Effects of polyunsaturated fatty acids on prostaglandin synthesis and cyclooxygenase-mediated DNA adduct formation by heterocyclic aromatic amines in human adenocarcinoma colon cells.

Dietary heterocyclic aromatic amines (HCA) and polyunsaturated fatty acids (PUFA) are both believed to play a role in colon carcinogenesis, and are both substrate for the enzyme cyclooxygenase (COX). In HCA-7 cells, highly expressing isoform COX-2, we investigated the effects of PUFA on prostaglandin synthesis and DNA adduct formation by the HCA 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). Furthermore, we studied the role of COX, COX-2 in particular, and cytochrome P4501A2 (CYP1A2) by using the enzyme inhibitors indomethacin (IM), NS-398, and phenethyl isothiocyanate (PEITC), respectively. COX-mediated formation of prostaglandin E2 (PGE2) from linoleic acid (LA) showed that HCA-7 cells can convert LA into arachidonic acid (AA). Alternatively, eicosapentaenoic acid (EPA) was found to compete with AA for COX. Strongly decreased PGE2 levels by addition of IM demonstrated involvement of COX in PUFA metabolism. Both IM and NS-398 inhibited adduct formation by HCA to nearly the same extent, indicating involvement of COX-2 rather than COX-1, while CYP1A2 activity in HCA-7 cells was demonstrated by addition of PEITC. Overall, inhibiting effects were stronger for PhIP than for IQ. HCA-DNA adduct formation was stimulated by addition of PUFA, although high PUFA concentrations partly reduced this stimulating effect. Finally, similar effects for n-3 and n-6 fatty acids suggested that adduct formation may not be the crucial mechanism behind the differential effects of PUFA on colon carcinogenesis that have been described. These results show that COX, and COX-2 in particular, can play a substantial role in HCA activation, especially in extrahepatic tissues like the colon. Furthermore, the obvious interactions between PUFA and HCA in COX-2 expressing cancer cells may be important in modulating colorectal cancer risk.

Methodologies for bulky DNA adduct analysis and biomonitoring of environmental and occupational exposures.

It is undisputed that DNA adduct formation is one of the key processes in early carcinogenesis. Therefore, analysis of DNA adduct levels may be one of the best tools available to characterize exposure to complex mixtures of genotoxic chemicals as occurring in different environmental and occupational exposure settings. However, from an analytical point of view the detection and quantification of DNA adducts is a challenging enterprise as extremely high sensitivity and selectivity are required. The entire spectrum of chromatographic techniques, including thin-layer chromatography (TLC), gas and liquid chromatography as well as capillary electrophoresis has been used in combination with different detection systems, all with their own specific characteristics. Among the various combinations of techniques, the TLC-(32)P-postlabeling combination appears to meet best with criteria of sensitivity and requirements of minimal amounts of material. Recent developments in the application of capillary electrophoresis in combination with either immunochemical or mass spectrometric detection techniques may offer new and promising approaches, with higher selectivity as compared to TLC-(32)P postlabeling. The applicability of these new techniques in biomonitoring studies aiming at the exposure and risk assessment of low and chronic exposures remains to be determined. In this paper we compare and discuss the advantages and limitations of different techniques used in DNA adduct analysis, with specific emphasis on those adducts formed by the polycyclic aromatic hydrocarbons and heterocyclic aromatic amines.