[Alternative methods to animal testing, present and future]. / Les méthodes alternatives à l'expérimentation animale, présent et futur.

Alternative methods to animal testing are used in fundamental and clinical research, for the realization of studies for regulatory purposes, and also screening operations in the development of new molecules. They are based on in vitro (cell models) or in silico (mathematical models) replacement methods. They have been largely promoted by the 3Rs rule (Replace, Reduce, Refine) which aims at regulating animal experimentation. For biomedical research, these different methods are valuable tools for better understanding the physiology of organisms and the mechanisms of the effects of chemicals and physical agents on them.

Title: Les méthodes alternatives à l'expérimentation animale, présent et futur. Abstract: Les méthodes alternatives à l'expérimentation animale sont utilisées en recherche fondamentale et clinique, pour la réalisation d'études à visée réglementaire et d'opérations de criblage en matière de développement de nouvelles molécules. Elles reposent sur des procédures de remplacement in vitro (modèles cellulaires) ou in silico (modèles mathématiques). Les méthodes alternatives ont été largement promues par la règle des 3R (Remplacer, Réduire, Raffiner) qui vise à encadrer l'expérimentation animale. Dans le domaine de la recherche, ces différentes méthodes sont des outils précieux qui permettent de mieux comprendre la physiologie des organismes et les mécanismes d'action des agents chimiques et physiques sur ces derniers.

The COVID-19 pandemic and global environmental change: Emerging research needs.

The outbreak of COVID-19 raised numerous questions on the interactions between the occurrence of new infections, the environment, climate and health. The European Union requested the H2020 HERA project which aims at setting priorities in research on environment, climate and health, to identify relevant research needs regarding Covid-19. The emergence and spread of SARS-CoV-2 appears to be related to urbanization, habitat destruction, live animal trade, intensive livestock farming and global travel. The contribution of climate and air pollution requires additional studies. Importantly, the severity of COVID-19 depends on the interactions between the viral infection, ageing and chronic diseases such as metabolic, respiratory and cardiovascular diseases and obesity which are themselves influenced by environmental stressors. The mechanisms of these interactions deserve additional scrutiny. Both the pandemic and the social response to the disease have elicited an array of behavioural and societal changes that may remain long after the pandemic and that may have long term health effects including on mental health. Recovery plans are currently being discussed or implemented and the environmental and health impacts of those plans are not clearly foreseen. Clearly, COVID-19 will have a long-lasting impact on the environmental health field and will open new research perspectives and policy needs.

Suitability of human and mammalian cells of different origin for the assessment of genotoxicity of metal and polymeric engineered nanoparticles.

Nanogenotoxicity is a crucial endpoint in safety testing of nanomaterials as it addresses potential mutagenicity, which has implications for risks of both genetic disease and carcinogenesis. Within the NanoTEST project, we investigated the genotoxic potential of well-characterised nanoparticles (NPs): titanium dioxide (TiO2) NPs of nominal size 20 nm, iron oxide (8 nm) both uncoated (U-Fe3O4) and oleic acid coated (OC-Fe3O4), rhodamine-labelled amorphous silica 25 (Fl-25 SiO2) and 50 nm (Fl-50 SiO) and polylactic glycolic acid polyethylene oxide polymeric NPs - as well as Endorem® as a negative control for detection of strand breaks and oxidised DNA lesions with the alkaline comet assay. Using primary cells and cell lines derived from blood (human lymphocytes and lymphoblastoid TK6 cells), vascular/central nervous system (human endothelial human cerebral endothelial cells), liver (rat hepatocytes and Kupffer cells), kidney (monkey Cos-1 and human HEK293 cells), lung (human bronchial 16HBE14o cells) and placenta (human BeWo b30), we were interested in which in vitro cell model is sufficient to detect positive (genotoxic) and negative (non-genotoxic) responses. All in vitro studies were harmonized, i.e. NPs from the same batch, and identical dispersion protocols (for TiO2 NPs, two dispersions were used), exposure time, concentration range, culture conditions and time-courses were used. The results from the statistical evaluation show that OC-Fe3O4 and TiO2 NPs are genotoxic in the experimental conditions used. When all NPs were included in the analysis, no differences were seen among cell lines - demonstrating the usefulness of the assay in all cells to identify genotoxic and non-genotoxic NPs. The TK6 cells, human lymphocytes, BeWo b30 and kidney cells seem to be the most reliable for detecting a dose-response.

Toxicity screenings of nanomaterials: challenges due to interference with assay processes and components of classic in vitro tests.

Given the multiplicity of nanoparticles (NPs), there is a requirement to develop screening strategies to evaluate their toxicity. Within the EU-funded FP7 NanoTEST project, a panel of medically relevant NPs has been used to develop alternative testing strategies of NPs used in medical diagnostics. As conventional toxicity tests cannot necessarily be directly applied to NPs in the same manner as for soluble chemicals and drugs, we determined the extent of interference of NPs with each assay process and components. In this study, we fully characterized the panel of NP suspensions used in this project (poly(lactic-co-glycolic acid)-polyethylene oxide [PLGA-PEO], TiO2, SiO2, and uncoated and oleic-acid coated Fe3O4) and showed that many NP characteristics (composition, size, coatings, and agglomeration) interfere with a range of in vitro cytotoxicity assays (WST-1, MTT, lactate dehydrogenase, neutral red, propidium iodide, (3)H-thymidine incorporation, and cell counting), pro-inflammatory response evaluation (ELISA for GM-CSF, IL-6, and IL-8), and oxidative stress detection (monoBromoBimane, dichlorofluorescein, and NO assays). Interferences were assay specific as well as NP specific. We propose how to integrate and avoid interference with testing systems as a first step of a screening strategy for biomedical NPs.

Toxicity evaluation of engineered nanoparticles for medical applications using pulmonary epithelial cells.

There are a multitude of nanoparticles (NPs) which have shown great potentials for medical applications. A few of them are already used for lung therapeutic and diagnostic purposes. However, there are few toxicological studies which determine possible adverse pulmonary responses. It is thus important to propose in vitro screening strategies to evaluate the pulmonary toxicity of NPs used in nanomedicine. Our goal was to determine the cellular effects of several biomedical NPs with different physico-chemical characteristics (chemical nature, size and coating) to establish suitable tests and useful benchmark NPs. The effects of poly(lactic-co-glycolic acid) (PLGA), silica, iron oxide and titanium dioxide NPs were studied using human bronchial (16HBE) and alveolar epithelial cells (A549). We evaluated cytotoxicity, reactive oxygen species (ROS) production and pro-inflammatory response in both cell lines. We demonstrated that PLGA NPs are good candidates for negative control NPs and SiO2 NPs were revealed to be the best benchmark NPs. Coating of Fe3O4 with sodium oleate, a known biocompatible compound, led to an unexpected increase in cytotoxicity. Moreover, 16HBE cells are more sensitive than A549 cells and propidium iodide uptake is a more sensitive cytotoxicity test than WST-1. The measurement of oxidative stress does not systematically allow us to predict cellular responses and different other cellular endpoints should also be addressed. We conclude that a battery of assays and cell lines are necessary to accurately evaluate the pulmonary effects of NPs and that PLGA and SiO2 NPs are suitable candidates respectively for negative and positive controls.

Prior lung inflammation impacts on body distribution of gold nanoparticles.

INTRODUCTION: Gold- (Au-) based nanomaterials have shown promising potential in nanomedicine. The individual health status is an important determinant of the response to injury/exposure. It is, therefore, critical to evaluate exposure to Au-nanomaterials with varied preexisting health status. OBJECTIVE: The goal of this research was to determine the extent of extrapulmonary translocation from healthy and inflamed lungs after pulmonary exposure to AuNPs. Male BALB/c mice received a single dose of 0.8 mg · kg(-1) AuNPs (40 nm) by oropharyngeal aspiration 24 hours after priming with LPS (0.4 mg · kg(-1)) through the same route. Metal contents were analyzed in different organs by inductively coupled plasma-mass spectrometry (ICP-MS). RESULTS: Oropharyngeal aspiration resulted in high metal concentrations in lungs (P < 0.001); however, these were much lower after pretreatment with LPS (P < 0.05). Significantly higher concentrations of Au were detected in heart and thymus of healthy animals, whereas higher concentrations of Au NPs were observed in spleen in LPS-primed animals. CONCLUSIONS: The distribution of AuNPs from lungs to secondary target organs depends upon the health status, indicating that targeting of distinct secondary organs in nanomedicine needs to be considered carefully under health and inflammatory conditions.

Deciphering the mechanisms of cellular uptake of engineered nanoparticles by accurate evaluation of internalization using imaging flow cytometry.

BACKGROUND: The uptake of nanoparticles (NPs) by cells remains to be better characterized in order to understand the mechanisms of potential NP toxicity as well as for a reliable risk assessment. Real NP uptake is still difficult to evaluate because of the adsorption of NPs on the cellular surface. RESULTS: Here we used two approaches to distinguish adsorbed fluorescently labeled NPs from the internalized ones. The extracellular fluorescence was either quenched by Trypan Blue or the uptake was analyzed using imaging flow cytometry. We used this novel technique to define the inside of the cell to accurately study the uptake of fluorescently labeled (SiO2) and even non fluorescent but light diffracting NPs (TiO2). Time course, dose-dependence as well as the influence of surface charges on the uptake were shown in the pulmonary epithelial cell line NCI-H292. By setting up an integrative approach combining these flow cytometric analyses with confocal microscopy we deciphered the endocytic pathway involved in SiO2 NP uptake. Functional studies using energy depletion, pharmacological inhibitors, siRNA-clathrin heavy chain induced gene silencing and colocalization of NPs with proteins specific for different endocytic vesicles allowed us to determine macropinocytosis as the internalization pathway for SiO2 NPs in NCI-H292 cells. CONCLUSION: The integrative approach we propose here using the innovative imaging flow cytometry combined with confocal microscopy could be used to identify the physico-chemical characteristics of NPs involved in their uptake in view to redesign safe NPs.

Internalization of SiO2 nanoparticles by alveolar macrophages and lung epithelial cells and its modulation by the lung surfactant substitute Curosurf.

Because of an increasing exposure to environmental and occupational nanoparticles (NPs), the potential risk of these materials for human health should be better assessed. Since one of the main routes of entry of NPs is via the lungs, it is of paramount importance to further characterize their impact on the respiratory system. Here, we have studied the uptake of fluorescently labeled SiO2 NPs (50 and 100 nm) by epithelial cells (NCI-H292) and alveolar macrophages (MHS) in the presence or absence of pulmonary surfactant. The quantification of NP uptake was performed by measuring cell-associated fluorescence using flow cytometry and spectrometric techniques in order to identify the most suitable methodology. Internalization was shown to be time and dose dependent, and differences in terms of uptake were noted between epithelial cells and macrophages. In the light of our observations, we conclude that flow cytometry is a more reliable technique for the study of NP internalization, and importantly, that the hydrophobic fraction of lung surfactant is critical for downregulating NP uptake in both cell types.

Nano-titanium dioxide modulates the dermal sensitization potency of DNCB.

We determined the ability of a model nanoparticle (NP) (titanium dioxide, TiO(2)) to modulate sensitization induced by a known potent dermal sensitizer (dinitrochlorobenzene) using a variant of the local lymph node assay called lymph node proliferation assay.BALB/c mice received sub-cutaneous injections of vehicle (2.5 mM sodium citrate), TiO(2) NPs (0.004, 0.04 or 0.4 mg/ml) or pigment particles (0.04 mg/ml) both stabilized in sodium citrate buffer at the base of each ear (2x50µl), before receiving dermal applications (on both ears) of 2,4-Dinitrochlorobenzene (DNCB) (2x25µl of 0.1%) or its vehicle (acetone olive oil - AOO (4:1)) on days 0, 1 and 2. On day 5, the stimulation index (SI) was calculated as a ratio of (3)HTdR incorporation in lymphocytes from DNBC-treated mice and AOO-treated controls. In a second experiment the EC(3)-value for DNCB (0 to 0.1%) was assessed in the absence or presence of 0.04 mg/ml TiO(2). In a third experiment, the lymphocyte subpopulations and the cytokine secretion profile were analyzed after TiO(2) (0.04 mg/ml) and DNCB (0.1%) treatment. Injection of NPs in AOO-treated control mice did not have any effect on lymph node (LN) proliferation. DNCB sensitization resulted in LN proliferation, which was further increased by injection of TiO(2) NPs before DNCB sensitization. The EC(3) of DNCB, with prior injection of vehicle control was 0.041%, while injection with TiO(2) decreased the EC(3) of DNCB to 0.015%. TiO(2) NPs pre-treatment did not alter the lymphocyte subpopulations, but significantly increased the level of IL-4 and decreased IL-10 production in DNCB treated animals.In conclusion, our study demonstrates that administration of nano-TiO(2) increases the dermal sensitization potency of DNCB, by augmenting a Th(2) response, showing the immunomodulatory abilities of NPs.

Autocrine effect of EGFR ligands on the pro-inflammatory response induced by PM(2.5) exposure in human bronchial epithelial cells.

Human exposure to PM(2.5) (particulate matter with an aerodynamic diameter below 2.5 µm) is known to be responsible for airway inflammation and may also induce airway remodelling. In respiratory epithelial cells exposed to PM(2.5), releases of pro-inflammatory cytokines such as granulocyte macrophage-colony stimulating factor (GM-CSF) and growth factor ligands of the epidermal growth factor receptor (EGFR) are increased. The present study aimed at determining the involvement of EGFR ligands by autocrine effects in PM(2.5)-induced GM-CSF release. PM(2.5) exposure triggers GM-CSF release by human bronchial epithelial (HBE) cells. This release is dependent on EGFR activation by ligand binding as it is inhibited by AG1478, an inhibitor of EGFR tyrosine kinase activity as well as by a neutralizing anti-EGFR antibody. The use of conditioned medium from cells previously exposed to PM(2.5) demonstrates that PM(2.5)-exposed cells release soluble EGFR ligands able to induce GM-CSF release by an autocrine manner. It was further demonstrated by inhibiting tumour-necrosis factor-alpha converting enzyme (TACE) that is involved in some EGFR ligand shedding. TAPI-2 and GM-6001, two TACE inhibitors, prevented the PM(2.5)-induced GM-CSF release as well as the silencing of TACE by siRNA. We provide evidence that the pro-inflammatory response induced by PM(2.5) exposure on HBE cells, results from an autocrine effect of EGFR ligands released by TACE activity. This autocrine loop by eliciting and sustaining inflammation could contribute to exacerbation of airway remodelling in respiratory-compromised individuals.

Nanoparticles: molecular targets and cell signalling.

Increasing evidence linking nanoparticles (NPs) with different cellular outcomes necessitate an urgent need for the better understanding of cellular signalling pathways triggered by NPs. Oxidative stress has largely been reported to be implicated in NP-induced toxicity. It could activate a wide variety of cellular events such as cell cycle arrest, apoptosis, inflammation and induction of antioxidant enzymes. These responses occur after the activation of different cellular pathways. In this context, three groups of MAP kinase cascades [ERK (extracellular signal-regulated kinases), p38 mitogen-activated protein kinase and JNK (c-Jun N-terminal kinases)] as well as redox-sensitive transcription factors such as NFκB and Nrf-2 were specially investigated. The ability of NPs to interact with these signalling pathways could partially explain their cytotoxicity. The induction of apoptosis is also closely related to the modulation of signalling pathways induced by NPs. Newly emerged scientific areas of research are the studies on interactions between NPs and biological molecules in body fluids, cellular microenvironment, intracellular components or secreted cellular proteins such as cytokines, growth factors and enzymes and use of engineered NPs to target various signal transduction pathways in cancer therapy. Recently published data present the ability of NPs to interact with membrane receptors leading to a possible aggregation of these receptors. These interactions could lead to a sustained modulation of specific signalling in the target cells or paracrine and even "by-stander" effects of the neighbouring cells or tissues. However, oxidative stress is not sufficient to explain specific mechanisms which could be induced by NPs, and these new findings emphasize the need to revise the paradigm of oxidative stress to explain the effects of NPs.

Polycyclic aromatic hydrocarbon components contribute to the mitochondria-antiapoptotic effect of fine particulate matter on human bronchial epithelial cells via the aryl hydrocarbon receptor.

BACKGROUND: Nowadays, effects of fine particulate matter (PM2.5) are well-documented and related to oxidative stress and pro-inflammatory response. Nevertheless, epidemiological studies show that PM2.5 exposure is correlated with an increase of pulmonary cancers and the remodeling of the airway epithelium involving the regulation of cell death processes. Here, we investigated the components of Parisian PM2.5 involved in either the induction or the inhibition of cell death quantified by different parameters of apoptosis and delineated the mechanism underlying this effect. RESULTS: In this study, we showed that low levels of Parisian PM2.5 are not cytotoxic for three different cell lines and primary cultures of human bronchial epithelial cells. Conversely, a 4 hour-pretreatment with PM2.5 prevent mitochondria-driven apoptosis triggered by broad spectrum inducers (A23187, staurosporine and oligomycin) by reducing the mitochondrial transmembrane potential loss, the subsequent ROS production, phosphatidylserine externalization, plasma membrane permeabilization and typical morphological outcomes (cell size decrease, massive chromatin and nuclear condensation, formation of apoptotic bodies). The use of recombinant EGF and specific inhibitor led us to rule out the involvement of the classical EGFR signaling pathway as well as the proinflammatory cytokines secretion. Experiments performed with different compounds of PM2.5 suggest that endotoxins as well as carbon black do not participate to the antiapoptotic effect of PM2.5. Instead, the water-soluble fraction, washed particles and organic compounds such as polycyclic aromatic hydrocarbons (PAH) could mimic this antiapoptotic activity. Finally, the activation or silencing of the aryl hydrocarbon receptor (AhR) showed that it is involved into the molecular mechanism of the antiapoptotic effect of PM2.5 at the mitochondrial checkpoint of apoptosis. CONCLUSIONS: The PM2.5-antiapoptotic effect in addition to the well-documented inflammatory response might explain the maintenance of a prolonged inflammation state induced after pollution exposure and might delay repair processes of injured tissues.

Carbon black and titanium dioxide nanoparticles elicit distinct apoptotic pathways in bronchial epithelial cells.

BACKGROUND: Increasing environmental and occupational exposures to nanoparticles (NPs) warrant deeper insight into the toxicological mechanisms induced by these materials. The present study was designed to characterize the cell death induced by carbon black (CB) and titanium dioxide (TiO2) NPs in bronchial epithelial cells (16HBE14o- cell line and primary cells) and to investigate the implicated molecular pathways. RESULTS: Detailed time course studies revealed that both CB (13 nm) and TiO2(15 nm) NP exposed cells exhibit typical morphological (decreased cell size, membrane blebbing, peripheral chromatin condensation, apoptotic body formation) and biochemical (caspase activation and DNA fragmentation) features of apoptotic cell death. A decrease in mitochondrial membrane potential, activation of Bax and release of cytochrome c from mitochondria were only observed in case of CB NPs whereas lipid peroxidation, lysosomal membrane destabilization and cathepsin B release were observed during the apoptotic process induced by TiO2 NPs. Furthermore, ROS production was observed after exposure to CB and TiO2 but hydrogen peroxide (H2O2) production was only involved in apoptosis induction by CB NPs. CONCLUSIONS: Both CB and TiO2 NPs induce apoptotic cell death in bronchial epithelial cells. CB NPs induce apoptosis by a ROS dependent mitochondrial pathway whereas TiO2 NPs induce cell death through lysosomal membrane destabilization and lipid peroxidation. Although the final outcome is similar (apoptosis), the molecular pathways activated by NPs differ depending upon the chemical nature of the NPs.

Effect of engineered nanoparticles on vasomotor responses in rat intrapulmonary artery.

Pulmonary circulation could be one of the primary vascular targets of finest particles that can deeply penetrate into the lungs after inhalation. We investigated the effects of engineered nanoparticles on vasomotor responses of small intrapulmonary arteries using isometric tension measurements. Acute in vitro exposure to carbon nanoparticles (CNP) decreased, and in some case abolished, the vasomotor responses induced by several vasoactive agents, whereas acute exposure to titanium dioxide nanoparticles (TiO(2)NP) did not. This could be attributed to a decrease in the activity of those vasoactive agents (including PGF(2)(alpha), serotonin, endothelin-1 and acetylcholine), as suggested when they were exposed to CNP before being applied to arteries. Also, CNP decreased the contraction induced by 30 mM KCl, without decreasing its activity. After endoplasmic reticulum calcium stores depletion (by caffeine and thapsigargin), CaCl(2) addition induced a contraction, dependent on Store-Operated Calcium Channels that was not modified by acute CNP exposure. Further addition of 30 mM KCl elicited a contraction, originating from activation of Voltage-Operated Calcium Channels that was diminished by CNP. Contractile responses to PGF(2)(alpha) or KCl, and relaxation to acetylcholine were modified neither in pulmonary arteries exposed in vitro for prolonged time to CNP or TiO(2)NP, nor in those removed from rats intratracheally instilled with CNP or TiO(2)NP. In conclusion, prolonged in vitro or in vivo exposure to CNP or TiO(2)NP does not affect vasomotor responses of pulmonary arteries. However, acute exposure to CNP decreases contraction mediated by activation of Voltage-Operated, but not Store-Operated, Calcium Channels. Moreover, interaction of some vasoactive agents with CNP decreases their biological activity that might lead to misinterpretation of experimental data.

Carbon black and titanium dioxide nanoparticles induce pro-inflammatory responses in bronchial epithelial cells: need for multiparametric evaluation due to adsorption artifacts.

The initiation of an inflammatory process is the main adverse effect observed following the exposure of the airway epithelium to nanoparticles (NPs). This study was designed to explore the pro-inflammatory potential of two different NPs of similar size but of different compositions (CB 13 nm and TiO(2) 15 nm) on a human bronchial epithelial cell line (16HBE14o-). The expression of granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL-6), and tumor necrosis factor alpha (TNFalpha) was evaluated in terms of mRNA, intracellular proteins, and released cytokines. Exposure to NPs induced a dose-dependent expression of all these cytokines, depending upon the chemical composition of NPs. The released cytokines appeared to be an inaccurate methodology to evaluate the pro-inflammatory response. Indeed, NPs adsorbed cytokines, and the binding was dependent on the nature of both the cytokine and NPs. Furthermore, addition of fetal calf serum or bovine serum albumin improved the detection of cytokines but also reduced cellular responses. Use of different detergents (Tween, Triton, and NP40) demonstrated limited efficiency to desorb cytokines from NPs. Thus, this study demonstrated the pro-inflammatory potential for CB and TiO(2) NP but underlines the methodological artifacts faced during the in vitro evaluation of cytokine release that necessitates a multiparametric evaluation.

Oxidative stress and proinflammatory effects of carbon black and titanium dioxide nanoparticles: role of particle surface area and internalized amount.

The ubiquitous presence of nanoparticles (NPs) together with increasing evidence linking them to negative health effects points towards the need to develop the understanding of mechanisms by which they exert toxic effects. This study was designed to investigate the role of surface area and oxidative stress in the cellular effects of two chemically distinct NPs, carbon black (CB) and titanium dioxide (TiO(2)), on the bronchial epithelial cell line (16HBE14o-). CB and TiO(2) NPs were taken up by 16HBE cells in a dose-dependent manner and were localized within the endosomes or free in the cytoplasm. Oxidative stress produced inside the cell by NPs was well correlated to the BET surface area and endocytosis of NPs. Contrary to intracellular conditions only CB NPs produced reactive oxygen species (ROS) under abiotic conditions. Exposure of cells to NPs resulted in an increased granulocyte macrophage colony stimulating factor (GM-CSF) mRNA expression and secretion. Inflammatory effects of NPs were dependent on the surface area and were mediated through oxidative stress as they were inhibited by catalase. It can be concluded that NP induced oxidative stress and pro-inflammatory responses are well correlated not only with the BET (Brunauer, Emmett and Teller) surface of the individual NPs but also with the internalized amount of NPs. Differences of even few nanometers in primary particle size lead to significant changes in inflammatory and oxidative stress responses.

Role of Paris PM(2.5) components in the pro-inflammatory response induced in airway epithelial cells.

Particulate matter (PM) is suspected to play a role in environmentally-induced pathologies. Due to its complex composition, the contribution of each PM components to PM-induced biological effects remains unclear. Four samples of Paris PM(2.5) having different polyaromatic hydrocarbons and metals contents were compared with each other and with their respective aqueous and organic extracts used alone or in combination. The four PM(2.5) samples similarly induced granulocyte macrophage-colony stimulating factor (GM-CSF) release, a pro-inflammatory cytokine, by human bronchial epithelial cells. It results from the activation of upstream signalling pathways and the modulation of the cellular redox state that is different according to PM(2.5) samples. The PM-aqueous extracts contained soluble metals involved in hydroxyl radical production in abiotic conditions. However they slightly contributed to the intracellular reactive oxygen species production and GM-CSF release by comparison with organic extracts. Organic compounds transactivated the xenobiotic responsive element (XRE) and antioxidant responsive element (ARE), leading to increased cytochrome P450 1A1 expression and NADPH-quinone oxydoreductase-1 expression respectively but to different extend according to PM samples underlying differences in their bioavailability. Our study underlines that chemical composition of particles per se is insufficient to predict cellular effects and that the interaction and the bioavailability of the various components were critical.

Arylamine N-acetyltransferase activity in bronchial epithelial cells and its inhibition by cellular oxidants.

Bronchial epithelial cells express xenobiotic-metabolizing enzymes (XMEs) that are involved in the biotransformation of inhaled toxic compounds. The activities of these XMEs in the lung may modulate respiratory toxicity and have been linked to several diseases of the airways. Arylamine N-acetyltransferases (NAT) are conjugating XMEs that play a key role in the biotransformation of aromatic amine pollutants such as the tobacco-smoke carcinogens 4-aminobiphenyl (4-ABP) and beta-naphthylamine (beta-NA). We show here that functional human NAT1 or its murine counterpart Nat2 are present in different lung epithelial cells i.e. Clara cells, type II alveolar cells and bronchial epithelial cells, thus indicating that inhaled aromatic amines may undergo NAT-dependent biotransformation in lung epithelium. Exposure of these cells to pathophysiologically relevant amounts of oxidants known to contribute to lung dysfunction, such as H(2)O(2) or peroxynitrite, was found to impair the NAT1/Nat2-dependent cellular biotransformation of aromatic amines. Genetic and non genetic impairment of intracellular NAT enzyme activities has been suggested to compromise the important detoxification pathway of aromatic amine N-acetylation and subsequently to contribute to an exacerbation of untoward effects of these pollutants on health. Our study suggests that oxidative/nitroxidative stress in lung epithelial cells, due to air pollution and/or inflammation, could contribute to local and/or systemic dysfunctions through the alteration of the functions of pulmonary NAT enzymes.