Gene‐ and tissue‐specificity of mutation in Big Blue® rats treated with the hepatocarcinogen N‐hydroxy‐2‐acetylaminofluorene†

Abstract

In a previous study, we found that treating transgenic Big Blue rats with the hepatocarcinogen N-hydroxy-2-acetylaminofluorene (N-OH-AAF) produced the same major DNA adduct in the target liver and the nontarget spleen lymphocytes and bone marrow cells, induced lacI mutants in the liver, and induced much lower frequencies of lacI and hprt mutants in spleen lymphocytes. In the present study, sequence analysis was conducted on lacI DNA and hprt cDNA from the mutants, to determine the mutational specificity of N-OH-AAF in the rat. All the mutation spectra from N-OH-AAF-treated rats differed significantly from corresponding mutation profiles from untreated animals (P = 0.02 to P < 0.0001). Although there were similarities among the mutational patterns derived from N-OH-AAF-treated rats (e.g., G:C --> T:A transversion was the most common mutation in all mutation sets), there were significant differences in the patterns of basepair substitution and frameshift mutation between the liver and spleen lymphocyte lacI mutants (P = 0.02) and between the spleen lymphocyte lacI and hprt mutants (P = 0.04). Also, multiplex PCR analysis of genomic DNA from the hprt mutants indicated that 12% of mutants from treated rats had major deletions in the hprt gene; no corresponding incidence of large deletions was evident among lacI mutations. All the mutation profiles reflect the general mutational specificity of the major DNA adduct formed by N-OH-AAF. The differences between N-OH-AAF mutation in the endogenous gene and transgene can be partially explained by the structures of the two genes. The tissue-specificity of the mutation spectra may contribute to targeting tumor formation to the liver. Environ. Mol. Mutagen. 37:203-214, 2001. Published 2001 Wiley-Liss, Inc.

Role of Human N‐Acetyltransferase 2 Genetic Polymorphism on Aromatic Amine Carcinogen‐Induced DNA Damage and Mutagenicity in a Chinese Hamster Ovary Cell Mutation Assay

“…These results are similar to our studies for PhIP, IQ, and MeIQx (Metry et al 2010) and to previous studies with PhIP and IQ (Wu et al 1997). Also, in a study with transgenic Big Blue rats dosed with N-hydroxy-2-acetylaminoflourene, G:C to T: A transversions predominated (Chen et al 2001).…”

“…The majority of induced mutations for both aromatic amines were missense/nonsense single-base substitutions, in which over 80% were at G:C base pairs. Also, in a study with transgenic Big Blue rats dosed with N-hydroxy-2-acetylaminoflourene, G:C to T: A transversions predominated (Chen et al 2001). Furthermore, when adding previously published heterocyclic amine-induced mutations (Metry et al 2010), positions 134-135 (GpG), 208-209 (string of G's), 418-419 (GpG), and 589 were frequent mutation locations.…”

Spontaneous mutant frequency and mutation spectrum for gene A of ΦX174 grown in E. coli

“…The removal of the two hot-spot target sites from the gene A mutation spectrum left all of 23 mutations in the G:C3 A:T transition category. This is also the most prevalent base substitution class for other transgenes recovered from untreated tissues: the lacI gene isolated from spleen and liver [Chen et al, 2001], the cII gene and lacI gene isolated from liver and colon [Kohara et al, 2001;Chen et al, 2002;Yamada et al 2002], the lacZ plasmid-based transgene isolated from spleen, brain, heart, liver, and small intestine [Dollé et al, 2002;Louro et al, 2002], the lacZ gene in bacteriophage isolated from spleen, liver, heart, brain, and testis [Ono et al, 1999[Ono et al, , 2000, and the gpt transgene isolated from bone marrow [Masumura et al, 1999]. However, in most of these studies, a majority of the G:C3 A:T transitions occurred at CpG sites, which implicates the deamination of 5-methylcytosine.…”

ACB-PCR measurement of K-ras codon 12 mutant fractions in livers of Big Blue(R) rats treated with N-hydroxy-2-acetylaminofluorene

“…Liver tissue from a previous study (17,18) was used for the ACB-PCR assays. Male 6-week-old Big Blue Ò rats were purchased from Taconic Farms (Germantown, NY).…”

“…With the goal of using ACB-PCR data to inform species extrapolation in cancer risk assessment, we developed assays for quantifying rat K-ras codon 12 GGT!GTT and GGT!GAT mutations in order to compare the role of K-ras mutations in rat models to human disease. The utility of the developed ACB-PCR assays were then demonstrated in the measurement of mutations in rat liver genomic DNA isolated from a previous study of Big Blue Ò rats treated with N-hydroxy-2-acetylaminofluorene (N-OH-AAF) (17,18). This approach also provides a direct comparison between the mutational responses in a transgenic reporter locus and an endogenous cancer-associated gene.…”