The association of maternal polyunsaturated fatty acids during pregnancy with social competence and problem behaviours at 7 years of age: The MEFAB cohort.

BACKGROUND: The prenatal exposure to maternal n-6 and n-3 polyunsaturated fatty acids (PUFAs) might influence the development of social competence and internalizing and externalizing behaviours of the child, because of the numerous functions of PUFAs within the nervous system. METHODS: To analyse the association of selected maternal PUFAs (i.e., AA, EPA, DHA, total n-6, total n-3, and the n-6:n-3 ratio) measured during gestation with childhood social competence and problem behaviours, we examined 311 mother-child pairs from the Maastricht Essential Fatty Acid Birth (MEFAB) cohort. For each woman, PUFA-specific changes in relative concentrations were calculated by identifying the best-fitting curve of PUFA concentration by linear splines of gestational age. The associations of changes in maternal PUFAs in early and late pregnancy with childhood social competence, total problems, internalizing and externalizing behaviours, measured with the Child Behaviour Checklist 4/18 at age 7, were investigated with linear regression analyses adjusted for maternal and children's socio-demographic characteristics. RESULTS: In late gestation (i.e., from gestational week 30), an increase in AA was associated with higher social competence, while a decrease in total n-6 was associated with lower externalizing behaviours. No other significant associations were found. DISCUSSION: In this prospective study, increasing maternal AA and decreasing total n-6 were associated with improved social competence and externalizing behaviours, respectively, in 7-year old children. Nonetheless, the clinical significance of the identified associations is modest and further investigations are warranted to clarify the relationship between maternal AA and total n-6 during pregnancy and childhood social and behavioural development.

Sirtuin 1 genetic variation, energy balance and colorectal cancer risk by sex and subsite in the Netherlands Cohort Study.

Sirtuin 1 (SIRT1) is an energy-sensing protein, which may affect tumorigenesis. We used SIRT1 variants as time-independent indicators of SIRT1 involvement in carcinogenesis and we studied two tagging SIRT1 variants in relation to colorectal cancer (CRC) risk. We also evaluated known energy balance-related CRC risk factors within SIRT1 genotype strata. The Netherlands Cohort Study includes 120,852 individuals and has 20.3 years follow-up (case-cohort: nsubcohort = 5000; nCRC cases = 4667). At baseline, participants self-reported weight, weight at age 20, height, trouser/skirt size reflecting waist circumference, physical activity, and early life energy restriction. SIRT1 rs12778366 and rs10997870 were genotyped in toenail DNA available for ~75% of the cohort. Sex- and subsite-specific Cox hazard ratios (HRs) showed that the rs12778366 CC versus TT genotype decreased CRC and colon cancer risks in women (HRCRC = 0.53, 95% confidence interval: 0.30-0.94) but not men. Multiplicative interactions were observed between SIRT1 variants and energy balance-related factors in relation to CRC endpoints, but the direction of associations was not always conform expectation nor specific to one genotype stratum. In conclusion, these results support SIRT1 involvement in colon cancer development in women. No conclusions could be made regarding a modifying effect of SIRT1 variants on associations between energy balance-related factors and CRC risk.

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.

Altered gene expression profiles in the lungs of benzo[a]pyrene-exposed mice in the presence of lipopolysaccharide-induced pulmonary inflammation.

Patients with inflammatory lung diseases are often additionally exposed to polycyclic aromatic hydrocarbons like B[a]P and B[a]P-induced alterations in gene expression in these patients may contribute to the development of lung cancer. Mice were intra-nasally treated with lipopolysaccharide (LPS, 20µg/mouse) to induce pulmonary inflammation and subsequently exposed to B[a]P (0.5mg/mouse) by intratracheal instillation. Gene expression changes were analyzed in mouse lungs by RNA microarrays. Analysis of genes that are known to be involved in the cellular response to B[a]P indicated that LPS significantly inhibited gene expression of various enzymes linked to B[a]P metabolism, which was confirmed by phenotypic analyses of enzyme activity. Ultimately, these changes resulted in higher levels of B[a]P-DNA adducts in the lungs of mice exposed to B[a]P with prior LPS treatment compared to the lungs of mice exposed to B[a]P alone. Using principle component analysis (PCA), we found that of all the genes that were significantly altered in their expression, those that were able to separate the different exposure conditions were predominantly related to immune-response. Moreover, an overall analysis of differentially expressed genes indicated that cell-cell adhesion and cell-cell communication was inhibited in lungs of mice that received both B[a]P and LPS. Our results indicate that pulmonary inflammation increased the genotoxicity of B[a]P via inhibition of both phase I and II metabolism. Therefore, inflammation could be a critical contributor to B[a]P-induced carcinogenesis in humans.

Polyunsaturated fatty acid levels at birth and child-to-adult growth: Results from the MEFAB cohort.

BACKGROUND: Prenatal exposure to polyunsaturated fatty acids (PUFAs) may influence childhood growth. However, available evidence mostly derived from short-term studies is inconsistent. OBJECTIVE: To assess whether fetal PUFA exposure is associated with height and body mass index (BMI), a common measure of adiposity, from 6 months to 23 years of age. METHODS: In the MEFAB cohort, we assessed cord blood phospholipid n-3 and n-6 PUFA levels, reflecting fetal exposure in late pregnancy. For 250 (45.2% females) participants, we collected a total of 1770 (n= 802 for females) repeated growth measurements from infancy to young adulthood. We examined sex-specific associations of PUFAs with height and BMI at different developmental ages (infant: 6 months; toddler: 2 years; pre-schooler: 4 years; school-aged child: 7 years; adolescent: 12 years; and young adult: 23 years) using fractional polynomial mixed models adjusted for important covariates. RESULTS: Higher n-3 PUFA levels were associated with higher infant length in males (ß= 0.44cm [95% CI: 0.07, 0.82] per SD increase), whereas, for females, higher n-6 PUFA concentrations were associated with lower length in infancy (ß= -0.69cm [95% CI: -1.08, -0.30] per SD increase). A higher ratio of n-3 to n-6 PUFAs was associated with higher infant length in both sexes (ß= 0.40cm [95% CI: 0.01, 0.78] and 0.42cm [95% CI: 0.05, 0.79] per unit increase for males and females, respectively). These associations were not detectable later in childhood and young adulthood. No associations with BMI were found at any time point examined. CONCLUSIONS: Our findings suggest a small sex-specific influence of PUFA status at birth on length in infancy, but this does not persist in later life up to young adulthood. PUFA status at birth does not seem to affect BMI from infancy till young adulthood.

Associations between maternal long-chain polyunsaturated fatty acid concentrations and child cognition at 7 years of age: The MEFAB birth cohort.

INTRODUCTION: Concentrations of the fish fatty acids EPA and DHA are low among Dutch women of reproductive age. As the human brain incorporates high concentrations of these fatty acids in utero, particularly during third trimester of gestation, these low EPA and DHA concentrations may have adverse consequences for fetal brain development and functioning. METHODS: Analyses were conducted using longitudinal observational data of 292 mother-child pairs participating in the MEFAB cohort. Maternal AA, DHA, and EPA were determined in plasma phospholipids - obtained in three trimesters - by gas-liquid chromatography. Cognitive function was assessed at 7 years of age, using the Kaufman Assessment Battery for Children, resulting in three main outcome parameters: sequential processing (short-term memory), simultaneous processing (problem-solving skills), and the mental processing composite score. Spline regression and linear regression analyses were used to analyse the data, while adjusting for potential relevant covariates. RESULTS: Only 2% of the children performed more than one SD below the mental processing composite norm score. Children with lower test scores (<25%) were more likely to have a younger mother with a higher pre-gestational BMI, less likely to be breastfed, and more likely to be born with a lower birth weight, compared to children with higher test scores (≥25%). Fully-adjusted linear regression models did not show associations of maternal AA, DHA, or EPA status during any of the pregnancy trimesters with childhood sequential and simultaneous processing. CONCLUSION: Maternal fatty acid status during pregnancy was not associated with cognitive performance in Dutch children at age 7.

Effect of interleukin (IL)-8 on benzo[a]pyrene metabolism and DNA damage in human lung epithelial cells.

It has been well established that inflammation and concurrent mutagenic exposures drive the carcinogenic process in a synergistic way. To elucidate the role of the inflammatory cytokine IL-8 in this process, we studied its effect on the activation and deactivation of the chemical mutagen benzo[a]pyrene B[a]P in the immortalized pulmonary BEAS-2B cell line. After 24h incubation with B[a]P in the presence or absence of IL-8, the B[a]P induced cytochrome P450 1A1 and 1B1 (CYP1A1 and CYP1B1) gene expression and CYP1A1 enzyme activity was significantly higher in the presence of the cytokine. Consistent with these findings, we observed higher concentration of the metabolite B[a]P-7,8-diol under concurrent IL-8 treatment conditions. Interestingly, we also found higher concentrations of unmetabolized B[a]P. To explain this, we examined the downstream effects of IL-8 on NADPH oxidases (NOXes). IL-8 lowered the intracellular NADPH level, but this effect could not explain the changes in B[a]P metabolism. IL-8 also significantly depleted intracellular glutathione (GSH), which also resulted in enhanced levels of unmetabolized B[a]P, but increased concentrations of the metabolite B[a]P-7,8-diol. No differences in B[a]P-DNA adducts level were found between B[a]P and B[a]P combined with IL-8, and this might be due to a 3-fold increase in nucleotide excision repair (NER) after IL-8 treatment. These findings suggest that IL-8 increased the formation of B[a]P-7,8-diol despite an overall delayed B[a]P metabolism via depletion of GSH, but DNA damage levels were unaffected due to an increase in NER capacity.

Acidic cellular microenvironment modifies carcinogen-induced DNA damage and repair.

Chronic inflammation creates an acidic microenvironment, which plays an important role in cancer development. To investigate how low pH changes the cellular response to the carcinogen benzo[a]pyrene (B[a]P), we incubated human pulmonary epithelial cells (A549 and BEAS-2B) with nontoxic doses of B[a]P using culturing media of various pH's (extracellular pH (pHe) of 7.8, 7.0, 6.5, 6.0 and 5.5) for 6, 24 and 48 h. In most incubations (pHe 7.0-6.5), the pH in the medium returned to the physiological pH 7.8 after 48 h, but at the lowest pH (pHe < 6.0), this recovery was incomplete. Similar changes were observed for the intracellular pH (pHi). We observed that acidic conditions delayed B[a]P metabolism and at t = 48 h, and the concentration of unmetabolized extracellular B[a]P and B[a]P-7,8-diol was significantly higher in acidic samples than under normal physiological conditions (pHe 7.8) for both cell lines. Cytochrome P450 (CYP1A1/CYP1B1) expression and its activity (ethoxyresorufin-O-deethylase activity) were repressed at low pHe after 6 and 24 h, but were significantly higher at t = 48 h. In addition, a DNA repair assay showed that the incision activity was ~80% inhibited for 6 h at low pHe and concomitant exposure to B[a]P. However, at t = 48 h, the incision activity recovered to more than 100% of the initial activity observed at neutral pHe. After 48 h, higher B[a]P-DNA adduct levels and γ-H2AX foci were observed at low pH samples than at pHe 7.8. In conclusion, acidic pH delayed the metabolism of B[a]P and inhibited DNA repair, ultimately leading to increased B[a]P-induced DNA damage.

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.

Body size, physical activity, genetic variants in the insulin-like growth factor pathway and colorectal cancer risk.

Insulin-like growth factors (IGFs) have been associated with growth, body size, physical activity and colorectal cancer (CRC). We hypothesized that variants in IGF-related genes increase the CRC susceptibility associated with a larger body size and a lack of physical activity. We assessed this in The Netherlands Cohort Study. Participants (n = 120852) completed a baseline questionnaire on diet and cancer. ~75% returned toenail clippings. Using a case-cohort approach and 16.3 years of follow-up, toenail DNA from 3768 subcohort members and 2580 CRC cases was genotyped. We aggregated unfavorable alleles (potentially increasing CRC risk) for 18 single nucleotide polymorphisms in 8 genes into a sum score. The sum score (in tertiles) and an IGF1 19-CA repeat polymorphism (19/19, 19/non-19 and non-19/non-19 repeats) in combination with body size (mostly in tertiles) and (non-)occupational physical activity (>12, 8-12 and <8 kJ/min in the job and >90, >60-90, >30-60 and ≤30 min/day) were analyzed by Cox regression. Increasingly higher hazard ratios (HRs) for CRC were observed for a larger adult body mass index, larger trouser size and tallness in the presence of more unfavorable alleles in men. HRs (95% confidence intervals) for joint effects were 1.55 (1.06-2.25), 1.78 (1.29-2.46) and 1.48 (1.01-2.17), respectively. In women, variant repeat alleles halved CRC risk irrespective of body size and physical activity. Almost no interactions tested significant. To conclude, a larger body size was a CRC risk factor in men in the presence of an accumulation of unfavorable alleles in IGF-related genes, but interactions were generally nonsignificant.

Environmental carcinogens and mutational pathways in atherosclerosis.

Atherosclerosis is associated with DNA damage in both circulating and vessel-wall cells and DNA adducts derived from exposure to environmental mutagens are abundant in atherosclerotic vessels. Environmental chemical carcinogens identified as risk factor for atherosclerosis include polycyclic aromatic hydrocarbons (benzo(a)pyrene, dimethylbenz(a)anthracene, beta-naphthoflavone, pyrene, 3-methylcolanthrene), arsenic, cadmium, 1,3-butadiene, cigarette smoke. Accordingly, polymorphisms of genes encoding for phase I/II metabolic reaction and DNA repair are risk factor for cardiovascular diseases, although their role is negligible as compared to other risk factors. The pathogenic relevance of mutation-related molecular damage in atherosclerosis has been demonstrated in experimental animal models involving the exposure to chemical mutagens. The relevance of mutation-related events in worsening atherosclerosis prognosis has been demonstrated in human clinical studies mainly as referred to mitochondrial DNA damage. Atherosclerosis is characterized by the occurrence of high level of oxidative damage in blood vessel resulting from both endogenous and exogenous sources. Mitochondrial damage is a main endogenous source of oxidative stress whose accumulation causes activation of intrinsic apoptosis through BIRC2 inhibition and cell loss contributing to plaque development and instability. Environmental physical mutagens, including ionizing radiation, are a risk factor for atherosclerosis even at the low exposure dose occurring in case of occupational exposure or the high exposure doses occurring during radiotherapy. Conversely, the role of exciting UV radiation in atherosclerosis is still uncertain. This review summarizes the experimental and clinical evidence supporting the pathogenic role of mutation-related pathway in atherosclerosis examining the underlying molecular mechanisms.

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.

DNA adducts in skin biopsies and 1-hydroxypyrene in urine of psoriasis patients and healthy volunteers following treatment with coal tar.

Coal tar ointments (CTO) are frequently used in the treatment of psoriasis and eczema, but CTO contain carcinogenic polycyclic aromatic hydrocarbons (PAH). PAH are absorbed and metabolized in the skin. In psoriasis, the skin barrier is altered and therefore, absorption and metabolism of PAH may differ from healthy skin. In this study, levels of urinary 1-hydroxypyrene and PAH-DNA adducts in the skin were studied in psoriatic patients and healthy volunteers. Three punch biopsies were taken from the lower back of 10 male volunteers and from a psoriatic plaque in 10 male patients. A surface of 6.25 cm(2) was treated with CTO. After 96 h CTO was removed and another three skin biopsies were collected from the treated area. DNA was isolated from skin biopsies and urine was collected during and after the exposure period. After 24h, a twofold lower 1-hydroxypyrene urinary excretion was observed in patients compared to healthy volunteers and after 48 h, this difference reached statistical significance (p<0.05). Over 96 h the median level of the sum of PAH-DNA adducts, analyzed by (32)P-post-labeling, increased from 3.5 before CTO administration to 21.1 adducts per 10(8) nucleotides in volunteers, and from 1.0 to 3.6 adducts per 10(8) nucleotides in patients. At 96 h, PAH-DNA levels were higher in healthy volunteers than in patients (p<0.05). Biomarkers for uptake, bioavailability and bioactivation of PAH were lower in patients compared to volunteers. These data suggest a lower risk of carcinogenic effects of CTO in psoriatic skin compared to healthy skin.

In vitro evaluation of baseline and induced DNA damage in human sperm exposed to benzo[a]pyrene or its metabolite benzo[a]pyrene-7,8-diol-9,10-epoxide, using the comet assay.

Exposure to genotoxins may compromise DNA integrity in male reproductive cells, putting future progeny at risk for developmental defects and diseases. To study the usefulness of sperm DNA damage as a biomarker for genotoxic exposure, we have investigated cellular and molecular changes induced by benzo[a]pyrene (B[a]P) in human sperm in vitro, and results have been compared for smokers and non-smokers. Sperm DNA obtained from five smokers was indeed more fragmented than sperm of six non-smokers (mean % Tail DNA 26.5 and 48.8, respectively), as assessed by the alkaline comet assay (P < 0.05). B[a]P-related DNA adducts were detected at increased levels in smokers as determined by immunostaining. Direct exposure of mature sperm cells to B[a]P (10 or 25 microM) caused moderate increases in DNA fragmentation which was independent of addition of human liver S9 mix for enzymatic activation of B[a]P, suggesting some unknown metabolism of B[a]P in ejaculates. In vitro exposure of samples to various doses of B[a]P (with or without S9) did not reveal any significant differences in sensitivity to DNA fragmentation between smokers and non-smokers. Incubations with the proximate metabolite benzo[a]pyrene-r-7,t-8-dihydrodiol-t9,10-epoxide (BPDE) produced DNA fragmentation in a dose-dependent manner (20 or 50 microM), but only when formamidopyrimidine DNA glycosylase treatment was included in the comet assay. These levels of DNA fragmentation were, however, low in relation to very high amounts of BPDE-DNA adducts as measured with (32)P postlabelling. We conclude that sperm DNA damage may be useful as a biomarker of direct exposure of sperm using the comet assay adapted to sperm, and as such the method may be applicable to cohort studies. Although the sensitivity is relatively low, DNA damage induced in earlier stages of spermatogenesis may be detected with higher efficiencies.

A profile of volatile organic compounds in breath discriminates COPD patients from controls.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is an inflammatory condition characterized by oxidative stress and the formation of volatile organic compounds (VOCs) secreted via the lungs. We recently developed a methodological approach able to identify profiles of VOCs in breath unique for patient groups. Here we applied this recently developed methodology regarding diagnosis of COPD patients. METHODS: Fifty COPD patients and 29 controls provided their breath and VOCs were analyzed by gas chromatography-mass spectrometry to identify relevant VOCs. An additional 16 COPD patients and 16 controls were sampled in order to validate the model, and 15 steroid naïve COPD patients were sampled to determine whether steroid use affects performance. FINDINGS: 1179 different VOCs were detected, of which 13 were sufficient to correctly classify all 79 subjects. Six of these 13 VOCs classified 92% of the subjects correctly (sensitivity: 98%, specificity: 88%) and correctly classified 29 of 32 subjects (sensitivity: 100%, specificity: 81%) from the independent validation population. Fourteen out of 15 steroid naïve COPD patients were correctly classified thus excluding treatment influences. INTERPRETATION: This is the first study distinguishing COPD subjects from controls solely based on the presence of VOCs in breath. Analysis of VOCs might be highly relevant for diagnosis of COPD.

Volatile organic compounds in exhaled breath as a diagnostic tool for asthma in children.

BACKGROUND: The correct diagnosis of asthma in young children is often hard to achieve, resulting in undertreatment of asthmatic children and overtreatment in transient wheezers. OBJECTIVES: To develop a new diagnostic tool that better discriminates between asthma and transient wheezing and that leads to a more accurate diagnosis and hence less undertreatment and overtreatment. A first stage in the development of such a tool is the ability to discriminate between asthmatic children and healthy controls. The integrative analysis of large numbers of volatile organic compounds (VOC) in exhaled breath has the potential to discriminate between various inflammatory conditions of the respiratory tract. METHODS: Breath samples were obtained and analysed for VOC by gas chromatography-mass spectrometry from asthmatic children (n=63) and healthy controls (n=57). A total of 945 determined compounds were subjected to discriminant analysis to find those that could discriminate diseased from healthy children. A set of samples from both asthmatic and healthy children was selected to construct a model that was subsequently used to predict the asthma or the healthy status of a test group. In this way, the predictive value of the model could be tested. MEASUREMENTS AND MAIN RESULTS: The discriminant analyses demonstrated that asthma and healthy groups are distinct from one another. A total of eight components discriminated between asthmatic and healthy children with a 92% correct classification, achieving a sensitivity of 89% and a specificity of 95%. Conclusion The results show that a limited number of VOC in exhaled air can well be used to distinguish children with asthma from healthy children.

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.

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.