Harald J.J. Moonen

Antioxidant status associated with inflammation in sarcoidosis: a potential role for antioxidants.

RATIONALE Enhanced production of reactive oxygen species (ROS), capable of reducing endogenous defense levels and enhancing inflammation, is suggested to play a role in sarcoidosis. Antioxidant supplementation might offer protection against such ROS-mediated damage. A promising candidate for antioxidant supplementation is the flavonoid quercetin. AIM To determine the antioxidant and inflammatory status in sarcoidosis. Furthermore, the potential of quercetin to mitigate the occurring inflammation will be assessed. METHODS Non-smoking sarcoidosis patients and healthy controls matched for age, gender and dietary behavior were enrolled (NCT-00512967). Measurements included assessment of total plasma antioxidant capacity, vitamin C, uric acid, glutathione, basal and LPS-induced levels of tumor necrosis factor alpha (TNFalpha), interleukin (IL)-8 and -10 as well as the effect of quercetin on these levels. RESULTS Compared to their controls, the sarcoidosis patients displayed significantly lower total plasma antioxidant capacity, decreased levels of vitamin C, uric acid and glutathione and increased levels of basal TNFalpha and IL-8. Quercetin significantly decreased ex vivo LPS-induced TNFalpha- and IL-8 production in a concentration-dependent manner in both groups. Interestingly, this quercetin effect was more pronounced in sarcoidosis patients. DISCUSSION The endogenous antioxidant defense was significantly reduced in sarcoidosis, indicating that oxidative stress underlies the pathology of this disease. Furthermore, the inflammatory status was significantly enhanced in sarcoidosis. Finally, our results regarding the effect of quercetin on cytokine production imply that sarcoidosis patients might benefit from antioxidant supplementation not only by empowering the relatively low protection against ROS but also by reducing inflammation.

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.

Generation of free radicals and induction of DNA adducts by activation of heterocyclic aromatic amines via different metabolic pathways in vitro

Food‐derived heterocyclic aromatic amines (HCAs) have proved to be carcinogenic in both rodents and nonhuman primates. Two different metabolic pathways are suggested for the metabolic activation of HCA. The hepatic pathway proceeds via a two‐step process involving N‐hydroxylation by cytochrome P4501A2 and subsequent O‐acetylation by N‐acetyltransferase‐2. An alternative pathway may be of particular interest in extrahepatic tissues and proceeds via one‐electron oxidation catalyzed by prostaglandin H synthase (PHS), rendering free‐radical metabolites. In this study, we investigated the metabolic activation of two HCAs, 2‐amino‐3‐methylimidazo[4,5‐f]quinoline (IQ) and 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP), by two different enzyme systems in vitro, generating different primary and secondary reactive metabolites. Rat liver S9 mix and PHS were used as the activating system and represent the hepatic and extrahepatic pathways, respectively. Electron‐spin resonance spectroscopy showed that both IQ and PhIP exerted inhibiting effects on PHS‐mediated formation of hydroxyl radicals during the conversion of arachidonic acid to prostaglandins. Evidence for the formation of HCA free radicals was presented in an indirect way by the formation of glutathione‐derived thiyl radicals, with purified PHS as the activating system. Activation by S9 mix did not result in the formation of detectable radical metabolites, showing that the two metabolic routes primarily led to the formation of different metabolites. In all electron‐spin resonance experiments, IQ appeared to be more effective than PhIP. In contrasts, DNA adduct analysis by means of 32P‐postlabeling showed similar adduct patterns for S9 and PHS in single‐stranded and double‐stranded salmon testes DNA after incubation with PhIP, indicating the ultimate formation of a common reactive intermediate. For IQ, activation by PHS led to an additional adduct spot that was not present after S9 activation. Furthermore, activation of IQ resulted in higher adduct levels compared with PhIP for both activation pathways. Overall, adduct levels were higher in single‐stranded DNA than double‐stranded DNA. Our results showed that the hepatic and extrahepatic pathways resulted in different primary metabolites, while the ultimate formation of a similar reactive intermediate for PhIP, possibly an arylnitrenium ion, suggested that both pathways could play an important role in the onset of carcinogenesis.

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.