Inflammation and the chemical carcinogen benzo[a]pyrene: Partners in crime.

Exposure to benzo[a]pyrene (B[a]P) is known to play a role in lung carcinogenesis and the underlying processes can be modified by the presence of inflammation. The inflammatory process can for instance enhance the concentration of reactive metabolites that bind to DNA and may also diminish DNA repair. Additionally, during the inflammatory process mediators are released that create a microenvironment which is suitable for further stimulation of cancer development. Various transcriptional pathways are activated by inflammation, including pathways that are mediated via nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), signal transducer and activator of transcription 3 (STAT-3), and hypoxia-inducible factor-1 (HIF-1). Crosstalk between these pathways and the aryl hydrocarbon receptor (AhR) occurs at multiple levels and thereby boosts B[a]P induced carcinogenesis. This review focuses on inflammatory mediators, including cytokines, chemokines and extracellular enzymes that modulate molecular events in B[a]P induced cancers.

Association between prenatal and current exposure to selected LCPUFAs and school performance at age 7.

INTRODUCTION: Long-chain polyunsaturated fatty acids (LCPUFAs) are important for brain functioning and might, thus, influence cognition and school performance. However, research investigating LCPUFAs relationships with school performance is limited. The objective of this study was to determine the association between levels of the LCPUFAs docosahexaenoic acid (DHA), arachidonic acid (AA), eicosapentaenoic acid (EPA) and n-6 docosapentaenoic acid (Osbond acid, ObA) at study entry, 22 weeks of pregnancy, 32 weeks of pregnancy, at partus, in umbilical cord plasma and child's plasma at age 7 and school performance scores at age 7. METHODS: Data from the Maastricht Essential Fatty Acid Birth cohort (MEFAB) were used for this study. Fatty acid levels of plasma phospholipids were measured in maternal blood plasma at study entry, 22 weeks of pregnancy, 32 weeks of pregnancy and partus. Childs fatty acid levels of plasma phospholipids were measured a in umbilical cord blood plasma, and in blood plasma of the child at age 7. Scores on national standardised tests for spelling, reading and arithmetic at age 7 were obtained via the school (scores were available for 149, 159 and 155 children, respectively). Associations between LCPUFA levels and school performance scores were analysed with categorical regression analyses with correction for covariates (smoking, maternal education, sex, breastfeeding, maternal intelligence, birth weight and BMI at age 7). RESULTS: Significant (p<0.001) associations between DHA level at age 7 and both reading (ß=0.158) and spelling (ß=0.146) were found. Consistent significant negative associations were observed between all maternal DHA plasma levels and arithmetic scores at age 7 (all p<0.001, all ß<-0.019). Additional significant negative associations were observed between maternal LCPUFA plasma levels at study entry and both reading and spelling scores at age 7; these associations were less consistent. CONCLUSION: Plasma DHA levels at age 7 were positively associated with reading and spelling scores at age 7. Consistent significant negative associations between maternal plasma DHA levels and arithmetic scores of the child at age 7 were found. Although this is an observational study, which cannot proof causality, the consistent negative associations observed between maternal plasma DHA levels and the arithmetic scores of the children at age 7 calls upon prudence when considering DHA supplementation during pregnancy.

Inflammation-associated extracellular ß-glucuronidase alters cellular responses to the chemical carcinogen benzo[a]pyrene.

Neutrophils infiltrate tissues during inflammation, and when activated, they release ß-glucuronidase. Since inflammation is associated with carcinogenesis, we investigated how extracellular ß-glucuronidase changed the in vitro cellular response to the chemical carcinogen benzo(a)pyrene (B[a]P). For this we exposed human liver (HepG2) and lung (A549) cells to B[a]P in the presence or absence of ß-glucuronidase. ß-Glucuronidase reduced B[a]P-induced expression of CYP1A1 and CYP1B1 at 6 h after exposure, which did not depend on ß-glucuronidase activity, because the inhibitor D-saccharic acid 1,4-lactone monohydrate did not antagonize the effect of ß-glucuronidase. On the other hand, the inhibitory effect of ß-glucuronidase on CYP expression was dependent on signalling via the insulin-like growth factor receptor (IGF2R, a known receptor for ß-glucuronidase), because co-incubation with the IGF2R inhibitor mannose-6-phosphate completely abolished the effect of ß-glucuronidase. Extracellular ß-glucuronidase also reduced the formation of several B[a]P metabolites and B[a]P-DNA adducts. Interestingly, at 24 h of exposure, ß-glucuronidase significantly enhanced CYP expression, probably because ß-glucuronidase de-glucuronidated B[a]P metabolites, which continued to trigger the aryl hydrocarbon receptor (Ah receptor) and induced expression of CYP1A1 (in both cell lines) and CYP1B1 (in A549 only). Consequently, significantly higher concentrations of B[a]P metabolites and DNA adducts were found in ß-glucuronidase-treated cells at 24 h. DNA adduct levels peaked at 48 h in cells that were exposed to B[a]P and treated with ß-glucuronidase. Overall, these data show that ß-glucuronidase alters the cellular response to B[a]P and ultimately enhances B[a]P-induced DNA adduct levels.

Benzo[a]pyrene-induced transcriptomic responses in primary hepatocytes and in vivo liver: toxicokinetics is essential for in vivo-in vitro comparisons.

The traditional 2-year cancer bioassay needs replacement by more cost-effective and predictive tests. The use of toxicogenomics in an in vitro system may provide a more high-throughput method to investigate early alterations induced by carcinogens. Recently, the differential gene expression response in wild-type and cancer-prone Xpa (-/-) p53 (+/-) primary mouse hepatocytes after exposure to benzo[a]pyrene (B[a]P) revealed downregulation of cancer-related pathways in Xpa (-/-) p53 (+/-) hepatocytes only. Here, we investigated pathway regulation upon in vivo B[a]P exposure of wild-type and Xpa (-/-) p53 (+/-) mice. In vivo transcriptomics analysis revealed a limited gene expression response in mouse livers, but with a significant induction of DNA replication and apoptotic/anti-apoptotic cellular responses in Xpa (-/-) p53 (+/-) livers only. In order to be able to make a meaningful in vivo-in vitro comparison we estimated internal in vivo B[a]P concentrations using DNA adduct levels and physiologically based kinetic modeling. Based on these results, the in vitro concentration that corresponded best with the internal in vivo dose was chosen. Comparison of in vivo and in vitro data demonstrated similarities in transcriptomics response: xenobiotic metabolism, lipid metabolism and oxidative stress. However, we were unable to detect cancer-related pathways in either wild-type or Xpa (-/-) p53 (+/-) exposed livers, which were previously found to be induced by B[a]P in Xpa (-/-) p53 (+/-) primary hepatocytes. In conclusion, we showed parallels in gene expression responses between livers and primary hepatocytes upon exposure to equivalent concentrations of B[a]P. Furthermore, we recommend considering toxicokinetics when modeling a complex in vivo endpoint with in vitro models.

Maternal intake of quercetin during gestation alters ex vivo benzo[a]pyrene metabolism and DNA adduct formation in adult offspring.

Variation in xenobiotic metabolism cannot entirely be explained by genetic diversity in metabolic enzymes. We suggest that maternal diet during gestation can contribute to variation in metabolism by creating an in utero environment that shapes the offspring's defence against chemical carcinogens. Therefore, pregnant mice were supplemented with the natural aryl hydrocarbon receptor (AhR) agonist quercetin (1 mmol quercetin/kg feed) until delivery. Next, it was investigated whether the adult offspring at the age of 12 weeks had altered biotransformation of the environmental pollutant benzo[a]pyrene (B[a]P). In utero quercetin exposure resulted in significantly enhanced gene expression of Cyp1a1, Cyp1b1, Nqo1 and Ugt1a6 in liver of foetuses at Day 14.5 of gestation. Despite cessation of supplementation after delivery, altered gene expression persisted into adulthood, but in a tissue- and gender-dependent manner. Expression of Phase I enzymes (Cyp1a1 and Cyp1b1) was up-regulated in the liver of adult female mice in utero exposed to quercetin, whereas expression of Phase II enzymes (Gstp1, Nqo1 and Ugt1a6) was predominantly enhanced in the lung tissue of female mice. Epigenetic mechanisms may contribute to this adapted gene expression, as the repetitive elements (SINEB1) were hypomethylated in liver of female mice prenatally exposed to quercetin. Studies on ex vivo metabolism of B[a]P by lung and liver microsomes showed that the amount of B[a]P-9,10-dehydrodiol, B[a]P-7,8-dihydrodiol and 3-hydroxy-B[a]P did not change, but the amount of unmetabolised B[a]P was significantly lower after incubation with lung microsomes from offspring that received quercetin during gestation. Moreover, ex vivo B[a]P-induced DNA adduct formation was significantly lower for liver microsomes of offspring that were exposed to quercetin during gestation. These results suggest that prenatal diet leads to persistent alterations in Phase I and II enzymes of adult mice and may affect cancer risk.

Development of accurate classification method based on the analysis of volatile organic compounds from human exhaled air.

Analysis of exhaled air leads to the development of fast accurate and non-invasive diagnostics. A comprehensive analysis of the entire range of volatile organic compounds (VOCs) in exhaled air samples will enable the identification of VOCs unique for certain patient groups. This study demonstrates proof of principle of our developed method tested on a smoking/non-smoking study population. Thermal desorption and gas chromatography coupled to time-of-flight mass spectrometry were used to analyse exhaled air samples. The VOC profiles obtained from each individual were combined into one final database based on similarity of mass spectra and retention indexes (RI), which offers the possibility for a reliable selection of compounds of interest. As proof of principle we correctly classified all subjects from population of smoking (N=11) and non-smoking (N=11) based on the VOC profiles available in their exhaled air. Support vector machine (SVM) analysis identified 4 VOCs as biomarkers of recent exposure to cigarette smoke: 2,5-dimethyl hexane, dodecane, 2,5-dimethylfuran and 2-methylfuran. This approach contributes to future development of fast, accurate and non-invasive diagnostics of inflammatory diseases including pulmonary diseases.

Effects of plant sterol and stanol ester consumption on lipid metabolism, antioxidant status and markers of oxidative stress, endothelial function and low-grade inflammation in patients on current statin treatment.

OBJECTIVE: The present study was designed to examine for the first time, side-by-side, the effects of plant sterol and stanol consumption on lipid metabolism and markers of antioxidant status, oxidative stress, endothelial dysfunction and low-grade inflammation in subjects on stable statin-treatment. DESIGN: Double-blind, randomized, placebo-controlled, intervention trial. SETTING: University. SUBJECTS: Forty-five patients on current statin treatment were recruited via newspaper advertisements. Data of 41 patients were used in statistical analysis. INTERVENTION: Subjects consumed margarine with no added plant sterols or stanols for 4 weeks and were then divided into three groups of 15 subjects. For the next 16 weeks, one group continued with the control margarine and the other two groups with either a plant sterol- or stanol (2.5 g/day)-enriched margarine. Blood was sampled at the end of the run-in and intervention periods. RESULTS: Plant sterol and stanol consumption significantly (P=0.026) reduced low-density lipoprotein (LDL) cholesterol by 0.34 mmol/l (95% confidence interval (CI), -0.67 to -0.04 mmol/l). No effects were shown on enzymatic and non-enzymatic antioxidants and markers of oxidative modification of lipids and DNA. In addition, no effect was found on soluble adhesion molecules, C-reactive protein and monocyte chemotactic protein-1 concentrations. CONCLUSIONS: We conclude that 16 weeks of plant sterol or stanol consumption did not affect markers of antioxidant status, oxidative stress, endothelial dysfunction and low-grade inflammation in patients on stable statin treatment, despite a significant reduction of LDL cholesterol.