Lead promotes abasic site accumulation and co-mutagenesis in mammalian cells by inhibiting the major abasic endonuclease Ape1.
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.
McNeill
Daniel R
DR
Laboratory of Molecular Gerontology, GRC, National Institute on Aging, IRP, NIH, Baltimore, Maryland 21224-6825, USA.
Wong
Heng-Kuan
HK
Narayana
Avinash
A
Wilson
David M
DM
D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D004249:DNA Damage; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000037:antagonists & inhibitors*; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D007854:Lead / Q000633:toxicity*; D016296:Mutagenesis; D009153:Mutagens / Q000633:toxicity; D017392:Thiobarbituric Acid Reactive Substances / Q000378:metabolism
D009153:Mutagens; D017392:Thiobarbituric Acid Reactive Substances; D007854:Lead; D007041:Hypoxanthine Phosphoribosyltransferase; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase