S-nitrosoglutathione reductase deficiency increases mutagenesis from alkylation in mouse liver

Abstract

In human hepatocellular carcinoma (HCC) and many other cancers, somatic point mutations are highly prevalent, yet the mechanisms critical in their generation remain poorly understood. S-nitrosoglutathione reductase (GSNOR), a key regulator of protein S-nitrosylation, is frequently deficient in human HCC. Targeted deletion of the GSNOR gene in mice can reduce the activity of the DNA repair protein O (6)-alkylguanine-DNA alkyltransferase (AGT) and promote both carcinogen-induced and spontaneous HCC. In this study, we report that following exposure to the environmental carcinogen diethylnitrosamine, the mutation frequency of a transgenic reporter in the liver of GSNOR-deficient mice (GSNOR(-/-)) is significantly higher than that in wild-type control. In wild-type mice, diethylnitrosamine treatment does not significantly increase the frequency of the transition from G:C to A:T, a mutation deriving from diethylnitrosamine-induced O (6)-ethylguanines that are normally repaired by AGT. In contrast, the frequency of this transition from diethylnitrosamine is increased ~20 times in GSNOR(-/-) mice. GSNOR deficiency also significantly increases the frequency of the transversion from A:T to T:A, a mutation not affected by AGT. GSNOR deficiency in our experiments does not significantly affect either the frequencies of the other diethylnitrosamine-induced point mutations or hepatocyte proliferation. Thus, GSNOR deficiency, through both AGT-dependent and AGT-independent pathways, significantly raises the rates of specific types of DNA mutations. Our results demonstrate a critical role for GSNOR in maintaining genomic integrity in mice and support the hypothesis that GSNOR deficiency is an important cause of the widespread mutations in human HCC.

Fig. 1.

Mutational spectra at the cII locus in the livers of wild-type and GSNOR^−/− Big Blue mice. Shown are the nucleotide sequence changes in the untreated (green) and DEN-treated (red) wild-type (above the reference cII sequence) or GSNOR^−/− (below the sequence) mice. Translational start and stop codons are outlined. +G and −G indicate single-nucleotide insertion and deletion, respectively, in the underlined G repeats; −A indicates single nucleotide deletion in the underlined A repeats.

Fig. 2.

Overall mutation frequency from DEN treatment is increased by GSNOR deficiency. (A) The frequency of spontaneous cII mutants in the livers of four untreated GSNOR^−/− mice is not significantly different from that in four wild-type (WT) controls (P = 0.2). (B) Mutant frequency in the livers of four GSNOR^−/− mice is significantly higher than that in four wild-type controls after DEN challenge (P = 0.006). The mutant frequencies of the DEN-treated wild-type and GSNOR^−/− mice are significantly higher than those in the unchallenged wild-type (P = 0.003) and GSNOR^−/− (P = 0.001) mice, respectively. Each dot represents the data from a single mouse; bar represents the group mean.

Fig. 3.

GSNOR deficiency increases the frequency of G:C to A:T transitions from DEN treatment. The frequency of cII mutants containing G:C to A:T transitions is from the liver of untreated (A) or DEN-treated (B) wild-type and GSNOR^−/− mice (n = 4 in each group). The frequency is significantly higher in DEN-treated GSNOR^−/− mice than in DEN-treated wild-type mice (P = 0.02).

Fig. 4.

GSNOR deficiency increases the frequency of A:T to T:A transversions from DEN treatment. The frequencies of cII mutants containing A:T to T:A transversions are from the livers of untreated (A) or DEN-treated (B) wild-type and GSNOR^−/− mice (n = 4 in each group). The frequency is significantly higher in DEN-treated GSNOR^−/− mice than in wild-type control (P = 0.003).

Fig. 5.

Mutations not significantly affected by GSNOR deficiency. The data (mean + SD) are from DEN-treated GSNOR^−/− and wild-type mice (n = 4 in each group).