The Toxicological Effect And Molecular Mechanism Of PM₁₀ And Typical PAHs On Central Nervous System

According to the estimates from World Health Organization (WHO), ambient air pollution in both cities and rural areas caused 3.7 million premature deaths worldwide per year in 2012 with 88% occurring in middle-and low-income countries, and this mortality is due to exposure to small particulate matter of 10 microns or less in diameter (PM10). The chemical composition of particulate matter varied with geographic location, seasons, sources, and closely with the toxic effects. Epidemiological studies show that particulate matter not only produced adverse effects on cardiorespiratory, but increased incidence of cerebrovascular diseases. Especially, relevant results suggested a statistically significant association between levels of particulate matter and morbidity/hospitalization for ischemic stroke and Alzheimer’s disease events in a number of polluted cities. Morever, in the indoor and outdoor air pollution caused deaths, the rate of stroke reached 34% and 40%, respectively. Inflammation easily occurred in the lungs and brain tissues of people who lived in serious polluting areas, and contributed to neuronal degenerative diseases. Therefore, long-term effect of exposure to low concentrations of atmospheric particulate matter-induced health problems is particularly outstanding. Then we proposed the topic research, to discuss the toxicological effect and molecular mechanism of PM10 and typical PAHs on central nervous system.1. In this study, we treated male Wistar rats with PM10 collected from heating period at different concentrations (0.3,1,3 and 10 mg/kg body weight (bw)), and investigated neuronal insults induced by PM10. Then we used htoxylin eosin (HE) and terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) staining to observe pathologic changes; cytokines expression were determined by enzyme-linked immunosorbent assay (ELISA); the messenger RNA (mRNA) and protein levels of endothelial function-, inflammation-, apoptosis-, synaptic plasticity-related factors were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot; synaptic ultrastructure was observed by transmission electron microscopy (TEM). The results indicated that mild pathological abnormal occurred after 15-d exposure (five times with three days each, a total of five times), followed by the balance between endothelin-1 (ET-1) and endothelial nitric oxide synthase (eNOS) was broken, the levels of inflammatory markers interleukin 1β (IL-1β), tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX-2), induced nitric oxide synthase (iNOS), and intercellular cell adhesion molecule (ICAM-1) were changed. Also, the sample up-regulated bax/bcl-2 ratio and p53 expression, and induced neuronal apoptosis. Moreover, PM10 exposure significantly stimulated the expression of synaptic plasticity-related markers synaptophysin (SYP), postsynaptic density protein 95 (PSD-95) and NMDA receptor subunit (NR2B), up-rcgulated the levels of phosphorylated extracellular signal-regulated kinase (p-ERK) and phosphorylated cAMP response element binding protein (p-CREB), presented obvious dose-effect relationship. What’s more, the variation of synapses characteristics in the rat hippocampus further confirmed the above results. It implicates that PM10 exerted injuries to rats’brain, and the mechanisms might be involved in endothelial dysfunction and inflammatory responses.2. Seasonal production activities will lead to different PM10 composition, and then various health effects may be produced. In the present study, we treated primary cultured cortical neuron, oxygen glucose deprivation (OGD) model and male Wistar rats (the animal experiments continued for 15-d exposure, five times with three days each, a total of five times) with PM10 samples collected from different seasons (spring, summer, autumn, winter), clarified an association between season-dependent PM10 pollution in atmospheric environment and increased risk of ischemia-like injuries. Then we used TUNEL staining and immunofluorescent staining (IF) to observe pathologic change; cytokines expression were determined by ELISA; the mRNA and protein levels of endothelial function-, inflammation-, apoptosis-, synaptic plasticity-related factors were detected by RT-PCR and Western blot, synaptic ultrastructure was observed by TEM. Moreover, correlative analysis between the ischemia-like injuries and physicochemical characteristics of different season samples was conducted. Our results indicated that PM10 exposure significantly stimulated inflammatory cytokine (iNOS, COX-2 and ICAM-1) release, up-regulated the levels of phosphorylated Ca2+/calmodulin dependent protein kinase Ⅱ a (p-CaMK Ⅱ a) and p-CREB, and elevated the expression of immediately early gene (c-jun and c-fos). The increases on OGD models were susceptive, and the actions were season-dependent. In addition, seasonal PM10 induced histopathological injury and neuronal apoptosis in the brain, caused endothelial dysfunction, inflammation, and sequent injury outcome. Correlative analysis implied that winter PM10, characterized by higher levels of polycyclic aromatic hydrocarbons (PAHs), caused brain injuries similar to that of cerebral ischemia, and its exposure in atmospheric environment might contribute to the development and progression of ischemic injuries in the brain.3. As a representative substance of PAHs, Benzoapyrene (BaP) is a widely distributed environmental contaminant. Besides its well-documented immunotoxicity and carcinogenicity, more recent data implicated the neurotoxicity of BaP, especially on adverse neurobehavioral effects and cognitive function. Here, by using primary cultured cortical neuron in vitro and male rat in vivo models (the animal experiments continued for 7-d exposure, once a day, a total of seven times), we confirmed neuronal insults induced by BaP and its metabolite Benzoapyrene diol epoxide (BPDE). Then we used TUNEL staining to observe neuronal apoptosis; 2’7’-dichlorofluorescin diacetate (DCFH-DA) fluorescent probe method was used to detect reactive oxygen species (ROS) level; prostaglandin (PGs) and cyclic adenosine monophosphate (cAMP) expression were determined by ELISA; the protein level of cyclooxygenase (COX-1/COX-2), caspase-3 and EP2/4 receptors were detected by Western blot. The results indicated that BaP and BPDE induced-neurotoxic effects were likely modulated through stimulating COX-2 elevation. Also, the BaP-caused neuronal insults were significantly inhibited by antioxidant agents, COX-2 inhibitor (NS398), EP2 and EP4 antagonists (AH6809 and AH23848) and protein kinase A (PKA) inhibitors (KT5720 and H89), which implying the pathway:the action of BaP on elevating COX-2 was firstly initiated by free radical attack, then followed by activating the production of COX-2-derived prostaglandin E2 (PGE2) and functioning of its EP2/EP4 receptors, and finally stimulating cyclic adenosine monophosphate/protein kinase A (cAMP/PKA).This study illustrates the neuronal insults induced by PM10 and BaP, and the molecular mechanism involved in oxidative damage, endothelial dysfunction, inflammation, apoptosis and synaptic plasticity, which will help set up biomarkers for test and risk evaluation, and open up therapeutic approaches for treating, ameliorating, or preventing brain injuries resulting from PM10 exposure in atmospheric polluting environment.