An applied synchronous fluorescence spectrophotometric assay to study benzo[a]pyrene-diolepoxide-DNA adducts

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Abstract
Synchronous scanning fluorescence with a fixed wavelength difference (Δλ) of 34 nm between excitation and emission was used to quantitate benzo[a]pyrene-diol epoxide (BPDE)-DNA adducts. Fluorescence emission maxima occurred at 382 nm for BPDE-DNA and at 379 nm for benzo[a]pyrene-tetrols and -triol, which are hydrolysis products of BPDE. Similarly, the peak for pyrene was at 372 nm and for 1-nitropyrene at 386 nm. The minimum detectable amount of BPDE-moieties in in vitro modified BPDE-DNA, after hydrolysis in HCl, was 20 fmol in 100 μg of DNA, which is equivalent to 1 adduct per 1.4 × 10 7 nucleotides. The correlation of fluorescence intensity and the amount of BPDE-moieties was linear between 20 fmol and 1 pmol. DNA isolated from human lymphoblasts incubated with BDPE had the same fluorescence peak and the correlation between the fluorescence intensity and the amount of BPDE in the incubation mixture was linear. Among the DNA-samples from peripheral blood lymphocytes of 30 aluminum plant workers, only one sample was found to contain a peak similar to BPDE-DNA. None of the DNA-samples from 10 persons not occupationally exposed were positive. Measurement of BPDE-DNA adducts by synchronous fluorescence spectrophotometry should be useful in monitoring human exposure to benzo[a]pyrene.

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