汞作业所致工人的遗传效应

目的探讨汞作业所致工人的遗传效应。方法选择荧光灯生产厂汞作业工人20名为接触组,无汞接触的其他从业人员20名为对照组,取外周血淋巴细胞,通过微核试验、彗星试验、hprt和TCR基因突变试验进行检测。结果接触组平均微核率(MNR)、平均微核细胞率(MCR)分别为(5.90±0.91)‰和(5.30±0.81)‰,对照组的MNR和MCR分别为(1.50±0.47)‰和(1.30±0.31)‰,两组差异有统计学意义(P<0.01)。接触组和对照组的平均尾长(MTL)分别为(3.16±0.31)和(0.99±0.07)μm,两组平均尾相(MTM)分别为1.63±0.22和0.39±0.03,差异有统计学意义(P<0.01)。接触组的hprt和TCR平均突变率与对照组比较,差异无统计学意义(P>0.05)。结论汞作业可致工人不良的遗传效应。     

Mutational specificity of 2-amino-3-methylimidazo-[4,5-f]quinoline in the hprt locus of CHO-K1 cells

2-Amino-3-methylimidazo[4,5-f]quinoline (IQ), a food carcinogen formed in cooked meats, can induce gene mutation at the hprt locus of CHO-K1 cells in the presence of hepatic 59 mix. To elucidate the molecular nature of IQ-induced mutation, we characterized the entire coding region of the hypoxanthine phosphoribosyl-transferase gene of 23 independent mutants derived from IQ-treated CHO cells by direct sequencing of polymerase chain reaction-amplified cDNA. Ten of the 23 IQ-induced mutants examined contained single base substitutions; one mutant had three single-base substitutions. Among the base substitutions, G·C→CG (six of 13) and A·T→CG (three of 13) transversions predominated. Most of the base-substitution mutations occurred preferentially at a middle G and had a dA in their 3′ ends. Of the 13 other mutations (56.5%), 12 missing one or more complete exons were splice-site mutations, and one mutant had a partial deletion of an exon. A high frequency of complete exon deletion (11 of 12) in exons 2–5 was observed. Interestingly, 75% of the mutants (nine of 12) with splice-site mutations were induced by IQ only at higher concentrations (300–500 μM). This was probably due to the occurrence of GC base-substitution mutations that affected hprt mRNA splicing, especially at the intron-exon boundaries. © 1995 Wiley-Liss, Inc.     

Lack of Direct Involvement of 8-Hydroxy-2'-deoxyguanosine in Hypoxanthine-guanine Phosphoribosyltransferase Mutagenesis in V79 Cells Treated with N,N'-Bis(2-hydroxyperoxy-2-methoxyethyl)-l,4,5,8-naphthalenetetracarboxylic-diimide (NP-III) or Riboflavin

The object of this study is to investigate the relationship between a typical product of oxidative DNA damage, 8-hydroxy-2'-deoxyguanosine (8OHdG), and mutagenesis in V79 cells through a molecular analysis of hypoxanthine-guanine phosphoribosyltransferase ( hprt ) gene mutants. We performed a direct sequencing analysis of the cDNA of mutants obtained after treatment with N,N'-bis(2-hydroxyperoxy-2-methoxyethyl)-l,4,5,8-naphthalenetetracarboxylic-diimide (NP-III) or riboflavin, each of which induces the formation of 8OHdG in cellular DNA upon UVA irradiation. The frequency of mutation after both treatments was no more than 2 to 5 times the control value. A considerable number of the mutants could not be amplified by RT-PCR, and this was also the case for the control mutants. Among the mutants analyzed, deletions and a TA→Ã transversion occurred predominantly. The reasons for the weak association of induction of 8OHdG with frequency of mutation and the possible mechanism of oxidative-stress-derived mutagenesis are discussed.     

hprt基因突变对宫颈癌放疗损伤的评估

目的：探讨hprt基因突变对宫颈癌放疗损伤的评估作用。方法：取15例宫颈鳞癌放疗患者放疗前及放疗累积剂量为10～60Gy时的外周静脉血,采用多核细胞法检测hprt基因突变率。结果：在照射累积剂量为30、40和50Gy时,hprt基因突变率在照射后升高,与照射前相比,差别有统计学意义（P〈0.05）。当累积剂量达60 Gy时,hprt基因突变率虽然较照射前升高,但差别无统计学意义（P〉0.05）。在0～40 Gy,hprt基因突变率基本呈线形上升,且突变率与累积剂量间呈现较好的剂量-效应关系（r=0.900,P〈0.05）。回归方程为y=0.459＋0.014x。结论：在累积剂量0～40 Gy范围内,hprt基因突变率在放疗后明显升高,呈现良好的剂量-效应关系,可用于评价放疗造成的损伤,是具有应用潜质的生物剂量计。     

Analysis of solvent control and 1-nitrosopyrene-induced Chinese hamster ovary cell mutants by Southern and northern blots and the polymerase chain reaction.

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

The popular herbal antimalarial, extract of Cryptolepis sanguinolenta, is potently cytotoxic.

The aqueous root extract of Cryptolepis sanguinolenta (CSE) is a popular antimalarial in West African ethnomedicine. Cryptolepine (CLP), the major alkaloid of the plant, is a cytotoxic DNA intercalator that has promise as an anticancer agent. To date the aqueous root extract, the traditional antimalarial formulation, has not been evaluated for toxicity. In this study, we have examined the in vitro toxicity of CSE and CLP using V79 cells, a Chinese hamster lung fibroblast frequently used to assess genetic toxicity, and a number of organ-specific human cancer cell lines. CSE and CLP caused a dose- and time-dependent reduction in viability of the V79 cell line. Flow cytometric analysis of CSE- and CLP-treated (24 h) asynchronously growing V79 cells using propidium iodide (PI) staining revealed an accumulation of cells (up to 55%) in the sub-G1 phase of the cell cycle, indicative of cell death. The V79 cells and almost all the organ-specific human cancer cell lines exposed to CSE and CLP were profoundly growth inhibited, as measured in a clonogenicity assay. In a V79 cell mutation assay (hprt gene), CSE (5-50 microg/ml) only induced mutation at the highest dose employed (mutation frequency approximately 4 and 38 mutant clones per 10(6) cells for control and CSE, respectively), but CLP (0.5-5.0 microM) was not mutagenic. These results indicate that CSE and CLP are very cytotoxic and may be weak mammalian mutagens and/or clastogens. The poor genotoxicity of CSE and CLP coupled with their potent cytotoxic action support their anticancer potential.     

Analysis of human HPRT deletion mutations with X-linked probes and pulsed field gel electrophoresis.

Because the human hprt gene is used in numerous mutation studies, it is important to fully characterize this gene. Therefore, our laboratory has undertaken to map the region around the hprt gene at band q26 of the human X chromosome. Utilizing hprt mutant T-cell clones isolated using the hprt clonal assay, which have deletions of all or part of the hprt gene, we have ordered 5 anonymous probes previously known to map in Xq26. Results suggest that this region includes between 460 kb and 18 Mb of DNA, which is at least 10 times the size of the hprt gene itself (43 kb). Pulsed field gel analysis of the region is underway to determine the exact distances between each of the anonymous probes and hprt and to determine deletion sizes in the mutant T-cell clones.     

放疗对肿瘤患者外周血淋巴细胞hprt基因位点的影响

目的了解放疗对肿瘤患者外周血淋巴细胞hprt基因位点的影响，探讨hprt基因突变分析作为放疗致机体辐射损伤监测的可行性。方法采用多核细胞法研究放疗对人外周血淋巴细胞hprt基因位点的影响及其突变频率的变化。结果放疗可诱发人外周血淋巴细胞hprt基因位点突变，突变频率随放疗累积剂量的增加而升高。在同一剂量点，鼻咽癌病人与乳腺癌病人的hprt基因突变频率统计学上没有显著差异。肿瘤病人的hprt基因突变频率均较正常对照组高。结论hprt基因位点突变频率分析可作为放疗致机体辐射损伤监测的生物学标志。     

Analysis of point mutations in the hprt gene of cancer patients treated with radioimmunoglobulin therapy

The mutagenic impact of various environmental and therapeutic agents can now be directly assayed in humans by the T-lymphocyte cloning assay. We have previously reported that following radioimmu-noglobulin therapy, cancer patients exhibited increased mutant frequency at the hprt locus and an increased yield of large intergenic deletions compared to unexposed controls. Here we report the results of the analysis of 26 independent hprt mutations in nine cancer patients who underwent radioimmunoglobulin therapy. The majority of mutations (52%) had lost exon sequences from the mRNA. The remaining mutations were 20% small deletions and frameshifts and 28% base substitutions. The type of mutations observed were similar to those seen in unexposed controls. The site distribution of the mutations, however, indicates that some sequence contexts may be more sensitive to radiation mutagenesis than others. © 1995 Wiley-Liss, Inc.     

氯化镉对V79细胞hprt基因位点突变频率的影响及锌的作用

目的了解氯化镉（CdCl2）对中国仓鼠肺成纤维细胞（V79细胞）hprt基因位点突变频率的影响及锌的拮抗作用。探讨镉的遗传毒性机制。方法采用克隆形成法了解CdCl2对V79细胞的慢性毒性作用;在此基础上采用克隆法研究不同浓度CACl2对V79细胞hprt基因位点突变频率的影响,并采用生理浓度的氯化锌（ZnCl2）与CdCl2同时作用后,观察锌对于镉致突变效应的影响。同时观察不同浓度CdCl2预先染毒24h后,对过氧化氢（H。（）。）所致hprt基因位点突变效应的影响及锌的拮抗作用。结果克隆形成实验中,CdCl2对V79细胞的毒性随染毒浓度增加而增高,呈线性关系;CdCl2可以引起V79细胞hprt基因位点突变频率增加,在0．1和8μmol／L染毒浓度处分别有两个峰值。CdCl2与H2O2联合作用表现为协同效应。ZnCl2可以拮抗这种效应。结论在本实验条件下,CdCl2可以导致V79细胞hprt基因位点突变,并可使其他致突变物的致突变性增强,而锌瓦‘以拮抗此效应。