Multiplex polymerase chain reaction-based analysis of T-cell receptor gamma gene rearrangements for the determination of T-lymphocyte clonality.

Determination of the frequency of mutations at hprt or other loci in human lymphocytes provides a useful biomarker for human exposure to mutagens. One problem, however, is distinguishing between unique mutants and sibling mutants arising as progeny of an earlier mutant cell. We have developed a multiplex polymerase chain reaction (PCR)-based method to analyze T-cell receptor (TCR) gamma gene rearrangements for determination of T-cell clonality in mutational spectrum analysis. PCR primers for different subgroups of the V gene segment of the TCR gamma gene were selected at different sites in the TCR gamma gene so that the size of PCR products could define which V subgroup was involved in rearranged TCR gamma genes; gamma genes involving different V and J subgroups could be determined directly by PCR. Mutant T-lymphocytes with rearranged TCR gamma genes containing the same V and J subgroups were analyzed using PCR-based denaturing polyacrylamide gel electrophoresis. All of the 161 hprt mutant clones analyzed contained rearranged TCR gamma genes. Rearrangements among all subgroups of the V and J gene segments of the TCR gamma gene could be detected. VgammaI and Jgamma1/2 subgroups were involved in 69 and 71% of rearranged TCR gamma genes, respectively. This PCR-based analysis of TCR gamma gene rearrangements provides a simple and comprehensive method for identifying the clonality of mutant T-lymphocytes in human hprt mutant lymphocyte assay and mutational spectrum analysis.

Antimutagenic effects of centchroman--a contraceptive and a candidate drug for breast cancer in multiple mutational assays.

Centchroman (CC), a non-steroidal oral contraceptive and a candidate drug for breast cancer, has been reported to exhibit partial to complete remission of lesions in 40.5% of breast cancer patients. The potent anti-oestrogenic activity, negligible side-effects and anti-breast cancer activity of CC prompted us to evaluate the antimutagenic effects of this compound in a bacterial mutagenicity assay and CHO/HPRT and AS52/GPT mutation assays in vitro and in vivo in female Swiss albino mice as measured by both sister chromatid exchange (SCE) and chromosome aberrations (CA) against three known positive mutagen compounds, dimethylbenz[a]anthracene (DMBA), cyclophosphamide (CP) and mitomycin C (MMC). Antimutagenicity assays in Salmonella strains TA97a, TA100, TA98 and TA102 were carried out against commonly used known positive mutagens, sodium azide, 4-nitro-o-phenylenediamine, cumine hydroperoxide, 2-aminofluorene and danthron. A significantly reduced number of bacterial histidine revertant colonies was observed in the plates treated with 0.1, 1, 5 and 10 microg/plate CC and a positive compound when compared with bacterial plates treated with the respective positive compound alone. Ethyl methanesulfonate (EMS), a commonly used positive mutagen for CHO/HPRT and AS52/GPT gene mutation assays, was used for antimutagenicity assay in these cells. CC exhibited protective effects against the mutagenicity of EMS in these two mammalian cell mutation assays, CHO/HPRT and AS52/GPT. In the in vivo studies, pretreatment with CC reduced DMBA-induced SCE and CA and CP- and MMC-induced CA when compared with the group treated only with the positive compounds. These results indicate that CC can reduce the mutagenic effects of known genotoxic compounds.

Co-mutagenicity of coumarin (1,2-benzopyrone) with aflatoxin B1 and human liver S9 in mammalian cells.

Coumarin (1,2-benzopyrone), a natural dietary constituent and drug currently under evaluation for treatment of certain cancers and lymphedema, reduces polycyclic aromatic hydrocarbon-induced neoplasms in rodents. Because most rodents metabolize coumarin through 3,4-epoxidation, whereas 7-hydroxylation predominates in humans, their suitability as a model for coumarin effects in humans has been questioned. We examined coumarin chemoprotection against the promutagen and dietary contaminant aflatoxin B1 with human liver S9 bioactivation in the Chinese hamster ovary cell/hypoxanthine-guanine phosphoribosyltransferase mutation assay. Coumarin in the absence of aflatoxin B1 was not mutagenic or cytotoxic up to 500 microM. When included with either 1 or 10 microM aflatoxin B1, coumarin produced a dose-dependent increase in mutant frequency and cytotoxicity. At concentrations greater than 50 microM, coumarin stimulated human liver S9 bioactivation of aflatoxin B1 to the mutagenic 8,9-epoxide. This increase was 12- and fivefold at 500 microM coumarin with 1 and 10 microM aflatoxin B1, respectively, compared with incubations with aflatoxin B1 alone. These findings differ from previous results with liver S9 from other species, and indicate that coumarin co-mutagenicity with aflatoxin B1 and human liver S9 is through increased aflatoxin B1 bioactivation.

PCR-directed DNA sequencing of "nondeletion" HPRT-mutants induced by bleomycin in CHO K1-BH4 cells.

Bleomycin is one of the radiomimetic antibiotics which induces DNA double-strand breaks by highly specific free radical attack on deoxyribose moieties in DNA. Earlier, we have shown that bleomycin induces a high proportion of large deletions involving one or more exons in the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus in a Chinese hamster ovary (CHO) cell line CHO K1-BH4, in which no spontaneously occurring large deletions were detected by a polymerase chain reaction (PCR)-based deletion screening assay. Here we report the molecular nature of another class of mutants in which we did not observe any abnormal exon pattern. We refer to these mutants as the "nondeletion" type. Since bleomycin is a reactive oxygen species (ROS)-generating agent, we also studied whether the change of intracellular levels of ROS may affect the bleomycin-induced mutation spectra. We therefore also investigated the hprt mutation spectra induced by bleomycin with pretreatment by TRIEN (triethylenetetramine), a superoxide dismutase (SOD) inhibitor, and TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl), a SOD mimic. Analysis of these three bleomycin-induced "nondeletion" mutation spectra revealed that 5'-GTC-3' or 5'-GCC-3' sequences were the hot spots for single basepair deletions. Other types of mutation include abnormal cDNA or no cDNA amplification on the hprt locus. Due to the small sample size, we are unable to draw a definitive conclusion about the effects of TRIEN and TEMPOL on bleomycin-induced spectrum of "nondeletion" type hprt mutations.

Coumarin chemoprotection against aflatoxin B1-induced gene mutation in a mammalian cell system: a species difference in mutagen activation and protection with chick embryo and rat liver S9.

Coumarin (1,2-benzopyrone), a natural food constituent, prevents polycyclic aromatic hydrocarbon-induced neoplasms in rats and mice, but has not been studied with other chemical carcinogens. We examined coumarin chemoprotection against aflatoxin B1 using the 6-thioguanine resistance mutation assay in two different Chinese hamster ovary cell lines (K1BH4 and AS52) with liver S9 from rats and 19-day-old chick embryos for aflatoxin B1 bioactivation. Laboratory rodents metabolize coumarin through 3-hydroxylation, whereas 7-hydroxylation predominates in chick embryos and humans. Chick embryo liver S9 was approximately 25-fold more effective in activating aflatoxin B1 to the mutagenic and cytotoxic metabolite(s) than rat liver S9. Coumarin added at 50 and 500 microM with chick embryo liver S9 reduced the mutant frequency of 1 microM aflatoxin B1 by 40 and 85%, respectively. Coumarin up to 500 microM had no effect on aflatoxin B1 mutagenicity with rat liver S9. When liver S9 from chick embryos pretreated with coumarin was used for aflatoxin B1 bioactivation, mutant frequency and cytotoxicity were decreased compared to liver S9 from vehicle-treated controls. Liver S9 from coumarin-treated rats did not significantly affect mutant frequency or cytotoxicity. HPLC analysis of chick embryo liver S9 incubated with 1 microM aflatoxin B1 showed a dose-dependent decrease by coumarin of aflatoxin B1 activation to the 8,9-epoxide ranging from 70% of controls at 5 microM coumarin to 4% of controls at 500 microM coumarin. In contrast, coumarin produced a dose-dependent increase in 20 microM aflatoxin B1 activation by rat liver S9, reaching twice the control levels at 500 microM coumarin. These findings, using a mammalian cell system as a mutagenic endpoint, demonstrate marked species differences in chemoprotection by coumarin.

Genetic and cytogenetic markers of exposure to high-linear energy transfer radiation.

It has been suggested that the more closely spaced, clustered DNA breaks seen with high-LET radiations are more likely to result in chromosome intrachanges than in chromosome interchanges. We determined whether analysis of the spectra of chromosome aberrations or Hprt gene mutations in CHO-K1 cells could detect a shift to more chromosome intrachanges and therefore distinguish exposure to high-LET radiation from exposure to low-LET radiation, and whether alterations in the processing of DNA breaks would influence this process. Both the frequency and type of chromosome aberrations and Hprt gene mutations were determined in CHO-K1 and xrs-5 cells exposed to 60Co gamma rays or 212Bi alpha particles. Xrs-5 cells are a radiosensitive derivative of CHO-K1 cells that are defective in rejoining of DNA double-strand breaks. The ratio of dicentrics to centric rings (F ratio) was significantly lower in CHO-K1 and xrs-5 cells exposed to alpha particles, consistent with a shift to more chromosome intrachanges with higher LET. In contrast, the frequency of large interstitial deletions at the Hprt locus, determined by multiplex polymerase chain reaction-based exon deletion analysis, was the same for both gamma-ray- and alpha-particle-exposed cells in each of the cell lines. Thus the F ratio can distinguish high-LET from low-LET radiations, and the end point is not influenced by differences in the processing of DNA double-strand breaks. The analysis of the spectrum of Hprt mutations, however, appears unable to discriminate low LET from high LET.

[Molecular analysis of spontaneous and arsenite-induced mutations at the gpt locus in Chinese hamster ovary cells].

In this study, we examined the mutagenicity of sodium arsenite at the xathine-guanine phosphoribosyl transferase locus (gpt) in a pSV2 gpt-transformed CHO cell line AS52. The chemical induced a dose-dependent increase of mutant frequency at the locus. Nested PCR analysis revealed that the majority of arsenite-induced AS52 mutants had totally deleted the gpt gene. For the spontaneous, 50 mumol/L arsenite--and 100 mumol/L arsenite--induced mutants in AS52 cells, the percentages of total deletion of the gpt gene were 36.00%, 54.72% and 66.67% respectively. The PCR products of gpt gene from nine 100 mumo/L sodium bisulfite-induced mutants were analyzed with direct DNA sequence technology. The frameshift mutation was identified in two mutants (2/9). No genomic alteration could be found in the structural gene examined in the other 7 mutants, where their molecular alterations probably located in the promoter region.

Polymerase chain reaction-based deletion analysis of spontaneous and arsenite-enhanced gpt mutants in CHO-AS52 cells.

In this study, we have examined the mutagenicity of sodium arsenite at the xanthine-guanine phosphoribosyltransferase locus (ypt) in a pSV2 gpt-transformed CHO cell line, AS52. Our results provide very weak evidence for arsenite as a gene mutagen because the chemical at high doses and at high cytotoxicity enhances barely a doubling of mutant frequency (MF) and a doubling of the gpt gene deletion frequency compared to controls. We suggest that the increase in MF in arsenite-treated cells results from arsenic, as comutagen, enhancing the induction effect of any unknown endogenous or exogenous factors on the spontaneous mutagenesis of AS52 cells. Nested PCR analysis mutants has a total deletion of the gpt gene. For the spontaneous, 50 microM arsenite- and 100 microM arsenite-enhanced spontaneous mutants in AS52 cells, the percentages of total deletion of the gpt gene are 36.00%, 54.72% and 66.67%, respectively. We suggest that a high proportion of the gene deletion in arsenite-enhanced mutants may be due to the high cytotoxicity of the chemical.

Molecular markers of ionizing radiation-induced gene mutations in mammalian cells.

We have isolated independent Chinese hamster ovary (CHO) cell mutants at the hypoxanthine guanine phosphoribosyltransferase (hprt) locus from untreated, 60Co gamma-ray-exposed, and 212Bi alpha-exposed cells and identified the molecular changes underlying the mutation determined by multiplex polymerase chain reaction (PCR)-based exon deletion analysis. Both the parental CHO-K1 cells and the X-ray-sensitive mutant xrs-5 cells were studied. The radiosensitive xrs-5 cells are defective in DNA double-strand break rejoining ability and in V(D)J recombination, which can be complemented by Ku protein. Of the 71 spontaneous CHO-K1 hprt mutants analyzed, 78% showed no change in exon number or size, 20% showed loss of one to eight exons (partial deletion), and 3% showed loss of all nine hprt exons (total deletion). Exposure of CHO-K1 cells to 6 Gy of gamma rays, which reduced survival levels to 10%, produced a high deletion spectrum with 45% of the 20 mutants analyzed showing a loss of one to eight exons and 30% showing total deletion. Exposure to an equitoxic dose of alpha radiation from 212Bi, a 220Rn daughter, resulted in a spectrum similar to the gamma-ray spectrum in that 75% of the 49 mutants analyzed were deletions. To alpha radiation, however, tended to produce larger intragenic deletions than gamma radiation. Of the 92 spontaneous xrs-5 mutants analyzed for deletions, 43% showed a loss of one to eight exons and 14% showed total deletion. This suggests that, in certain regions of the hprt gene, base alterations can be converted into large deletions and alteration in the Ku protein complex can influence this type of mutational process. Exposure to alpha radiation (10% survival) to xrs-5 cells resulted in a deletion spectrum similar to that seen in CHO-K1 cells. Of the 49 mutants analyzed, 43% showed on change in exon number or size, 16% showed a loss of one to eight exons, and 41% showed total deletion. While the defect in xrs-5 cells has a profound effect on spontaneous mutant spectra, this defect does not appear to affect alpha-induced mutation spectra.

The molecular nature of spontaneous mutations at the hprt locus in the radiosensitive CHO mutant xrs-5.

The radiosensitive mutant xrs-5, a derivative of the Chinese hamster ovary (CHO) K1 cell, is defective in DNA double-strand break rejoining ability and in V(D)J recombination. The radiosensitivity and defective repair phenotype are complemented by the 80-kDa subunit of the Ku protein. We determined the nature of the mutations that develop spontaneously at the hprt locus in this cell line using both multiplex PCR deletion screening and DNA sequencing. Ninety-two independent spontaneous mutants were analyzed and the results were compared to the mutation spectrum of 64 previously analyzed hprt spontaneous mutants isolated from the parental CHO-K1 cell line. More than 50% of the spontaneous xrs-5 mutants had lost one or more exons while less than 25% of spontaneous CHO-K1 mutants had lost one or more exons. Most of the deletions in xrs-5 cells involved the loss of multiple exons while single exon deletions predominated in CHO-K1. There was also a nonrandom distribution of breakpoints in both CHO-K1 and xrs-5. Most of the deletion breakpoints were 3' to exon 9, around exons 4-6, or near exon 1. Although the frequency of base substitutions was lower in xrs-5, the spectrum of base substitutions was qualitatively similar to that of CHO-K1. There was no significant difference in the spontaneous mutant frequency in xrs-5 and CHO-K1. The results suggest that in certain regions of the hprt gene, base alterations can be converted to large deletions, and that alterations in the Ku protein complex can influence this process.

Molecular nature of spontaneous mutations at the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus in Chinese hamster ovary cells.

The hypoxanthine-guanine phosphoribosyltransferase (hprt) locus has been widely used as a selectable genetic marker for studies of mammalian cell mutagenesis. We report here the spontaneous mutation spectrum at the hprt locus in 64 independently isolated mutants of Chinese hamster ovary (CHO) cells. All nine hprt exons were simultaneously analyzed via multiplex polymerase chain reaction (PCR) for rapid detection of gene deletions or insertions. Structural point mutations were identified by direct sequence analysis of the PCR amplified cDNA. The molecular nature of RNA splicing errors and insertions was analyzed by solid-phase direct exon sequencing. Single base substitutions were found in 24 mutants (38%), of which 21 were missense and 3 were nonsense mutations. Transversions were about twice as frequent as transitions. Fifteen mutants (23%) had deletions involving either intragenic small fragments (2), single exons (9), or multiple exons (4). The majority of deletion breakpoints (71%) were located in regions surrounding exons 4, 5, and 6. RNA splicing mutations were observed in 15 mutants (23%) and affected exons 3-8; most (6/15) resulted in the loss of exon 7. Two insertion mutants, one with a 209 bp insert in exon 4 and the other with a 88 bp insert accompanied by a 24 bp deletion in exon 6, represent novel mutations reported for the first time in spontaneous mutants of the mammalian hprt gene.

Multiplex polymerase chain reaction-based deletion analysis of spontaneous, gamma ray- and alpha-induced hprt mutants of CHO-K1 cells.

Independent Chinese hamster ovary (CHO)-K1 cell mutants at the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus were isolated from untreated, 60Co gamma ray- and 212Bi alpha-exposed cells and the genetic changes underlying the mutation determined by multiplex polymerase chain reaction (PCR)-based exon deletion analysis. In the 71 spontaneous mutants analyzed, 77.5% of the clones showed no change in exon number or size, 15.5% showed a loss of a single exon, 4.2% showed a loss of 2-8 exons, and 2.8% showed loss of all nine hprt exons (total gene deletion). Exposure to 6 Gy of gamma rays, which reduced survival levels to 10%, produced a significantly different deletion spectrum that was shifted toward deletions with 45% of the 20 mutants analyzed showing a loss of a single exon and 30% showing a loss of all nine exons. Exposure to 2 Gy alpha radiation from 212Bi, a 220Rn daughter, a dose which also reduced survival levels to about 10%, resulted in a deletion spectrum similar to the gamma-ray spectrum in that more than 75% of the 49 mutants analyzed were deletions. The alpha spectrum, however, was significantly different from both the spontaneous and gamma spectra with 55.1% of the alpha mutants showing a loss of all nine exons, 10.2% showing loss of a single exon, and 14.3% showing loss of 2-8 exons. Thus, alpha-radiation appears to produce larger intragenic deletions than gamma radiation. The results suggest that intragenic deletion size should be considered when low- and high linear energy transfer (LET) mutation spectra are compared.

Adriamycin induces large deletions as a major type of mutation in CHO cells.

Adriamycin (ADR), a commonly used cancer chemotherapy antibiotic, exhibits a variety of genotoxicities. In this study, we have examined the mutagenicity of ADR at the hypoxanthine-guanine phosphoribosyltransferase gene (hprt) in Chinese hamster ovary (CHO) cells and the xanthine-guanine phosphoribosyltransferase locus (gpt) in a pSV2gpt-transformed CHO cell line, AS52. Although ADR induced a dose-dependent increase of mutant frequency at both loci, it was more mutagenic to the gpt gene than to the hprt locus. Multiplex PCR analysis revealed that 35% of the 103 independent ADR-induced HPRT-deficient mutants carried large deletions. Among these deletion mutants, 33% were total gene deletions, 22% affected multiple exons, and 42% involved a single exon, of which most (9/15) were exon 1. The majority (63%) of ADR-induced AS52 mutants had a total deletion of the gpt gene. These observations indicate that ADR induces large deletions as a major type of gene mutation in mammalian cells, suggesting the involvement of reactive oxygen species as one mutagenic pathway in the mutagenesis of ADR.

Modulative effects of metabolic effectors on benzo(a)pyrene-induced cytotoxicity and mutagenicity in mammalian cells.

Conjugation and detoxification of mixed function oxidase (MFO)-mediated benzo(a)pyrene [B(a)P] metabolites with glucuronic acid and glutathione (GSH) are major pathways of B(a)P elimination and ultimately excretion in vivo. We have studied the effects of uridine diphosphate alpha-D-glucuronic acid (UDPGA) and GSH, a cofactor for the synthesis of glucuronide and GSH conjugates, respectively, on B(a)P-induced cytotoxicity and mutagenicity in mammalian cells. The S9-mix used in the Chinese hamster ovary cell/hypoxanthine-guanine phosphoribosyltransferase (CHO/HPRT) mutational assay was supplemented with either UDPGA, GSH, or GSH plus purified GSH-S-transferases (GSHTs), to study modulation of glucuronide and GSH detoxification mechanisms on B(a)P-induced cytotoxic and mutagenic effects. We found that the addition of UDPGA to S9-mix reduces cytotoxicity induced by either B(a)P or B(a)P 6-OH but not by B(a)P 7,8-diol [B(a)P-diol]. The reduction of B(a)P and B(a)P 6-OH-induced cytotoxicity by glucuronide conjugation is likely due to elimination of cytotoxic phenols and quinones. The addition of GSH to the S9-mix resulted in a reduction of B(a)P- and B(a)P-diol-induced cytotoxicity. GSH plus GSHT reduced B(a)P-induced cytotoxicity and mutagenicity. GSH inhibited the mutagenicity at low concentrations of B(a)P-diol. GSH plus GSHTs inhibited the cytotoxicity and mutagenicity of B(a)P-diol at concentrations not affected by GSH alone. These studies demonstrate that mechanisms of detoxification can affect the biological activity of B(a)P and B(a)P-diol as profoundly as bioactivation by the MFO system. Future research should address studies of mutagenicity modulation by metabolic effectors at both the molecular (DNA sequence) and cellular (quantitative mutagenesis) level.

Polymerase chain reaction-directed DNA sequencing of bleomycin-induced "nondeletion"-type, 6-thioguanine-resistant mutants in Chinese hamster ovary cell derivative AS52: effects of an inhibitor and a mimic of superoxide dismutase.

Bleomycin-induced, 6-thioguanine-resistant, "non deletion" mutants pretreated with or without either TRIEN (triethylenetetramine), a superoxide dismutase (SOD) inhibitor, or TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl), a SOD mimic, were analyzed by polymerase chain reaction (PCR)-directed DNA sequencing in a Chinese hamster ovary (CHO) cell derivative, AS52. Among the 23 bleomycin-induced mutants, six have 3-bp 5'-TGA-3' deletions in the region of 366-371, five have single-base deletions, seven have base substitutions, three have insertions, and two have possible translocations. Among the 16 bleomycin-induced mutants pretreated with TRIEN, six have the 5'-TGA-3' deletion (366-371), two have single-base deletions, one has a 13-bp deletion, four have single-base substitutions, one has a double-base substitution, and two have insertions. Among the 17 bleomycin-induced mutants pretreated with TEMPOL, six have the same TGA deletions, two have single-base deletions, two have single-base insertions, four have single-base substitutions, one mutant has a 12-bp deletion, one has a 13-bp deletion, and one mutant shows no detectable change in its coding region in the DNA sequence. A possible shift from a ROS-mediated mutational spectrum to a spontaneous mutational spectrum by TRIEN further indicates that reactive oxygen species play an important role in bleomycin mutagenesis in mammalian cells.

Polymerase chain reaction-based deletion screening of bleomycin induced 6-thioguanine-resistant mutants in Chinese hamster ovary cells: the effects of an inhibitor and a mimic of superoxide dismutase.

Bleomycin-induced 6-thioguanine-resistant mutants pretreated with or without TRIEN (triethylenetetramine), a superoxide dismutase (SOD) inhibitor, or TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl), an SOD mimic, were analyzed by polymerase chain reaction (PCR)-based deletion screening in a Chinese hamster ovary (CHO) clone K1-BH4 and its derivative AS52 cells. As we proposed earlier, TRIEN would decrease and TEMPOL would increase the intracellular level of hydroxyl radical leading to a higher and lower recovery of deletion mutants. We found that the proportion of the deletion mutants induced by bleomycin at the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus in K1-BH4 cells was 45.5% (25/55). The proportion of deletion HPRT- mutants induced by bleomycin pretreated with TRIEN was 31.0% (9/29) and with TEMPOL was 50.0% (14/28). The proportion of deletion mutants induced by bleomycin on the xanthine-guanine phosphoribosyltransferase (gpt) gene in AS52 cells was 61.0% (36/59). The proportion of deletion GPT- mutants induced by bleomycin pretreated with TRIEN was 56.8% (21/37) and with TEMPOL was 61.4% (27/44). The trend of the change of the proportion of bleomycin-induced deletion mutants as affected by TRIEN and by TEMPOL provides molecular evidence for the involvement of reactive oxygen species (ROS) in bleomycin mutagenesis in mammalian cells, in which deletion is a major type of induced mutation.

Quantitative and molecular analyses of genetic risk: a study with ionizing radiation.

Mammalian cells in culture have been used to study the genetic effects of physical and chemical agents. We have used Chinese hamster ovary (CHO) cells, clone K1-BH4, to quantify mutations at the X-linked, large (35 kb) hypoxanthine-guanine phosphoribosyltransferase (hprt) locus (the CHO/HPRT assay) induced by environmental agents. By transfecting an hprt-deletion mutant CHO cell line with the plasmid vector pSV2gpt, we isolated a transformant, AS52. AS52 cells carry a single functional copy of an autosomal, small (456 bp) xanthine-guanine phosphoribosyltransferase (gpt) gene (the bacterial equivalent of the mammalian hprt gene; AS52/GPT assay). We found that ionizing radiations such as X-rays and neutrons and oxidative genotoxic chemicals such as Adriamycin, bleomycin, hydrogen peroxide, and potassium superoxide are much more mutagenic to the gpt gene in AS52 cells than to the hprt locus in K1-BH4 cells. The hypermutability of the gpt gene probably results from a higher recovery of multilocus deletion mutants in AS52 cells than in K1-BH4 cells, rather than a higher yield of induced mutants. These results demonstrate that the use of the hprt locus alone could lead to an underestimate of the genetic risk of these agents. Analyses of the mutation spectrum using a polymerase chain reaction-based deletion screening and DNA sequencing procedure showed that a high proportion of HPRT- and GPT- mutants induced by X-rays carry deletion mutations. Thus, both the mutant frequency and mutation spectrum need to be considered in assessing the genetic risk of ionizing radiation and oxidative genotoxic chemicals.

Multiplex DNA amplification and solid-phase direct sequencing for mutation analysis at the hprt locus in Chinese hamster cells.

We report here the development of multiplex in vitro DNA amplification and solid-phase direct exon sequencing for the analysis of mutations at the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus in Chinese hamster cells. 18 representative HPRT-deficient mutants, derived either spontaneously, or after exposure to UV light or ionizing radiation, were analyzed. All 9 hprt exons were simultaneously amplified via the polymerase chain reaction (PCR) for rapid deletion detection. 5 mutants involve single- or multiple-exon deletions. Altered multiplex PCR patterns were detected in mutants Bsp-040, Bsp-065 and BGR-606. Subsequent direct sequence analysis reveals that Bsp-040 and Bsp-065 carry a 52-bp and a 13-bp intragenic DNA deletion in exon 3, respectively. BGR-606 contains a 223-bp insertion accompanied by a 10-bp deletion of intron sequence within exon 4 fragment. Other subtle DNA alterations identified by direct exon sequence analysis include single-base substitutions, small deletions and insertions, and RNA splicing mutations.

Mutagenicity and cytotoxicity of 2-methoxyethanol and its metabolites in Chinese hamster cells (the CHO/HPRT and AS52/GPT assays).

2-Methoxyethanol (ethylene glycol monomethyl ether) (EGME), is one of the most commonly used solvents for industrial and consumer products. Although the solvent has been shown to be a reproductive toxin the genotoxic activities of EGME especially its metabolites, have not been adequately investigated. The mutagenicity and cytotoxicity of EGME and its major metabolites, methoxyacetaldehyde (MALD) and methoxyacetic acid (MAA) in Chinese hamster ovary (CHO) cells were therefore examined by us. We have determined the mutagenicity of these compounds at the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus in CHO-K1-BH4 cells (CHO/HPRT assay) and the xanthine-guanine phosphoribosyl transferase (gpt) locus in CHO AS52 cells (AS52/GPT assay). The results show that these chemicals are not mutagenic to the hprt locus in CHO-K1-BH4 cells either with or without rat liver S9 mix as the metabolic activating system. With AS52 cells, only MALD is mutagenic in the absence of S9. It induced a dose-dependent mutagenic response. A dose-dependent cytotoxicity was induced by all compounds in both cell lines. MALD is the most and EGME is the least cytotoxic compounds. Our study shows that a metabolite of EGME, MALD, is highly cytotoxic and likely induces deletion-type mutations in AS52 cells. The genotoxic effect of EGME is, therefore, dependent upon its metabolism and its detection is dependent upon the assays used.

Effects of an inhibitor and a mimic of superoxide dismutase on bleomycin mutagenesis in Chinese hamster ovary cells.

We have investigated the roles of reactive oxygen species (ROS) in bleomycin (BLM)-induced gene mutations in Chinese hamster ovary (CHO) cells using a superoxide dismutase (SOD) inhibitor, triethylenetetramine (TRIEN), and a SOD mimic, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEM-POL), to lower and increase intracellular 'SOD activity', respectively. Pretreatment of CHO cells with TRIEN (1 mM) for 1 h enhanced the mutagenic response of BLM (5-50 micrograms/ml, 1 h treatment) in the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus in CHO cell clone K1-BH4 (CHO/HPRT assay) and the xanthine-guanine phosphoribosyltransferase (gpt) gene in a CHO-K1 cell derivative AS52 (AS52/GPT assay). Pretreatment with TEMPOL (1 mM) for 1 h decreased the BLM (20-100 micrograms/ml, 1 h treatment) mutagenicity in the AS52/GPT assay. The mutagenic response of BLM appears to be modulated by the intracellular level of 'SOD activity' and hence the intracellular level of ROS. These data provide further evidence for the involvement of ROS in bleomycin mutagenesis in mammalian cells.