Impact of more intense smoking parameters and flavor variety on toxicant levels in emissions of a Heated Tobacco Product.

INTRODUCTION: IQOS HEETS are promoted as reduced risk alternatives to cigarettes. Although some studies have investigated the chemical composition of HEETS emissions, little is known on whether toxicant levels in such emissions are affected by different puffing parameters and flavor varieties. This has important implications when assessing actual human exposure, since IQOS users develop a specific and personalized puffing behavior and may use different HEETS variants. METHODS: This study measured the levels of nicotine, Total Particulate Matter (TPM), carbonyl compounds and tobacco-Specific Nitrosamines (TSNAs) in the emissions of nine differently flavored HEETS and two cigarettes (1R6F and Marlboro Red, MR). Emissions from Yellow HEETS, 1R6F and MR were collected using the World Health Organization Intense (WHOI) smoking regime and four more intense smoking regimes. RESULTS: Yellow HEETS aerosol contained lower levels of toxicants compared to 1R6F and MR smoke. More intense smoking regimes increased carbonyls release in cigarette smoke, whereas only higher puff frequency led to lower levels of toxicants in Yellow HEETS aerosol. Some HEETS varieties exhibited higher levels of formaldehyde and TSNAs in their aerosol compared to Yellow HEETS. CONCLUSIONS: Puff frequency was identified as the only smoking parameter that significantly lowered the release of almost all toxicants in Yellow HEETS, whereas a combination of higher puff volume and puff duration led to increased levels of some carbonyls. Differences in toxicants levels between various commercially-available HEETS have important implications when assessing their health impact, as their consumption might induce different toxicant exposure and health effects. IMPLICATIONS: HEETS release about half as much nicotine and substantially lower levels of toxicants compared to cigarettes. Literature data showed that puffing intensity is increased in cigarette smokers switching to HEETS, maybe in reaction to these lower nicotine levels. Our results show a differential impact of increased puff frequency, puff duration and puff volume in the release of toxicants from HEETS. Thus, industry-independent studies on puff topography are critical to make choices for the most relevant puffing regime for HTP regulation. Regulators should consider evaluating the health impact of multiple HEETS varieties, as the tobacco filler composition significantly affects the release of certain toxicants.

The use of exhaled air analysis in discriminating interstitial lung diseases: a pilot study.

BACKGROUND: Fibrotic Interstitial lung diseases (ILD) are a heterogeneous group of chronic lung diseases characterized by diverse degrees of lung inflammation and remodeling. They include idiopathic ILD such as idiopathic pulmonary fibrosis (IPF), and ILD secondary to chronic inflammatory diseases such as connective tissue disease (CTD). Precise differential diagnosis of ILD is critical since anti-inflammatory and immunosuppressive drugs, which are beneficial in inflammatory ILD, are detrimental in IPF. However, differential diagnosis of ILD is still difficult and often requires an invasive lung biopsy. The primary aim of this study is to identify volatile organic compounds (VOCs) patterns in exhaled air to non-invasively discriminate IPF and CTD-ILD. As secondary aim, the association between the IPF and CTD-ILD discriminating VOC patterns and functional impairment is investigated. METHODS: Fifty-three IPF patients, 53 CTD-ILD patients and 51 controls donated exhaled air, which was analyzed for its VOC content using gas chromatograph- time of flight- mass spectrometry. RESULTS: By applying multivariate analysis, a discriminative profile of 34 VOCs was observed to discriminate between IPF patients and healthy controls whereas 11 VOCs were able to distinguish between CTD-ILD patients and healthy controls. The separation between IPF and CTD-ILD could be made using 16 discriminating VOCs, that also displayed a significant correlation with total lung capacity and the 6 min' walk distance. CONCLUSIONS: This study reports for the first time that specific VOC profiles can be found to differentiate IPF and CTD-ILD from both healthy controls and each other. Moreover, an ILD-specific VOC profile was strongly correlated with functional parameters. Future research applying larger cohorts of patients suffering from a larger variety of ILDs should confirm the potential use of breathomics to facilitate fast, non-invasive and proper differential diagnosis of specific ILDs in the future as first step towards personalized medicine for these complex diseases.

The disturbed redox-balance in pulmonary fibrosis is modulated by the plant flavonoid quercetin.

Idiopathic pulmonary fibrosis (IPF) is characterized by a disturbed pulmonary redox balance associated with inflammation. To restore this balance, antioxidants are often suggested as therapy for IPF but previous clinical trials with these compounds and their precursors have not been successful in the clinic. The exogenous antioxidant quercetin, which has a versatile antioxidant profile and is effective in restoring a disturbed redox balance, might be a better candidate. The aim of this study was to evaluate the protective effect of quercetin on oxidative and inflammatory markers in IPF. Here, we demonstrate that IPF patients have a significantly reduced endogenous antioxidant defense, shown by a reduced total antioxidant capacity and lowered glutathione and uric acid levels compared to healthy controls. This confirms that the redox balance is disturbed in IPF. Ex vivo incubation with quercetin in blood of both IPF patients and healthy controls reduces LPS-induced production of the pro-inflammatory cytokines IL-8 and TNFα. This anti-inflammatory effect was more pronounced in the blood of the patients. Our pro-fibrotic in vitro model, consisting of bleomycin-triggered BEAS-2B cells, shows that quercetin boosts the antioxidant response, by increasing Nrf2 activity, and decreases pro-inflammatory cytokine production in a concentration-dependent manner. Collectively, our findings implicate that IPF patients may benefit from the use of quercetin to restore the disturbed redox balance and reduce inflammation.

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.

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.

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.

[Pharmacogenetics in the treatment of tobacco addiction]. / Farmacogenetica bij de behandeling van tabaksverslaving.

BACKGROUND: Smokers vary in their genetic susceptibility to become addicted to smoking and probably also in their reaction to smoking cessation pharmacotherapies. AIM: To provide an overview of the developments on the pharmacogenetics of the treatment of tobacco addiction. METHOD: Review article describing the biological processes associated with tobacco addiction, and the influence of genetic variants on smoking behavior and the efficacy of smoking cessation therapies. RESULTS: Several (combinations of) genetic variants in smoking-related genes influence nicotine dependence. Moreover, several genetic variants in smoking- and treatment-related genes seem to influence the efficacy of smoking cessation therapies which are distinctive for the different forms of pharmacotherapy, especially when they have a different mechanism-of-action. CONCLUSION: Much progress has been made in unraveling the (pharmaco)genetics of tobacco addiction, but much still remains to be done before genetically tailored smoking cessation therapy can be implemented in clinical practice.

Differences in Treatment Response in Bronchial Epithelial Cells from Idiopathic Pulmonary Fibrosis (IPF) Patients: A First Step towards Personalized Medicine?

Idiopathic pulmonary fibrosis (IPF) has a detrimental prognosis despite antifibrotic therapies to which individual responses vary. IPF pathology is associated with oxidative stress, inflammation and increased activation of SRC family kinases (SFK). This pilot study evaluates individual responses to pirfenidone, nintedanib and SFK inhibitor saracatinib, markers of redox homeostasis, fibrosis and inflammation, in IPF-derived human bronchial epithelial (HBE) cells. Differentiated HBE cells from patients with and without IPF were analyzed for potential alterations in redox and profibrotic genes and pro-inflammatory cytokine secretion. Additionally, the effects of pirfenidone, nintedanib and saracatinib on these markers were determined. HBE cells were differentiated into a bronchial epithelium containing ciliated epithelial, basal, goblet and club cells. NOX4 expression was increased in IPF-derived HBE cells but differed on an individual level. In patients with higher NOX4 expression, pirfenidone induced antioxidant gene expression. All drugs significantly decreased NOX4 expression. IL-6 (p = 0.09) and IL-8 secretion (p = 0.014) were increased in IPF-derived HBE cells and significantly reduced by saracatinib. Finally, saracatinib significantly decreased TGF-ß gene expression. Our results indicate that treatment responsiveness varies between IPF patients in relation to their oxidative and inflammatory status. Interestingly, saracatinib tends to be more effective in IPF than standard antifibrotic drugs.

Impact of sub-acute acrolein inhalation on the molecular regulation of mitochondrial metabolism in rat lung.

Nowadays, mitochondria are recognized as key players in the pathogenesis of a variety of smoking-associated lung diseases. Acrolein, a component of cigarette smoke, is a known driver of biological mechanisms underlying smoking-induced respiratory toxicity. The impact of sub-acute acrolein inhalation in vivo on key processes controlling mitochondrial homeostasis in cells of the airways however is unknown. In this study, we investigated the activity/abundance of a myriad of molecules critically involved in mitochondrial metabolic pathways and mitochondrial quality control processes (mitochondrial biogenesis and mitophagy) in the lungs of Sprague-Dawley rats that were sub-acutely exposed to filtered air or 3 ppm acrolein by whole-body inhalation (5 h/day, 5 days/week for 4 weeks). Acrolein exposure induced a general inflammatory response in the lung as gene expression analysis revealed an increased expression of Icam1 and Cinc1 (p < 0.1; p < 0.05). Acrolein significantly decreased enzyme activity of hydroxyacyl-Coenzyme A dehydrogenase (p < 0.01), and decreased Pdk4 transcript levels (p < 0.05), suggestive of acrolein-induced changes in metabolic processes. Investigation of constituents of the mitochondrial biogenesis pathways and mitophagy machinery revealed no pronounced alterations. In conclusion, sub-acute inhalation of acrolein did not affect the regulation of mitochondrial metabolism and quality control, which is in contrast to more profound changes after acute exposure in other studies.

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.

Acrolein inhalation acutely affects the regulation of mitochondrial metabolism in rat lung.

Exposure of the airways to cigarette smoke (CS) is the primary risk factor for developing several lung diseases such as Chronic Obstructive Pulmonary Disease (COPD). CS consists of a complex mixture of over 6000 chemicals including the highly reactive α,ß-unsaturated aldehyde acrolein. Acrolein is thought to be responsible for a large proportion of the non-cancer disease risk associated with smoking. Emerging evidence suggest a key role for CS-induced abnormalities in mitochondrial morphology and function in airway epithelial cells in COPD pathogenesis. Although in vitro studies suggest acrolein-induced mitochondrial dysfunction in airway epithelial cells, it is unknown if in vivo inhalation of acrolein affects mitochondrial content or the pathways controlling this. In this study, rats were acutely exposed to acrolein by inhalation (nose-only; 0-4 ppm), 4 h/day for 1 or 2 consecutive days (n = 6/group). Subsequently, the activity and abundance of key constituents of mitochondrial metabolic pathways as well as expression of critical proteins and genes controlling mitochondrial biogenesis and mitophagy were investigated in lung homogenates. A transient decreasing response in protein and transcript abundance of subunits of the electron transport chain complexes was observed following acrolein inhalation. Moreover, acrolein inhalation caused a decreased abundance of key regulators associated with mitochondrial biogenesis, respectively a differential response on day 1 versus day 2. Abundance of components of the mitophagy machinery was in general unaltered in response to acrolein exposure in rat lung. Collectively, this study demonstrates that acrolein inhalation acutely and dose-dependently disrupts the molecular regulation of mitochondrial metabolism in rat lung. Hence, understanding the effect of acrolein on mitochondrial function will provide a scientifically supported reasoning to shortlist aldehydes regulation in tobacco smoke.

Non-invasive breath collection in murine models using a newly developed sampling device.

Volatile organic compounds (VOCs) in exhaled breath have the potential to be used as biomarkers for screening and diagnosis of diseases. Clinical studies are often complicated by both modifiable and non-modifiable factors influencing the composition of VOCs in exhaled breath. Small laboratory animal studies contribute in obtaining fundamental insight in alterations in VOC composition in exhaled breath and thereby facilitate the design and analysis of clinical research. However, long term animal experiments are often limited by invasive breath collection methods and terminal experiments. To overcome this problem, a novel device was developed for non-invasive breath collection in mice using glass nose-only restrainers thereby omitting the need of anesthetics. C57Bl/6 J mice were used to test reproducibility and different air sampling settings for air-flow (ml min-1) and time (minutes). Exhaled air was collected on desorption tubes and analysed for VOCs by gas chromatography time-of-flight mass spectrometry (GC-tof-MS). In total 27 compounds were putatively identified and used to assess the variability of the VOC measurements in the breath collections. Best reproducibility is obtained when using an air flow of 185 ml min-1and a collection time of 20 min. Due to the non-invasive nature of breath collections in murine models, this device has the potential to facilitate VOC research in relation to disturbed metabolism and or disease pathways.

Breath analysis for the detection of digestive tract malignancies: systematic review.

BACKGROUND: In recent decades there has been growing interest in the use of volatile organic compounds (VOCs) in exhaled breath as biomarkers for the diagnosis of multiple variants of cancer. This review aimed to evaluate the diagnostic accuracy and current status of VOC analysis in exhaled breath for the detection of cancer in the digestive tract. METHODS: PubMed and the Cochrane Library database were searched for VOC analysis studies, in which exhaled air was used to detect gastro-oesophageal, liver, pancreatic, and intestinal cancer in humans, Quality assessment was performed using the QUADAS-2 criteria. Data on diagnostic performance, VOCs with discriminative power, and methodological information were extracted from the included articles. RESULTS: Twenty-three articles were included (gastro-oesophageal cancer n = 14, liver cancer n = 1, pancreatic cancer n = 2, colorectal cancer n = 6). Methodological issues included different modalities of patient preparation and sampling and platform used. The sensitivity and specificity of VOC analysis ranged from 66.7 to 100 per cent and from 48.1 to 97.9 per cent respectively. Owing to heterogeneity of the studies, no pooling of the results could be performed. Of the VOCs found, 32 were identified in more than one study. Nineteen were reported as cancer type-specific, whereas 13 were found in different cancer types. Overall, decanal, nonanal, and acetone were the most frequently identified. CONCLUSION: The literature on VOC analysis has documented a lack of standardization in study designs. Heterogeneity between the studies and insufficient validation of the results make interpretation of the outcomes challenging. To reach clinical applicability, future studies on breath analysis should provide an accurate description of the methodology and validate their findings.

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.

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.

Genetic variation as a predictor of smoking cessation success. A promising preventive and intervention tool for chronic respiratory diseases?

Tobacco smoking continues to be the largest preventable cause of premature morbidity and mortality throughout the world, including chronic respiratory diseases such as asthma and chronic obstructive pulmonary disease. Although most smokers are highly motivated to quit and many smoking cessation therapies are available, cessation rates remain very low. Recent research strongly suggests that variation in genetic background is an important determinant of smoking behaviour and addiction. Since these genetic variants might also influence the response to smoking cessation pharmacotherapies, it is likely that assessment of genetic background could be a promising tool to guide selection of the most effective cessation treatment for an individual smoker. Recently, it has been shown that genetic variants in the dopaminergic system, opioid receptors, the bupropion-metabolising enzyme CYP2B6 and the nicotine-metabolising enzyme CYP2A6 may play an important role in predicting smoking cessation responses to nicotine replacement therapy and bupropion treatment. Despite the progress that has been made, several challenges will still have to be overcome before genetically tailored smoking cessation therapy can be implemented in standard clinical practice.

Comparing patterns of volatile organic compounds exhaled in breath after consumption of two infant formulae with a different lipid structure: a randomized trial.

Infant formulae have been used since decades as an alternative to or a complement to human milk. Human milk, the "gold standard" of infant nutrition, has been studied for its properties in order to create infant formulae that bring similar benefits to the infant. One of the characteristics of milk is the size of the lipid droplets which is known to affect the digestion, gastric emptying and triglyceride metabolism. In the current study a concept infant milk formula with large, phospholipid coating of lipid droplets (mode diameter 3-5 µm; NUTURIS, further described as "active"), was compared to a commercially available formula milk characterised by smaller lipid droplets, further described as "control" (both products derived from Nutricia). We investigated whether we could find an effect of lipid droplet size on volatile compounds in exhaled air upon ingestion of either product. For that purpose, exhaled breath was collected from a group of 29 healthy, non-smoking adult males before ingestion of a study product (baseline measurements, T0) and at the following time points after the test meal: 30, 60, 120, 180 and 240 min. Volatile organic compounds (VOCs) in breath were detected by gas chromatography-time-of-flight-mass spectrometry. Any differences in the time course of VOCs patterns upon intake of active and control products were investigated by regularised multivariate analysis of variance (rMANOVA). The rMANOVA analysis revealed statistically significant differences in the exhaled breath composition 240 min after ingestion of the active formula compared to control product (p-value < 0.0001), but did not show significant changes between active and control product at any earlier time points. A set of eight VOCs in exhaled breath had the highest contribution to the difference found at 240 minutes between the two formulas. A set of ten VOCs was different between baseline and the two formulae at T240 with p-value < 0.0001. To our knowledge this is the first study that shows the ability of VOCs in exhaled breath to monitor metabolic effects after ingestion of infant formulae with different lipid structure. The statistically significant differences in compound abundance found between active and control formula milk may be related to: (i) specific differences in the digestion, (ii) absorption of lipids and proteins and (iii) assimilation of the products in the gut.

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.

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.