Mutations induced by benzo[a]pyrene diolepoxide at the hprt locus in human T-lymphocytes in vitro

Human T-lymphocytes have been treated with benzo[a]pyrene diolepoxide (BPDE) in vitro and T-cell clones mutated in the hprt gene have been isolated. The mutant frequencies in BPDE-treated T-cell cultures were on average 24-fold higher than those of untreated cultures. Thus, BPDE is a potent inducer of gene mutation in this system. In order to examine which types of mutations are induced by BPDE in human cells, 41 spontaneous and 44 BPDE-induced mutant clones have been characterized using the Southern blot technique. In addition, rearrangements of the T-cell-receptor beta and gamma loci have been used to determine the proportion of isolated clones that are unique, and thus likely to represent independent mutational events. Out of 23 independent spontaneous mutants 4 had large hprt alterations that could be detected on Southern blots. Two of these alterations, deletions of exons 2-6, have been confirmed using PCR of hprt cDNA and direct sequencing of the PCR product. All 33 independent BPDE-induced mutants had normal hprt restriction patterns which indicates that BPDE is mainly a point mutagen in this system.     

Mutagenicity of 1,3-butadiene at the Hprt locus of T-lymphocytes following inhalation exposures of female mice and rats.

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

DNA damage induced by three major metabolites of 1,3-butadiene in human hepatocyte L02 cells

1,3-Butadiene (BD) is a carcinogenic air pollutant. Its bioactivation produces four major metabolites, i.e., 3,4-epoxy-1-butene (EB), 3,4-epoxy-1,2-butanediol (EBD), 1,2,3,4-diepoxybutane (DEB), and 3-butene-1,2-diol (BDD). Studies have been mostly focused on DEB due to its strong mutagenicity/carcinogenicity. In contrast, studies of genotoxicity of EB, EBD, and BDD have been limited. In particular, genotoxicity of EBD and BDD using strand breaks as the endpoint has not been investigated. To obtain a more complete understanding of BD toxicity, in the present study, we used comet assay to investigate DNA damage induced by EB, EBD, and BDD in human hepatocyte L02 cells, with the aim to determine their relative potencies, the types of DNA damage, and the possible pathway to form strand breaks. Using alkaline comet assay (pH>13), it was observed that EB and EBD caused similar concentration-dependent increases in DNA migration from 50 to 1000μM. However, BDD induced a statistically significant increase only at 1000μM, and the increase itself was very small. EBD was as potent as EB at lower concentrations (≤200μM), and was slightly less potent than EB at higher concentrations. The results indicated that these metabolites could generate strand breaks in cells with the rank order of the potencies being EB>≈EBD?BDD. All three compounds failed to cause statistically significant increases in DNA migration in pre-lysed cells, suggesting that they did not produce strand breaks through chemical pathways under our experimental conditions. By using comet assays at pH 11.9 and pH 9, it was demonstrated that EB and EBD generated both single-strand breaks (SSB) and alkali-labile sites, but BDD produced only SSB. To our knowledge, this is the first report to investigate EBD- and BDD-induced strand breaks in cells. The results implied that EBD could play an important role in toxicity of BD.     

Comparison of the mutations at Hprt exon 3 of T-lymphocytes from B6C3F1 mice and F344 rats exposed by inhalation to 1,3-butadiene or the racemic mixture of 1,2:3,4-diepoxybutane

Experiments were conducted to define the spectra of mutations occurring in Hprt exon 3 of T-cells isolated from spleens of female B6C3F1 mice and F344 rats exposed by inhalation to 1,3-butadiene (BD) or its reactive metabolite, (+/-)-diepoxybutane (DEB). Hprt mutant frequencies (Mfs) in BD-exposed (1250 ppm for 2 weeks or 625 ppm for 4 weeks; 6 h/day, 5 days/week) and DEB-exposed (2 or 4 ppm for 4 weeks or 5 ppm for 6 weeks; 6 h/day, 5 days/week) mice and rats were significantly increased over concurrent control values. Mutant T-cell colonies from control and treated animals were screened for mutations in Hprt exon 3 using PCR amplification of genomic DNA and denaturing gradient gel electrophoresis, followed by sequence analysis. Exon 3 mutations were found at the following frequencies: 20/394 (5%) in control mice, 56/712 (8%) in BD-exposed mice, 59/1178 (5%) in BD-exposed rats, 66/642 (10%) in DEB-exposed mice, and 51/732 (7%) in DEB-exposed rats. Mutations in exposed animals included base substitutions, small deletions (1 to 74 bp), and small insertions (1 to 8 bp), with base substitutions predominating. Among the types of base substitutions observed in mice, the proportions of G.C-->A.T transitions (p=0.035, Fisher's Exact Test) and G.C-->C.G transversions (p=0.05) were significantly different in control vs. BD-exposed animals. Given the small number of exon 3 mutants analyzed, there was a high degree of overlap in the mutational spectra between BD-exposed mice and rats, between BD- and DEB-exposed mice, and between BD- and DEB-exposed rats in terms of the sites with base substitutions, the mutations found at those mutated sites, the relative occurrence of the most frequently observed base substitutions, and the occurrence of a consistent strand bias for the most frequently observed base substitutions. The spectra data suggest that adduction of both G.C and A.T bps is important in the induction of in vivo mutations by BD metabolites in exposed mice and rats.     

Alterations of the glutathione-redox balance induced by metals in CHO-K1 cells

The effects of cadmium (Cd(2+)), mercury (Hg(2+)), lead (Pb(2+)), copper (Cu(2+)) and nickel (Ni(2+)) on the glutathione (GSH)-redox cycle were assessed in CHO-K1 by the neutral red uptake inhibition (NR) assay (NR(6.25), NR(12.5) and NR(25)). Mercury proved to be the most and lead the least toxic of the metals tested. The effects on GSH content and intracellular specific activities of enzymes involved in the GSH-redox balance were measured after a 24-h exposure. Total GSH content increased significantly in cultures exposed to the lowest metal concentration assayed (NR(6.25)), but fell to below control values when exposed to concentrations equivalent to NR(25). Oxidised glutathione content dropped significantly at NR(6.25), while somewhat higher values were obtained for cultures exposed to higher doses. Glutathione peroxidase (Gpx) activities were 1.2-, 1.5-, 1.6-, 2.0- and 2.5-fold higher than untreated controls for cadmium, copper, mercury, nickel and lead, respectively, at concentrations equivalent to NR(6.25). Gpx activity declined at metal concentrations equivalent to NR(12.5) and NR(25). Glutathione reductase activity remained almost unchanged except at low doses of mercury, nickel and lead. Glutathione-S-transferase activity decreased at rising metal concentrations. The results suggest that a homeostatic defence mechanism was activated when cells were exposed to doses equivalent to NR(6.25) while the ability of the cells to respond weakened as the dose increased. A close relationship was also observed between metal cytotoxicity, total GSH content and the dissociation energy of the sulphur-metal bonds. These facts confirm the involvement of antioxidant defence mechanisms in the toxic action of these ions.     

Mutations induced by X-rays at the HPRT locus in cultured Chinese hamster cells are mostly large deletions

We investigated the molecular basis of 19 X-ray-induced HPRT-deficient mutants of V79 Chinese hamster cells with Southern hybridisation techniques. 12 of those mutants suffer from a big deletion (greater than 10 kb) of HPRT DNA sequences. Cytological studies of chromosome preparations of those 12 deletion mutants showed that in at least 3 of these mutants part of the long arm of the X-chromosome was lost. After correction for spontaneous arising mutations we estimate that at least 70-80% of X-ray-induced mutations are caused by large deletions.     

Mutagenicity of the racemic mixtures of butadiene monoepoxide and butadiene diepoxide at the Hprt locus of T-lymphocytes following inhalation exposures of female mice and rats.

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

Sulfhydryl compounds inhibit the cyto- and geno-toxicity of o-phenylphenol metabolites in CHO-K1 cells

The effects of cysteine and reduced glutathione (GSH) on the genotoxicity of o-phenylphenol (OPP) and its metabolites, phenylhydroquinone (PHQ) and phenylbenzoquinone (PBQ), were examined using the frequency of sister-chromatid exchanges (SCEs) and chromosome aberrations in CHO-K1 cells as parameters. Cytotoxic (cell-progression delay) and cytogenetic effects induced by a 3-h treatment with OPP, PHQ (100 micrograms/ml) or PBQ (50 micrograms/ml) with S9 mix after a 27-h expression time were inhibited by cysteine or GSH (3-10 mM). Materials corresponding to the cysteine or GSH adducts were found by HPLC in each incubation mixture. In the culture without S9 mix, PHQ and PBQ showed severe cytotoxicity since no metaphases could be obtained at doses over 25 and 5 micrograms/ml, respectively, and the sulfhydryl compounds inhibited the toxicity by the formation of adducts with PBQ and by inhibiting the formation of PBQ in the case of PHQ. With PHQ, the sulfhydryl compounds appeared to inhibit autooxidation. However, the sulfhydryl compounds did not inhibit the cytotoxic and cytogenetic effects caused by OPP in the cell mixture without S9 mix, but on the contrary intensified them. No adduct formation was detected in the incubation solution. On the basis of these results, it is considered that electrophilic quinone (PBQ) and/or semiquinone (phenylsemiquinone, PSQ) radicals, capable of binding to nucleophilic small molecules (such as cysteine and GSH) or (biological) macromolecules, are produced from metabolite PHQ in metabolic oxidation of OPP, and induce cyto- and geno-toxic effects in the cells. The cyto- and geno-toxic effects of OPP itself to the cells are clearly independent of any electrophilic radical reaction.     

Next generation tools for high-throughput promoter and expression analysis employing single-copy knock-ins at the Hprt1 locus

We have engineered a set of useful tools that facilitate targeted single copy knock-in (KI) at the hypoxanthine guanine phosphoribosyl transferase 1 (Hprt1) locus. We employed fine scale mapping to delineate the precise breakpoint location at the Hprt1(b-m3) locus allowing allele specific PCR assays to be established. Our suite of tools contains four targeting expression vectors and a complementing series of embryonic stem cell lines. Two of these vectors encode enhanced green fluorescent protein (EGFP) driven by the human cytomegalovirus immediate-early enhancer/modified chicken beta-actin (CAG) promoter, whereas the other two permit flexible combinations of a chosen promoter combined with a reporter and/or gene of choice. We have validated our tools as part of the Pleiades Promoter Project (http://www.pleiades.org), with the generation of brain-specific EGFP positive germline mouse strains.     

Sister-chromatid exchanges induced by 1.3-butadiene and its epoxides in CHO cells

Sister-chromatid exchanges (SCEs) were analyzed in CHO cells after pulse treatment with 1,3-butadiene, 3,4-epoxy-1-butene (monoepoxybutene) and 1,2:3,4-diepoxybutane (diepoxybutane). A weak dose effect was observed after exposure to 1,3-butadiene but only in the presence of S9 mix. Monoepoxybutene and diepoxybutane were highly effective in inducing SCEs at concentrations of 0.1-1 microM both in the presence and in the absence of S9 mix. At higher concentrations the response was more pronounced without S9 mix.     

Age-, gender-, and species-dependent mutagenicity in T cells of mice and rats exposed by inhalation to 1,3-butadiene

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

Molecular analysis of mutations induced by N-ethyl-N-nitrosourea at the HPRT locus in mouse lymphoma cells

The molecular basis of 29 N-ethyl-N-nitrosourea (ENU)-induced HPRT-deficient mutants of mouse lymphoma cells (GRSL 13-2) was investigated using nucleic acid blot hybridization techniques. DNA from all 29 mutants showed normal restriction patterns on Southern blots when probed with HPRT cDNA, but 10 mutants differed from wild-type cells in their cytoplasmic HPRT mRNA level. In 5 mutants we found 10-25% of the normal amount of HPRT mRNA, whereas in another 5 mutants no HPRT mRNA could be detected at all. These mutants do not seem to be induced by hypermethylation of regulatory sequences of the HPRT gene, since they could not be reverted to an HPRT-proficient phenotype by treatment of the cells with 5-azacytidine.     

Mutations induced by glyoxal and methylglyoxal in mammalian cells.

To investigate the mutation spectra of glyoxal and methylglyoxal in mammalian cells, we analyzed mutations in a bacterial suppressor tRNA (supF) gene in the shuttle vector plasmid pMY189. The cytotoxicity and the mutation frequency increased according to the doses of glyoxal and methylglyoxal. The majority of glyoxal-induced mutations (65%) were base-pair substitutions, in which G:C-->C:G transversions were predominant. In the mutants induced by methylglyoxal, multi-base deletions were predominant (50%), followed by base-pair substitutions (35%), in which G:C-->C:G and G:C-->T:A transversions were predominant.     

Induction of chromosome aberrations and sister-chromatid exchanges in CHO-K1 cells by o-phenylphenol

o-Phenylphenol (OPP), is used in Japan as a fungicide in food additives for citrus fruits. The induction of chromosome aberrations and sister-chromatid exchanges (SCEs) by OPP in cultured Chinese hamster ovary (CHO-K1) cells was studied. Cells were exposed to various concentrations of OPP ranging from 50 to 175 micrograms/ml for 3 h, and further incubated for 27 and 42 h. These incubation periods are almost equal to 2 and 3 cell cycles. SCEs and chromosome aberrations were induced by OPP at concentrations of 100, 125 and 150 micrograms/ml after the incubation for 27 h. For chromosome aberrations, chromatid breaks and exchanges there was a dose-dependent increase. Diplochromosomes due to endoreduplication were also caused by the same concentrations of OPP in a dose-dependent manner. After incubation for 42 h, chromosome aberrations were also increased by OPP at concentrations of 100 and 125 micrograms/ml, but the frequencies of SCEs were not significantly different from those of the control. These results suggest that OPP has a cytogenetic toxicity, and that the DNA damage resulting in SCEs induced by OPP is relatively short-lived and can be repaired during the longer incubation time.     

Micronuclei, DNA single-strand breaks and DNA-repair activity in mice exposed to 1,3-butadiene by inhalation

We investigated single-strand breaks and endonuclease III-sensitive sites in DNA along with gamma-irradiation-specific DNA-repair activity in hepatocytes and frequencies of micronuclei in polychromatic bone-marrow erythrocytes of male NMRI mice (2 months old, weight 30-35 g) during sub-acute inhalation exposure to 1,3-butadiene (28 days, 500 mg/m3) and up to 28 days after the exposure. Concentrations of 1,3-butadiene in blood, an indicator of internal exposure, moderately increased during the exposure period. The most interesting finding was that gamma-irradiation-specific DNA-repair activity gradually increased during exposure, being significantly higher compared with control levels on days 7 and 28 of exposure (P = 0.005 and 0.035, respectively), reaching a maximum on day 1 after the termination of exposure (P = 0.003) and then returning to control levels. A significant correlation between gamma-irradiation-specific DNA-repair activity and the concentration of 1,3-butadiene in blood (R = 0.866, P = 0.050) supports a possible induction of DNA-repair activity by the exposure to 1,3-butadiene and formation of its metabolites. The initial increase in micronucleus frequency (micronuclei per 1000 cells) in the exposed mice continuously decreased from 20.4 +/- 5.1 (day 3) to 15.1 +/- 3.2 (day 28) within the exposure period, and subsequently from 12.4 +/- 5.1 to 4.6 +/- 1.6 in the period following termination of the 1,3-butadiene exposure, while micronucleus frequencies in control animals were significantly lower (from 1.7 +/- 1.5 to 4.2 +/- 0.8).     

Temporal, spatial and tissue-specific expression of a myogenin-lacZ transgene targeted to the Hprt locus in mice.

A lacZ transgene, expressed by the myogenin promoter, was introduced into the mouse hypoxanthine phosphoribosyltransferase (Hprt) locus by gene targeting in embryonic stem cells. Embryos between E10.5-E18.5 days were analyzed for expression of the transgene after staining for beta-galactosidase activity. Transgene expression was restricted to the skeletal muscle lineages reflecting a similar temporal and spatial pattern previously demonstrated for the endogenous myogenin gene. Additionally, a second transgene, MC1tk, showed expression in 87% of the clones when targeted to Hprt. This strategy, called targeted transgenesis, provides control for analyzing promoter sequences and for comparing various transgenes expressed by the same promoter.     

Mutations induced at the white and vermilion loci in Drosophila melanogaster

The white and vermilion loci in D. melanogaster were selected as target genes for the study of the mutational specificity of ionizing radiation and N-ethyl-N-nitrosourea (ENU) in a whole organism. Analysis of X-ray- and neutron-induced white mutants by a combination of genetic and molecular techniques showed that ionizing radiation induces primarily break-type mutations against a repair-proficient background, the majority of these alterations being deletions. Both very large multi-locus deficiencies and deletions of only a few base pairs were observed. These small deletions are flanked by repeats of 2-3 nucleotides, one copy of which is retained at the new junction. Presumably these small repeats are involved in the generation of the X-ray-induced deletions. In excision-repair-deficient mus201D1 flies, the frequency of whole-body white mutants recovered after X-ray irradiation is the same as in the wild-type strain. The percentage of mosaic mutations, however, is enhanced by a factor 3-4. Analysis by blot hybridization of ENU-induced white mutants strongly indicates that most mutations are due to base-pair changes. This was confirmed by sequence analysis of 25 ENU-induced vermilion mutants. In all mutants the alterations are due to base-pair changes, the majority being GC to AT transitions (61%).