In vitro genotoxicity of lead acetate: induction of single and double DNA strand breaks and DNA–protein cross-links

Abstract

Lead is present in the natural and occupational environment and is reported to interact with DNA, but the mechanism of this interaction is not fully understood. Using the alkaline comet assay we showed that lead acetate at 1–100 μM induced DNA damage in isolated human lymphocytes measured the change in the comet tail length. At 1 and 10 μM we observed an increase in the tail length, whereas at 100 μM a decrease was seen. The former effect could follow from the induction of DNA strand breaks and/or alkali-labile sites (ALS), the latter from the formation of DNA–DNA and/or DNA–protein cross-links. No difference was observed between tail length for the alkaline and pH 12.1 versions of the assay, which indicates that strand breaks and not ALS are responsible for the tail length increase induced by lead. The neutral version of the test revealed that lead acetate induced DNA double-strand breaks at all concentrations tested. The presence of spin traps, 5,5-dimethyl-pyrroline N-oxide (DMPO) and N- tert-butyl-α-phenylnitrone (PBN) did not influence the level of DNA damage induced by lead. Post-treatment of the lead-damaged DNA (at 100 μM treatment concentration) by endonuclease III (Endo III) and formamidopyrimidine-DNA glycosylase (Fpg), enzymes recognizing oxidized DNA bases, as well as 3-methyladenine-DNA glycosylase II, an enzyme recognizing alkylated bases, gave rise to a significant increase in the extent of DNA damage. Proteinase K caused an increase in comet tail length, suggesting that lead acetate might cross-link DNA with nuclear proteins. Vitamin A, E, C, calcium chloride and zinc chloride acted synergistically on DNA damage evoked by lead. The results obtained suggest that lead acetate may induce single-strand breaks (SSB) and double-strand breaks (DSB) in DNA as well as DNA–protein cross-links. The participation of free radicals in DNA-damaging potential of lead is not important and it concerns other reactive species than could be trapped by DMPO or PBN.