Nitrative stress, oxidative stress and plasma endothelin levels after inhalation of particulate matter and ozone.

BACKGROUND: While exposure to ambient air contaminants is clearly associated with adverse health outcomes, disentangling mechanisms of pollutant interactions remains a challenge. OBJECTIVES: We aimed at characterizing free radical pathways and the endothelinergic system in rats after inhalation of urban particulate matter, ozone, and a combination of particles plus ozone to gain insight into pollutant-specific toxicity mechanisms and any effect modification due to air pollutant mixtures. METHODS: Fischer 344 rats were exposed for 4 h to a 3 × 3 concentration matrix of ozone (0, 0.4, 0.8 ppm) and EHC-93 particles (0, 5, 50 mg/m(3)). Bronchoalveolar lavage fluid (BALF), BAL cells, blood and plasma were analysed for biomarkers of effects immediately and 24 h post-exposure. RESULTS: Inhalation of ozone increased (p < 0.05) lipid oxidation products in BAL cells immediately post-exposure, and increased (p < 0.05) total protein, neutrophils and mature macrophages in the BALF 24 h post-exposure. Ozone increased (p < 0.05) the formation of reactive oxygen species (ROS), assessed by m-, p-, o-tyrosines in BALF (Ozone main effects, p < 0.05), while formation of reactive nitrogen species (RNS), indicated by 3-nitrotyrosine, correlated with dose of urban particles (EHC-93 main effects or EHC-93 × Ozone interactions, p < 0.05). Carboxyhemoglobin levels in blood exhibited particle exposure-related increase (p < 0.05) 24 h post recovery. Plasma 3-nitrotyrosine and o-tyrosine were increased (p < 0.05) after inhalation of particles; the effect on 3-nitrotyrosine was abrogated after exposure to ozone plus particles (EHC-93 × Ozone, p < 0.05). Big endothelin-1 (BET-1) and ET-1 were increased in plasma after inhalation of particles or ozone alone, but the effects appeared to be attenuated by co-exposure to contaminants (EHC-93 × Ozone, p < 0.05). Plasma ET levels were positively correlated (p < 0.05) with BALF m- and o-tyrosine levels. CONCLUSIONS: Pollutant-specific changes can be amplified or abrogated following multi-pollutant exposures. Oxidative and nitrative stress in the lung compartment may contribute to secondary extra-pulmonary ROS/RNS formation. Nitrative stress and endothelinergic imbalance emerge as potential key pathways of air pollutant health effects, notably of ambient particulate matter.

Na+-dependent sources of intra-axonal Ca2+ release in rat optic nerve during in vitro chemical ischemia.

The contribution of intracellular stores to axonal Ca2+ overload during chemical ischemia in vitro was examined by confocal microscopy. Ca2+ accumulation was measured by fluo-4 dextran (low-affinity dye, KD approximately 4 microM) or by Oregon Green 488 BAPTA-1 dextran (highaffinity dye, KD approximately 450 nM). Axonal Na+ was measured using CoroNa Green. Ischemia in CSF containing 2 mM Ca2+ caused an approximately 3.5-fold increase in fluo-4 emission after 30 min, indicating a large axonal Ca2+ rise well into the micromolar range. Axonal Na+ accumulation was enhanced by veratridine and reduced, but not abolished, by TTX. Ischemia in Ca2+-free (plus BAPTA) perfusate resulted in a smaller but consistent Ca2+ increase monitored by Oregon Green 488 BAPTA-1, indicating release from intracellular sources. This release was eliminated in large part when Na+ influx was reduced by replacement with N-methyl-D-glucamine (NMDG+; even in depolarizing high K+ perfusate), Li+, or by the application of TTX and significantly increased by veratridine. Intracellular release also was reduced significantly by neomycin or 1-(6-[(17beta-methoxyestra-1,3,5 [10]-trien-17-yl) amino] hexyl)-1H-pyrrole-2,5-dione (U73122 [GenBank]) (phospholipase C inhibitors), heparin [inositol trisphosphate (IP3) receptor blocker], or 7-chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one (CGP37157; mitochondrial Na+/Ca2+ exchange inhibitor) as well as ryanodine. Combining CGP37157 with U73122 [GenBank] or heparin decreased the response more than either agent alone and significantly improved electrophysiological recovery. Our conclusion is that intra-axonal Ca2+ release during ischemia in rat optic nerve is mainly dependent on Na+ influx. This Na+ accumulation stimulates three distinct intra-axonal sources of Ca2+: (1) the mitochondrial Na+/Ca2+ exchanger driven in the Na+ import/Ca2+ export mode, (2) positive modulation of ryanodine receptors, and (3) promotion of IP3 generation by phospholipase C.