Frédéric Dionnet

Development of a new in vitro system for continuous in vitro exposure of lung tissue to complex atmospheres: Application to diesel exhaust toxicology

The purpose of this study was the development of a new incubation system that can allow continuous exposure of lung tissue to complex atmospheres as a tool for the assessment of aerial environmental lung toxicology. To assess the pertinence of this new exposure system, we studied the impact of diesel engine exhausts as a complex atmosphere containing both gaseous and particulate fractions and have been able to discriminate between the toxicological impacts of the gaseous phase and particulate matter from diesel exhausts. Continuous flow-through rotating chambers with controlled pO2, pCO2, and hygrometry have been designed in which lung slices are positioned in rolling inserts that allow free access of atmosphere to the exposed lung tissue. Under control conditions, cell viability was preserved for at least 48 h as assessed by intracellular ATP, GSH, and K+ levels and slice O2 consumption levels. Short-term exposure (1 h) to diesel whole exhausts did not affect intracellular potassium or slice O2 consumption, while intracellular ATP and GSH levels were markedly decreased. Exposure to filtered exhausts showed less marked effects on both ATP and GSH levels. Superoxide dismutase activity was decreased in a similar way by both total and filtered exhausts while Se+-dependent glutathione peroxidase activity was induced by filtered exhausts to a larger extent than after total exhaust exposure, showing different response patterns of lung tissue after exposure to whole or filtered exhausts. In conclusion, this newly designed model opens a promising area in in vitro environmental lung toxicology testing.

Evaluation of cerium oxide and cerium oxide based fuel additive safety on organotypic cultures of lung slices

Among the various technological proposals under consideration to limit diesel exhaust particles (DEP) emissions, the use of a particulate filter in conjunction with a fuel-borne catalyst is currently the primary focus and likely to be introduced over the coming years. One specific catalyst is a cerium-based compound Envirox™ developed by Oxonica (Kidlington, UK) and used as a fuel additive to reduce particulate matter (PM) emissions from diesel engines. Prior to any commercialization it is necessary to undertake an assessment of any potential adverse health effects resulting from exposure to cerium oxide particles especially via the inhalation route, i.e., on the lung. Since the composition of engine exhaust emissions from additized fuel might be different to those emissions from un-additized diesel fuel, the second phase of the current investigation compared the potential biological impact of engine emissions from cerium oxide additized fuel to that of a reference fuel. An experimental procedure developed in our laboratory using an organotypic culture of lung slices was used. Biological endpoints were chosen in order to evaluate cell viability (ATP, intracellular GSH), pro-inflammatory reaction (TNF alpha) and anti-oxidant enzyme activity (total GPx, Mn SOD, catalase). No impact of nanoparticulate cerium oxide aerosol on lung tissue biological parameters was observed with one exception, an increased catalase activity which was not associated with a concomitant loss in cell viability. It is therefore concluded, on the basis of this study and from the prospective potential exposure to cerium oxide, that a very low prospective risk is associated with the expected dissemination of cerium oxide in the atmosphere. Concerning the biological impact of Envirox™ additized-diesel fuel, the observed effects are of very limited incidence in that the observed trend on organotypic culture viability and TNF alpha is beneficial, while a limited oxidant activity is observed through catalase induction. Emissions from diesel fuel additized with the tested cerium oxide catalyst do not induce any increase in the known adverse biological effects caused by diesel fuel engine emissions alone in our experimental set-up.

Biphasic culture of rat lung slices for pharmacotoxicological evaluation of complex atmospheres

The relevance to the in vivo situation of in vitro toxicity studies of complex atmospheres has frequently been limited by the procedures used for the exposure of the biological samples. We have evaluated from on-road measurements the size distribution pattern and the subsequent respiratory tract deposition rates of particulate matter from urban atmospheric aerosols, which are in the range of 110 and 3 pg/cm2 per min for tracheobronchial and alveolar areas, respectively. Continuous flow-through rotating chambers and a specific design for exhaust sampling and dilution with controlled adjustment of pO2 and pCO2 to 20% and 5%, respectively, have been developed to expose biphasic air/liquid organotypic cultures of rat lung slices to continuous flows of diluted exhausts from diesel engines with preservation of the physicochemical properties of the exhaust. The size distribution of the particulate matter and the bioavailability of pollutants were preserved, thus allowing us to closely mimic in vitro the in vivo atmosphere/tissue interactions that occur mainly through diffusion mechanisms. The toxicity response profile has been assessed in terms of tissue viability, oxidative stress, DNA injury, and the early phase of inflammatory reaction. Exhaust filtration, addition to fuel of rapeseed methyl ester, and preincubation of lung tissue with soy isoflavones modulated the toxicity response profile of exhausts. The importance of preserving both particulate matter size distribution and adsorbed pollutant bioavailability, which could not be ascertained using more classical in vitro approaches, is discussed and should be considered a prerequisite for further developments of in vitro studies to modelize in vivo inhalation of complex atmospheres.

Prevalidation of in vitro continuous flow exposure systems as alternatives to in vivo inhalation safety evaluation experimentations: outcome from MAAPHRI-PCRD5 research program.

Diesel engine emission aerosol-induced toxicity patterns were compared using both in vitro (organotypic cultures of lung tissue) and in vivo experimentations mimicking the inhalation situation with continuous aerosol flow exposure designs. Using liquid media resuspended diesel particles, we show that toxic response pattern is influenced by the presence of tensioactive agent in the medium which alter particle-borne pollutant bioavailability. Using continuous aerosol exposure in vitro, we show that with high sulfur fuel (300ppm) in the absence of oxidation catalysis, particulate matter was the main toxic component triggering DNA damage and systemic inflammation, while a very limited oxidant stress was evidenced. In contrast, with ultra-low sulfur fuel in the presence of strong diesel oxidation catalysis, the specific role of particulate matter is no longer evidenced and the gas phase then becomes the major component triggering strong oxidant stress, increased NO(2) being the most probable trigger. In vivo, plasma tumor necrosis factor alpha (TNFalpha), lung superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx) activity levels varied in agreement with in vitro observations. Diesel emission treatment with oxycat provokes a marked systemic oxidant stress. Again NO(2) proved to account for a major part of these impacts. In conclusion, similar anti-oxidant responses were observed in in vitro and in vivo experiments after diesel emission aerosol continuous flow exposures. The lung slice organotypic culture model-exposed complex aerosol appears to be a very valuable alternative to in vivo inhalation toxicology experimentations in rodents.

Comparative study of diesel and biodiesel exhausts on lung oxidative stress and genotoxicity in rats

The contribution of diesel exhaust to atmospheric pollution is a major concern for public health, especially in terms of occurrence of lung cancers. The present study aimed at addressing the toxic effects of a repeated exposure to these emissions in an animal study performed under strictly controlled conditions. Rats were repeatedly exposed to the exhaust of diesel engine. Parameters such as the presence of a particle filter or the use of gasoil containing rapeseed methyl ester were investigated. Various biological parameters were monitored in the lungs to assess the toxic and genotoxic effects of the exposure. First, a transcriptomic analysis showed that some pathways related to DNA repair and cell cycle were affected to a limited extent by diesel but even less by biodiesel. In agreement with occurrence of a limited genotoxic stress in the lungs of diesel-exposed animals, small induction of γ-H2AX and acrolein adducts was observed but not of bulky adducts and 8-oxodGuo. Unexpected results were obtained in the study of the effect of the particle filter. Indeed, exhausts collected downstream of the particle filter led to a slightly higher induction of a series of genes than those collected upstream. This result was in agreement with the formation of acrolein adducts and γH2AX. On the contrary, induction of oxidative stress remained very limited since only SOD was found to be induced and only when rats were exposed to biodiesel exhaust collected upstream of the particle filter. Parameters related to telomeres were identical in all groups. In summary, our results point to a limited accumulation of damage in lungs following repeated exposure to diesel exhausts when modern engines and relevant fuels are used. Yet, a few significant effects are still observed, mostly after the particle filter, suggesting a remaining toxicity associated with the gaseous or nano-particular phases.

Mutagenicity of diesel engine exhaust in the Ames / Salmonella assay using a direct exposure method

The aim of this study was to investigate the potential mutagenic activity of diesel engine exhaust in the Ames/Salmonella assay using a direct aerosol exposure system. So, TA 98 and TA 100 strains, with or without added S9 mix, were exposed to diesel emissions after varying degrees of filtration. Variants of these two strains, deficient in nitroreductase (TA 98NR and TA 100NR) or over-expressing O-Acetyl Transferase (YG 1024 and YG 1029), were also exposed to total (unfiltered) diesel exhaust to highlight the putative mutagenicity of any nitro-PAHs present in these emissions. Mutagenic activity of the diesel exhaust was demonstrated on Salmonella typhimurium, strains TA 100 and variants TA 100 NR and YG1029. The use of a particle filter did not modify the genotoxicity of the diesel emissions, indicating a major contribution of the gas phase to the mutagenicity of these diesel emissions. The prominent role of the particulate-associated nitro-polycyclic aromatic hydrocarbons (nitro-PAHs) claimed by some authors working on diesel exhaust organic extracts was not confirmed by our results with native diesel exhaust exposure. Our results show that the gas phase is potentially more mutagenic than the particles alone.

Impact of after-treatment devices and biofuels on diesel exhausts genotoxicity in A549 cells exposed at air-liquid interface

Using an air-liquid interface (ALI) device in dynamic conditions, we evaluated the efficiency of fuel after-treatment strategies (diesel oxidation catalysis, DOC, and diesel particulate filter, DPF, devices) and the impact of 7% and 30% rapeseed methyl esters (RME) blending on oxidative stress and genotoxicity induced in A549 lung cells after 3h exposure to whole Diesel exhausts. Oxidative stress was studied using assays of ROS production, glutathione level, catalase and superoxide-dismutase (SOD) activities. No oxidative stress and no clear differences on cytotoxicity patterns between biodiesel and standard Diesel exhausts were found. A weak but significant genotoxicity (8-oxodGuo adducts) and, for standard Diesel only, a DNA damage response (DDR) as evidenced by ƔH2AX foci, remained after DOC+DPF flowing. All together, these data could contribute to the improvement of the after treatment strategies and to health risk assessment of current diesel exhausts.