T.M.C.M. de Kok

Induction of DNA adducts by several polychlorinated biphenyls.

It is known that lower‐chlorinated biphenyls are metabolically activated to electrophilic quinoid species capable of binding to DNA. Also, certain metabolites are capable of redox cycling, thereby increasing oxidative stress in biological systems. In the present study, we tested mono‐, di‐, tri‐, tetra‐, penta‐, hexa‐, and heptachlorinated biphenyls for their ability to bind with DNA and to induce oxidative DNA damage. We present additional evidence that several PCB congeners form DNA adducts after metabolic activation, which can be detected by the nuclease P1‐ or butanol‐enrichment procedures of the 32P‐postlabeling technique. Butanol and nuclease P1 enrichments showed different adduct recoveries, depending on the level of chlorination of the biphenyls. Application of the nuclease P1 enrichment showed that the incubation of 2‐chloro‐; 3,4‐dichloro‐; 2,4,4′‐trichloro‐; 3,4,5‐trichloro‐; and 2,2′,5,5′‐tetrachlorobiphenyl with calf thymus DNA and liver microsomes from rats treated with phenobarbital, followed by oxidation with a peroxidase, produced five to eight different DNA adducts. For these lower‐chlorinated biphenyls, butanol enrichment generally showed a lower recovery. For some higher substituted congeners (3,3′,4,4′,5‐pentachloro‐, 2,2′,3,4,4′,5′‐hexachloro‐, 2,2′,4,4′,5,5′‐hexachloro‐, and 2,2′,3,4,4′,5,5′‐heptachlorobiphenyl), after butanol enrichment a single dominant spot was observed, which was absent in the nuclease P1 procedure. After incubation of calf thymus DNA with either higher‐ or lower‐chlorinated PCB congeners, we were not able to detect significantly increased levels of oxidative DNA damage above background levels, measured as 8‐oxo‐7,8‐dihydro‐2′deoxyguanosine. In view of the carcinogenicity of PCB mixtures in animals and the ability of PCB metabolites to bind covalently to DNA, rats were orally treated with a mixture of PCBs (Aroclor 1242). PCB‐DNA adduct levels were analyzed in PCB target organs: liver, thymus, glandular stomach, spleen, testes, seminal vesicles and prostate DNA. In vivo PCB‐DNA adducts could not be detected by either the butanol‐ or by the NP1‐enrichment procedure in rat target tissue DNA. Also, no differences in oxidative DNA damage could be observed between PCB‐treated rats and controls. These results indicate a lack of DNA reactivity of PCB mixtures in vivo. Environ. Mol. Mutagen. 36:79–86, 2000.

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.

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.

Ah receptor agonist activity in frequently consumed food items

The aryl hydrocarbon receptor (AhR) receives much attention for its role in the toxicity of dioxins and dioxin-like polychlorinated biphenyls. However, many other compounds have also been reported to bind and activate AhR, of which natural food components are of special interest from a human health perspective. Using the dioxin receptor–chemical-activated luciferase gene expression (DR CALUX®) bioassay, extracts from many food items frequently consumed in the Netherlands were screened to estimate the intake of natural AhR agonists (NAhRAs). Using the prototypical AhR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as standard, it was estimated that the daily intake of NAhRAs might be considerably higher than the reported intake of dioxins and dioxin-like polychlorinated biphenyls. Potatoes, cruciferous vegetables, bread, hamburgers, and grapefruit juice contained most NAhRAs. Food preparation and acid treatment can show a significant effect on AhR activation. The interaction of natural and xenobiotic AhR agonists should be taken into account when performing risk–benefit analysis of both types of compounds.

Dietary acrylamide intake and the risk of colorectal cancer with specific mutations in KRAS and APC

Acrylamide, a probable human carcinogen, is present in heat-treated carbohydrate-rich foods. Epidemiological studies have not shown a clear association between acrylamide intake and colorectal cancer (CRC) risk. This may be due to the molecular heterogeneity in colorectal tumors, which was not taken into consideration before. Since the acrylamide metabolite glycidamide induces specific DNA mutations in rodents, we investigated whether acrylamide is associated with CRC risk characterized by mutations in Kirsten-ras (KRAS) and adenomatous polyposis coli (APC); key genes in colorectal carcinogenesis. This case-cohort analysis, within the Netherlands Cohort Study on diet and cancer, was based on 7.3 years of follow-up. Acrylamide intake was assessed with a food frequency questionnaire. Mutation analysis of codons 1286-1520 in exon 15 in APC and codons 12 and 13 in exon 1 in KRAS was performed on tumor tissue of 733 cases. Hazard ratios (HR) were calculated using Cox proportional hazards analysis. Among men, acrylamide intake was statistically significantly associated with an increased risk of particularly tumors with an activating KRAS mutation {HR fourth versus first quartile: 2.12 [95% confidence interval (CI): 1.16-3.87], P trend: 0.01}. Among women, acrylamide intake was statistically significantly associated with a decreased risk of particularly tumors with a truncating APC mutation (fourth versus first quartile: 0.47 (95% CI: 0.23-0.94), P trend: 0.02), but only in the highest quartile of intake. This is the first study to show that acrylamide might be associated with CRC with specific somatic mutations, differentially in men and women. More research is needed to corroborate or refute these findings.

Radicals in the church

To the Editors: Public health is seriously threatened by exposure to ambient and indoor particulate matter (PM). Epidemiological studies have demonstrated that long-term exposure is associated with lung cancer and other pulmonary diseases 1. In vitro studies have demonstrated the involvement of polycyclic aromatic hydrocarbons (PAH), inflammation and oxidative stress in these adverse health effects 2–4. As free radicals are also known to play a crucial role in these processes, we developed an electron spin resonance (ESR)-based methodology to quantify the radical-generating capacity of PM. We applied this methodology in order to evaluate the effect of burning candles and incense, known sources of indoor air PM …