Abstract
B27
Introduction: Benzo[a]pyrene B[a]P is a member of a family of polycyclic aromatic hydrocarbons that are ubiquitous environmental carcinogens. B[a]P is metabolically activated to the proximate carcinogen B[a]P-7,8-dihydrodiol which is converted to a catechol by Aldo-Keto Reductases (AKRs). The catechol metabolite then undergoes two sequential 1-electron oxidations to give B[a]P-7,8-dione with the concomitant generation of reactive oxygen species (ROS). B[a]P-7,8-dione is then reduced back to the catechol establishing a futile redox cycle, which continually produces ROS that in turn can cause the formation of 8-oxo-2’-deoxyguanosine (8-oxo-dGuo) adducts in DNA. >Experimental Procedure: It is difficult to quantify true cellular 8-oxo-dGuo levels due to artifact formation during isolation and hydrolysis of the DNA from the cells and tissue samples by the presence of trace amounts of transition metal ions in the buffers that are used. To avoid this problem a cold guanidine thiocyanate non-phenolic method was used to extract DNA from cells and desferal was used in Chelex-treated buffers to remove transition metal ions. This prevented the artifactual formation of 8-oxo-dGuo from Fenton Chemistry. Immunoaffinity purification was employed in order to remove interfering substances that co-eluted with 8-oxo-dGuo during subsequent chromatography. Stable isotope dilution liquid chromatography/electrospray ionization/multiple reaction monitoring/mass spectrometry (LC/ESI/MRM/MS) was used to provide maximal specificity and sensitivity. Human bronchoalveolar cells were treated with B[a]P-7,8-dihydrodiol and 8-oxo-dGuo present in the DNA was quantified. >Results: The 8-oxo-dGuo levels were increased after B[a]P-7,8-dihydrodiol treatment compared to untreated cells and DMSO treated cells. The 8-oxo-dGuo levels were increased in dose dependent manner. However, when the cells were pre-treated with the COMT inhibitor, R041-0961, a significant increase in 8-oxo-dGuo levels was observed. The COMT inhibitor alone had no effect on 8-oxo-dGuo formation. B[a]P-7,8-dione formation in the cells indicated that 8-oxo-dGuo was formed by the ROS generated during the two electron oxidation of the B[a]P-7,8-catechol. >Conclusion: These data confirm that the new LC-MS-based method efficiently eliminated artifact formation that occurred during the isolation and hydrolysis of DNA. This made it possible to detect elevated 8-oxo-dGuo levels on exposure of H358 cells to B[a]P-7,8-dihydrodiol. The formation of B [a]P-7,8-dione in the cells also supports the proposed formation of ROS during the oxidation of the catechol metabolite. The increased level of 8-oxo-dGuo on pretreatment of the COMT inhibitor, suggests that the catechol metabolite is normally detoxified by conversion to a methyl ether derivative. Supported by NIH grants 1R01CA130038 and 5P30ES013508.
Sixth AACR International Conference on Frontiers in Cancer Prevention Research-- Dec 5-8, 2007; Philadelphia, PA