Minimum information for reporting on the TEER (trans-epithelial/endothelial electrical resistance) assay (MIRTA).

Standard information reporting helps to ensure that assay conditions and data are consistently reported and to facilitate inter-laboratory comparisons. Here, we present recommendations on minimum information for reporting on the TEER (trans-epithelial/endothelial electrical resistance) assay (MIRTA). The TEER assay is extensively used to evaluate the health of an epithelial/endothelial cell culture model and as an indicator of the potential toxicity of a test substance. This publication is the result of an international collaboration─called the RespTox (Respiratory Toxicity) Collaborative─through which twelve laboratories shared their protocols for assessing the barrier function of respiratory epithelial cells using the TEER assay following exposure to substances. The protocols from each laboratory were reviewed to identify general steps for performing the TEER assay, interlaboratory differences between steps, the rationale for differences, whether these differences impact results or cross-laboratory comparisons between TEER measurements. While the MIRTA recommendations are focused on respiratory epithelial cell systems, these recommendations can be adapted for other cell systems that form barriers. The use of these recommendations will support data transparency and reproducibility, reduce challenges in data interpretation, enable cross-laboratory comparisons, help assess study quality, and facilitate the incorporation of the TEER assay into national and international testing guidance.

The role of fuel type and combustion phase on the toxicity of biomass smoke following inhalation exposure in mice.

The characteristics of wildland fire smoke exposures which initiate or exacerbate cardiopulmonary conditions are unclear. We previously reported that, on a mass basis, lung toxicity associated with particulate matter (PM) from flaming smoke aspirated into mouse lungs is greater than smoldering PM. In this study, we developed a computer-controlled inhalation system which can precisely control complex biomass smoke emissions from different combustion conditions. This system was used to examine the toxicity of inhaled biomass smoke from peat, eucalyptus, and oak fuels generated under smoldering and flaming phases with emissions set to the same approximate concentration of carbon monoxide (CO) for each exposure (60-110 ppm), resulting in PM levels of ~ 4 mg/m3 for flaming and ~ 40 mg/m3 for smoldering conditions. Mice were exposed by inhalation 1 h/day for 2 days, and assessed for lung toxicity at 4 and 24 h after the final exposure. Peat (flaming and smoldering) and eucalyptus (smoldering) smoke elicited significant inflammation (neutrophil influx) in mouse lungs at 4 h with the peat (flaming) smoke causing even greater lung inflammation at 24-h post-exposure. A significant alteration in ventilatory timing was also observed in mice exposed to the peat (flaming) and eucalyptus (flaming and smoldering) smoke immediately after each day of exposure. No responses were seen for exposures to similar concentrations of flaming or smoldering oak smoke. The lung toxicity potencies (neutrophil influx per PM mass) agreed well between the inhalation and previously reported aspiration studies, demonstrating that although flaming smoke contains much less PM mass than smoldering smoke, it is more toxic on a mass basis than smoldering smoke exposure, and that fuel type is also a controlling factor.

The dynamicity of acute ozone-induced systemic leukocyte trafficking and adrenal-derived stress hormones.

Ozone exposure induces neuroendocrine stress response, which causes lymphopenia. It was hypothesized that ozone-induced increases in stress hormones will temporally follow changes in circulating granulocytes, monocytes- and lymphocyte subpopulations. The goal of this study was to chronicle the changes in circulating stress hormones, cytokines, and leukocyte trafficking during 4 h exposure to ozone. Male Wistar Kyoto rats were exposed to air or ozone (0.4 or 0.8 ppm) for 0.5, 1, 2, or 4 h. After each time point, circulating stress hormones, cytokines, and lung gene expression were assessed along with live and apoptotic granulocytes, monocytes (classical and non-classical), and lymphocytes (B, Th, and Tc) in blood, thymus, and spleen using flow cytometry. Circulating stress hormones began to increase at 1 h of ozone exposure. Lung expression of inflammatory cytokines (Cxcl2, Il6, and Hmox1) and glucocorticoid-responsive genes (Nr3c1, Fkbp5 and Tsc22d3) increased in both a time- and ozone concentration-dependent manner. Circulating granulocytes increased at 0.5 h of ozone exposure but tended to decrease at 2 and 4 h, suggesting a rapid egress and then margination to the lung. Classical monocytes decreased over 4 h of exposure periods (∼80 % at 0.8 ppm). B and Tc lymphocytes significantly decreased after ozone exposure at 2 and 4 h. Despite dynamic shifts in circulating immune cell populations, few differences were measured in serum cytokines. Ozone neither increased apoptotic cells nor altered thymus and spleen lymphocytes. The data show that ozone-induced increases in adrenal-derived stress hormones precede the dynamic migration of circulating immune cells, likely to the lung to mediate inflammation.

Exacerbation of ozone-induced pulmonary and systemic effects by ß2-adrenergic and/or glucocorticoid receptor agonist/s.

Agonists of ß2 adrenergic receptors (ß2AR) and glucocorticoid receptors (GR) are prescribed to treat pulmonary diseases. Since ozone effects are mediated through the activation of AR and GR, we hypothesized that the treatment of rats with relevant therapeutic doses of long acting ß2AR agonist (LABA; clenbuterol; CLEN) and/or GR agonist (dexamethasone; DEX) would exacerbate ozone-induced pulmonary and systemic changes. In the first study, male 12-week-old Wistar-Kyoto rats were injected intraperitoneally with vehicle (saline), CLEN (0.004 or 0.02 mg/kg), or DEX (0.02 or 0.1 mg/kg). Since dual therapy is commonly used, in the second study, rats received either saline or combined CLEN + DEX (each at 0.005 or 0.02 mg/kg) one day prior to and on both days of exposure (air or 0.8ppm ozone, 4 hr/day x 2-days). In air-exposed rats CLEN, DEX or CLEN + DEX did not induce lung injury or inflammation, however DEX and CLEN + DEX decreased circulating lymphocytes, spleen and thymus weights, increased free fatty acids (FFA) and produced hyperglycemia and glucose intolerance. Ozone exposure of vehicle-treated rats increased bronchoalveolar lavage fluid protein, albumin, neutrophils, IL-6 and TNF-α. Ozone decreased circulating lymphocytes, increased FFA, and induced hypeerglycemia  and glucose intolerance. Drug treatment did not reverse ozone-induced ventillatory changes, however, lung effects (protein and albumin leakage, inflammation, and IL-6 increase) were exacerbated by CLEN and CLEN + DEX pre-treatment in a dose-dependent manner (CLEN > CLEN + DEX). Systemic effects induced by DEX and CLEN + DEX but not CLEN in air-exposed rats were analogous to and more pronounced than those induced by ozone. These data suggest that adverse air pollution effects might be exacerbated in people receiving LABA or LABA plus glucocorticoids.

Inhalation of Simulated Smog Atmospheres Affects Cardiac Function in Mice.

The health effects of individual criteria air pollutants have been well investigated. However, little is known about the health effects of air pollutant mixtures that more realistically represent environmental exposures. The present study was designed to evaluate the cardiac effects of inhaled simulated smog atmospheres (SA) generated from the photochemistry of either gasoline and isoprene (SA-G) or isoprene (SA-Is) in mice. Four-month-old female mice were exposed for 4 h to filtered air (FA), SA-G, or SA-Is. Immediately and 20 h after exposure, cardiac responses were assessed with a Langendorff preparation using a protocol consisting of 20 min of global ischemia followed by 2 h of reperfusion. Cardiac function was measured by index of left-ventricular developed pressure (LVDP) and cardiac contractility (dP/dt) before ischemia. Pre-ischemic LVDP was lower in mice immediately after SA-Is exposure (52.2 ± 5.7 cm H2O compared to 83.9 ± 7.4 cm H2O after FA exposure; p = 0.008) and 20 h after SA-G exposure (54.0 ± 12.7 cm H2O compared to 79.3 ± 7.4 cm H2O after FA exposure; p = 0.047). Pre-ischemic left ventricular contraction dP/dtmax was lower in mice immediately after SA-Is exposure (2025 ± 169 cm H2O/sec compared to 3044 ± 219 cm H2O/sec after FA exposure; p < 0.05) and 20 h after SA-G exposure (1864 ± 328 cm H2O/sec compared to 2650 ± 258 cm H2O/sec after FA exposure; p = 0.05). In addition, SA-G reduced the coronary artery flow rate 20 h after exposure compared to the FA control. This study demonstrates that acute SA-G and SA-Is exposures decrease LVDP and cardiac contractility in mice, indicating that photochemically-altered atmospheres affect the cardiovascular system.

Beta-2 Adrenergic and Glucocorticoid Receptor Agonists Modulate Ozone-Induced Pulmonary Protein Leakage and Inflammation in Healthy and Adrenalectomized Rats.

We have shown that acute ozone inhalation activates sympathetic-adrenal-medullary and hypothalamus-pituitary-adrenal stress axes, and adrenalectomy (AD) inhibits ozone-induced lung injury and inflammation. Therefore, we hypothesized that stress hormone receptor agonists (ß2 adrenergic-ß2AR and glucocorticoid-GR) will restore the ozone injury phenotype in AD, while exacerbating effects in sham-surgery (SH) rats. Male Wistar Kyoto rats that underwent SH or AD were treated with vehicles (saline + corn oil) or ß2AR agonist clenbuterol (CLEN, 0.2 mg/kg, i.p.) + GR agonist dexamethasone (DEX, 2 mg/kg, s.c.) for 1 day and immediately prior to each day of exposure to filtered air or ozone (0.8 ppm, 4 h/day for 1 or 2 days). Ozone-induced increases in PenH and peak-expiratory flow were exacerbated in CLEN+DEX-treated SH and AD rats. CLEN+DEX affected breath waveform in all rats. Ozone exposure in vehicle-treated SH rats increased bronchoalveolar lavage fluid (BALF) protein, N-acetyl glucosaminidase activity (macrophage activation), neutrophils, and lung cytokine expression while reducing circulating lymphocyte subpopulations. AD reduced these ozone effects in vehicle-treated rats. At the doses used herein, CLEN+DEX treatment reversed the protection offered by AD and exacerbated most ozone-induced lung effects while diminishing circulating lymphocytes. CLEN+DEX in air-exposed SH rats also induced marked protein leakage and reduced circulating lymphocytes but did not increase BALF neutrophils. In conclusion, circulating stress hormones and their receptors mediate ozone-induced vascular leakage and inflammatory cell trafficking to the lung. Those receiving ß2AR and GR agonists for chronic pulmonary diseases, or with increased circulating stress hormones due to psychosocial stresses, might have altered sensitivity to air pollution.