The Organic Selenium Compound Selenomethionine Modulates Bleomycin-Induced DNA Damage and Repair in Human Leukocytes

The objective of this work was to evaluate the effects of selenomethionine (SeMet) on the induction, repair, and persistence of DNA damage in human leukocytes challenged with bleomycin (BLM). Comet assay was used to determine DNA strand breaks and hOGG1 for the specific recognition of oxidative damage. Leukocytes were (A) stimulated with phytohemagglutinin, (B) damaged with BLM, and (C) incubated to allow DNA repair. Comet assay was performed after each phase. SeMet (50 μM) was supplemented either during phase A, B, or C, or AB, or ABC. Treatment with SeMet decreased BLM-induced stand breaks when added during phase AB. Results obtained after the repair period indicate that SeMet favors repair of DNA damage especially when applied during phase AB. The comparison between DNA damage before and after repair showed that BLM-induced damage was repaired better in the presence of SeMet. Our results showed antigenotoxic effect of SeMet on BLM-induced DNA and also on repair and persistence of this damage when applied before and simultaneously with BLM.     

Molecular characterization of methyl methanesulphonate (MMS)-induced HPRT mutations in V79 cells

Spontaneous and methyl methanesulphonate-induced HPRT-deficient mutants were analysed for changes in the hprt gene structure using multiplex polymerase chain reaction. The PCR amplification pattern of 21 MMS-induced mutations revealed one total deletion of the hprt coding exons and one small deletion within exon 5, while 19 mutants showed the V79 wild-type pattern. Molecular analysis of 30 spontaneous mutations revealed no mutants with amplification patterns which differed from those of wild-type cells. We further analysed MMS-induced mutants in a different V79 cell line with a high (40%) spontaneous deletion frequency. MMS caused a dose-dependent increase in the mutant frequency but the incidence of deletions was reduced to 6% at 2 × 10⁻⁴ M and to 13% at 5 × 10⁻⁴ M indicating that mainly point mutations were induced. The repair inhibitor cytosine arabinoside (araC) enhanced mutation induction by MMS but did not change the proportion of deletions in the mutation spectrum. The results indicate that different V79 cell lines spontaneously produce different amounts of deletion mutations. The frequency of MMS-induced deletions does not depend on the frequency of spontaneous deletions in a given cell line. The MMS-induced mutation spectrum seems to be unchanged even at high concentrations with a strong cytotoxic effect. Deletions are not increased as a consequence of araC-inhibited repair of MMS-inducd lesions.     

Large deletions are tolerated at the hprt locus of in vivo derived human T-lymphocytes.

A cloning assay was used to recover hprt- T-lymphocytes from adult human males. Analysis of crude cellular extracts by polymerase chain reactions (PCRs) demonstrated that 7% (16/218) of the hprt mutations were due to total deletion of the hprt gene. 14 of the 16 mutants were examined by PCR for the presence of flanking DNA to determine the extent of the deletions. The deletion mutation in 13 mutants was at least 350 kb with 5 of these deletions being at least 700 kb. The largest deletions were greater than 15 times the size of the hprt gene. Therefore, large deletions are tolerated at the hprt locus of human T-lymphocytes.     

The nature of mutants induced by ionising radiation in cultured hamster cells. III. Molecular characterization of HPRT-deficient mutants induced by gamma-rays or alpha-particles showing that the majority have deletions of all or part of the hprt gene

DNA from 58 independent HPRT-deficient mutants of V79 hamster cells induced by ionising radiation was analysed by Southern blot hybridization to a full-length hamster hprt cDNA. About half of the gamma-ray-induced mutants (20/43) were apparently total gene deletions, because they lacked all functional hprt gene sequences hybridizing to the cDNA probe. Another 10 mutants showed various partial deletions and/or rearrangements of the hprt gene. The remaining 13 mutants showed no detectable change in comparison to the structure of the normal gene, which correlated well with previous characterization of these mutants indicating that most carry point mutations in the hprt gene. However, it is probable that some of these point mutations occurred spontaneously rather than being radiation-induced. A smaller number of alpha-particle induced mutants gave similar results: out of a total of 15 mutants, 6 appeared to be total gene deletions, 5 had partial deletions and/or rearrangements, and 4 had no detectable changes. Thus, 70% or more of radiation-induced HPRT-deficient mutants arise through large genetic changes, especially deletions of all or part of the hprt gene. This result is to be contrasted with data published previously by ourselves and others indicating that the majority of spontaneous and ethyl methanesulphonate-induced mutations of hprt and similar genes arise by point mutation.     

DNA strand specificity for UV-induced mutations in mammalian cells.

The influence of DNA repair on the molecular nature of mutations induced by UV light (254 nm) was investigated in UV-induced hprt mutants from UV-sensitive Chinese hamster cells (V-H1) and the parental line (V79). The nature of point mutations in hprt exon sequences was determined for 19 hprt mutants of V79 and for 17 hprt mutants of V-H1 cells by sequence analysis of in vitro-amplified hprt cDNA. The mutation spectrum in V79 cells consisted of single- and tandem double-base pair changes, while in V-H1 cells three frameshift mutations were also detected. All base pair changes in V-H1 mutants were due to GC----AT transitions. In contrast, in V79 all possible classes of base pair changes except the GC----CG transversion were present. In this group, 70% of the mutations were transversions. Since all mutations except one did occur at dipyrimidine sites, the assumption was made that they were caused by UV-induced photoproducts at these sites. In V79 cells, 11 out of 17 base pair changes were caused by photoproducts in the nontranscribed strand of the hprt gene. However, in V-H1 cells, which are completely deficient in the removal of pyrimidine dimers from the hprt gene and which show a UV-induced mutation frequency enhanced seven times, 10 out of 11 base pair changes were caused by photoproducts in the transcribed strand of the hprt gene. We hypothesize that this extreme strand specificity in V-H1 cells is due to differences in fidelity of DNA replication of the leading and the lagging strand. Furthermore, we propose that in normal V79 cells two processes determine the strand specificity of UV-induced mutations in the hprt gene, namely preferential repair of the transcribed strand of the hprt gene and a higher fidelity of DNA replication of the nontranscribed strand compared with the transcribed strand.     

Strand-specific mutation spectra in repair-proficient and repair-deficient hamster cells

The mutation spectrum induced by UV light has been determined at the hprt locus for both cultured normal (AA8) and UV-sensitive (UV-5) Chinese hamster ovary cells to investigate the effect of DNA repair on the nature of induced mutations. DNA base-pair changes of 23 hprt mutants of AA8 and of 28 hprt mutants of UV-5 were determined by sequence analysis of in vitro amplified hprt cDNA. Almost all mutants in AA8 carried single-base substitutions, transitions and transversions accounting for 38% and 62% of the base changes, respectively. In contrast, in repair-deficient cells (UV-5) tandem and nontandem double mutations represented a considerable portion of the mutations observed (30%), whereas the vast majority of base-pair substitutions were GC greater than AT transitions (87%). Moreover, 5 splice mutants and 2 frameshift mutations were found in the UV-5 collection. In almost all mutants analyzed base changes were located at dipyrimidine sites where UV photoproducts could have been formed. In AA8 the photolesions causing mutations were predominantly located in the nontranscribed strand whereas a strong bias for mutation induction towards photolesions in the transcribed strand was found in UV-5. We hypothesize that preferential removal of lesions from the transcribed strand of the hprt gene accounts for the observed DNA strand specificity of mutations in repair-proficient cells. Furthermore, differences in the degree of misincorporation opposite a lesion for lagging and leading strand DNA synthesis may dictate the pattern of UV-induced mutations in the absence of DNA repair.     

Molecular analysis of spontaneous and ethyl methanesulphonate-induced mutations of the hprt gene in hamster cells

Independent spontaneous or ethyl methanesulphonate (EMS)-induced mutants lacking HPRT enzyme activity were analysed for changes in hprt gene structure. Of 21 spontaneous mutants, 6 had total gene deletions, 2 had partial gene deletions, and 13 were indistinguishable from wild-type by Southern analysis. In contrast a sample of 23 EMS-induced mutants, each of which showed potentially interesting characteristics (e.g. high reversion frequency, X-chromosome rearrangement), showed no detectable hprt gene changes. RNA isolated from 59 mutants with presumptive point mutations (13 spontaneous, 46 EMS-induced) was analysed on dot blots for changes in the amount of hprt mRNA. A wide range of mRNA levels was found, from mutants with undetectable amounts to those with more than wild-type amounts. However, Northern blots of all these mutant RNAs revealed only one (EMS-induced) mutation with a change in hprt mRNA size. Taken with our previously-published data on these mutants, it is argued that they represent a broad range of mutational types, and that the hprt gene mutation system provides a sensitive means of distinguishing mutational spectra of different DNA-damaging agents.     

Alteration of cadmium-induced mutational spectrum by catalase depletion in Chinese hamster ovary-K1 cells.

Previously, we have demonstrated that cadmium acetate significantly induces hprt mutation frequency in Chinese hamster ovary (CHO)-K1 and that 3-amino-1,2,4-triazole (3AT), a catalase inhibitor, potentiates the mutagenicity of cadmium [Chem. Res. Toxicol. 9 (1996) 1360-1367]. In this study, we investigate the role of intracellular peroxide in the molecular nature of mutations induced by cadmium. Using 2',7'-dichlorofluorescin diacetate and fluorescence spectrophotometry, we have shown that cadmium dose-dependently increased the amounts of intracellular peroxide and the levels were significantly enhanced by 3AT. Furthermore, we have characterized and compared the hprt mutation spectra in 6-thioguanine-resistant mutants derived from CHO-K1 cells exposed to 4 microM of cadmium acetate for 4h in the absence and presence of 3AT. The mutation frequency induced by cadmium and cadmium plus 3AT was 11- and 16-fold higher than that observed in untreated populations (2.2 x 10(-6)), respectively. A total of 40 and 51 independent hprt mutants were isolated from cadmium and cadmium plus 3AT treatments for mRNA-polymerase chain reaction (PCR), genomic DNA-PCR and DNA sequencing analyses. 3AT co-administration significantly enhanced the frequency of deletions induced by cadmium. Cadmium induced more transversions than transitions. In contrast, 3AT co-administration increased the frequency of GC-->AT transitions and decreased the frequencies of TA-->AT and TA-->GC transversions. Together, the results suggest that intracellular catalase is important to prevent the formation of oxidative DNA damage as well as deletions and GC-->AT transitions upon cadmium exposure.     

Molecular analysis of mitochondrial DNA mutations from bleomycin-treated rats

In our previous studies, we have shown the mutagenicity of bleomycin (BLM) at the nuclear hprt locus. In the present study we have analyzed mutagenic effects of BLM in mitochondrial DNA (mtDNA) using short extension-PCR (SE-PCR) method for detection of low-copy deletions. Fisher 344 rats were treated with a single dose of BLM and total DNA preparations from splenic lymphocytes were processed in SE-PCR assay. Spontaneous deletions were typically flanked by direct repeats (78.5%), while the in BLM-treated group, direct repeats were found in only 46.6% of breakpoints. The ratio between deletions based on direct repeats and random sequence deletions changed from 3.67 in control group to 0.87 in BLM-treated animals, which corresponds to an approximate 1.7-fold increase in the deletion mutation frequency. Furthermore, 62.5% of deletions not flanked by direct repeats in the treated group contained cleavage sites for BLM. The localization of breakpoints was not entirely random. We have found four clusters containing deletions from both groups indicative of deletion hot spots. The results indicate that BLM exposure may be associated with the induction of mtDNA mutations, and suggest the utility of SE-PCR method for evaluating drug-induced genotoxicity.     

Strand specificity for UV-induced DNA repair and mutations in the Chinese hamster HPRT gene.

DNA excision repair modulates the mutagenic effect of many genotoxic agents. The recently observed strand specificity for removal of UV-induced cyclobutane dimers from actively transcribed genes in mammalian cells could influence the nature and distribution of mutations in a particular gene. To investigate this, we have analyzed UV-induced DNA repair and mutagenesis in the same gene, i.e. the hypoxanthine phosphoribosyl-transferase (hprt) gene. In 23 hprt mutants from V79 Chinese hamster cells induced by 2 J/m2 UV we found a strong strand bias for mutation induction: assuming that pre-mutagenic lesions occur at dipyrimidine sequences, 85% of the mutations could be attributed to lesions in the nontranscribed strand. Analysis of DNA repair in the hprt gene revealed that more than 90% of the cyclobutane dimers were removed from the transcribed strand within 8 hours after irradiation with 10 J/m2 UV, whereas virtually no dimer removal could be detected from the nontranscribed strand even up to 24 hr after UV. These data present the first proof that strand specific repair of DNA lesions in an expressed mammalian gene is associated with a strand specificity for mutation induction.     

The aprt heterozygote/hemizygote system for screening mutagenic agents allows detection of large deletions

Frequencies of mutation at the hprt and aprt loci in various CHO cell lines were measured after exposure of the cells to ionizing radiation. In D423 and AA8-16, which are aprt+/- heterozygotes, the ratio of hprt- mutants to aprt- mutants ranged from 0.11 to 0.36. In D422 and AA8-5, which are aprt+/0 cell lines in which only one copy of the gene and its flanking sequences is present these ratios were greater than 5. In contrast, chemical mutagenesis generated mutations at both loci, in all cell lines, at equal frequencies. Southern blot analysis of DNA from hprt- and aprt- mutants of one of the aprt+/- heterozygous lines showed some apparently unaltered genes, some rearrangements and some complete deletions of hprt among hprt- mutants, but only complete deletions of aprt-linked sequences among aprt- mutants. These results strongly suggest that X-ray-induced mutational events are frequently larger than 40 kb (the length of the hprt gene) and that the difference among the frequencies observed at the two loci in the two types of cell lines were due to the presence of essential sequences close the respective target genes. The combined use of these cell lines in screening environmental mutagens should allow qualitative as well as quantitative analysis of the mutagenic potential of environmental agents.     

Molecular characterisation of camptothecin-induced mutations at the hprt locus in Chinese hamster cells

The capacity of the topoisomerase I inhibitor camptothecin (CPT) to induce single locus mutations at the hypoxanthine-guanine phosphoribosyltransferase (hprt) gene and the DNA changes underlying induced mutations were analysed in Chinese hamster ovary cells. Camptothecin treatments increased hprt mutations up to 50-fold over the spontaneous levels at highly cytotoxic doses. Genomic DNA was isolated from 6-thioguanine resistant clones and subjected to multiplex PCR to screen for gross alterations in the gene structure. The molecular analysis revealed that deletion mutants represented 80% of the analysed clones, including total hprt deletion, multiple and single exon deletions. Furthermore, a fraction of the analysed clones showed deletions of more than one exon that were characterised by the absence of non-contiguous exons. These data show that single locus mutations induced by camptothecin are characterised by large deletions or complex rearrangements rather than single base substitutions and suggest that the recombinational repair of camptothecin-induced strand breaks at replication fork may be involved in the generations of these alterations at the chromatin structure level.     

The effect of dose rate on X-radiation-induced mutant frequency and the nature of DNA lesions in mouse lymphoma L5178Y cells

The induction of mutants at the heterozygous tk locus by X radiation was found to be dose-rate dependent in L5178Y-R16 (LY-R16) cells, but very little dose-rate dependence was observed in the case of strain L5178Y-S1 (LY-S1), which is deficient in the repair of DNA double-strand breaks. Induction of mutants by X radiation at the hemizygous hprt locus was dose-rate independent for both strains. These results are in agreement with the hypothesis that the majority of X-radiation-induced TK-/- mutants harbor multilocus deletions caused by the interaction of damaged DNA sites. Repair of DNA lesions during low-dose-rate X irradiation would be expected to reduce the probability of lesion interaction. The results suggest that in contrast to the TK-/- mutants, the majority of mutations at the hprt locus in these strains of L5178Y cells are caused by single lesions subject to dose-rate-independent repair. The vast majority of the TK-/- mutants of strain LY-R16 showed loss of the entire active tk allele, whether the mutants arose spontaneously or were induced by high-dose-rate or low-dose-rate X irradiation. The proportion of TK-/- mutants with multilocus deletions (in which the products of both the tk gene and the closely linked gk gene were inactivated) was higher in the repair-deficient strain LY-S1 than in strain LY-R16. However, even though the mutant frequency decreased with dose rate, the proportion of mutants showing inactivation of both the tk and gk genes increased with a decrease in dose rate. The reason for these apparently conflicting results concerning the effect of DNA repair on the induction of extended lesions is under investigation.     

Herpes simplex virus type 2 mutagenesis: characterization of mutants induced at the hprt locus of nonpermissive XC cells.

In a previous report, herpes simplex virus type 2 (HSV-2) was shown to increase the frequency of mutation at the hypoxanthine phosphoribosyltransferase (hprt) locus of nonpermissive rat XC cells (L. Pilon, A. Royal, and Y. Langelier, J. Gen. Virol. 66:259-265, 1985). A series of 17 independent mutants were isolated after viral infection together with 12 spontaneous noninfected mutants to characterize the nature of the mutations induced by the virus at the molecular level. The DNA of the mutants isolated after viral infection was probed with cloned HSV-2 fragments representing the entire genome. In these mutants, no authentic HSV-2 hybridization could be detected. This was indicative of a mechanism of mutagenesis which did not require the permanent integration of viral sequences in the host genome. The structure of the hprt gene was determined by the method of Southern (J. Mol. Biol. 98:503-517, 1975), and the level of hprt mRNA was analyzed by Northern blots. Except for the identification of one deletion mutant in each of the two groups, the HPRT- clones showed no evidence of alteration in their hprt gene. A total of 7 of 12 spontaneous mutants and 11 of 15 mutants isolated from the infected population transcribed an hprt mRNA of the same size and abundance as did the wild-type cells. Thus, the majority of the mutants seemed to have a point mutation in their hprt structural gene. Interestingly, the proportion of the different types of mutations was similar in the two groups of mutants. This analysis revealed that HSV-2 infection did not increase the frequency of rearrangements but rather that it probably induced a general increase of the level of mutations in the cells. This type of response is thought to be compatible with the biology of the virus, and the possible mechanisms by which HSV-2 induces somatic mutations in mammalian cells are discussed.     

The effect of folate deficiency on the hprt mutational spectrum in Chinese hamster ovary cells treated with monofunctional alkylating agents.

Folic acid deficiency acts synergistically with alkylating agents to increase DNA strand breaks and mutant frequency at the hprt locus in Chinese hamster ovary (CHO) cells. To elucidate the mechanism of this synergy, molecular analyses of hprt mutants were performed. Recently, our laboratory showed that folate deficiency increased the percentage of clones with intragenic deletions after exposure to ethyl methanesulfonate (EMS) but not N-nitroso-N-ethylurea (ENU) compared to clones recovered from folate replete medium. This report describes molecular analyses of the 37 hprt mutant clones obtained that did not contain deletions. Folate deficient cells treated with EMS had a high frequency of G>A transitions at non-CpG sites on the non-transcribed strand, particularly when these bases were flanked on both sides by G:C base pairs. Thirty-three percent of these mutations were in the run of six G's in exon 3. EMS-treated folate replete cells had a slightly (but not significantly) lower percentage of G>A transitions, and the same sequence specificity. Treatment of folate deficient CHO cells with ENU resulted in predominantly T>A transversions and C>T transitions relative to the non-transcribed strand. These findings suggest a model to explain the synergy between folate deficiency and alkylating agents: (1) folate deficiency causes extensive uracil incorporation into DNA; (2) greatly increased utilization of base excision repair to remove uracil and to correct alkylator damage leads to error-prone DNA repair. In the case of EMS, this results in more intragenic deletions and G:C to A:T mutations due to impaired ligation of single-strand breaks generated during base excision repair and a decreased capacity to remove O6-ethylguanine. In the case of ENU additional T>A transversions and C>T transitions are seen, perhaps due to mis-pairing of O2-ethylpyrimidines. Correction of folate deficiency may reduce the frequency of these types of genetic damage during alkylator therapy.     

UV-induced hprt mutations in a UV-sensitive hamster cell line from complementation group 3 are biased towards the transcribed strand.

The molecular nature of 254 nm ultraviolet light (UV)-induced mutations at the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus in UV24 Chinese hamster ovary (CHO) cells, which are defective in nucleotide excision repair, was determined. Sequence analysis of 19 hprt mutants showed that single base substitutions (9 mutants) and tandem base changes (7 mutants) dominated the UV mutation spectrum in this cell line. Sixty-five percent of the base substitutions were GC greater than AT transitions, whereas the rest consisted of transitions and transversions at AT base pairs. Analysis of the distribution of dipyrimidine sites over the two DNA strands, where the photoproducts causing these mutations presumably were formed, showed that 12 out of 14 mutations were located in the transcribed strand of the hprt gene. A similar strand distribution of mutagenic photoproducts as in UV24 has previously been found in two other UV-sensitive Chinese hamster cell lines (V-H1 and UV5), indicating that under defective nucleotide excision repair conditions the induction of mutations is strongly biased towards lesions in the transcribed strand of the hprt gene. A plausible explanation for this phenomenon is that during DNA replication large differences exist in the error rate with which DNA polymerase(s) bypass lesions in the templates for the leading and lagging strand, respectively.     

Molecular analysis of spontaneous mutations at the gpt locus in Chinese hamster ovary (AS52) cells

AS52 cells are Chinese hamster ovary (CHO) cells that carry a single functional copy of the bacterial gpt gene and allow the isolation of 6-thioguanine-resistant (6TGr)mutants arising from mutation at the chromosally integrated gpt locus. The gpt locus in AS52 cells is extremely stable, giving rise to 6TGr mutants at frequencies comparable to the endogenous CHO hprt locus. In this study, we describe the spectrum of spontaneous mutations observed in AS52 cells by Southern blot and DNA sequence analyses. Using the polymerase chain reaction (PCR) and the Thermus aquaticus (Taq) polymerase, we have enzymatically amplified 6TGr mutant gpt sequences in vitro. The PCR product was then sequenced without further cloning manipulations to directly identify gpt structural gene mutations. Deletions predominant among the 62 spontaneous 6TGr-AS52 mutant clones analyzed in this study. Of these, 79% (49/62) of the mutations were identified as deletions either by Southern blotting, PCR amplification or DNA sequence analysis. Among these deletions is a predominant 3-base deletion that was observed in 31% (19/62) of the mutants. These data provide a basis for future comparisons of induced point mutational spectra derived in the AS52 cell line, and demonstrate the utility of PCR in the generation of DNA sequence spectra derived from chromosomally integrated mammalian loci.     

Molecular analysis of hprt mutants induced by 2-cyanoethylene oxide in human lymphoblastoid cells

The mutagenic epoxide metabolite of acrylonitrile, 2-cyanoethylene oxide (ANO), was used to treat human TK6 lymphoblasts (150 microM x 2 h ANO). A collection of hypoxanthine-phosphoribosyltransferase (hprt) mutants was isolated and characterized by dideoxy sequencing of cloned hprt cDNA. Base-pair substitution mutations in the hprt coding region were observed in 19/39 of hprt mutants: 11 occurred at AT base pairs and 8 at GC base pairs. Two -1 frameshift mutations involving GC bases were also observed. Approximately half (17/39) of the hprt mutants displayed the complete loss of single and multiple exons from hprt cDNA, as well as small deletions, some extending from exon/exon junctions. Southern blot analysis of 5 mutants with single exon losses revealed no visible alterations. Analysis of 1 mutant missing exons 3-6 in its hprt mRNA revealed a visible deletion in the corresponding region in its genomic DNA. The missing exon regions of 4 mutants (one each with exons 6, 7 and 8 loss and one mutant with a 17-base deletion of the 5' region of exon 9) were PCR amplified from genomic DNA and analyzed by Southern blot using exon-specific probes. The exons missing from the hprt mRNA were present in the genomic hprt sequence. DNA sequencing of the appropriate intron/exon regions of hprt genomic DNA from a mutant with exon 8 loss and a mutant exhibiting aberrant splicing in exon 9 revealed point mutations in the splice acceptor site of exon 8 (T----A) and exon 9 (A----G), respectively.     

Inherited susceptibility to bleomycin-induced micronuclei: correlating polymorphisms in GSTT1, GSTM1 and DNA repair genes with mutagen sensitivity

Susceptibility to DNA damage varies among individuals and sensitivity to bleomycin (BLM) may reflect the inter-individual differences. BLM sensitivity in part may be explained by inherited differences in DNA repair genes. We investigated the association between genetic polymorphisms in the GSTT1, GSTM1, XPD, XRCC1 and XRCC3 genes and the levels of spontaneous and BLM-induced DNA damage in peripheral blood lymphocytes from 200 healthy, unexposed individuals. The investigation of BLM sensitivity on cancer- or disease-free subjects and not occupationally exposed to known mutagen represents the strengths of the present study, as the detection of genetic damage is not biased by any disease- and occupational-related factor. The micronucleus (MN) assay was used to detect the spontaneous and BLM-induced genetic damage whereas, genotype analysis was carried out using methods based on polymerase chain reaction. Poisson regression analysis showed that subject's age, gender and smoking status had no effect on the spontaneous and BLM-induced MN frequencies. Genotype analysis revealed a clear association between GSTT1-null and XPD polymorphisms and both spontaneous and BLM-induced MN frequencies, whereas the effect of the XRCC1 polymorphism was marginally significant only with regard to spontaneous MN frequency. Genotype analysis did not reveal a clear association between the other studied SNPs (GSTM1 and XRCC3) and MN frequencies. Poisson regression analysis revealed no association between the score of protective alleles and the frequency of spontaneous MN. However, an increased number of protective alleles was significantly associated with a lower frequency of BLM-induced MN (P=0.0003). This finding highlights the genetic basis for BLM sensitivity, which could be a valid and useful surrogate for identifying genotypes that might increase susceptibility in population exposed to carcinogens. Further investigations in a large sample size and including more SNPs, reflecting the complexity of DNA repair machinery, might lead to the identification of a genetic profile responsible for the susceptibility to genotoxicants, with a far-reaching long-term impact on primary prevention and early detection of disease associated genes.     

用多引物PCR分析γ射线引起人外周血淋巴细胞hprt基因突变

正常人外周血用~（６０）Ｃｏ射线分别进行１～８Ｇｙ照射后,在含６－ＴＧ的培养基上克隆和筛选人淋巴细胞ｈｐｒｔ突变细胞。细胞的突变频率与γ射线的照射剂量呈正相关．获得４２个ｈｐｒｔ突变细胞株,用８对ｈｐｒｔ寡核苷酸引物进行多聚酶链反应（ＰＣＲ）从细胞粗提物中分别扩增ｈｐｒｔ各个外显子,分析突变细胞的ｈｐｒｔ基因突变,约６０％（２５／４２）的ｈｐｒｔ基因突变为基因缺失,其中１３个突变是ｈｐｒｔ基因全部缺失,而１２个是部分ｈｐｒｔ基因外显子缺失,约有４０％（１７／４２）的突变无明显的ＰＣＲ扩增变化．同时显示ｈｐｒｔ基因外显子的缺失突变与辐射剂量有关,实验结果提示,此方法有可能用于估计辐射剂量和进行辐射远后效果观察,进而揭示辐射致突的分子机理．