Sequence specific mutations induced by N-nitrosodimethylamine at two marker loci in metabolically competent human lymphoblastoid cells.

N-Nitrosodimethylamine (NDMA) is a potent mutagen and animal carcinogen to which many people are exposed through the consumption of contaminated food and the use of tobacco products. Although the mutational specificity of NDMA has been studied in bacteria, little is known about the specific types of mutations induced by NDMA in the human genome. Knowledge of the mutational spectrum of NDMA in human genes may help to substantiate the role of NDMA in the etiology of human cancers. In the current study, the mutational spectrum of NDMA was characterized at the tk and hprt loci, in human lymphoblastoid cells capable of metabolically activating NDMA. A number of patterns were observed among NDMA-induced mutations. At both marker loci, G:C-->A:T transitions dominated the mutational spectrum of NDMA, which were indicative of the mutagenicity of the O6meG lesion. In addition, the majority of G:C-->A:T mutations occurred at guanines 3' to another guanine. Almost all of these mutations originated on the non-transcribed strand, which suggests that transcription-coupled repair influenced the distribution of G:C-->A:T transitions at the tk and hprt loci. Furthermore, the observation of hotspots for G:C-->A:T mutations, within both loci, suggests that differential repair kinetics may exist, and consequently affect the distribution of mutations. Finally, a comparison of the site specificity of G:C-->A:T mutations at the tk and hprt loci, indicated that the gene used for mutational analysis influenced the site specificity of NDMA-induced mutations, and possibly reflects the number of 5'-GG-3' sites in the tk and hprt loci that when mutated would yield a mutant phenotype.

The influence of cellular apoptotic capacity on N-nitrosodimethylamine-induced loss of heterozygosity mutations in human cells.

The induction of loss of heterozygosity (LOH) by the environmental carcinogen N-nitrosodimethylamine (NDMA), and the factors that influence the recovery of LOH mutations were studied in two directly related human lymphoblastoid cell lines, AHH-1 (h2E1.v2) and MCL-5. Initially, the NDMA-induced mutation frequency at the heterozygous tk locus in AHH-1 cells was observed to be 5-fold higher in AHH-1 compared with MCL-5. Molecular analysis of NDMA-induced TK- mutants indicated that the induced mutant fraction attributable to small intragenic mutations was similar in both cell lines. However, the induced mutant fraction, because of LOH, was 18-fold greater in AHH-1. In addition, LOH mutations were more extensive among TK- mutants derived from AHH-1 cells. We hypothesized that the increased recovery of large LOH mutations in AHH-1 cells could be attributable to reduced apoptotic capacity, as it has been reported that AHH-1 cells carry a heterozygous mutation in the p53 locus, whereas MCL-5 cells are homozygous wild-type. Analysis of the kinetics of apoptosis showed that the apoptotic response of the AHH-1 cell line was diminished and delayed compared with MCL-5. Based on the analyses presented here, and several recent reports, it is suggested that the recovery of LOH mutations in p53 deficient cell lines is affected not only by abnormalities in cellular apoptotic response, but also involves a number of p53-mediated responses to DNA damage.

Extensive loss of heterozygosity accounts for differential mutation rate on chromosome 17q in human lymphoblasts.

In order to investigate the influence of loss of heterozygosity (LOH) events on mutation rate, we studied two closely related human lymphoblastoid cell lines, AHH-1 (h2E1.v2) and MCL-5, which are heterozygous at the tk locus (chromosome 17q23-25). Although they have similar mutant fractions at the hprt locus, the mutant fraction and rate at tk is four to five times higher in AHH-1. Analysis of 58 spontaneous TK- mutants from AHH-1 and MCL-5 showed that the occurrence of LOH events was more frequent (23/24) in AHH-1 than MCL-5 (16/34). A set of five microsatellite polymorphism loci was used to map the extent of LOH along chromosome 17q. In AHH-1 cells, 15/23 of the LOH events encompassed at least 35% of the sex-averaged genetic length of chromosome 17q (98 cM). Additionally, the next most extensive category of LOH accounted for 5/23 TK- mutants, and encompassed at least 17 cM. In contrast, LOH events observed in MCL-5 are very restricted in extent; only one LOH tract extended as far as 4 cM from tk. The higher mutation rate at tk in AHH-1 can, therefore, be entirely attributed to the recovery of chromosomal scale LOH in viable, normal growth TK- mutants. Furthermore, these data demonstrate that the regional potential for LOH is likely to be an important determinant of mutation rate for loci within that chromosomal segment.

Role of oxygen radicals in the chromosomal loss and breakage induced by the quinone-forming compounds, hydroquinone and tert-butylhydroquinone.

The mechanisms by which two quinone-forming compounds, hydroquinone (HQ) and tert-butyl-hydroquinone (tBHQ), induce chromosomal loss and breakage in a prostaglandin H synthase-containing V79 cell line have been investigated using the cytokinesis-block micronucleus assay with CREST antibody staining. Increased frequencies of CREST-positive micronuclei (indicating chromosome loss) and CREST-negative micronuclei (indicating chromosome breakage) were observed following exposure of cells to HQ and tBHQ. The formation of micronuclei by HQ, but not tBHQ, was dependent on arachidonic acid supplementation, indicating activation by prostaglandin H synthase. Since the oxidation of hydroquinones can result in the generation of oxygen radicals, the contribution of oxygen radicals to the formation of chromosomal alterations induced by HQ and tBHQ was investigated. In the presence of a superoxide-generating system consisting of hypoxanthine and xanthine oxidase, a significant increase in micronucleated cells was observed. These induced micronuclei consisted exclusively of CREST-negative micronuclei and their formation was completely inhibited by pretreatment with catalase. Catalase also significantly inhibited the CREST-negative micronuclei induced by HQ and tBHQ. In addition, glutathione treatment inhibited both CREST-positive and negative micronuclei induced by these phenolic compounds. These results indicate that both chromosome loss and breakage are induced by these two quinone-forming agents. Reactive oxygen species contribute to the chromosomal breakage induced by HQ and tBHQ but the observed chromosomal loss appears to result from other mechanisms such as an interference of quinone metabolites with spindle formation.