ABSTRACT
Acrolein (Acr) is a major component in cigarette smoke and a ubiquitous environmental pollutant. It is also formed as a product of lipid peroxidation. Following ring closure via the Michael addition, Acr modifies deoxyguanosine (dG) in DNA by forming cyclic 1,N(2)-propanodeoxyguanosine adducts (OHPdG). The reactions of Acr with dG yield, depending on the direction of ring closure, two regioisomers, α- and γ-OHPdG, in approximately equal amounts. However, previous (32)P-postlabeling studies showed that the γ isomers were detected predominantly in the DNA of rodent and human tissues. Because of the potential differential biological activity of the isomeric OHPdG adducts, it is important to confirm and study the chemical basis of the regioselective formation of γ isomers in vivo. In this study, it is confirmed that γ-OHPdG adducts are indeed the major isomers formed in vivo as evidenced by a LC-MS/MS method specifically developed for Acr-derived dG adducts. Furthermore, we have shown that the formation of γ-isomers is increased in the presence of amino-containing compounds, including amino acids, proteins, and cell lysates. A product of Acr and arginine that appears to mediate the regioselective formation of γ isomers was identified, but its structure was not fully characterized due to its instability. This study demonstrates that intracellular amino-containing compounds may influence the regiochemistry of the formation of OHPdG adducts and reveals a mechanism for the preferential formation of γ-OHPdG by Acr in vivo.
Subject(s)
Acrolein/chemistry , Amino Acids/chemistry , DNA Adducts/chemistry , Deoxyguanosine/analogs & derivatives , Proteins/chemistry , Animals , Borohydrides/chemistry , Cell Line , Chromatography, High Pressure Liquid , DNA/chemistry , Deoxyguanosine/chemistry , Histones/chemistry , Humans , Liver/chemistry , Oxidation-Reduction , Proteins/metabolism , Rats , Serum Albumin, Bovine/chemistry , Smoking , Spermidine/chemistry , StereoisomerismABSTRACT
We show that naturally occurring isothiocyanates (ITCs) sensitize human non-small cell lung cancer cells to cisplatin. Moreover, the structure of the ITC side chain moiety is important for sensitization. In NCI-H596 cells, 20 microM benzyl isothiocyanate (BITC) and phenethyl isothiocyanate (PEITC) enhance the efficacy of various concentrations of cisplatin, but sulforaphane (SFN) does not. Reducing the concentration of BITC and PEITC to 10 microM still allows for the sensitization of cells to cisplatin. Neither cellular platinum accumulation nor DNA platination account for this increased cytotoxicity. BITC and PEITC deplete beta-tubulin, but SFN does not; this correlates with and may be important for sensitization.
Subject(s)
Antineoplastic Agents/pharmacology , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/pathology , Cisplatin/pharmacology , Isothiocyanates/pharmacology , Lung Neoplasms/drug therapy , Antineoplastic Agents/chemistry , Carcinoma, Non-Small-Cell Lung/metabolism , Cell Line, Tumor , Cell Survival/drug effects , Cisplatin/chemistry , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Humans , Isothiocyanates/chemistry , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Molecular Structure , Sensitivity and SpecificityABSTRACT
Oxidation of polyunsaturated fatty acids (PUFAs) releases alpha,beta-unsaturated aldehydes that modify deoxyguanosine (dG) to form cyclic 1,N(2)-propanodeoxyguanosine adducts. One of the major adducts detected in vivo is acrolein (Acr)-derived 1,N(2)-propanodeoxyguanosine (Acr-dG). We used a chemical model system to examine the effects of 4 antioxidants known to inhibit fatty acid oxidation on the formation of Acr-dG and 8-oxodeoxyguaonsine (8-oxodG) from the PUFA docosahexaenoic acid (DHA) under oxidative conditions. We found that epigallocatechin gallate (EGCG) and dihydrolipoic acid (DHLA) inhibit both Acr-dG and 8-oxodG formation. In contrast, ascorbic acid and alpha-tocopherol actually increase Acr-dG at high concentrations and do not show a concentration-dependant inhibition of 8-oxodG. We also studied their effects on blocking Acr-dG formation directly from Acr. EGCG and DHLA can both effectively block Acr-dG formation, but ascorbic acid and alpha-tocopherol show weak or little effect. These results highlight the complexity of antioxidant mechanisms and also reveal that EGCG and DHLA are effective at suppressing lipid peroxidation-induced Acr-dG and 8-oxodG formation as well as blocking the reaction of dG with Acr.