Molecular characterization of mutations in the hprt gene of normal human skin keratinocytes treated with N-ethyl-N-nitrosourea: Influence of O6-alkylguanine alkyltransferase

O⁶-Alkylguanine-DNA alkyltransferase (AGT) is responsible for repairing the O⁶-alkylguanine lesion in DNA. There is wide variation in the levels of AGT between organ and cell types, which appears to correlate with cell and tissue type sensitivity to the mutagenic and carcinogenic effects of alkylating agents. In order to investigate the role of AGT in modulating the frequency and types of mutations induced in one type of normal human parenchymal cells, we examined the types and frequency of mutations in the hypoxanthine (guanine) phosphoribosyltransferase (hprt) gene in 116 mutants derived from two N-ethyl-N-nitrosourea (ENU)-treated normal human skin keratinocyte cell lines. O⁶-Benzylguanine (O⁶-BZ; 5 μM × 2 hours) was used to specifically inhibit AGT activity before ENU treatment (0 to 5 mM × 1 hour). O⁶-BZ increased both the cytotoxic and mutagenic effects of ENU by 1.8- and 3- to 5-fold, respectively. In both treatment groups, most of the mutations were base substitutions (72%). The proportion of GC to AT transitions in the O⁶-BZgroup (14/31) was twice that in the group treated with ENU alone, consistent with the loss of AGT activity in these cells. There was no strand specificity of GC to AT and AT to GC transitions in both groups. Base transversions accounted for 28% of total base substitutions. A lower than expected proportion of AT to TA transversions were observed in both cell lines, which decreased in the O⁶-BZ pretreated group. A strand bias was observed for GC to TA and AT to TA transversions. Most of the G to A and G to T base substitutions had one or more purines flanking 3′ to the mutated deoxyguanosines. There were more deletion mutants with the deletion of exon 1, 4, 6, and 8 in the BZ group than in the control group. These data, characterizing the mutational spectra of ENU in normal human keratinocytes treated in vitro, indicate that GC to AT and AT to GC transition mutations predominate in these cells depleted or not depleted of AGT. Environ. Mol. Mutagen. 29:168–179, 1997. © 1997 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。     

丙烯酰胺诱导人白血病HL-60和NB_4细胞hprt基因的分子突变谱

为了研究丙烯酰胺的遗传毒理作用 ,采用单细胞克隆培养 ,双向筛选计数 ,多重PCR扩增与电泳分析 ,研究了诱导HL 6 0和NB4 两种细胞hprt基因突变率及分子突变谱 .发现只有丙烯酰胺高剂量组 (70 0mg·L- 1)才对两种细胞有明确的致hprt基因突变作用 ;丙烯酰胺诱发突变主要由点突变和缺失两部分组成 (40 .0 %～ 6 6 .7% ,33.3%～ 6 0 .0 % ) ,而自发突变几乎全是点突变 (90 .0 %以上 ) ,两种细胞均无全基因缺失型 ;缺失突变可以发生于hprt基因上的每个外显子 (除外显子 7/ 8以外 ) ,较集中于基因的 3′末端 ,且诱发突变中绝大多数是点突变与单个外显子缺失 (93.3% ,86 .1% ) ,两种细胞情况类似 .结果提示 ,丙烯酰胺具有较弱的诱导hprt基因突变的作用 ,且诱发突变与自发突变的分子图谱不一样 ,这可能与其作用机理有关     

Mutagenicity in vitro of 3,4-dichloro-5-hydroxy-2(5H)-furanone (mucochloric acid), a chlorine disinfection by-product in drinking water

The mutagenicity of chlorinated humic drinking waters is accounted for mainly by a single contaminant, 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), as assessed in Salmonella typhimurium strain TA100. In the present study, 3,4-dichl-oro-5-hydroxy-2(5H)-furanone (mucochloric acid, MA), another drinking water contaminant much less potent as a mutagen in TA100 than MX, was tested in Chinese hamster ovary (CHO) cells for the induction of mutation at the hypoxanthine phosphoribosyl transferase (hprl) locus to 6-thioguanine resistance (TG'). Unexpectedly, MA induced TG mutants in CHO cells with a potency comparable to that reported previously for MX. In subsequent experiments with S. typhimurium, the presence of pKM1O1 plasmid in strain TA100 increased susceptibility to the mutagenicity of MA, but much less than to that of MX, relative to the parental strain TA1535 lacking pKMlOl. The difference between the two compounds in TA100 thus appears to be due to a higher enhancement of the mutagenicity of MX than that of MA by pKM101 mediated error-prone DNA repair. © 1995 Wiley-Liss, Inc.     

Nitrogen mustard-mediated mutagenesis in human T-lymphocytes in vitro

The cytotoxic and mutagenic effect of the bifunctional alkylating agent nitrogen mustard (HN₂) was examined. Primary human lymphocytes were exposed to graded doses of HN₂ in vitro and relative survival was determined. Mutation induction at the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus was measured by cloning the exposed T-cells in microtitre plates in the presence and absence of 6-thioguanine (TG). The IC₅₀-value determined for 30 min exposure to HN₂ was 1.34 μM. The mutant frequencies (MF) in exposed T-cell cultures were 10-fold (2 μM HN₂) to 32-fold (4 μM HN₂) higher than those of unexposed cultures (median values). Nitrogen mustard-mediated mutagenesis is discussed in terms of the current ideas about DNA damage and repair. Received: 14 May 1996 / Accepted: 28 August 1996     

NATb/NAT1*4 promotes greater arylamine N-acetyltransferase 1 mediated DNA adducts and mutations than NATa/NAT1*4 following exposure to 4-aminobiphenyl

N‐acetyltransferase 1 (NAT1) is a phase II metabolic enzyme responsible for the biotransformation of aromatic and heterocyclic amine carcinogens such as 4‐aminobiphenyl (ABP). NAT1 catalyzes N‐acetylation of arylamines as well as the O‐acetylation of N‐hydroxylated arylamines. O‐acetylation leads to the formation of electrophilic intermediates that result in DNA adducts and mutations. NAT1 is transcribed from a major promoter, NATb, and an alternative promoter, NATa, resulting in mRNAs with distinct 5′‐untranslated regions (UTR). NATa mRNA is expressed primarily in the kidney, liver, trachea, and lung while NATb mRNA has been detected in all tissues studied. To determine if differences in 5′‐UTR have functional effect upon NAT1 activity and DNA adducts or mutations following exposure to ABP, pcDNA5/FRT plasmid constructs were prepared for transfection of full‐length human mRNAs including the 5′‐UTR derived from NATa or NATb, the open reading frame, and 888 nucleotides of the 3′‐UTR. Following stable transfection of NATb/NAT1*4 or NATa/NAT1*4 into nucleotide excision repair (NER) deficient Chinese hamster ovary cells, N‐acetyltransferase activity (in vitro and in situ), mRNA, and protein expression were higher in NATb/NAT1*4 than NATa/NAT1*4 transfected cells (P < 0.05). Consistent with NAT1 expression and activity, ABP‐induced DNA adducts and hypoxanthine phosphoribosyl transferase mutants were significantly higher (P < 0.05) in NATb/NAT1*4 than in NATa/NAT1*4 transfected cells following exposure to ABP. These differences observed between NATa and NATb suggest that the 5′‐UTRs are differentially regulated. © 2011 Wiley Periodicals, Inc.     

Molecular analysis of hprt mutations generated in Chinese hamster ovary EM9 cells by uranyl acetate, by hydrogen peroxide, and spontaneously

Naturally occurring uranium and depleted uranium (DU) are believed to be health hazards by virtue of both their chemical and radiological properties. The mechanism(s) behind uranium's chemotoxic effects has yet to be elucidated. Previous work has shown that DU, as uranyl acetate (UA), was mutagenic at the hypoxanthine (guanine) phosphoribosyltransferase (hprt) locus in XRCC1-deficient CHO EM9 cells. The purpose of the current study was to characterize the mutations induced by UA at the hprt locus of CHO EM9 cells and compare the mutation spectrum of UA with those of hydrogen peroxide and spontaneous mutations in the same line. The hypothesis being tested was that if DU as UA is chemically genotoxic then the mutation spectrum induced by the heavy metal should be distinct from that produced spontaneously or by H2O2. A total of 59 UA-induced, 38 spontaneous, and 45 H2O2-induced mutations were identified. Base substitutions comprised 29%, 42%, and 16% of UA, spontaneous, and H2O2 mutants, respectively. The frequency of G --> T or C --> A substitutions was not significantly different in spontaneous or H2O2-induced mutants than in UA-induced mutants, suggesting a possible role for 8-oxodG damage in UA mutagenesis. However, the observation that UA produced significantly more major genomic rearrangements (multiexon insertions and deletions) than occurred spontaneously suggests the possibility that DNA strand breaks or crosslinks could also be UA-induced mutagenic lesions. The unique mutation spectrum elicited by exposure to UA suggests that UA generates mutations in ways that are different from spontaneous and free radical as well as radiological mechanisms.     

Molecular analyses of in vivo hprt mutations in human T-lymphocytes: IV. Studies in newborns

In order to characterize in vivo gene mutations that occur during fetal development, molecular analyses were undertaken of mutant 6-thioguanine resistant T-lymphocytes isolated from placental cord blood samples of 13 normal male newborns. These mutant T-cells were studied to define hypoxanthine-guanine phosphoribosyltransferase (hprt) gene structural alterations and to determine T-cell receptor (TCR) gene rearrangement patterns. Structural hprt alterations, as shown by Southern blot analyses, occurred in 85% of these mutant clones. These alterations consisted mostly of deletion of exons 2 and 3. These findings contrast with the 10-20% of gross structural alterations (i.e., those visible on Southern blots) occurring randomly across the entire gene previously reported for T-cell mutants isolated from normal young adults. Iterative analyses of hprt structural alterations and TCR gene rearrangement patterns show that approximately one-third of the newborn derived mutants may have originated as pre- or intrathymic hprt mutations. This too contrasts with previous findings in adults where the background in vivo hprt mutations appeared to originate in postthymic T-lymphocytes.     

Molecular analysis of hprt mutant lymphocytes from 1, 3-butadiene-exposed workers

1,3-Butadiene (BD) has been shown to be a potent animal carcinogen and a probable human carcinogen, yet the molecular mechanisms of BD genotoxicity and carcinogenicity still are not fully understood. Our hypothesis is that metabolites of BD induce specific structural changes in the human hprt gene like those observed in vitro in TK6 cells and in vivo in the mouse. Characteristic mutations in BD-exposed subjects can be identified and used as biomarkers for monitoring genotoxic effects associated with BD exposure. Molecular analysis of hprt mutant lymphocytes from BD-exposed workers and unexposed control subjects was carried out to identify changes in the structure of the hprt gene. A multiplex polymerase chain reaction (PCR) assay was used to detect exon deletions in 360 hprt mutant clones. We determined that exon deletions were significantly more frequent (P < 0.05) in BD-exposed workers (17.5%) than in control subjects (9.7%). Sequence analysis of hprt cDNA from 175 independent mutants indicated that the distribution of the types of mutations was different between the workers and the unexposed control subjects. There was a significant increase in -1 frameshift mutations in BD-exposed workers, predominantly in repeated DNA sequences, and single-base substitutions were decreased to 66% in the workers compared to 83% in the control subjects (P < 0.05). In addition to the spectral changes, hprt clonal assays revealed an elevation in mutant frequency in the lymphocytes of workers (N = 10) when compared with that in unexposed control subjects (N = 11; P < 0. 05). There also was a twofold increase of A:T --> T:A transversions in BD-exposed workers (16% in BD-exposed workers compared to 8% in controls, P = 0.25). Some of the BD-associated changes in mutational spectra observed in our study have the potential for application in monitoring genotoxic effects related to butadiene exposure.     

Induction of 6-thioguanine-resistant lymphocytes in Fischer 344 rats following in vivo exposure to N-ethyl-N-nitrosourea and cyclophosphamide

We have developed a limiting dilution clonal assay for determining the frequency of 6-thioguanine-resistant (TGr) lymphocytes produced in rats by in vivo exposure to genotoxic agents. Spleen lymphocytes were isolated from female Fischer 344 rats and were cultured with 1 microgram/ml of phytohemagglutinin (PHA) for 40 hr. Northern blot analysis revealed that this procedure resulted in increased hprt and beta-actin mRNA synthesis. Conditions for optimum cloning were established by culturing four PHA-primed lymphocytes/well in 96-well round-bottom microtiter plates containing a medium supplemented with interleukin-2. These cultures also contained autologous and/or TK6 feeder cells inactivated with different doses of irradiation. Lymphocyte cloning efficiencies (CEs) were highest in plates containing both irradiated TK6 cells (5 x 10(3) cells/well; 90 Gy) and irradiated autologous feeder cells (5 x 10(4) cells/well; 50 Gy). CE did not depend on the number of primed lymphocytes/well when four or fewer target cells/well were cloned. To measure the effects of chemical mutagens on the frequency of TGr lymphocytes, rats were given a single i.p. injection of 0-150 mg/kg of N-ethyl-N-nitrosourea (ENU), a direct-acting alkylating agent, or 0-50 mg/kg of cyclophosphamide (CP), an indirect acting alkylating agent. Lymphocytes were isolated, primed, and cloned at 4 weeks after CP treatment and at 1, 2, 4 and 6 weeks after ENU treatment. CE in these cultures ranged from 12% to 27%. Cultures were also established for measuring CE in the presence of 6-thioguanine (TG) and these contained 5 x 10(3) irradiated TK6 cells and 5 x 10(4) primed rat lymphocytes/well. The frequency of TGr lymphocytes was calculated by correcting the CE in the presence of TG with the CE measured in its absence. ENU exposure produced a higher frequency of TGr lymphocytes than CP, but both chemicals produced a dose-dependent increase in TGr cells. In addition, the frequency of ENU-induced TGr lymphocytes increased with time after treatment. The TGr cells are presumed to be hprt mutants, but further analysis at the DNA level is required to establish this. The dose-dependent responses obtained with both ENU and CP treatments suggest that rat lymphocytes are sensitive to direct- and indirect-acting alkylating agents administered in vivo and that the rat lymphocyte assay is a useful complement to the in vivo/in vitro mouse assay for determining the mutagenicity of environmental toxicants.