Emmanuel Minet

Metabolic characterization of cell systems used in in vitro toxicology testing: lung cell system BEAS-2B as a working example.

The bioactivation of pro-toxicants is the biological process through which some chemicals are metabolized into reactive metabolites. Therefore, in vitro toxicological evaluation should ideally be conducted in cell systems retaining adequate metabolic competency and relevant to the route of exposure. The respiratory tract is the primary route of exposure to inhaled pro-toxicants and lung-derived BEAS-2B cell line has been considered as a potentially suitable model for in vitro toxicology testing. However, its metabolic activity has not been characterized. We performed a gene expression analysis for 41 metabolism-related genes and compared the profile with liver- and lung-derived cell lines (HepaRG, HepG2 and A549). To confirm that mRNA expression was associated with the corresponding enzyme activity, we used a series of metabolic substrates of CYPs (CYP1A1/1B1, CYP1A2, CYP2A6/2A13 and CYP2E1) known to bioactivate inhaled pro-toxicants. CYP activities were compared between BEAS-2B, HepaRG, HepG2, and A549 cells and published literature on primary bronchial epithelium cells (HBEC). We found that in contrast to HBEC, BEAS-2B and A549 have limited CYP activity which was in agreement with their CYP gene expression profile. Control cell lines such as HepG2 and HepaRG were metabolically active for the tested CYPs. We recommend that similar strategies can be used to select suitable cell systems in the context of pro-toxicant assessment.

Urinary biomarkers of smokers’ exposure to tobacco smoke constituents in tobacco products assessment: a fit for purpose approach

Abstract There are established guidelines for bioanalytical assay validation and qualification of biomarkers. In this review, they were applied to a panel of urinary biomarkers of tobacco smoke exposure as part of a “fit for purpose” approach to the assessment of smoke constituents exposure in groups of tobacco product smokers. Clinical studies have allowed the identification of a group of tobacco exposure biomarkers demonstrating a good doseresponse relationship whilst others such as dihydroxybutyl mercapturic acid and 2-carboxy-1-methylethylmercapturic acid – did not reproducibly discriminate smokers and non-smokers. Furthermore, there are currently no agreed common reference standards to measure absolute concentrations and few inter-laboratory trials have been performed to establish consensus values for interim standards. Thus, we also discuss in this review additional requirements for the generation of robust data on urinary biomarkers, including toxicant metabolism and disposition, method validation and qualification for use in tobacco products comparison studies.

Urinary excretion of the acrylonitrile metabolite 2-cyanoethylmercapturic acid is correlated with a variety of biomarkers of tobacco smoke exposure and consumption

Acrylonitrile is an IARC class 2B carcinogen present in cigarette smoke. Urinary 2-cyanoethylmercapturic acid (CEMA) is an acrylonitrile metabolite and a potential biomarker for acrylonitrile exposure. The objective of this work was to study the dose response of CEMA in urine of non-smokers and smokers of different ISO tar yield cigarettes. We observed that smokers excreted >100-fold higher amounts of urinary CEMA than non-smokers. The CEMA levels in smokers were significantly correlated with ISO tar yield, daily cigarette consumption, and urinary biomarkers of smoke exposure. In conclusion, urinary CEMA is a suitable biomarker for assessing smoking-related exposure to acrylonitrile.

CYP1A1/1B1 and CYP2A6/2A13 activity is conserved in cultures of differentiated primary human tracheobronchial epithelial cells

BACKGROUND The respiratory tract is the primary route of exposure to inhaled toxicants such as environmental pollutants and tobacco smoke. Metabolic activation of xenobiotics is a contributor to the onset of lung diseases. Enzymes such as CYP1A1/1B1 and CYP2A6/2A13 activate polycyclic aromatic hydrocarbons and nitrosamines, respectively. Yet, few in vitro models retaining both adequate morphology and metabolic activities are currently available to investigate smoke toxicity. OBJECTIVE We characterised the expression and activity of the toxicologically relevant metabolic enzymes CYP1A1/1B1 and CYP2A6/2A13 in polarised primary tracheobronchial epithelial cells cultured at the air-liquid interface. Metabolic activity was compared with NCI-H292 and A549, two commonly used lung epithelial cell models. RESULTS We report that CYP activity and inducibility is conserved in polarised primary tracheobronchial epithelial cells for 7- and 28-days cultured at the air-liquid interface. In comparison, NCI-H292 cells did not show CYP2A6/2A13 activity whilst A549 cells did not display significant metabolic activity for CYP1A1/1B1 or CYP2A6/2A13. CONCLUSION Primary tracheobronchial epithelial cells retain both a polarised morphology and significant metabolic activity over a prolonged period of time. On the other hand, although A549 cells and NCI-H292 cells have been extensively used as lung models for toxicological assessment, they lack critical metabolic activation capability.

Targeted omics analyses, and metabolic enzyme activity assays demonstrate maintenance of key mucociliary characteristics in long term cultures of reconstituted human airway epithelia

3D reconstituted respiratory epithelia have emerged as better in vitro models for toxicological testing compared to cell lines due to the conservation of key morphological features and functions. MucilAir™ is a commercially available human airway epithelia system that can potentially maintain functional attributes for up to a year, however, detailed mucociliary characteristics and xenobiotic metabolism relevant to inhaled pro-toxicant bioactivation is lacking. Here, we assessed in MucilAir™ some key biomarkers that are characteristic of the respiratory epithelia including morphology, function and xenobiotics metabolism. The end points that were measured included targeted proteomics using a panel of 243 airway surface liquid (ASL) proteins, cilia beat frequency (CBF), a qRT-PCR screen of xenobiotic metabolizing enzymes, and CYP2A6/13, CYP1A1/1B1 activity. Comparison of ASL proteomics with human sputum identified key proteins common to both matrices, but present at different levels. Xenobiotic metabolism gene profiling demonstrated strong similarities with the normal human lung and did not reveal any consistent changes when assessed over a 6 month period. Inducibility and activity of CYP1A1/1B1 and activity of CYP2A6/2A13 were present at one month in culture and maintained in one tested MucilAir™ donor for several months. In conclusion, MucilAir™ presented important morphological and metabolic characteristics of a mucociliary epithelium in short and long term culture. MucilAir™ is therefore a potentially useful model to test repeated sub-cytotoxic doses of toxicants.

Quantification of plasma microRNAs in a group of healthy smokers, ex-smokers and non-smokers and correlation to biomarkers of tobacco exposure

Abstract The stability of circulating miRNAs, their non-invasive sampling techniques and deregulation in diseases make them potential candidate biomarkers of biological effect. Here, we profiled the level of 84 plasma miRNAs in 30 smokers, 20 non-smokers and 20 ex-smokers. A robust statistical strategy was applied with replicate samples to account for reproducibility of the results. We identified differential expression of miR-124 and let-7a between the smoking and control groups. We further explored the dose–response relationship of miR-124 and let-7a with two biomarkers of tobacco exposure and found that this relationship was affected by adjustments based on age, pack-year and gender.

Assessing modified risk tobacco and nicotine products: Description of the scientific framework and assessment of a closed modular electronic cigarette

ABSTRACT Cigarette smoking causes many human diseases including cardiovascular disease, lung disease and cancer. Novel tobacco products with reduced yields of toxicants compared to cigarettes, such as tobacco‐heating products, snus and electronic cigarettes, hold great potential for reducing the harms associated with tobacco use. In the UK several public health agencies have advocated a potential role for novel products in tobacco harm reduction. Public Health England has stated that “The current best estimate is that e‐cigarettes are around 95% less harmful than smoking” and the Royal College of Physicians has urged public health to “Promote e‐cigarettes widely as substitute for smoking”. Health related claims on novel products such as ‘reduced exposure’ and ‘reduced risk’ should be substantiated using a weight of evidence approach based on a comprehensive scientific assessment. The US FDA, has provided draft guidance outlining a framework to assess novel products as Modified Risk Tobacco Products (MRTP). Based on this, we now propose a framework comprising pre‐clinical, clinical, and population studies to assess the risk profile of novel tobacco products. Additionally, the utility of this framework is assessed through the pre‐clinical and part of the clinical comparison of a commercial e‐cigarette (Vype ePen) with a scientific reference cigarette (3R4F) and the results of these studies suggest that ePen has the potential to be a reduced risk product. HighlightsDescription of a scientific framework to assess the risk profile of next generation products relative to cigarettes.Multi‐disciplinary studies comparing a commercial e‐cigarette (Vype ePen) with a scientific reference cigarette (3R4F).The test e‐cigarette aerosol is compositionally simpler, containing reduced levels of toxicants relative to cigarette smoke.Results from a range of multi‐disciplinary studies showed reduced responses for the test e‐cigarette relative to cigarettes.

A comparative in vitro kinetic study of [14C]-eugenol and [14C]-methyleugenol activation and detoxification in human, mouse, and rat liver and lung fractions

Eugenol is a natural alkenylbenzene compound used in a variety of consumer products. There is limited evidence for the carcinogenicity of eugenol to experimental animals. However, in vitro tests for the genotoxic potential of eugenol have on occasion reported a positive result. In contrast, the structurally related alkenylbenzene methyleugenol is consistently reported as genotoxic and carcinogenic in vitro and in vivo. Metabolism of eugenol and methyleugenol has been documented but comparative kinetics have never been conducted in multiple species. Thus, we decided to investigate the bioactivation and detoxification kinetics of eugenol and methyleugenol in human, rat, and mouse to further assess their differential toxicity. The formation of a 1′-hydroxy proximate carcinogen, and a cytotoxic quinone methide, two key toxic metabolites for alkenylbenzenes, were quantified in hepatic and pulmonary microsomes and S9 fractions. Kinetic constants appKm, appVmax, and CLint were measured for these reactions. We report that methyleugenol generates a significant amount of the 1′-hydroxy proximate carcinogen while eugenol glucuronidation prevents the formation of both 1′-hydroxyeugenol and the quinone methide. Comparative kinetics highlighted key metabolic differences between human, mouse, and rat, providing a mechanistic insight into the bioactivation and detoxification of alkenylbenzenes in each species.

Nicotine, cotinine, and β-nicotyrine inhibit NNK-induced DNA-strand break in the hepatic cell line HepaRG.

Recent in vitro work using purified enzymes demonstrated that nicotine and/or a nicotine metabolite could inhibit CYPs (CYP2A6, 2A13, 2E1) involved in the metabolism of the genotoxic tobacco nitrosamine NNK. This observation raises the possibility of nicotine interaction with the mechanism of NNK bioactivation. Therefore, we hypothesized that nicotine or a nicotine metabolite such as cotinine might contribute to the inhibition of NNK-induced DNA strand breaks by interfering with CYP enzymes. The effect of nicotine and cotinine on DNA strand breaks was evaluated using the COMET assay in CYP competent HepaRG cells incubated with bioactive CYP-dependent NNK and CYP-independent NNKOAc (4-(acetoxymethylnitrosoamino)-1-(3-pyridyl)-1-butanone). We report a dose-dependent reduction in DNA damage in hepatic-derived cell lines in the presence of nicotine and cotinine. Those results are discussed in the context of the in vitro model selected.

A correlation study applied to biomarkers of internal and effective dose for acrylonitrile and 4-aminobiphenyl in smokers.

Abstract The urinary metabolites 2-cyanoethylmercapturic acid and 4-aminobiphenyl have been correlated with tobacco smoke exposure. Similarly, 2-cyanoethylvaline and 4-aminobiphenyl haemoglobin adducts have been used as biomarkers of effective dose for the exposure to acrylonitrile and 4-aminobiphenyl, respectively. Each pair of biomarkers is derived from the same parent chemical; however, the correlation between the urinary and the haemoglobin biomarkers has not been investigated. Using clinical study samples, we report a weak correlation between urinary and haemoglobin biomarkers due to different accumulation and elimination rates. Time course analysis showed that a reduction in exposure was paralleled by a delayed reduction in haemoglobin adducts.