Lead promotes abasic site accumulation and co‐mutagenesis in mammalian cells by inhibiting the major abasic endonuclease Ape1

Abstract

Lead is a widespread environmental toxin, found in contaminated water sources, household paints, and certain occupational settings. Classified as a probable carcinogen by the International Agency for Research on Cancer (IARC), lead promotes mutagenesis when combined with alkylating and oxidizing DNA-damaging agents. We previously reported that lead inhibits the in vitro repair activity of Ape1, the major endonuclease for repairing mutagenic and cytotoxic abasic sites in DNA. We investigated here whether lead targets Ape1 in cultured mammalian cells. We report a concentration-dependent inhibition of apurinic/apyrimidinic (AP) site incision activity of Chinese hamster ovary (CHO) AA8 whole cell extracts by lead. In addition, lead exposure results in a concentration-dependent accumulation of AP sites in the genomic DNA of AA8 cells. An increase in the oxidative base lesion 8-oxoguanine was observed only at high lead levels (500 microM), suggesting that non-specific oxidation plays little role in the production of lead-related AP lesions at physiological metal concentrations--a conclusion corroborated by "thiobarbituric acid reactive substances" assays. Notably, Ape1 overexpression in AA8 (hApe1-3 cell line) abrogated the lead-dependent increase in AP site steady-state levels. Moreover, lead functioned cooperatively to promote a further increase in abasic sites with agents known to generate AP sites in DNA (i.e., methyl methansulfonate (MMS) and hydrogen peroxide (H2O2), but not the DNA crosslinking agent mitomycin C. Hypoxanthine guanine phosphoribosyltransferase (hprt) mutation analysis revealed that, whereas lead alone had no effect on mutation frequencies, mutagenesis increased in MMS treated, and to a greater extent lead/MMS treated, AA8 cells. With the hApe1-3 cell line, the number of mutant colonies in all treatment groups was found to be equal to that of the background level, indicating that Ape1 overexpression reverses MMS- and lead-associated hprt mutagenesis. Our studies in total indicate that Ape1 is a member of an emerging group of DNA surveillance proteins that are inhibited by environmental heavy metals, and suggest an underlying mechanism by which lead promotes co-carcinogenesis.

Human Skin Cells Are More Sensitive than Human Lung Cells to the Cytotoxic and Cell Cycle Arresting Impacts of Particulate and Soluble Hexavalent Chromium

“…However, lead can interfere with the repair of DNA damage. One study reported lead inhibits theapurinic/apyrimidinic endonuclease (APE1), an important repair protein in the DNA base excision repair pathway [34]. Gastaldo et al, showed lead inhibits the non-homologous DNA end-joining (NHEJ) double strand break repair process by inhibiting DNA-PK activity [35].…”

Human Embryonic Stem Cells Have Enhanced Repair of Multiple Forms of DNA Damage

“…8-Hydroxydeoxyguanosine (8-oxoG) was quantified as previously described [34]: DNA was extracted using a salting out procedure [35,36]. Specifically, cell pellets were resuspended in lysis buffer (0.5 M Tris-HCl, pH 8, 20 mM EDTA, 10 mM NaCl, 1% SDS, and 0.5 mg/ml proteinase K) and incubated overnight at 37°C.…”

A Dominant-Negative Form of the Major Human Abasic Endonuclease Enhances Cellular Sensitivity to Laboratory and Clinical DNA-Damaging Agents

“…Cells were then challenged with 0.4 mmol/L MMS for 1 h at 37jC or received no treatment. Cells were harvested and processed as outlined by McNeill et al (56). In brief, abasic site quantification consisted of isolating the chromosomal DNA from MMS-treated or untreated cells using the GetpureDNA kit according to the manufacturer's specifications.…”