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

Abstract

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 O 6 meG lesion. In addition, the majority of G:C→A:T mutations occurred at guanines 3Ј 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 5Ј-GG-3Ј sites in the tk and hprt loci that when mutated would yield a mutant phenotype.

Mutant frequency and mutational spectra in the Tk and Hprt genes of N‐ethyl‐N‐nitrosourea‐treated mouse lymphoma cells†

“…No frameshift mutations were found in ENU-induced Tk or Hprt mutants. A previous study [Dobo et al, 1998] demonstrated that the methylating agent N-nitrosodimethylamine also produced similar mutational spectra in the TK and HPRT genes of human lymphoblasts. In addition, the types of mutations are consistent with the DNA adducts induced by ENU and other alkylating agents.…”

Evaluating the mutagenicity of N-nitrosodimethylamine in 2D and 3D HepaRG cell cultures using error-corrected next generation sequencing

“…Since the HepaRG cell line is derived from a single donor with unknown AAG levels, this mutation may not be common to all human hepatic cells. Intriguingly, NDMA induced primarily G:C → A:T transitions in human B lymphoblastoid cell lines AHH-1 and MCL-5, both transfected with the human CYP2E1 gene (Dobo et al 1998 ). It is worth noting that the mutational spectrum in this study was derived from phenotypically selected mutants at two reporter genes, TK and HPRT , whereas our study analyzed a larger portion of the genome using a non-selection method for identifying mutations, and this may produce a broader assessment of NDMA-induced mutations in metabolically competent human cells.…”