrations above 0.4 mM. A comparison with previous in vivo data obtained by the same assays in T-lymphocytes of styrene-exposed workers suggests that chronic, low dose exposure to styrene in the work environment may be more efficient in inducing persistent DNA adducts and HPRT mutation than acute, short-term exposure.

Biomarkers of styrene exposure in lamination workers: levels of O6-guanine DNA adducts, DNA strand breaks and mutant frequencies in the hypoxanthine guanine phosphoribosyltransferase gene in T-lymphocytes.

Occupational exposure to styrene was studied in nine workers of a hand lamination plant in Bohemia. Personal dosimeters were used to monitor the styrene workplace exposure, and the levels of styrene in blood and mandelic acid in urine were measured. Blood samples were taken at four occasions during a 7 month period to determine styrene-specific O6-guanine DNA adducts in lymphocytes and granulocytes, DNA strand breaks and hypoxanthine guanine phosphoribosyltransferase (HPRT) mutant frequency in T-lymphocytes. Seven administrative employees in the same factory (factory controls) and eight persons in a research laboratory (laboratory controls) were used as referents. DNA adduct levels determined by the 32P-postlabelling method in lymphocytes of laminators were remarkably constant and significantly higher (P < 0.0001) than in factory controls at all four sampling times. HPRT mutant frequencies (MF) measured by the T-cell cloning assay were higher in the laminators (17.5 x 10(-6), group mean) than in the factory controls (15.7 x 10(-6), group mean) at three of the four sampling times, but the differences were not statistically significant. However, a statistically significant (P = 0.021) difference between MF in the laminators (18.0 x 10(-6), group mean) and laboratory controls (11.8 x 10(-6), group mean) was observed at sampling time 4 (the only sampling time when this latter group was studied). This result indicates that styrene exposure may induce gene mutation in T-cells in vivo. DNA strand breaks were studied by the 'Comet assay' at the fourth sampling time. The laminators were found to have significantly higher levels of DNA strand breaks than the factory controls (P = 0.032 for tail length, TL; P = 0.007 for percentage of DNA in tail, T%; and P = 0.020 for tail moment, TM). A statistically significant correlation was also found between the levels of lymphocyte DNA adducts and all three DNA strand break parameters (TL P = 0.046; T% P = 0.026 and TM P = 0.034). On the contrary, no significant correlations were found between DNA adduct levels and the HPRT mutant frequencies or between the mutant frequencies and DNA strand breaks. Taken together, these results add further support to the genotoxic and possibly mutagenic effects of styrene exposure in vivo. However, no simple quantitative relationship seems to exist between the levels of styrene-induced DNA damage and frequency of HPRT mutation in T-lymphocytes.

Mutations induced in the hypoxanthine phosphoribosyl transferase gene by three urban air pollutants: acetaldehyde, benzo[a]pyrene diolepoxide, and ethylene oxide.

Provisional mutational spectra at the hypoxanthine phosphoribosyl transferase (HPRT) locus in vitro have been worked out for acetaldehyde (AA) and benzo[a]pyrene diolepoxide (BPDE) in human (T)-lymphocytes and for ethylene oxide (EtO) in human diploid fibroblasts using Southern blotting and polymerase chain reaction (PCR)-based DNA sequencing techniques. The results indicate that large genomic deletions are the predominating hprt mutations caused by AA and EO, whereas BPDE induces point mutations that are mainly GC > TA transversions. The mutational spectra induced by the three agents are clearly different from the background spectrum in human T-cells. Thus, the hprt locus is a useful target for the study of chemical-specific mutational events that may help identify causes of background mutation in human cells in vivo.

Molecular analysis of ethylene oxide-induced mutations at the HPRT locus in human diploid fibroblasts.

Ethylene oxide (EtO)-induced mutations in the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene were characterized in 28 independently derived 6-thioguanine-resistant human diploid fibroblast clones using polymerase chain reaction-based techniques and Southern blot analysis. Sequence analysis revealed one single base pair deletion and 13 base substitutions, nine of which were transversions: five AT-->TA, three GC-->TA and one GC-->CG. Four mutants were found to have GC-->AT transitions. Seven of the point mutations caused splicing errors. Six occurred in splice site sequences and one created a new splice acceptor site 16 bp upstream of exon 9. Three splice mutations were localized at the same site in the splice donor sequence of intron 8. Fourteen mutants had large HPRT gene deletions. In seven mutants the entire HPRT gene was deleted. The remaining deletion mutants had a truncated HPRT gene, where one or several exons were lost. These results show that EtO induces many different kinds of HPRT mutations, among which as many as 50% are large deletions.

Differences between survival, mutagenicity and DNA replication in MMS- and MNU-treated V79 hamster cells.

After treatment with methyl methanesulfonate (MMS) or N-methyl-N-nitrosourea (MNU), the mutagenicity and survival of Chinese hamster V79 cells were investigated, as well as the inhibition of daughter DNA synthesis and, using the DNA unwinding technique and hydroxylapatite chromatography, the character of the newly synthesized DNA was studied. It was found that different cytotoxicity and mutagenicity of MMS and MNU was accompanied by different types of DNA synthesis inhibition. The treatment with the former compound resulted in a longer inhibition of DNA synthesis, while the treatment with the latter showed that as early as 2 h after exposure the percentage of nascent DNA increased. Shortly after the exposure to both alkylating agents, the newly synthesized DNA contained a higher number of gaps than control DNA, in dependence on the concentration used. During culturing after treatment, the character of nascent DNA in MMS-treated cells gradually returned to that of control DNA, while MNU-treated cells, for the whole time of our study, synthesized DNA with a larger number of gaps than control DNA. We suggest that the character of nascent daughter DNA reflects the occurrence of lesions in parental DNA. These are repaired within a shorter time in MMS- than in MNU-treated cells. The long-term persistence of lesions in the DNA of MNU-treated cells might be one of the factors responsible not only for the higher cytotoxic but also for the many times higher mutagenic effect of this alkylating agent.