Role of inflammatory cells and adenosine in lung ischemia reoxygenation injury using a model of lung donation after cardiac death.

AIM: The objective of this study is to analyze the role of inflammation in the lung ischemia reperfusion (IR) injury and determine the protective role of adenosine in an in vitro lung transplantation model. MATERIALS AND METHODS: We used a hybrid model of lung donor after cardiac death, with warm ischemia in corpo of varying duration (2 h, 4 h) followed by in vitro lung slices culture for reoxygenation (1 h, 4 h and 24 h), in the presence or not of lymphocytes and of adenosine. To quantify the inflammatory lesions, we performed TNFα, IL2 assays, and histological analysis. RESULTS: In this model of a nonblood perfused system, the addition of lymphocytes during reoxygenation lead to higher rates of TNFα and IL2 after 4 h than after 2 h of warm ischemia (P < .05). These levels increased with the duration of reoxygenation and were maximum at 24 h (P < .05). In the presence of adenosine TNFα and IL2 decreased. After 2 h of warm ischemia, we observed a significant inflammatory infiltration, alveolar thickening and a necrosis of the bronchiolar cells. After 4 h of warm ischemia, alveolar cells necrosis was associated. CONCLUSION: This model showed that lymphocytes increased the inflammatory response and the histological lesions after 4 h of warm ischemia and that adenosine could have an anti-inflammatory role with potential reconditioning action when used in the pneumoplegia solution.

Organotypic lung culture: A new model for studying ischemia and ex vivo perfusion in lung transplantation.

AIM: Donors after cardiac death (DCD) in lung transplantation is considered as a solution for organ shortage. However, it is characterized by warm ischemic period, which could be involved in severe Ischemia-Reperfusion lesion (IR) with early graft dysfunction. We describe a new hybrid model combining in vivo ischemia followed by in vitro reoxygenation using organ-specific culture. MATERIAL AND METHODS: A hybrid model using in vivo ischemic period followed by in vitro lung slice reoxygenation was set up in rat to mimic DCD in lung transplantation with in vitro perfusion. Different markers (bioenergetics, oxidant stress assays, and histology) were measured to evaluate the viability of lung tissue after different ischemic times (I-0, I-1, I-2, I-4, I-15 hours) and reoxygenation times (R-0, R-1, R-4, R-24 hours). RESULTS: No differences were found in cell viability, ATP concentrations, extracellular LDH assays or histology, demonstrating extensive viability of up to 4 hours in lung tissue warm ischemia. We found oxidative stress mainly during the ischemic period with no burst at reoxygenation. Cytosolic anti-oxidant system was involved first (I-0,I-1,I-2) followed by mitochondrial anti-oxidant system for extensive ischemia (I-4). Histological features showed differences in this model of ischemia-reoxygenation between bronchial epithelium and lung parenchymal cells, with epithelium regeneration after 2 hours of warm ischemia and 24 hours of perfusion. CONCLUSION: The results of our hybrid model experiment suggest extensive lung viability of up to 4 hours ischemia. Our model could be an interesting tool to evaluate ex vivo reconditioning techniques after different in vivo lung insults.

Precision cut lung slices as an efficient tool for in vitro lung physio-pharmacotoxicology studies.

1.We review the specific approaches for lung tissue slices preparation and incubation systems and the research application fields in which lung slices proved to be a very efficient alternative to animal experimentation for biomechanical, physiological, pharmacological and toxicological approaches. 2.Focus is made on air-liquid interface dynamic organ culture systems that allow direct tissue exposure to complex aerosol and that best mimic in vivo lung tissue physiology. 3.A compilation of research applications in the fields of vascular and airway reactivity, mucociliary transport, polyamine transport, xenobiotic biotransformation, chemicals toxicology and complex aerosols supports the concept that precision cut lung slices are a very efficient tool maintaining highly differentiated functions similar to in vivo lung organ when kept under dynamic organ culture. They also have been successfully used for lung gene transfer efficiency assessment, for lung viral infection efficiency assessment, for studies of tissue preservation media and tissue post-conditioning to optimize lung tissue viability before grafting. 4.Taken all together, the reviewed studies point to a great interest for precision cut lung slices as an efficient and valuable alternative to in vivo lung organ experimentation.

Caenorhabditis elegans: a model to monitor bacterial air quality.

BACKGROUND: Low environmental air quality is a significant cause of mortality and morbidity and this question is now emerging as a main concern of governmental authorities. Airborne pollution results from the combination of chemicals, fine particles, and micro-organisms quantitatively or qualitatively dangerous for health or for the environment. Increasing regulations and limitations for outdoor air quality have been decreed in regards to chemicals and particles contrary to micro-organisms. Indeed, pertinent and reliable tests to evaluate this biohazard are scarce. In this work, our purpose was to evaluate the Caenorhaditis elegans killing test, a model considered as an equivalent to the mouse acute toxicity test in pharmaceutical industry, in order to monitor air bacterial quality. FINDINGS: The present study investigates the bacterial population in dust clouds generated during crop ship loading in harbor installations (Rouen harbor, Normandy, France). With a biocollector, airborne bacteria were impacted onto the surface of agar medium. After incubation, a replicate of the colonies on a fresh agar medium was done using a velvet. All the replicated colonies were pooled creating the "Total Air Sample". Meanwhile, all the colonies on the original plate were isolated. Among which, five representative bacterial strains were chosen. The virulence of these representatives was compared to that of the "Total Air Sample" using the Caenorhaditis elegans killing test. The survival kinetic of nematodes fed with the "Total Air Sample" is consistent with the kinetics obtained using the five different representatives strains. CONCLUSIONS: Bacterial air quality can now be monitored in a one shot test using the Caenorhaditis elegans killing test.

New exposure system to evaluate the toxicity of (scooter) exhaust emissions in lung cells in vitro.

A constantly growing number of scooters produce an increasing amount of potentially harmful emissions. Due to their engine technology, two-stroke scooters emit huge amounts of adverse substances, which can induce adverse pulmonary and cardiovascular health effects. The aim of this study was to develop a system to expose a characterized triple cell coculture model of the human epithelial airway barrier, to freshly produced and characterized total scooter exhaust emissions. In exposure chambers, cell cultures were exposed for 1 and 2 h to 1:100 diluted exhaust emissions and in the reference chamber to filtered ambient air, both controlled at 5% CO(2), 85% relative humidity, and 37 degrees C. The postexposure time was 0-24 h. Cytotoxicity, used to validate the exposure system, was significantly increased in exposed cell cultures after 8 h postexposure time. (Pro-) inflammatory chemo- and cytokine concentrations in the medium of exposed cells were significantly higher at the 12 h postexposure time point. It was shown that the described exposure system (with 2 h exposure duration, 8 and 24 h postexposure time, dilution of 1:100, flow of 2 L/min as optimal exposure conditions) can be used to evaluate the toxic potential of total exhaust emissions.

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.

Hazard and risk assessment of a nanoparticulate cerium oxide-based diesel fuel additive - a case study.

Envirox is a scientifically and commercially proven diesel fuel combustion catalyst based on nanoparticulate cerium oxide and has been demonstrated to reduce fuel consumption, greenhouse gas emissions (CO(2)), and particulate emissions when added to diesel at levels of 5 mg/L. Studies have confirmed the adverse effects of particulates on respiratory and cardiac health, and while the use of Envirox contributes to a reduction in the particulate content in the air, it is necessary to demonstrate that the addition of Envirox does not alter the intrinsic toxicity of particles emitted in the exhaust. The purpose of this study was to evaluate the safety in use of Envirox by addressing the classical risk paradigm. Hazard assessment has been addressed by examining a range of in vitro cell and cell-free endpoints to assess the toxicity of cerium oxide nanoparticles as well as particulates emitted from engines using Envirox. Exposure assessment has taken data from modeling studies and from airborne monitoring sites in London and Newcastle adjacent to routes where vehicles using Envirox passed. Data have demonstrated that for the exposure levels measured, the estimated internal dose for a referential human in a chronic exposure situation is much lower than the no-observed-effect level (NOEL) in the in vitro toxicity studies. Exposure to nano-size cerium oxide as a result of the addition of Envirox to diesel fuel at the current levels of exposure in ambient air is therefore unlikely to lead to pulmonary oxidative stress and inflammation, which are the precursors for respiratory and cardiac health problems.

Mutagenicity of benzyl chloride in the Salmonella/microsome mutagenesis assay depends on exposure conditions.

Benzyl chloride (BCl) is a clear yellowish, volatile liquid that is widely used as an intermediate for the production of benzyl alcohol and benzyl compounds used in perfumery, dyes and pharmaceuticals. In previous studies BCl has shown weak and inconsistent mutagenicity in the Salmonella/microsome mutagenesis assay (Ames test). The aim of the present study was to investigate the potential mutagenic activity of BCl using modifications of the standard Ames test in order to adapt the method to the volatile nature of the test compound. Tests were performed using (a) the standard plate-incorporation method, (b) incubation of the treated plates in closed containers, (c) a vaporization-diffusion method to expose Ames test plates to volatilised BCl and (d) the pre-incubation method. Using the standard plate-incorporation method, BCl showed no (in the absence of metabolic activation) or very weak (in the presence of metabolic activation) mutagenic activity in Salmonella typhimurium tester strain TA 100. The use of the pre-incubation method did not improve detection of mutagenic activity of BCl. The use of closed containers significantly increased the response, but the most marked response was obtained by testing BCl in volatilised form in the vaporization-diffusion method. Using the latter approach there appeared to be less toxicity of the BCl treatments to the tester bacteria. Our findings suggest that BCl may show greater mutagenic activity in the gaseous phase. This work underlines the importance of using appropriate methods for the evaluation of volatile compounds. The modifications described here are easy to realize in practice and should prove useful for the investigation of other volatile materials or atmospheric contaminants.

Inhalation of diluted diesel engine emission impacts heart rate variability and arrhythmia occurrence in a rat model of chronic ischemic heart failure.

Both increase in cardiac arrhythmia incidence and decrease in heart rate variability (HRV) have been described following human and experimental animal exposures to air pollutants. However, the potential causal relationship between these two factors remains unclear. Incidence of ventricular arrhythmia and HRV were evaluated during and after a 3 h period of Diesel engine exhaust exposure in ten healthy and ten chronic ischemic heart failure (CHF, 3 months after coronary ligation) Wistar rats using implantable ECG telemetry. Air pollutants were delivered to specifically designed whole body individual exposure chambers at particulate matter concentrations similar to those measured inside cabins of cars inserted in congested urban traffic. Recordings were obtained from unrestrained and unsedated vigil rats. Immediate decrease in RMSSD was observed in both healthy (6.64 +/- 2.62 vs. 4.89 +/- 1.67 ms, P < 0.05) and CHF rats (8.01 +/- 0.89 vs. 6.6 +/- 1.37 ms, P < 0.05) following exposure. An immediate 200-500% increase in ventricular premature beats was observed in CHF rats only. Whereas HRV progressively returned to baseline values within 2.5 h after exposure start, the proarrhythmic effect persisted as late as 5 h after exposure termination in CHF rats. Persistence of ventricular proarrhythmic effects after HRV normalization suggests that HRV reduction is not the mechanism of cardiac arrhythmias in this model. Our methodological approach, closely reflecting the real clinical situations, appeared to be a unique tool to provide further insight into the pathophysiological mechanisms of traffic related airborne pollution health impact.

Oryctes monoceros trapping with synthetic pheromone and palm material in Ivory Coast.

Oryctes monoceros is the most serious pest in coconut plantations, causing up to 40% damage in tropical Africa, especially in Ivory Coast. With a view to reducing pest populations by olfactory trapping, field trials were carried out to assess the efficiency of a synthetic aggregation pheromone: ethyl 4-methyloctanoate (1), 4-methyloctanoic acid (2), a related volatile produced by males, and decaying palm material, either oil palm empty fruit bunches (EFB) or pieces of coconut wood (CW) of various ages. Vertical polyvinyl chloride tube traps (2 x 0.16 m with two openings in the upper half), embedded in the soil, were more efficient than 30-L pail traps 1.5 m above ground. EFB, which were inactive alone, synergized captures with synthetic pheromone. CW was more effective than EFB in comparative trials. Compound 2 did not catch any beetles when assessed with EFB, and reduced catches by 1 + EFB when tested at >10% with the pheromone. Trapping over 6 mo in 2002 and 2003 in a 19-ha coconut plot inside a 4,000-ha oil palm estate reduced damage from 3.8% in 2001 to 0.5% in 2002, then to 0.2% in 2003. Damage was 0.0% in 2004 with routine trapping using 32 traps, which caught 3369 beetles in 9 mo. The results are discussed in relation to other Dynastid palm pests and coconut protection in Ivory Coast.

Interactions between acetoin, a plant volatile, and pheromone in Rhynchophorus palmarum: behavioral and olfactory neuron responses.

Aggregation of Rhynchophorus palmarum weevils on host plants is mediated by a male pheromone (rhynchophorol: R) and host-plant volatiles (PVs) acting in synergy. Synthetic PV blends synergizing pheromone contain acetoin (A) and ethyl acetate (EtAc). R, A, and EtAc are detected by specialized olfactory receptor neurons (ORNs). In addition, particular types of ORNs are tuned to both A and R. To specify the role played by acetoin in pheromone perception, we recorded the responses of ORNs to 100 ng of A or R presented either separately or mixed. Behavioral responses to R, A, and EtAc were studied in a four-armed olfactometer and by field trapping. We screened 59 R-, A-, and AR-tuned ORNs by recording specific responses to odors presented either separately or mixed. Stimulations by blends elicited complex response profiles from the three ORN types: some gave synergistic responses, others were inhibited, and the remainder responded as though both odors were detected independently. Several gave either a weak or no response to a first stimulation by R, but responded clearly to a second stimulation after an intercalary stimulation by A. In the olfactometer, both sexes were more attracted to a blend of A + R (1 + 0.01 ng/sec) than to pure compounds, whereas EtAc did not enhance response to R. Pheromone-baited traps (1 mg/day) containing PV blends (650 mg/day) based on an ethanol/EtAc blend (1:1), plus either 5 or 10% A, or a more complex reference blend, or sugarcane (natural pheromone synergist), caught similar numbers of weevils and about twice as many insects as a control ethanol/EtAc blend. Traps with only pheromone caught about 10 times fewer insects. Behavioral results support the role of acetoin as a pheromone synergist for R. palmarum, and electrophysiological data provide evidence of modulation of peripheral sensory responses to pheromone by acetoin. Sexual dimorphism was observed neither at the ORN nor at the behavioral levels.

Assessment of a new cell culture perfusion apparatus for in vitro chronic toxicity testing. Part 1: technical description.

In vitro models for chronic toxicity, defined as a recurring exposure to compounds over a prolonged period of time, are still underrepresented in drug evaluation processes. The classical approach to cell culture is not readily suitable to long term repetitive applications. Therefore, we assessed the use of a commercially available perfusion cell culture apparatus in its applicability to chronic renal toxicity testing and describe the technical aspects of adopting the perfusion cell culture system to our purposes. It was apparent that there is a subtle dynamic difference between human renal proximal tubular cells cultured under perfusion and static conditions as illustrated by the accumulation of lactate dehydrogenase (LDH) and the secondary metabolism of resazurin to hydroresorufin, which occurred only under static conditions. The major achievement was the standardisation of the handling of this system with regard to cell cultivation, pH regulation, temperature regulation, and reproducibility of common toxicity endpoints.

Assessment of a new cell culture perfusion apparatus for in vitro chronic toxicity testing. Part 2: toxicological evaluation.

The goal of replacement, refinement and reduction of animal testing is critically dependent on the development and assessment of novel in vitro methodologies and the further development of existing methodologies. Here, we evaluated the use of a modified perfusion cell culture apparatus for application to chronic in vitro nephrotoxicity testing using DMSO, SDS, paracetamol and cyclosporine A as test compounds. Renal epithelial monolayers were cultured on microporous growth supports and exposed to test compounds under static or perfusion conditions. Alamar Blue reduction, gamma-glutamyl transpeptidase activity (GGT), lactate dehydrogenase activity (LDH) and remnant protein were used to assay cell toxicity. There was no significant difference in IC(50) values between static and perfusion cultures up to 72 hours exposure. However, the perfusion system allowed continuous real-time monitoring of plasma membrane damage, which gives important information of time, duration and scale of toxicity. The complexity of the system restrains its use to low-throughput analysis. However, the real and theoretical advantages of this and similar systems merit further investigations.

Male aggregation pheromone of date palm fruit stalk borer Oryctes elegans.

Laboratory and field investigations were carried out to characterize the chemical communication system of the date palm fruit stalk borer, Oryctes elegans, and to develop pheromone-based trapping in Eastern Iran. Adults of both sexes feeding on date palm pieces attracted conspecifics, whereas date palm alone was minimally attractive. Males were twice as attractive as females. More beetles were captured at the palm crown than at ground level. Odors from adults feeding on sugarcane were sampled and analyzed by gas chromatography and mass spectrometry. Whereas females did not emit sex specific volatiles, males emitted a blend of 4-methyloctanoic acid (1: major component) and ethyl 4-methyloctanoate (2), occasionally mixed with minor components: 4-methyloctanyl acetate (3), methyl 4-methyloctanoate (4), 4-methyloctanol (5), and nonanyl acetate (6). Electroantennography and field trapping experiments demonstrated that compound 1 is an essential component of the male aggregation pheromone of O. elegans. It was barely attractive by itself but synergistic with fresh date palm odor. It attracted many more beetles than any of compounds 2-6. The addition of one or several of compounds 2-6 to 1 did not improve trap captures. During the course of 2 years, we captured 4000 beetles, with a weekly average of 6.3 beetles/trap, and were able to monitor the seasonal flight of O. elegans. Our results provide the basis for developing mass trapping for control of this pest.

Activity of male pheromone of Melanesian rhinoceros beetle Scapanes australis.

Laboratory and field investigations were carried out to investigate the nature and role of the male pheromone emitted by the Dynast beetle Scapanes australis and to develop a mass trapping technique against this major coconut pest in Papua New Guinea. We report the biological data obtained from natural and synthetic pheromone, previously described as an 84:12:4 (w/w) mixture of 2-butanol (1), 3-hydoxy-2-butanone (2), and 2,3-butanediol (3). EAG recordings from natural and synthetic pheromone and a pitfall olfactometer were poorly informative. In contrast, extensive field trapping trials with various synthetic pheromone mixtures and doses showed that 1 and 2 (formulated in polyethylene sachets in 90:5 v/v ratio) were necessary and sufficient for optimum long-range attraction. Beetles were captured in traps baited with racemic 1 plus 2, with or without a stereoisomer mixture of 3 (2.5- to 2500-mg/day doses). Plant pieces, either sugarcane or coconut, enhanced captures by the synthetic pheromone, which was active alone. Traps with the pheromone caught both sexes in a 3:2 female-male ratio. A pheromone-based mass trapping led to the capture of 2173 beetles in 14 traps surrounding 40 ha of a cocoa-coconut plantation. The captures followed a log-linear decrease during the 125-week trapping program. The role of the male pheromone and its potential for crop protection are discussed.

Isoflavones protect against diesel engine exhaust injury in organotypic culture of lung tissue.

The wide range of potential health beneficial effects of isoflavones, including a chemoprentive action, have prompted us to study the potential benefits of genistein and daidzein in an experimental model of environmental pollution impact on lung tissue. A diesel engine placed was used to generate reproducible emissions including both gaseous and particulate matters that are commonly found in urban atmospheres. Isoflavones were added to culture medium of rat lung slices 2 h prior to their exposure to pollutants for 3 h. Intracellular ATP and GSH levels, TNFα production, nucleosome assay and TUNEL labeling were monitored. Isoflavones showed almost total in vitro protection against inflammatory and pro-apoptotic responses in lung slices. Isoflavones 0.3 and 1 µmol/l protected against exhaust induced GSH depletion. Isoflavones 0.3 µmol/l appeared to exert the most beneficial effects. In conclusion, this study points out the potential interest of soy isoflavones consumption in polluted areas. Further studies should be undertaken to verify that similar effects could be obtained after in vivo administration of isoflavones.