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 µM), 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 (H 2 O 2 )), 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. Published 2006 Wiley‐Liss, Inc. †
Many environmental metals are co-carcinogens, eliciting their effects via inhibition of DNA repair. Apurinic/apyrimidinic (AP) endonuclease 1 (Ape1) is the major mammalian abasic endonuclease and initiates repair of this cytotoxic/mutagenic lesion by incising the DNA backbone via a Mg(2+)-dependent reaction. In this study we examined the effects of arsenite [As(III)], cadmium [Cd(II)], cobalt [Co(II)], iron [Fe(II)], nickel [Ni(II)], and lead [Pb(II)] at concentrations ranging from 0.3 to 100 microM on the incision activity of Ape1 in the presence of 1 mM MgCl(subscript)2(/subscript). Pb(II) and Fe(II) inhibited Ape1 activity at each of the concentrations tested, with an IC(subscript)50(/subscript) (half-maximal inhibitory concentration) of 0.61 and 1.0 microM, respectively. Cd(II) also inhibited Ape1 activity but only at concentrations > 10 microM. No inhibition was seen with As(III), Co(II), or Ni(II). A similar inhibition pattern was observed with the homologous Escherichia coli protein, exonuclease III, but no inhibition was seen with the structurally distinct AP endonuclease E. coli endonuclease IV, indicating a targeted effect of Pb(II), Fe(II), and Cd(II) on the Ape1-like repair enzymes. Excess nonspecific DNA did not abrogate the metal inactivation, suggesting a protein-specific effect. Notably, Cd(II), Fe(II), and Pb(II) [but not As(III), Co(II), or Ni(II)] inhibited AP endonuclease activity in whole-cell extracts but had no significant effect on single nucleotide gap filling, 5'-flap endonuclease, and nick ligation activities, supporting the idea of selective inactivation of Ape1 in cells. Our results are the first to identify a potential DNA repair enzyme target for lead and suggest a means by which these prevalent environmental metals may elicit their deleterious effects.
The Cockayne syndrome B (CSB) protein—defective in a majority of patients suffering from the rare autosomal disorder CS—is a member of the SWI2/SNF2 family with roles in DNA repair and transcription. We demonstrate herein that purified recombinant CSB and the major human apurinic/apyrimidinic (AP) endonuclease, APE1, physically and functionally interact. CSB stimulates the AP site incision activity of APE1 on normal (i.e. fully paired) and bubble AP–DNA substrates, with the latter being more pronounced (up to 6-fold). This activation is ATP-independent, and specific for the human CSB and full-length APE1 protein, as no CSB-dependent stimulation was observed with Escherichia coli endonuclease IV or an N-terminal truncated APE1 fragment. CSB and APE1 were also found in a common protein complex in human cell extracts, and recombinant CSB, when added back to CSB-deficient whole cell extracts, resulted in increased total AP site incision capacity. Moreover, human fibroblasts defective in CSB were found to be hypersensitive to both methyl methanesulfonate (MMS) and 5-hydroxymethyl-2′-deoxyuridine, agents that introduce base excision repair (BER) DNA substrates/intermediates.
Abstract Previous studies using rodent cells indicate that a deficiency in XRCC1 results in reduced single‐strand break repair, increased sensitivity to DNA‐damaging agents, and elevated levels of sister chromatid exchange (SCE). Epidemiological studies have suggested an association of certain human XRCC1 polymorphisms with genetic instability and cancer susceptibility. However, investigations on the molecular functions of XRCC1 in human cells are limited. To determine the contributions of this nonenzymatic scaffold protein, we suppressed XRCC1 levels in several human cell lines using small interfering RNA (siRNA) technology. We report that XRCC1 down‐regulation in HeLa cells leads to a concomitant decrease in the DNA ligase 3 protein level and an impaired nick ligation capacity. In addition, depletion of XRCC1 resulted in a significantly increased sensitivity to the alkylating agent methyl methanesulfonate and the thymidine base analog 5‐hydroxymethyl‐2′‐deoxyuridine, a slightly increased sensitivity to ethyl methanesulfonate and 1,3‐ bis (2‐chloroethyl)‐1‐nitrosourea, and no change in the response to camptothecin. We also discovered that a 70–80% reduction in XRCC1 protein leads to an elevated level of SCE in both HeLa cells and normal human fibroblasts, but does not affect chromosome aberrations in the diploid fibroblasts. Last, XRCC1 siRNA transfection led to an ∼40% decrease in the survival of BRCA2‐deficient cells, supporting a model whereby the accumulation of unrepaired SSBs leads to the accumulation of cytotoxic DNA double strand breaks following replication fork collapse in cells defective in homologous recombination. Environ. Mol. Mutagen., 2007. Published 2007 Wiley‐Liss, Inc.
There is currently a great interest in the efficiency of micronutrients against age-associated disorders. The present study aimed to evaluate the efficacy of beta-carotene on the incidence of lymphoid neoplasia, a fatal pathology associated with OFI mouse ageing. Beta-carotene, given as a water-dispersible preparation to 8-month-old mice, on a four month follow-up study, significantly reduced the incidence of neoplasm (12.5% versus 50% for controls). Evaluation of the parameters of oxidative stress showed a highly-significant reduction of the antioxidant defenses in the liver of cancer mice when compared to healthy controls (78% decrease in GSH-Px activity and 47% decrease of the ratio GSH/GSSG). Liver GSH-Px activity was 35% higher in old than in young mice, which correlated with higher (41%) plasma Se level. In conclusion beta-carotene improved the antioxidant status of the mice, causing a 4.5-fold increase in the liver GSH/GSSG ratio, an effect which was probably responsible for the lowered incidence of neoplasia observed.
