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.

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.

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.

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.

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.

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.

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.

Exposure to genotoxic compounds alters in vitro cellular VOC excretion.

Genotoxic carcinogens significantly damage cells and tissues by targeting macromolecules such as proteins and DNA, but their mechanisms of action and effects on human health are diverse. Consequently, determining the amount of exposure to a carcinogen and its cellular effects is essential, yet difficult. The aim of this manuscript was to investigate the potential of detecting alterations in volatile organic compounds (VOCs) profiles in the in vitro headspace of pulmonary cells after exposure to the genotoxic carcinogens cisplatin and benzo[a]pyrene using two different sampling set-ups. A prototype set-up was used for the cisplatin exposure, whereas a modified set-up was utilized for the benzo[a]pyrene exposure. Both carcinogens were added to the cell medium for 24 h. The headspace in the culture flask was sampled to measure the VOC content using gas chromatography-time-of-flight-mass spectrometry. Eight cisplatin-specific VOCs and six benzo[a]pyrene-specific VOCs were discriminatory between treated and non-treated cells. Since the in vivo biological effects of both genotoxic compounds are well-defined, the origin of the identified VOCs could potentially be traced back to common cellular processes including cell cycle pathways, DNA damage and repair. These results indicate that exposing lung cells to genotoxins alters headspace VOC profiles, suggesting that it might be possible to monitor VOC changes in vivo to study drug efficacy or exposure to different pollutants. In conclusion, this study emphasizes the innovative potential of in vitro VOCs experiments to determine their in vivo applicability and discover their endogenous origin.

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.

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.

Biomarkers for visceral hypersensitivity in patients with irritable bowel syndrome.

BACKGROUND: Increased visceral sensitivity is observed in up to 60% of patients with Irritable Bowel Syndrome (IBS). Mucosal inflammation, altered neuroendocrine activity and intraluminal metabolic processes may contribute to the development of visceral hypersensitivity. Previously, we demonstrated that biomarkers, indicative for these biological processes, were altered in IBS patients compared to healthy controls. However, how these processes relate to visceral hypersensitivity is unknown. AIM: The aim of this study was to provide insight in biological processes associated with visceral hypersensitivity. Fecal and plasma biomarkers were measured in normosensitive and hypersensitive IBS patients. METHODS: A total of 167 IBS patients underwent a rectal barostat procedure to assess visceral sensitivity to pain. Based on the outcome, patients were classified into a normosensitive or hypersensitive group. Calprotectin, human ß-defensin 2 (HBD2), chromogranin A (CgA), and short chain fatty acids (SCFAs) were measured in feces, citrulline in plasma, and serotonin and its main metabolite 5-hydroxyindoleacetic acid (5-HIAA) in platelet-poor plasma. KEY RESULTS: Fecal markers and plasma citrulline were measured in 83 hypersensitive and 84 normosensitive patients, while platelet-poor plasma for the assessment of serotonin and 5-HIAA was available for a subgroup, i.e. 53 hypersensitive and 42 normosensitive patients. No statistically significant differences were found in concentrations of biomarkers between groups. Adjustment of the analyses for potential confounders, such as medication use, did not alter this conclusion. CONCLUSIONS & INFERENCES: Our findings do not support a role for the biological processes as ascertained by biomarkers in visceral hypersensitivity in IBS patients. This study is registered in the US National Library of Medicine (clinicaltrials.gov, NCT00775060).

Factors that influence the volatile organic compound content in human breath.

BACKGROUND: Thousands of endogenous and exogenous volatile organic compounds (VOCs) are excreted in each breath. Inflammatory and deviant metabolic processes affect the level of endogeneous VOCs, which can serve as specific biomarkers for clinical diagnosis and disease monitoring. Important issues that still need to be tackled are related to potential confounding factors like gender and age and endogenous and exogenous factors, like f.i. smoking. METHODS: The aim of this study was to systematically access the effect of endogenous and exogenous factors on VOC composition of exhaled breath. In the current study breath samples from 1417 adult participants from the LifeLines cohort, a general population cohort in the Netherlands, were collected and the total content of VOCs was measured using gas chromatography-time-of-flight-mass spectrometry. Breath samples were collected in Groningen and transferred to carbon tubes immediately. These samples were then shipped to Maastricht and measured in batches. VOCs profiles were correlated to 14 relevant characteristics of all participants including age, BMI, smoking and blood cell counts and metabolic parameters as well as to 16 classes of medications. RESULTS: VOCs profiles were shown to be significantly influenced by smoking behavior and to a lesser extent by age, BMI and gender. These factors need to be controlled for in breath analysis studies. We found no evidence whatsoever in this 1417 subjects' cohort that white blood cell counts, cholesterol or triglycerides levels have an influence on the VOC profile. Thus they may not have to be controlled for in exhaled breath studies. CONCLUSION: The large cohort of volunteers used here represents a unique opportunity to gauge the factors influencing VOCs profiles in a general population i.e. the most clinically relevant population. Classical clinical parameters and smoking habits clearly influence breath content and should therefore be accounted for in future clinical studies involving breath analysis.

The potential of volatile organic compounds for the detection of active disease in patients with ulcerative colitis.

BACKGROUND: To optimise treatment of ulcerative colitis (UC), patients need repeated assessment of mucosal inflammation. Current non-invasive biomarkers and clinical activity indices do not accurately reflect disease activity in all patients and cannot discriminate UC from non-UC colitis. Volatile organic compounds (VOCs) in exhaled air could be predictive of active disease or remission in Crohn's disease. AIM: To investigate whether VOCs are able to differentiate between active UC, UC in remission and non-UC colitis. METHODS: UC patients participated in a 1-year study. Clinical activity index, blood, faecal and breath samples were collected at each out-patient visit. Patients with clear defined active faecal calprotectin >250 µg/g and inactive disease (Simple Clinical Colitis Activity Index <3, C-reactive protein <5 mg/L and faecal calprotectin <100 µg/g) were included for cross-sectional analysis. Non-UC colitis was confirmed by stool culture or radiological evaluation. Breath samples were analysed by gas chromatography time-of-flight mass spectrometry and kernel-based method to identify discriminating VOCs. RESULTS: In total, 72 UC (132 breath samples; 62 active; 70 remission) and 22 non-UC-colitis patients (22 samples) were included. Eleven VOCs predicted active vs. inactive UC in an independent internal validation set with 92% sensitivity and 77% specificity (AUC 0.94). Non-UC colitis patients could be clearly separated from active and inactive UC patients with principal component analysis. CONCLUSIONS: Volatile organic compounds can accurately distinguish active disease from remission in UC and profiles in UC are clearly different from profiles in non-UC colitis patients. VOCs have demonstrated potential as new non-invasive biomarker to monitor inflammation in UC.

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.

Volatile organic compounds in breath as markers for irritable bowel syndrome: a metabolomic approach.

BACKGROUND: The diagnosis of irritable bowel syndrome (IBS) is challenging because of its heterogeneity and multifactorial pathophysiology. No reliable biomarkers of IBS have been identified so far. AIMS: In a case-control study, using a novel application of breath analysis to distinguish IBS patients from healthy controls based on the analysis of volatile organic compounds (VOCs). Subsequently, the diagnostic VOC-biomarker set was correlated with self-reported gastrointestinal (GI) symptoms of subjects of the Maastricht IBS clinical cohort and of a general population cohort, LifeLines DEEP. METHODS: Breath samples were collected from 170 IBS patients and 153 healthy controls in the clinical cohort and from 1307 participants in general population cohort. Multivariate statistics were used to identify the most discriminatory set of VOCs in the clinical cohort, and to find associations between VOCs and GI symptoms in both cohorts. RESULTS: A set of 16 VOCs correctly predicted 89.4% of the IBS patients and 73.3% of the healthy controls (AUC = 0.83). The VOC-biomarker set correlated moderately with a set of GI symptoms in the clinical (r = 0.55, P = 0.0003) and general population cohorts (r = 0.54, P = 0.0004). A Kruskal-Wallis test showed no influence from possible confounding factors in distinguishing IBS patients from healthy controls. CONCLUSIONS: A set of 16 breath-based biomarkers that distinguishes IBS patients from healthy controls was identified. The VOC-biomarker set correlated significantly with GI symptoms in two independent cohorts. We demonstrate the potential use of breath analysis in the diagnosis and monitoring of IBS, and a possible application of VOC analyses in a general population cohort.