Measurement of DNA Adducts as Biomarkers of Genotoxic Exposure to Benzo[A]Pyrene Using a LC-MS/MS Technique

Abstract

P-133 Introduction: Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants representing the first family of carcinogenic pollutants in professional environment. It is now well established that some of these molecules including the tumorigenic benzo[a]pyrene (BaP) are responsible for the apparition of lung, skin and bladder cancers. It is therefore of major importance to evaluate individual exposure to carcinogenic PAHs in order to improve health risk assessment. In addition to the classical determination of atmospheric concentrations of gaseous and particulate PAHs and 1-hydroxyprene excreted in urine, we are interested in measuring specific urinary metabolites and DNA adducts of BaP. Methods: A high performance liquid chromatography coupled to fluorescence detection (HPLC-Fluo) method has been set-up for the urinary determination of 3-hydroxybenzo[a]pyrene (3-OHBaP) and BaP tetrol, and compared to a HPLC coupled to tandem mass spectrometry (HPLC-MS/MS) method. This technique already used for the quantification of 8-oxo-7,8-dihydro-2′-désoxyguanosine was also developed for the measurement of several DNA adducts of BaP. Results: The limit of detection (LOD) obtained for 3-OHBaP is 0.4 ng/L of urine thanks to a double-step procedure of purification and concentration of urinary samples on Sep-Pak cartridges followed by an on-line extraction on the HPLC-Fluo system, and is not improved using a tandem mass spectrometric detection. The LOD of BaP tetrol is currently determined. HPLC-MS/MS allows a highly sensitive and specific detection and quantification of eight stable and depurinating DNA adducts of BaP. These include five DNA adducts of the carbocation pathway and three adducts arising from the BaP-diol-epoxyde pathway. The corresponding limits of detection range from 1 to 40 injected fmoles. Discussion and Conclusions: In the weeks coming, our developed analytical tools will be applied to measure BAP metabolites, DNA adducts and DNA oxidative damages in urinary samples obtained from volunteers professionally exposed to PAHs. The results should be of main importance to identify pertinent biomarkers in order to measure exposure to BaP and assess health risks of workers which may orientate preventing actions.