Quantitative detection of benzo[alpha]pyrene diolepoxide-DNA adducts by cryogenic laser induced fluorescence.

Abstract

In the present report, we describe a fluorescence-based method capable of measuring benzo[alpha]pyrene diolepoxide (BPDE) adducts in intact genomic DNA, with a sensitivity of a few hundreds copies per cell. The assay is based on cryogenic laser-induced fluorescence technology at liquid nitrogen temperatures, coupled with an intensified charge-coupled device camera, and incorporates several enhancements to existing methodologies. One important modification was the incorporation of terbium(III)nitrate pentahydrate, Tb(NO3)3, as an internal fluorescence standard to correct for differences in light scattering and fluctuations in instrument parameters. Since the fluorescence spectrum of Tb(NO3)3 does not overlap with those of BPDE-DNA adducts, use of this lanthanide salt markedly improved the sensitivity of cryogenic laser-induced fluorescence. The limit of quantification of the assay is 6.4 BPDE-DNA adducts/10(8) nucleotides, or 776 adducts/cell, using 22.5 micrograms of genomic DNA. This assay is rapid, highly sensitive, and economical and has been applied to monitor DNA adduct levels as a function of time after exposure to BPDE in repair-competent human lymphoblastoid AHH-1 and TK6 cells.