Comparative Effects of Mercury(II) and Cadmium on MutS Homolog 6(MSH6)-Mediated DNA Mismatch Binding Activities in Zebrafish (Danio rerio) Embryos.

MutS homolog 6 (MSH6) of the MSH2-MSH6 complex binds simple mispairs and small insertion-deletion loops (IDL) then initiates DNA mismatch repair in eukaryotes. We have shown the ability of Cd(2+) to downregulate msh2/msh6 expression in zebrafish embryos via oxidative stress. This study explored the effects of Cd(2+) and Hg(2+) on MSH6-mediated mismatch binding activities. MSH6-mediated G-T and IDL-specific binding activities were significantly inhibited at similar levels after exposing zebrafish embryos at 1 h postfertilization) to HgCl2 or CdCl2 at 1.0 to 2.5 µM for 9 h, but MSH6 synthesis was found to be less sensitive to Hg(2+) than to Cd(2+) . Real-time RT-PCR and in situ hybridization also detected a weaker susceptibility of MSH gene transcription to Hg(2+) . The weaker response of MSH gene activities to Hg(2+) correlated with the lower oxidative stress-inducing potential of Hg(2+) . Hence, Hg(2+) targets mismatch sensing capacity at protein function rather than at transcription level.

Cadmium(Cd)-induced oxidative stress down-regulates the gene expression of DNA mismatch recognition proteins MutS homolog 2 (MSH2) and MSH6 in zebrafish (Danio rerio) embryos.

DNA mismatch repair (MMR) of simple base mismatches and small insertion-deletion loops in eukaryotes is initiated by the binding of the MutS homolog 2 (MSH2)-MSH6 heterodimer to mismatched DNA. Cadmium (Cd) is a genotoxic heavy metal that has been recognized as a human carcinogen. Oxidant stress and inhibition of DNA repair have been proposed as major factors underlying Cd genotoxicity. Our previous studies indicated the ability of Cd to disturb the gene expression of MSH6 in zebrafish (Danio rerio) embryos. This study was undertaken to explore if Cd-induced oxidative stress down-regulated MSH gene activities. Following the exposure of zebrafish embryos at 1 h post fertilization (hpf) to sublethal concentrations of Cd at 3-5 µM for 4 or 9 h, a parallel down-regulation of MSH2, MSH6 and Cu/Zn superoxide dismutase (Cu/Zn-SOD) gene expression was detected by real-time RT-PCR and the expression levels were 40-50% of control after a 9-h exposure. Cd exposure also induced oxidative stress, yet no inhibition of catalase gene activity was observed. Whole mount in situ hybridization revealed a wide distribution of msh6 mRNA in the head regions of 10 hpf embryos and pretreatment of embryos with antioxidants butylhydroxytoluene (BHT), d-mannitol or N-acetylcysteine (NAC) at 1-10 µM restored Cd-suppressed msh6 expression. QPCR confirmed the protective effects of antioxidants on Cd-suppressed msh2/msh6 mRNA production. Down-regulated MSH gene activities reaching about 50% of control were also induced in embryos exposed to paraquat, a reactive oxygen species (ROS)-generating herbicide, or hydrogen peroxide at 200 µM. Hence, Cd at sublethal levels down-regulates msh2/msh6 expression primarily via ROS as signaling molecules. The transcriptional activation of human msh6 is known to be fully dependent on the specificity factor 1 (Sp1). Cd failed to inhibit the DNA binding activity of zebrafish Sp1 unless at lethal concentrations based on band shift assay, therefore excluding the involvement of Sp1 inactivation in Cd-induced MSH gene inhibition in zebrafish embryos.