Limited ability of DNA polymerase kappa to suppress benzo[a]pyrene‐induced genotoxicity in vivo

Abstract

DNA polymerase kappa (Polk) is a specialized DNA polymerase involved in translesion DNA synthesis. To understand the protective roles against genotoxins in vivo, we established inactivated Polk knock-in gpt delta (inactivated Polk KI) mice that possessed reporter genes for mutations and expressed inactive Polk. In this study, we examined genotoxicity of benzo[a]pyrene (BP) to determine whether Polk actually suppressed BP-induced genotoxicity as predicted by biochemistry and in vitro cell culture studies. Seven-week-old inactivated Polk KI and wild-type (WT) mice were treated with BP at doses of 5, 15, or 50 mg/(kg·day) for three consecutive days by intragastric gavage, and mutations in the colon and micronucleus formation in the peripheral blood were examined. Surprisingly, no differences were observed in the frequencies of mutations and micronucleus formation at 5 or 50 mg/kg doses. Inactivated Polk KI mice exhibited approximately two times higher gpt mutant frequency than did WT mice only at the 15 mg/kg dose. The frequency of micronucleus formation was slightly higher in inactivated Polk KI than in WT mice at the same dose, but it was statistically insignificant. The results suggest that Polk has a limited ability to suppress BP-induced genotoxicity in the colon and bone marrow and also that the roles of specialized DNA polymerases in mutagenesis and carcinogenesis should be examined not only by in vitro assays but also by in vivo mouse studies. We also report the spontaneous mutagenesis in inactivated Polk KI mice at young and old ages. Environ. Mol. Mutagen. 58:644-653, 2017. © 2017 Wiley Periodicals, Inc.

DNA polymerase kappa counteracts inflammation‐induced mutagenesis in multiple organs of mice

“…The number of experimental groups and numbers of mice in the experimental groups were limited by the availability of Pok −/− mice. However, the numbers of mice used in the study were consistent with previous studies (Hakura et al, ; Sonoda et al, ; Masumura et al, ). The overall study design was approved by the Institutional Animal Care and Use Committee of the test site Sunplanet Co. and the experiments were carried out according to the Sunplanet animal experimentation regulations.…”

“…Interestingly, similar enhancement of G:C to C:G mutation is observed in Polk −/− or Polk KO mice at old age (Stancel et al, ; Masumura et al, ). Spontaneous gpt MF increases about eight times at Week 52 in male and female Polk −/− mice and G:C to C:G mutation is about 30 and 60 times higher in liver of male and female Polk −/− mice, respectively, than in liver of corresponding Polk +/+ mice (Masumura et al, ). The similarity of the increase in G:C to C:G mutation led us to suggest that reactive oxygen and nitrogen species and lipid peroxidation products may contribute to the age‐related increase in gpt MF in Polk −/− mice.…”

“…The Polk −/− mice express catalytically inactive Polκ from its cognate promoter and possess a transgene gpt as a reporter for mutation in vivo (Takeiri et al, ). Despite the in vitro results that Polκ efficiently bypasses N 2 ‐dG adducts by BPDE in an error‐free manner, Polk −/− mice did not exhibit appreciably higher MF to BP than did Polk +/+ mice (Masumura et al, ). In this study, we compared carcinogenic sensitivity to BP between Polk −/− and Polk +/+ mice.…”

“…In addition, Polκ appears to be involved in nucleotide excision repair (Ogi et al, 2010), replication checkpoint (Betous et al, 2013), DNA synthesis through non-B structures (Betous et al, 2009) and repair of strand breaks in DNA (Zhang et al, 2013). Both knock-in mice whose catalytic activity of Polκ is specifically inactivated (hereafter we call the inactivated Polk knock-in gpt delta mice simply Polk −/− mice) (Takeiri et al, 2014;Masumura et al, 2017) and knockout mice (Stancel et al, 2009) are viable and exhibit spontaneous increase in mutant frequency (MF) in the liver at old age. Despite numerous in vitro studies, protective roles of Polκ against genotoxic agents in vivo (whole body) remain largely unknown.…”

“…To gain insights into in vivo protective roles, we previously examined sensitivity of Polk −/− mice to the genotoxic effects of BP via intragastric gavage route, and compared MF in colon with that of gpt delta mice (hereafter we call gpt delta mice Polk +/+ mice) (Nohmi et al, 1996;Nohmi et al, 2000;Masumura et al, 2017). BP was chosen because it is the most extensively characterized mutagen for Polκ (Ogi et al, 2002;Suzuki et al, 2002;Avkin et al, 2004;Niimi et al, 2009;Kanemaru et al, 2015) and induces high MF in colon of gavaged mice (Hakura et al, 1998;Hakura et al, 1999).…”