Benoit Nemery

Diesel Exhaust Particles in Lung Acutely Enhance Experimental Peripheral Thrombosis

Background—Pollution by particulates has consistently been associated with increased cardiovascular morbidity and mortality, but a plausible biological basis for this association is lacking. Methods and Results—Diesel exhaust particles (DEPs) were instilled into the trachea of hamsters, and blood platelet activation, experimental thrombosis, and lung inflammation were studied. Doses of 5 to 500 &mgr;g of DEPs per animal induced neutrophil influx into the bronchoalveolar lavage fluid with elevation of protein and histamine but without lactate dehydrogenase release. The same doses enhanced experimental arterial and venous platelet rich-thrombus formation in vivo. Blood samples taken from hamsters 30 and 60 minutes after instillation of 50 &mgr;g of DEPs yielded accelerated aperture closure (ie, platelet activation) ex vivo, when analyzed in the Platelet Function Analyser (PFA-100). The direct addition of as little as 0.5 &mgr;g/mL DEPs to untreated hamster blood significantly shortened closure time in vitro. Conclusions—The intratracheal instillation of DEPs leads to lung inflammation as well as a rapid activation of circulating blood platelets. The kinetics of platelet activation are consistent with the reported clinical occurrence of thrombotic complications after exposure to pollutants. Our findings, therefore, provide a plausible explanation for the increase in cardiovascular morbidity and mortality accompanying urban air pollution.

Size effect of intratracheally instilled particles on pulmonary inflammation and vascular thrombosis

Particulate air pollution is associated with cardiorespiratory effects and ultrafine particles (UFPs, diameter < 100 nm) are believed to play an important role. We studied the acute (1 h) effect of intratracheally instilled unmodified (60 nm), negatively charged carboxylate-modified (60 nm), or positively charged amine-modified (60 or 400 nm) polystyrene particles on bronchoalveolar lavage (BAL) indices and on peripheral thrombosis in hamster. The latter was assessed by measuring the extent of photochemically induced thrombosis in a femoral vein via transillumination. Unmodified and negative UFPs did not modify thrombosis and BAL indices. Positive UFPs increased thrombosis at 500 microg per animal (+ 341 +/- 96%) and at 50 microg per animal (+ 533 +/- 122%), but not at 5 microg per animal. Neutrophils, lactate dehydrogenase, and histamine were increased in BAL at all these doses but protein concentration was increased only at 500 microg per animal. Positive 400-nm particles (500 microg per animal) did not affect thrombosis, although they led to a neutrophil influx and an increase in BAL proteins and histamine. Using the Platelet Function Analyser (PFA-100), the platelets of hamsters were activated by the in vitro addition of positive UFPs and 400-nm particles to blood. We conclude that intratracheally administered positive ultrafine and 400-nm particles induce pulmonary inflammation within 1 h. Positive UFPs, but not the 400-nm particles enhance thrombosis. Hence, particle-induced lung inflammation and thrombogenesis can be partially uncoupled.

Putrescine and 5-hydroxytryptamine accumulation in rat lung slices: Cellular localization and responses to cell-specific lung injury

The cellular localization of putrescine (1,4-diaminobutane) and 5-hydroxytryptamine (5HT) following the accumulation of tritium-labeled putrescine (2.5 microM) or 5HT (0.5 microM) into rat lung slices was determined by autoradiography at the light microscope level. Putrescine labeling was found to occur in type II alveolar epithelial cells and in branchiolar nonciliated (Clara) cells, and possibly also in type I alveolar epithelial cells. The pattern of 5HT labeling was clearly different from that with putrescine, since the parenchyma was diffusely labeled with no preferential location in type II cells, but with strong labeling of the endothelium of large vessels and also the pleural mesothelium. The apparent kinetic parameters for the tissue uptake of [3H]putrescine (2.5 to 80 microM) and [14C]5HT (0.5 to 16 microM; both being simultaneously present in a 5 to 1 molar ratio) were studied in lung slices from normal rats and rats pretreated with O,S,S-trimethyl phosphorodithioate (OSSMe, 11 to 95 mg/kg, po), with paraquat (20 mg/kg, ip), or with alpha-naphthylthiourea (ANTU, 5 or 10 mg/kg, ip). OSSMe and paraquat were used as models for pulmonary epithelium-damaging agents, and ANTU was taken as a model for a pulmonary endothelium-damaging agent. The Vmax for the uptake of 5HT was significantly increased (without change in Km) following treatment with OSSMe and paraquat. Following ANTU treatment the Vmax for the uptake of 5HT was unchanged (5 mg/kg) or increased (10 mg/kg, Km also increased). These results indicate that in lung slices the response to lung injury may be associated with an increased accumulation of 5HT. The Vmax for the uptake of putrescine was significantly decreased (without change in Km) following treatment with OSSMe and paraquat. Following ANTU treatment the Vmax for the uptake of putrescine was unchanged (5 mg/kg) or decreased (10 mg/kg, no change in Km). These results suggest that a decreased putrescine uptake is a sensitive index of pulmonary epithelial damage.

Short-term exposure to particulate matter induces arterial but not venous thrombosis in healthy mice

Summary.  Background:  Epidemiological findings suggest an association between exposure to particulate matter (PM) and venous thrombo‐embolism. Objectives:  To investigate arterial vs. venous thrombosis, inflammation and coagulation in mice, (sub)acutely exposed to two types of PM. Methods:  Various doses (25, 100 and 200 μg per animal) of urban particulate matter (UPM) or diesel exhaust particles (DEP) were intratracheally (i.t.) instilled in C57Bl6/n mice and several endpoints measured at 4, 10 and 24 h. Mice were also repeatedly exposed to 100 μg per animal on three consecutive days with endpoints measured 24 h after the last instillation. Results:  Exposure to 200 μg per mouse UPM enhanced arterial thrombosis, but neither UPM nor DEP significantly enhanced venous thrombosis. Both types of PM induced dose‐dependent increases in broncho‐alveolar lavage fluid (BALF) total cell numbers (mainly neutrophils) and cytokines (IL‐6, KC, MCP‐1, RANTES, MIP‐1α), with peaks at 4 h and overall higher values for UPM than for DEP. Systemic inflammation was limited to increased serum IL‐6 levels, 4 h after UPM. Both types of PM induced similar and dose‐dependent but modest increases in factor (F)VII, FVIII and fibrinogen. Three repeated instillations did not or only modestly enhance the proinflammatory and procoagulant status. Conclusions:  Compared with DEP, UPM induced more pronounced pulmonary inflammation, but both particle types triggered similar and mild short‐term systemic effects. Hence, acute exposure to PM triggers activation of primary hemostasis in the mouse, but no substantial secondary hemostasis activation, resulting in arterial but not venous thrombogenicity.

Some aspects of the toxicology of trimethyl and triethyl phosphorothioates.

The pharmacokinetics (disposal curves) of trimethyl and triethyl phosphorothioates have been determined. The concentrations to which the lung has been exposed at the LD50 dose of different chemical structures have been compared with the dose administered to the animal; the variation of LD50 of different chemical structures is little reduced. The in vitro kinetics of the reaction of O,S,S-trimethyl phosphorodithioate or O,O,S-triethyl phosphorothioate with plasma cholinesterase and carboxylesterase and brain acetylcholinesterase have been determined. The relation between inhibition and circulating concentrations in vivo have been examined. Changes in Clara cells reported by others seem to be physiological rather than pathological. O,S,S-Trimethyl phosphorodithioate is metabolised by rat lung and liver slices and microsomes. From these studies and the effect of various pretreatments of the rats on toxicity to the lung, it is probable that the proximal toxin is produced in the lung by oxidative attack on the alkylthio moeity of the compounds.

Studies on the metabolism of the pneumotoxin O,S,S-trimethyl phosphorodithioate. I. Lung and liver microsomes

The metabolism of O,S,S-trimethyl phosphorodithioate (OSSMe), a pneumotoxic impurity in some organophosphorus insecticides, was investigated in rat lung and liver microsomal preparations, using OSSMe labelled with 3H or 14C on one of its thiolo-methyl (CH3S-) groups. Production of O,S-dimethyl phosphorothioate (OSMeO-) and binding of radioactivity to protein were NADPH-dependent and were shown to be, at least partly, cytochrome P-450-dependent processes in both lung and liver microsomes. Incubation with reduced glutathione prevented the binding of radioactivity without affecting OSMeO- production. The Km for the conversion of OSSMe to OSMeO- was 15-fold lower in lung (0.30 +/- 0.07 mM) than in liver (4.63 +/- 2.42 mM) microsomes. These results show that cytochrome P-450-dependent mixed-function oxidase is implicated in at least part of the metabolic activation of OSSMe, and suggest that the pulmonary isozyme(s) are more active at metabolizing OSSMe than hepatic isozymes. It is speculated, on the basis of literature data on other sulphur-containing chemicals, that the metabolic activation of OSSMe involves oxidation of a thiolo-sulphur, with subsequent formation of CH3-S-S-protein disulphides.

Cellular Specific Toxicity in the Lung

The architectural structure of the lung is designed to provide and protect a vast surface area within the chest cavity which allows the effective exchange of respired gases with the bloodstream. This means that the lung has numerous cell types with specific functions and when the cell types in the blood are taken into consideration, over forty individual cell types have been identified (Sorokin, 1970). Since the total cardiac output passes through the lung, the lung can be exposed to toxic xenobiotic compounds and their metabolites present in the blood. The lung is also exposed to gases, vapours and particles (if small enough) present in the inspired air. Even toxins present at very low concentrations in the atmosphere may present a risk to the lung, especially when one considers that the adult human lung respires approximately three tons of air per year (Mustafa and Tierney, 1978).

A germ-free status does not protect from the lethal effects of acute lung damage caused by o,s,s,-trimethyl phosphorodithioate

Abstract To investigate whether a normal resident microbiological flora of conventional rats influences the lethality of chemical-induced lung damage, the pneumotoxin O,S,S-trimethyl phosphorodithioate (OSSMe, 75 or 100 mg/kg, s.c.) was administered to age-matched conventional and germ-free male F344 rats. Microbiological and serological examinations confirmed the germ-free state of the germ-free rats and showed that no specific lung pathogens were present in the conventional rats. As in conventional rats, clinical symptoms and death of OSSMe-treated germ-free rats resulted from respiratory failure. The germ-free rats were not more resistant, but rather more susceptible to OSSMe than conventional rats. Increases in lung weight and histological examination of lung tissue 3 days after dosing with OSSMe (75 mg/kg, s.c.) showed no differences between germ-free and conventional rats. Despite alterations in their nasopharyngeal flora, death in the conventional rats was probably not caused by bacterial superinfection. The higher susceptibility of germ-free rats to OSSMe can be partly attributed to pharmacokinetic differences, since plasma levels of OSSMe decreased more slowly in germ-free than in conventional rats. It is concluded that germ-free rats are not protected from the lethal consequences of acute chemical-induced lung damage.