Immunogold electron microscopy in situ end-labeling (EM-ISEL): assay for biomaterial DNA damage detection.

ABSTRACT

We have evaluated a genotoxicity assay that combines in situ end-labeling, colloidal gold tagging and electron microscopy in order to adapt it to the measurement of in vitro biomaterial-induced genotoxicity. Human lymphocytes were cultured in semi-physiological medium which had been previously exposed to biomaterial extracts of commercially pure titanium following ISO standards. In order to visualize the location of induced DNA strand breaks, cells were then exposed to exonuclease III which partially digests and amplifies lesions by releasing nucleotides at free 3' hydroxyl ends from nicked double-stranded DNA. The resulting single-stranded DNA was allowed to hybridize with short oligonucleotides of random sequences including biotinylated dUTP. After random priming using Escherichia coli DNA polymerase I, incorporation of biotin-dUTP was detected by immunogold binding to the chromatin. Cells exposed to a mutagenic concentration of methyl methanesulfonate, as a positive control, showed a significantly higher and stronger gold staining than both titanium-exposed and unexposed specimens. This assay allows a precise localization and quantification of both in vitro DNA breakage and DNA repair. It could provide a powerful tool for rapid assessment of the genotoxic potential of new biomaterials.