The benefits and risks of beetroot juice consumption: a systematic review.

Beetroot juice (BRJ) has become increasingly popular amongst athletes aiming to improve sport performances. BRJ contains high concentrations of nitrate, which can be converted into nitric oxide (NO) after consumption. NO has various functions in the human body, including a vasodilatory effect, which reduces blood pressure and increases oxygen- and nutrient delivery to various organs. These effects indicate that BRJ may have relevant applications in prevention and treatment of cardiovascular disease. Furthermore, the consumption of BRJ also has an impact on oxygen delivery to skeletal muscles, muscle efficiency, tolerance and endurance and may thus have a positive impact on sports performances. Aside from the beneficial aspects of BRJ consumption, there may also be potential health risks. Drinking BRJ may easily increase nitrate intake above the acceptable daily intake, which is known to stimulate the endogenous formation of N-nitroso compounds (NOC's), a class of compounds that is known to be carcinogenic and that may also induce several other adverse effects. Compared to studies on the beneficial effects, the amount of data and literature on the negative effects of BRJ is rather limited, and should be increased in order to perform a balanced risk assessment.

Human 3D multicellular microtissues: An upgraded model for the in vitro mechanistic investigation of inflammation-associated drug toxicity.

Inflammation is one of the factors that may increase the sensitivity of hepatic cells to acetaminophen (APAP) induced toxicity. To investigate the mechanisms, we exposed 3-dimensional (3D) Human Liver Microtissues, a co-culture of primary human hepatocytes (PHH) and Kupffer cells (KCs), to 0, 0.5 (low), 5 (median) and 10 mM (high dose) APAP for 24 h, with/without lipopolysaccharide (LPS). Microarray-technology was used to evaluate the transcriptome changes. In the presence of LPS, the median-dose of APAP is sufficient to inhibit the expression of respiratory chain- and antioxidant-related genes, suggesting the involvement of reactive oxygen species (ROS) and oxidative stress. Furthermore, the median- and high-dose of APAP inhibited the expression of Fc fragment receptor (FcγR)-coding genes, regardless of the presence of LPS. The toll-like receptor 4 (TLR4) expression, however, was continuously elevated after the LPS/APAP co-exposures, which may result in reduced KC-phagocytosis and unbalanced cytokine patterns. Compared to the treatment with LPS only, LPS/APAP co-exposures induced the production of interleukin (IL)-8, a pro-inflammatory cytokine, but suppressed the secretion of IL-6, a cytokine regulating hepatic regeneration, along with the increase in APAP dosages. In addition to the disrupted mitochondrial functions, the presence of LPS exacerbated APAP toxicity. These findings suggest that 3D Microtissues are a suitable model for the mechanistic exploration of inflammation-associated drug toxicity.

The idiosyncratic drug-induced gene expression changes in HepG2 cells.

The inflammatory stress has been associated with an increase in susceptibility to idiosyncratic drug-induced liver injury (DILI). However, the molecular mechanisms of this inflammation-associated idiosyncratic drug hepatotoxicity remain unknown. We exposed HepG2 cells with high and low doses of three idiosyncratic (I) and three non-idiosyncratic (N) compounds, in the presence (I+ and N+) or absence (I- and N-) of a cytokine mix for 6, 12 and 24 h. To investigate the genome-wide expression patterns, microarray was performed using the Agilent 4×44K Whole Human Genome chips. The data presented in this DIB include the expression of genes participating in the ceramide metabolism, ER stress, apoptosis and cell survival pathways. The functions of these genes were illustrated in our associated article (Jiang et al., 2017) [1]. Raw and normalized gene expression data are available through NCBI GEO (accession number GSE102006).

Omics-based identification of the combined effects of idiosyncratic drugs and inflammatory cytokines on the development of drug-induced liver injury.

The mechanisms of idiosyncratic drug-induced hepatotoxicity remain largely unclear. It has demonstrated that the drug idiosyncrasy is potentiated in the context of inflammation and intracellular ceramides may play a role in this process. To study the mechanisms, HepG2 cells were co-treated with high and low doses of three idiosyncratic (I) and three non-idiosyncratic (N) compounds, with (I+ and N+) or without (I- and N-) a cytokine mix. Microarray, lipidomics and flow cytometry were performed to investigate the genome-wide expression patterns, the intracellular ceramide levels and the induction of apoptosis. We found that all I+ treatments significantly influenced the immune response- and response to stimulus-associated gene ontology (GO) terms, but the induction of apoptotic pathways, which was confirmed by flow cytometry, only appeared to be induced after the high-dose treatment. The ceramide signaling-, ER stress-, NF-kB activation- and mitochondrial activity-related pathways were biologically involved in apoptosis induced by the high-dose I+. Additionally, genes participating in ceramide metabolism were significantly altered resulting in a measurable increase in ceramide levels. The increases in ceramide concentrations may induce ER stress and activate the JNK pathway by affecting the expression of the related genes, and eventually trigger the mitochondria-independent apoptosis in hepatocytes. Overall, our study provides a potential mechanism to explain the role of inflammation in idiosyncratic drug reactions.

Prediagnostic transcriptomic markers of Chronic lymphocytic leukemia reveal perturbations 10 years before diagnosis.

BACKGROUND: B-cell lymphomas are a diverse group of hematological neoplasms with differential etiology and clinical trajectories. Increased insights in the etiology and the discovery of prediagnostic markers have the potential to improve the clinical course of these neoplasms. METHODS: We investigated in a prospective study global gene expression in peripheral blood mononuclear cells of 263 incident B-cell lymphoma cases, diagnosed between 1 and 17 years after blood sample collection, and 439 controls, nested within two European cohorts. RESULTS: Our analyses identified only transcriptomic markers for specific lymphoma subtypes; few markers of multiple myeloma (N = 3), and 745 differentially expressed genes in relation to future risk of chronic lymphocytic leukemia (CLL). The strongest of these associations were consistently found in both cohorts and were related to (B-) cell signaling networks and immune system regulation pathways. CLL markers exhibited very high predictive abilities of disease onset even in cases diagnosed more than 10 years after blood collection. CONCLUSIONS: This is the first investigation on blood cell global gene expression and future risk of B-cell lymphomas. We mainly identified genes in relation to future risk of CLL that are involved in biological pathways, which appear to be mechanistically involved in CLL pathogenesis. Many but not all of the top hits we identified have been reported previously in studies based on tumor tissues, therefore suggesting that a mixture of preclinical and early disease markers can be detected several years before CLL clinical diagnosis.

Dietary acrylamide intake and risk of breast cancer in the UK women's cohort.

BACKGROUND: No studies to date have demonstrated a clear association with breast cancer risk and dietary exposure to acrylamide. METHODS: A 217-item food frequency questionnaire was used to estimate dietary acrylamide intake in 33,731 women aged 35-69 years from the UK Women's Cohort Study followed up for a median of 11 years. RESULTS: In all, 1084 incident breast cancers occurred during follow-up. There was no evidence of an overall association between acrylamide intake and breast cancer (hazard ratio=1.08 per 10 µg day(-1), 95% CI: 0.98-1.18, P(trend)=0.1). There was a suggestion of a possible weak positive association between dietary acrylamide intake and premenopausal breast cancer after adjustment for potential confounders (hazard ratio=1.2, 95% CI: 1.0-1.3, P(trend)=0.008). There was no suggestion of any association for postmenopausal breast cancer (hazard ratio=1.0, 95% CI: 0.9-1.1, P(trend)=0.99). CONCLUSIONS: There is no evidence of an association between dietary acrylamide intake and breast cancer. A weak association may exist with premenopausal breast cancer, but requires further investigation.

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.

Cigarette smoking and KRAS oncogene mutations in sporadic colorectal cancer: results from the Netherlands Cohort Study.

Since a KRAS oncogene mutation is an early event in colorectal cancer development and cigarette smoking is thought to have an effect on early stages of colorectal tumorigenesis, smoking, especially long-term smoking, may be associated with the risk for colorectal cancer with KRAS oncogene mutations. In the Netherlands Cohort Study on diet and cancer (n=120,852 men and women), using a case-cohort design, adjusted incidence rate ratios (RR) and 95% confidence intervals (CI) were computed for colorectal tumors with wild-type and with mutated KRAS gene, and with specific G:C-->T:A or G:C-->A:T point mutations in KRAS, according to cigarette smoking status, frequency, duration, pack years, age at first exposure, years since cessation, inhalation and filter usage. After 7.3 years and excluding the first 2.3 years, 648 cases and 4083 sub-cohort members were included in the analyses. Ex-smokers, but not current smokers, were at increased risk for colorectal cancer with wild-type KRAS gene tumors when compared with never smokers, albeit not statistically significant (RR 1.26, 95% CI 0.96-1.66). This was not observed for KRAS mutated tumors when comparing ex-smokers with never smokers (RR 1.15, 95% CI 0.79-1.66). The highest category of smoking frequency (>20 cigarettes/day) and inhalation of smoke were associated with an increased risk for colorectal cancer with wild-type KRAS gene tumors, though not statistically significant, when compared with never smoking (frequency: RR 1.24, 95% CI 0.90-1.71 and inhalation: RR 1.25, 95% CI 0.94-1.67). These associations were strongest in men (ex-smokers: RR 1.79, 95% CI 1.00-3.20; frequency: RR 1.91, 95% CI 1.03-3.52; inhalation: RR 1.69, 95% CI 0.94-3.04). No associations were observed between any of the smoking characteristics and the risk for colorectal cancer with mutated KRAS gene tumors, nor where there any clear associations with tumors with specific G:C-->A:T transitions or G:C-->T:A transversions. These results suggest that, in contrast to the hypothesis, smoking does not increase the risk for colorectal tumors with a mutated KRAS gene. Some smoking characteristics, i.e. being an ex-smoker, frequency and inhalation, may be associated with risk for colorectal cancer characterized by the wild-type KRAS gene, especially in men.

Gene expression profiling in Caco-2 human colon cells exposed to TCDD, benzo[a]pyrene, and natural Ah receptor agonists from cruciferous vegetables and citrus fruits.

Cruciferous vegetables and citrus fruits are reported to possess health-beneficial properties, but also have been shown to contain natural aryl hydrocarbon receptor (AhR) agonists (NAhRAs). Binding to the AhR is widely assumed to activate the main pathway by which dioxins, like 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exert their toxicity. To establish whether or not activation of the AhR pathway by NAhRAs and dioxin-like substances results in similar cellular responses, gene expression profiles induced in Caco-2 cells were studied using microarray analysis. Cells were exposed to indolo[3,2-b]carbazole (ICZ), an acid reaction product from cruciferous vegetables, and to extracts of citrus pulp and grapefruit juice. Gene expression profiles induced by these NAhRAs were compared to those of the xenobiotic AhR agonists TCDD and benzo[a]pyrene (B[a]P). Over 20 genes were found more than 1.5 times up- or down-regulated by TCDD, and the expression of most of these genes was modulated in the same direction and to a similar extent by B[a]P and the NAhRAs. Results were confirmed by RT-PCR, and many of these genes may be involved in dioxin-related toxic effects. In conclusion, this in vitro study showed similar effects induced by NAhRAs, TCDD and B[a]P at the transcriptome level in a human intestinal cell line.

[Relationship between the composition of fine dust particles in the air and lung function in school children]. / Relatie tussen de samenstelling van fijn stofin de lucht en de longfunctie van schoolkinderen.

OBJECTIVE: To determine whether or not there is a relationship between the lung function of school children and the ability of fine dust particles in the air to generate radicals. DESIGN: Descriptive. METHOD: Six primary schools in locations with different traffic volumes were selected in Maastricht, the Netherlands. Air samples were taken in these schools over a period of 4 days; the concentration of fine dust was measured in the 6 pooled samples. Lung function tests were performed in children in the age of 8-13 and their parents filled out a questionnaire on the state of their children's health. RESULTS: An average of 66% of the children (184 girls and 158 boys, with an average age of 10 years (range: 8-13 years)) participated. The average FEV1 for the children from the 6 schools was not related with the total amount of fine dust particles in the air. However, a lower average FEV1 was associated with a higher radical-generating capacity in the air samples. No direct association was observed between the radical-generating capacity of the dust and the traffic intensity. CONCLUSION: There was a clear relationship between lung function and the radical-generating capacity of fine dust in the air. On the basis of these findings future guidelines could be based on chemical properties of the fine dust particles and not exclusively on the quantity of fine dust.

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.

Effects on cell proliferation, activator protein-1 and genotoxicity by fecal water from patients with colorectal adenomas.

BACKGROUND: The free water phase of feces (fecal water) may mediate the effects of diet on colon carcinogenesis. We examined the effects of fecal water from adenoma patients and controls on three parameters in colonocytes believed to be relevant to tumorigenesis, i.e. genotoxicity in intact cells and on isolated DNA, proliferative activity and activator protein-1 (AP-1) activity. METHODS: Genotoxicity in intact colonic cells was assayed using the single-cell gel electrophoresis assay ('comet' assay) and on isolated DNA using double-stranded DNA from the X-174 RF plasmid. Cell proliferation was assessed using the commercially available 'alamar blue' proliferation kit and AP-1 activity using cells transiently transfected with an AP-1-luciferase reporter construct. RESULTS: The data showed that lipid extracts of fecal water samples from the adenoma patients had a significantly higher capacity to induce cell proliferation than those from controls, and that this effect could be explained to a large extent by the concentrations of deoxycholic and chenodeoxycholic acids in the fecal water using regression models. No difference between patients and controls was observed for induction of AP-1 activity or induction of DNA strand breaks in intact cells. However, induction of DNA strand breaks in isolated DNA was significantly higher for the fecal waters from patients than for those from controls, which could be explained in part in a regression model by concentrations of lithocholic acid in fecal water and fecapentaene-12 in feces. CONCLUSIONS: Our results support the hypothesis that the biochemistry of fecal waters from adenoma patients and controls differs.

Analysis of oxidative DNA damage after human dietary supplementation with linoleic acid.

It has been hypothesized that oxygen radicals generated by peroxidation of dietary linoleic acid may induce genetic damage and thereby increase cancer risk. We examined the effect of dietary supplementation with linoleic acid on the levels of oxidative DNA damage in peripheral lymphocytes and on the blood plasma antioxidant potential. Thirty volunteers received during 6 weeks either a high dose of linoleic acid (15 g/day), an intermediate dose of linoleic acid (7.5 g/day) or an isocaloric supplement without linoleic acid (15 g palmitic acid/day). After the intervention, no significant increase in oxidative DNA damage, measured as relative amounts of 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodG) in DNA from peripheral lymphocytes, was observed in both high and intermediate linoleic acid-supplemented groups (increase of respectively 13 and 21%; P>0.05). Also, the differences between levels of oxidative DNA damage in the high or intermediate linoleic acid-supplemented group and the control group receiving palmitic acid (23% decrease) were not significant. Furthermore, no statistically significant differences were found between the total antioxidant capacities of blood plasma from the different experimental groups. Plasma levels of malondialdehyde, an important end-product of lipid peroxidation, were not increased after supplementation, nor were effects found on the plasma concentrations of retinol, alpha-tocopherol and beta-carotene. Despite the experimental design that excludes several forms of bias introduced in studies based on modulation of dietary composition, our results provide no indication of increased oxidative stress or genetic damage as a result of increased dietary intake of linoleic acid. Therefore, we see no scientific basis to reconsider the public health policy to stimulate the intake of polyunsaturated fatty acids aimed at the reduction of coronary heart diseases.

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.

A molecular dosimetry approach to assess human exposure to environmental tobacco smoke in pubs.

Although the involvement of environmental tobacco smoke (ETS) in human lung cancer is no longer a matter of dispute, the magnitude of its impact still is. This is mainly due to the inefficiency of methodology to assess exposure to ETS especially in public places. Setting a real life exposure condition (3 h stay in local pubs) and using a matched-control study design, we quantified smoke-related DNA adducts in induced sputum and peripheral blood lymphocytes (PBL) of healthy non-smokers (n = 15) before and after a single pub visit by means of the (32)P-post-labeling assay. For verification, we also measured a spectrum of polycyclic aromatic hydrocarbons (PAH) in the ambient air of the pubs by personal air monitors, and determined the plasma concentrations of nicotine and cotinine by gas chromatography/mass spectrometry. The ambient air concentrations of all PAH were several orders of magnitude higher than those already reported for other indoor environments. The plasma concentrations of both nicotine and cotinine increased significantly after the pub visit (P = 0.001 and P = 0.0007, respectively). Accordingly, the overall DNA adduct profile in induced sputum, but not in PBL, changed quantitatively and qualitatively after the pub visit. Of most significance was the formation of a distinct DNA adduct in induced sputum of three individuals consequent to ETS exposure. This adduct co-migrated with the standard (+/-)-anti-benzo[a]pyrene diol epoxide-DNA adduct, which is known to form at lung cancer mutational hotspots. We conclude that real life exposure to ETS can give rise to pro-mutagenic lesions in the lower airway, and this can be best investigated in a relevant surrogate matrix such as induced sputum.

A multi-biomarker approach to study the effects of smoking on oxidative DNA damage and repair and antioxidative defense mechanisms.

We investigated the effects of smoking-induced oxidative stress in healthy volunteers (21 smokers versus 24 non-smokers) by quantifying various markers of oxidative DNA damage and repair, and antioxidative defense mechanisms. Lymphocytic 7-hydroxy-8-oxo-2'-deoxyguanosine (8-oxo-dG) levels measured by high performance liquid chromatography with electrochemical detection, were significantly lower in smokers as compared with non-smokers (38.6 +/- 5.2 versus 50.9 +/- 4.6/10(6) dG, P = 0.05). The levels of oxidized pyrimidine bases in lymphocytes of smokers quantified by the endonuclease III-modified comet assay were non-significantly lower than those of non-smokers (% DNA in tail: 13 +/- 3 versus 14 +/- 2; tail length: 69 +/- 13 versus 96 +/- 10; tail moment: 6416 +/- 1220 versus 7545 +/- 1234). Urinary excretion levels of 8-hydroxy-2'-deoxyguanosine (8-OH-dG) assessed by enzyme-linked immunosorbent assay did not differ significantly between smokers and non-smokers (197 +/- 31 versus 240 +/- 33 ng/body mass index, P = 0.3). Overall DNA repair activity expressed as unscheduled DNA synthesis in blood leukocytes, was not significantly different between smokers and non-smokers (2.9 +/- 0.3 versus 3.3 +/- 0.3, P = 0.4). Plasma antioxidative capacity measured by the Trolox equivalent antioxidant capacity assay was slightly higher in smokers as compared with non-smokers (440 +/- 16 versus 400 +/- 15 microM Trolox equivalent, P = 0.09), and it was significantly related to lymphocytic 8-oxo-dG levels (r = 0.4, P = 0.001). Genotyping of human 8-OH-dG glycosylase/apurinic lyase and glutathione S-transferase M1 showed that a polymorphism in either or both of the two genes does not affect any of the quantified biomarkers. We conclude that oxidative stress imposed by cigarette smoking has a low impact upon certain pathways involved in DNA damage and the antioxidative defense system.

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 (32)P-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.

Evaluation of fecal mutagenicity and colorectal cancer risk.

Colorectal cancer is one of the most common internal malignancies in Western society. The cause of this disease appears to be multifactorial and involves genetic as well as environmental aspects. The human colon is continuously exposed to a complex mixture of compounds, which is either of direct dietary origin or the result of digestive, microbial and excretory processes. In order to establish the mutagenic burden of the colorectal mucosa, analysis of specific compounds in feces is usually preferred. Alternatively, the mutagenic potency of fecal extracts has been determined, but the interpretation of these more integrative measurements is hampered by methodological shortcomings. In this review, we focus on exposure of the large bowel to five different classes of fecal mutagens that have previously been related to colorectal cancer risk. These include heterocyclic aromatic amines (HCA) and polycyclic aromatic hydrocarbons (PAH), two exogenous factors that are predominantly ingested as pyrolysis products present in food and (partially) excreted in the feces. Additionally, we discuss N-nitroso-compounds, fecapentaenes and bile acids, all fecal constituents (mainly) of endogenous origin. The mutagenic and carcinogenic potency of the above mentioned compounds as well as their presence in feces, proposed mode of action and potential role in the initiation and promotion of human colorectal cancer are discussed. The combined results from in vitro and in vivo research unequivocally demonstrate that these classes of compounds comprise potent mutagens that induce many different forms of genetic damage and that particularly bile acids and fecapentaenes may also affect the carcinogenic process by epigenetic mechanisms. Large inter-individual differences in levels of exposures have been reported, including those in a range where considerable genetic damage can be expected based on evidence from animal studies. Particularly, however, exposure profiles of PAH and N-nitroso compounds (NOC) have to be more accurately established to come to a risk evaluation. Moreover, lack of human studies and inconsistency between epidemiological data make it impossible to describe colorectal cancer risk as a result of specific exposures in quantitative terms, or even to indicate the relative importance of the mutagens discussed. Particularly, the polymorphisms of genes involved in the metabolism of heterocyclic amines are important determinants of carcinogenic risk. However, the present knowledge of gene-environment interactions with regard to colorectal cancer risk is rather limited. We expect that the introduction of DNA chip technology in colorectal cancer epidemiology will offer new opportunities to identify combinations of exposures and genetic polymorphisms that relate to increased cancer risk. This knowledge will enable us to improve epidemiological study design and statistical power in future research.