Scanning ion conductance microscopy reveals differential effect of PM2.5 exposure on A549 lung epithelial and SH-SY5Y neuroblastoma cell membranes.

Numerous studies have linked a wide range of diseases including respiratory illnesses to harmful particulate matter (PM) emissions indoors and outdoors, such as incense PM and industrial PM. Because of their ability to penetrate the lower respiratory tract and the circulatory system, fine particles with diameters of 2.5 µm or less (PM2.5) are believed to be more hazardous than larger PMs. Despite the enormous number of studies focusing on the intracellular processes associated with PM2.5 exposure, there have been limited reports studying the biophysical properties of cell membranes, such as nanoscale morphological changes induced by PM2.5. Our study assesses the membrane topographical and structural effects of PM2.5 from incense PM2.5 exposure in real time on A549 lung carcinoma epithelial cells and SH-SY5Y neuroblastoma cells that had been fixed to preclude adaptive cell responses. The size distribution and mechanical properties of the PM2.5 sample were characterized with atomic force microscopy (AFM). Nanoscale morphological monitoring of the cell membranes utilizing scanning ion conductance microscopy (SICM) indicated statistically significant increasing membrane roughness at A549 cells at half an hour of exposure and visible damage at 4 h of exposure. In contrast, no significant increase in roughness was observed on SH-SY5Y cells after half an hour of PM2.5 exposure, although continued exposure to PM2.5 for up to 4 h affected an expansion of lesions already present before exposure commenced. These findings suggest that A549 cell membranes are more susceptible to structural damage by PM2.5 compared to SH-SY5Y cell membranes, corroborating more enhanced susceptibility of airway epithelial cells to exposure to PM2.5 than neuronal cells.

MAPK pathway activation by chronic lead-exposure increases vascular reactivity through oxidative stress/cyclooxygenase-2-dependent pathways.

Chronic exposure to low lead concentration produces hypertension; however, the underlying mechanisms remain unclear. We analyzed the role of oxidative stress, cyclooxygenase-2-dependent pathways and MAPK in the vascular alterations induced by chronic lead exposure. Aortas from lead-treated Wistar rats (1st dose: 10 µg/100g; subsequent doses: 0.125µg/100g, intramuscular, 30days) and cultured aortic vascular smooth muscle cells (VSMCs) from Sprague Dawley rats stimulated with lead (20µg/dL) were used. Lead blood levels of treated rats attained 21.7±2.38µg/dL. Lead exposure increased systolic blood pressure and aortic ring contractile response to phenylephrine, reduced acetylcholine-induced relaxation and did not affect sodium nitroprusside relaxation. Endothelium removal and L-NAME left-shifted the response to phenylephrine more in untreated than in lead-treated rats. Apocynin and indomethacin decreased more the response to phenylephrine in treated than in untreated rats. Aortic protein expression of gp91(phox), Cu/Zn-SOD, Mn-SOD and COX-2 increased after lead exposure. In cultured VSMCs lead 1) increased superoxide anion production, NADPH oxidase activity and gene and/or protein levels of NOX-1, NOX-4, Mn-SOD, EC-SOD and COX-2 and 2) activated ERK1/2 and p38 MAPK. Both antioxidants and COX-2 inhibitors normalized superoxide anion production, NADPH oxidase activity and mRNA levels of NOX-1, NOX-4 and COX-2. Blockade of the ERK1/2 and p38 signaling pathways abolished lead-induced NOX-1, NOX-4 and COX-2 expression. Results show that lead activation of the MAPK signaling pathways activates inflammatory proteins such as NADPH oxidase and COX-2, suggesting a reciprocal interplay and contribution to vascular dysfunction as an underlying mechanisms for lead-induced hypertension.

The Aging Lacrimal Gland of Female C57BL/6J Mice Exhibits Multinucleate Macrophage Infiltration Associated With Lipid Dysregulation.

Purpose: Loss of function of the lacrimal gland (LG), which produces the aqueous tear film, is implicated in age-related dry eye. To better understand this deterioration, we evaluated changes in lipid metabolism and inflammation in LGs from an aging model. Methods: LG sections from female C57BL/6J mice of different ages (young, 2-3 months; intermediate, 10-14 months; old,  ≥24 months) were stained with Oil Red-O or Toluidine blue to detect lipids. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis and western blotting of LG lysates determined differences in the expression of genes and proteins related to lipid metabolism. A photobleaching protocol to quench age-related autofluorescence was used in LG sections to evaluate changes in immunofluorescence associated with NPC1, NPC2, CTSL, and macrophages (F4/80, CD11b) with age using confocal fluorescence microscopy. Results: Old LGs showed increased lipids prominent in basal aggregates in acinar cells and in extra-acinar sites. LG gene expression of Npc1, Npc2, Lipa, and Mcoln2, encoding proteins involved in lipid metabolism, was increased with age. NPC1 was also significantly increased in old LGs by western blotting. In photobleached LG sections, confocal fluorescence microscopy imaging of NPC1, NPC2, and CTSL immunofluorescence showed age-associated enrichment in macrophages labeled to detect F4/80. Although mononuclear macrophages were detectable in LG at all ages, this novel multinucleate macrophage population containing NPC1, NPC2, and CTSL and enriched in F4/80 and some CD11b was increased with age at extra-acinar sites. Conclusions: Lipid-metabolizing proteins enriched in F4/80-positive multinucleated macrophages are increased in old LGs adjacent to sites of lipid deposition in acini.

Copper exposure for 30 days at a daily dose twice the recommended increases blood pressure and cardiac contractility.

Copper is an essential factor for the body's homeostasis. However, excess copper compromises organic functions. AIMS: We investigated the effects of copper exposure for 30 days on blood pressure (BP) and cardiac contractility and the putative involvement of nitric oxide (NO) and reactive oxygen species. MAIN METHODS: Wistar rats (12 weeks old, 280 g) were randomized to the treated group that was exposed for 30 days to copper (2000 µg/kg/day CuCl2) and the control (Ct) group that received intraperitoneal saline (0.9%). KEY FINDINGS: The blood concentration of copper was ~1.26 µg/mL in the copper-exposed group and ~0.024 µg/mL in the Ct group. The main metal accumulations occurred in the liver and kidneys. Copper exposure increased systolic BP (Cu: 141 ± 3 mmHg; Ct: 133 ± 3 mmHg) (tail cuff method), left ventricular systolic pressure and papillary muscle force. Calcium release from the sarcoplasmic reticulum was reduced. The contractile response to Ca2+ was increased by copper, and the effect was not altered by L-NAME. Copper increased contractions dependent on sarcolemmal Ca2+ influx, and this effect was not altered by L-NAME. The percentage response to isoproterenol decreased in the copper-exposed group, but L-NAME did not alter this reduction. Papillary force development at the peak and plateau of tetanic contractions also increased after copper exposure, but this effect was not altered by L-NAME. In situ detection of OH local production increased. SIGNIFICANCE: Copper increased BP and cardiac force, increased Ca2+ inflow, reduced Ca2+ reuptake by the sarcoplasmic reticulum, and increased OH local production. Copper exposure at doses considered tolerable affects cardiac contractility.

Sub-chronic lead exposure produces ß1-adrenoceptor downregulation decreasing arterial pressure reactivity in rats.

Lead is considered a causative factor for hypertension and other cardiovascular diseases. AIMS: To investigate the effects of sub-chronic lead exposure on blood pressure reactivity and cardiac ß1-adrenoceptor activity and to evaluate whether the effects found in vitro are similar to those found in vivo. MAIN METHODS: Male Wistar rats were randomly distributed into two groups: control rats (Ct) and rats administered drinking water containing 100ppm lead (Pb) for 30days. KEY FINDINGS: Blood pressure in the Pb rats increased starting from the first week of treatment until the end of the study [systolic blood pressure, Ct: 122±4 vs. Pb: 143±3mmHg; diastolic blood pressure, Ct: 63±4 vs. Pb: 84±4mmHg]. The heart rate was also increased (Ct: 299±11 vs. Pb: 365±11bpm), but the pressure reactivity to phenylephrine was decreased. Losartan and hexamethonium exhibited a greater reduction in blood pressure of Pb rats than in the Ct rats. Isoproterenol increased the left ventricular systolic and end-diastolic pressure, and heart rate only in Ct rats, suggesting that lead induced ß1-adrenoceptor downregulation. Indomethacin reduced the blood pressure and heart rate in the Pb rats, suggesting the involvement of cyclooxygenase-derived products (which are associated with reduced nitric oxide bioavailability) in this process. SIGNIFICANCE: These findings offer further evidence that the effects of sub-chronic lead exposure in vitro can be reproduced in vivo-even at low concentrations-thus triggering mechanisms for the development of hypertension. Therefore, lead should be considered an environmental risk factor for cardiovascular disease.