Preclinical Characterization of HPN536, a Trispecific, T-Cell-Activating Protein Construct for the Treatment of Mesothelin-Expressing Solid Tumors.

PURPOSE: Mesothelin (MSLN) is a glycophosphatidylinositol-linked tumor antigen overexpressed in a variety of malignancies, including ovarian, pancreatic, lung, and triple-negative breast cancer. Early signs of clinical efficacy with MSLN-targeting agents have validated MSLN as a promising target for therapeutic intervention, but therapies with improved efficacy are still needed to address the significant unmet medical need posed by MSLN-expressing cancers. EXPERIMENTAL DESIGN: We designed HPN536, a 53-kDa, trispecific, T-cell-activating protein-based construct, which binds to MSLN-expressing tumor cells, CD3ε on T cells, and to serum albumin. Experiments were conducted to assess the potency, activity, and half-life of HPN536 in in vitro assays, rodent models, and in nonhuman primates (NHP). RESULTS: HPN536 binds to MSLN-expressing tumor cells and to CD3ε on T cells, leading to T-cell activation and potent redirected target cell lysis. A third domain of HPN536 binds to serum albumin for extension of plasma half-life. In cynomolgus monkeys, HPN536 at doses ranging from 0.1 to 10 mg/kg demonstrated MSLN-dependent pharmacologic activity, was well tolerated, and showed pharmacokinetics in support of weekly dosing in humans. CONCLUSIONS: HPN536 is potent, is well tolerated, and exhibits extended half-life in NHPs. It is currently in phase I clinical testing in patients with MSLN-expressing malignancies (NCT03872206).

TriTACs, a Novel Class of T-Cell-Engaging Protein Constructs Designed for the Treatment of Solid Tumors.

T cells have a unique capability to eliminate cancer cells and fight malignancies. Cancer cells have adopted multiple immune evasion mechanisms aimed at inhibiting T cells. Dramatically improved patient outcomes have been achieved with therapies genetically reprogramming T cells, blocking T-cell inhibition by cancer cells, or transiently connecting T cells with cancer cells for redirected lysis. This last modality is based on antibody constructs that bind a surface antigen on cancer cells and an invariant component of the T-cell receptor. Although high response rates were observed with T-cell engagers specific for CD19, CD20, or BCMA in patients with hematologic cancers, the treatment of solid tumors has been less successful. Here, we developed and characterized a novel T-cell engager format, called TriTAC (for Trispecific T-cell Activating Construct). TriTACs are engineered with features to improve patient safety and solid tumor activity, including high stability, small size, flexible linkers, long serum half-life, and highly specific and potent redirected lysis. The present study establishes the structure/activity relationship of TriTACs and describes the development of HPN424, a PSMA- (FOLH1-) targeting TriTAC in clinical development for patients with metastatic castration-resistant prostate cancer.

Identifying the Transcriptional Response of Cancer and Inflammation-Related Genes in Lung Cells in Relation to Ambient Air Chemical Mixtures in Houston, Texas.

Atmospheric pollution represents a complex mixture of air chemicals that continually interact and transform, making it difficult to accurately evaluate associated toxicity responses representative of real-world exposure. This study leveraged data from a previously published article and reevaluated lung cell transcriptional response induced by outdoor atmospheric pollution mixtures using field-based exposure conditions in the industrialized Houston Ship Channel. The tested hypothesis was that individual and co-occurring chemicals in the atmosphere relate to altered expression of critical genes involved in inflammation and cancer-related processes in lung cells. Human lung cells were exposed at an air-liquid interface to ambient air mixtures for 4 h, with experiments replicated across 5 days. Real-time monitoring of primary and secondary gas-phase pollutants, as well as other atmospheric conditions, was simultaneously conducted. Transcriptional analysis of exposed cells identified critical genes showing differential expression associated with both individual and chemical mixtures. The individual pollutant identified with the largest amount of associated transcriptional response was benzene. Tumor necrosis factor (TNF) and interferon regulatory factor 1 (IRFN1) were identified as key upstream transcription factor regulators of the cellular response to benzene. This study is among the first to measure lung cell transcriptional responses in relation to real-world, gas-phase air mixtures.

Cardiovascular effects of diesel exhaust inhalation: photochemically altered versus freshly emitted in mice.

This study was designed to compare the cardiovascular effects of inhaled photochemically altered diesel exhaust (aged DE) to freshly emitted DE (fresh DE) in female C57Bl/6 mice. Mice were exposed to either fresh DE, aged DE, or filtered air (FA) for 4 hr using an environmental irradiation chamber. Cardiac responses were assessed 8 hr after exposure utilizing Langendorff preparation with a protocol consisting of 20 min of perfusion and 20 min of ischemia followed by 2 hr of reperfusion. Cardiac function was measured by indices of left-ventricular-developed pressure (LVDP) and contractility (dP/dt) prior to ischemia. Recovery of post-ischemic LVDP was examined on reperfusion following ischemia. Fresh DE contained 460 µg/m3 of particulate matter (PM), 0.29 ppm of nitrogen dioxide (NO2) and no ozone (O3), while aged DE consisted of 330 µg/m3 of PM, 0.23 ppm O3 and no NO2. Fresh DE significantly decreased LVDP, dP/dtmax, and dP/dtmin compared to FA. Aged DE also significantly reduced LVDP and dP/dtmax. Data demonstrated that acute inhalation to either fresh or aged DE lowered LVDP and dP/dt, with a greater fall noted with fresh DE, suggesting that the composition of DE may play a key role in DE-induced adverse cardiovascular effects in female C57Bl/6 mice.

Residential water-soluble organic gases: chemical characterization of a substantial contributor to indoor exposures.

Characterization of residential indoor air is important to understanding exposures to airborne chemicals. While it is well known that non-polar VOCs are elevated indoors, polar VOCs remain poorly characterized. Recent measurements showed that total polar water-soluble organic gas (WSOG) concentrations are also much higher indoors than directly outdoors (on average 15× greater at 13 homes, on a carbon-mass basis). This work aims to chemically characterize these WSOG mixtures. Acetic, lactic, and formic acids account for 41% on average (30-54% across homes), of the total WSOG-carbon collected inside each home. Remaining WSOGs were characterized via high-resolution positive-mode electrospray ionization mass spectrometry. In total, 98 individual molecular formulas were detected. On average 67% contained the elements CHO, 11% CHN, 11% CHON, and 11% contained sulfur, phosphorus, or chlorine. Some molecular formulas are consistent with compounds having known indoor sources such as diethylene glycol (m/z+ 117.091, C4H10O3), hexamethylenetetramine (m/z+ 141.113, C6H12N4), and methacrylamide (m/z+ 86.060, C4H7NO). Exposure pathways, potential doses, and implications are discussed.

Photochemical Cloud Processing of Primary Wildfire Emissions as a Potential Source of Secondary Organic Aerosol.

We investigated the gas-phase chemical composition of biomass burning (BB) emissions and their role in aqueous secondary organic aerosol (aqSOA) formation through photochemical cloud processing. A high-resolution time-of-flight chemical ionization mass spectrometer using iodide reagent ion chemistry detected more than 100 gas-phase compounds from the emissions of 30 different controlled burns during the 2016 Fire Influence on Regional and Global Environments Experiment (FIREX) at the Fire Science Laboratory. Compounds likely to partition to cloudwater were selected based on high atomic oxygen-to-carbon ratio and abundance. Water solubility was confirmed by detection of these compounds in water after mist chamber collection during controlled burns and analysis using ion chromatography and electrospray ionization interfaced to high-resolution time-of-flight mass spectrometry. Known precursors of aqSOA were found in the primary gaseous BB emissions (e.g., phenols, acetate, and pyruvate). Aqueous OH oxidation of the complex biomass burning mixtures led to rapid depletion of many compounds (e.g., catechol, levoglucosan, methoxyphenol) and formation of others (e.g., oxalate, malonate, mesoxalate). After 150 min of oxidation (approximatively 1 day of cloud processing), oxalate accounted for 13-16% of total dissolved organic carbon. Formation of known SOA components suggests that cloud processing of primary BB emissions forms SOA.

Occurrence of Staphylococcus aureus in swine and swine workplace environments on industrial and antibiotic-free hog operations in North Carolina, USA: A One Health pilot study.

Occupational exposure to swine has been associated with increased Staphylococcus aureus carriage, including antimicrobial-resistant strains, and increased risk of infections. To characterize animal and environmental routes of worker exposure, we optimized methods to identify S. aureus on operations that raise swine in confinement with antibiotics (industrial hog operation: IHO) versus on pasture without antibiotics (antibiotic-free hog operation: AFHO). We associated findings from tested swine and environmental samples with those from personal inhalable air samplers on worker surrogates at one IHO and three AFHOs in North Carolina using a new One Health approach. We determined swine S. aureus carriage status by collecting swab samples from multiple anatomical sites, and we determined environmental positivity for airborne bioaerosols with inhalable and impinger samplers and a single-stage impactor (ambient air) cross-sectionally. All samples were analyzed for S. aureus, and isolates were tested for antimicrobial susceptibility, absence of scn (livestock marker), and spa type. Seventeen of twenty (85%) swine sampled at the one IHO carried S. aureus at >1 anatomical sites compared to none of 30 (0%) swine sampled at the three AFHOs. All S. aureus isolates recovered from IHO swine and air samples were scn negative and spa type t337; almost all isolates (62/63) were multidrug resistant. S. aureus was recovered from eight of 14 (67%) ambient air and two (100%) worker surrogate personal air samples at the one IHO, whereas no S. aureus isolates were recovered from 19 ambient and six personal air samples at the three AFHOs. Personal worker surrogate inhalable sample findings were consistent with both swine and ambient air data, indicating the potential for workplace exposure. IHO swine and the one IHO environment could be a source of potential pathogen exposure to workers, as supported by the detection of multidrug-resistant S. aureus (MDRSA) with livestock-associated spa type t337 among swine, worker surrogate personal air samplers and environmental air samples at the one IHO but none of the three AFHOs sampled in this study. Concurrent sampling of swine, personal swine worker surrogate air, and ambient airborne dust demonstrated that IHO workers may be exposed through both direct (animal contact) and indirect (airborne) routes of transmission. Investigation of the effectiveness of contact and respiratory protections is warranted to prevent IHO worker exposure to multidrug-resistant livestock-associated S. aureus and other pathogens.

Effect of secondary organic aerosol from isoprene-derived hydroxyhydroperoxides on the expression of oxidative stress response genes in human bronchial epithelial cells.

Isoprene-derived secondary organic aerosol (SOA), which comprise a large portion of atmospheric fine particulate matter (PM2.5), can be formed through various gaseous precursors, including isoprene epoxydiols (IEPOX), methacrylic acid epoxide (MAE), and isoprene hydroxyhydroperoxides (ISOPOOH). The composition of the isoprene-derived SOA affects its reactive oxygen species (ROS) generation potential and its ability to alter oxidative stress-related gene expression. In this study we assess effects of isoprene SOA derived solely from ISOPOOH oxidation on human bronchial epithelial cells by measuring the gene expression changes in 84 oxidative stress-related genes. In addition, the thiol reactivity of ISOPOOH-derived SOA was measured through the dithiothreitol (DTT) assay. Our findings show that ISOPOOH-derived SOA alter more oxidative-stress related genes than IEPOX-derived SOA but not as many as MAE-derived SOA on a mass basis exposure. More importantly, we found that the different types of SOA derived from the various gaseous precursors (MAE, IEPOX, and ISOPOOH) have unique contributions to changes in oxidative stress-related genes that do not total all gene expression changes seen in exposures to atmospherically relevant compositions of total isoprene-derived SOA mixtures. This study suggests that amongst the different types of known isoprene-derived SOA, MAE-derived SOA are the most potent inducer of oxidative stress-related gene changes but highlights the importance of considering isoprene-derived SOA as a total mixture for pollution controls and exposure studies.

Variation in DNA-Damage Responses to an Inhalational Carcinogen (1,3-Butadiene) in Relation to Strain-Specific Differences in Chromatin Accessibility and Gene Transcription Profiles in C57BL/6J and CAST/EiJ Mice.

BACKGROUND: The damaging effects of exposure to environmental toxicants differentially affect genetically distinct individuals, but the mechanisms contributing to these differences are poorly understood. Genetic variation affects the establishment of the gene regulatory landscape and thus gene expression, and we hypothesized that this contributes to the observed heterogeneity in individual responses to exogenous cellular insults. OBJECTIVES: We performed an in vivo study of how genetic variation and chromatin organization may dictate susceptibility to DNA damage, and influence the cellular response to such damage, caused by an environmental toxicant. MATERIALS AND METHODS: We measured DNA damage, messenger RNA (mRNA) and microRNA (miRNA) expression, and genome-wide chromatin accessibility in lung tissue from two genetically divergent inbred mouse strains, C57BL/6J and CAST/EiJ, both in unexposed mice and in mice exposed to a model DNA-damaging chemical, 1,3-butadiene. RESULTS: Our results showed that unexposed CAST/EiJ and C57BL/6J mice have very different chromatin organization and transcription profiles in the lung. Importantly, in unexposed CAST/EiJ mice, which acquired relatively less 1,3-butadiene-induced DNA damage, we observed increased transcription and a more accessible chromatin landscape around genes involved in detoxification pathways. Upon chemical exposure, chromatin was significantly remodeled in the lung of C57BL/6J mice, a strain that acquired higher levels of 1,3-butadiene-induced DNA damage, around the same genes, ultimately resembling the molecular profile of CAST/EiJ. CONCLUSIONS: These results suggest that strain-specific changes in chromatin and transcription in response to chemical exposure lead to a "compensation" for underlying genetic-driven interindividual differences in the baseline chromatin and transcriptional state. This work represents an example of how chemical and environmental exposures can be evaluated to better understand gene-by-environment interactions, and it demonstrates the important role of chromatin response in transcriptomic changes and, potentially, in deleterious effects of exposure. https://doi.org/10.1289/EHP1937.

Gene Expression Profiling in Human Lung Cells Exposed to Isoprene-Derived Secondary Organic Aerosol.

Secondary organic aerosol (SOA) derived from the photochemical oxidation of isoprene contributes a substantial mass fraction to atmospheric fine particulate matter (PM2.5). The formation of isoprene SOA is influenced largely by anthropogenic emissions through multiphase chemistry of its multigenerational oxidation products. Considering the abundance of isoprene SOA in the troposphere, understanding mechanisms of adverse health effects through inhalation exposure is critical to mitigating its potential impact on public health. In this study, we assessed the effects of isoprene SOA on gene expression in human airway epithelial cells (BEAS-2B) through an air-liquid interface exposure. Gene expression profiling of 84 oxidative stress and 249 inflammation-associated human genes was performed. Our results show that the expression levels of 29 genes were significantly altered upon isoprene SOA exposure under noncytotoxic conditions (p < 0.05), with the majority (22/29) of genes passing a false discovery rate threshold of 0.3. The most significantly affected genes belong to the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) transcription factor network. The Nrf2 function is confirmed through a reporter cell line. Together with detailed characterization of SOA constituents, this study reveals the impact of isoprene SOA exposure on lung responses and highlights the importance of further understanding its potential health outcomes.

Assessment of biological responses of EpiAirway 3-D cell constructs versus A549 cells for determining toxicity of ambient air pollution.

CONTEXT: EpiAirway™ 3-D constructs are human-derived cell cultures of differentiated airway epithelial cells that may represent a more biologically relevant model of the human lung. However, limited information is available on their utility for exposures to air pollutants at the air-liquid interface (ALI). OBJECTIVE: To assess the biological responses of EpiAirway™ cells in comparison to the responses of A549 human alveolar epithelial cells after exposure to air pollutants at ALI. METHODS: Cells were exposed to filtered air, 400 ppb of ozone (O3) or a photochemically aged Synthetic Urban Mixture (SynUrb54) consisting of hydrocarbons, nitrogen oxides, O3 and other secondary oxidation products for 4 h. Basolateral supernatants and apical washes were collected at 9 and 24 h post-exposure. We assessed cytotoxicity by measuring lactate dehydrogenase (LDH) release into the culture medium and apical surface. Interleukin 6 (IL-6) and interleukin 8 (IL-8) proteins were measured in the culture medium and in the apical washes to determine the inflammatory response after exposure. RESULTS: Both O3 and SynUrb54 significantly increased basolateral levels of LDH and IL-8 in A549 cells. No significant changes in LDH and IL-8 levels were observed in the EpiAirway™ cells, however, IL-6 in the apical surface was significantly elevated at 24 h after O3 exposure. CONCLUSION: LDH and IL-8 are robust endpoints for assessing toxicity in A549 cells. The EpiAirway™ cells show minimal adverse effects after exposure suggesting that they are more toxicologically resistant compared to A549 cells. Higher concentrations or longer exposure times are needed to induce effects on EpiAirway™ cells.

A comparison of three dispersion media on the physicochemical and toxicological behavior of TiO2 and NiO nanoparticles.

UNLABELLED: Nanomaterials represent a burgeoning field of technological innovation. With the onset of environmental release and commercial product exposure associated with nanomaterial manufacture and proliferation, the concomitant effects on human health remain unknown and demand further investigation. Agglomeration of nanomaterials in biologically relevant media used in in vitro methods further complicates dosing in toxicological study. OBJECTIVE: to compare the effects of in vitro dispersion techniques on the physicochemical and toxicological dosimetry of TiO2 (<50 nm) and NiO (<20 nm) nanoparticles and some resulting toxicological endpoints to test for potential effects. METHODS: three media were prepared for A549 and 16hbe14o cells with varying concentrations of TiO2 and NiO nanoparticles. Physicochemical effects were analyzed with dynamic light scattering, ICP-MS, SEM, and TEM. Toxicological effects were determined after stimulation of cells with nanoparticles for 4 and 24h followed by analysis of inflammatory and oxidative stress markers with ELISA and RT-PCR. Our data show that dispersion media differentially affect physicochemical properties and toxicological endpoints. Therefore, we conclude that in vitro nanotoxicology models that use re-suspension methods of exposure yield inconsistent and misleading biological results due to physicochemical variation of particle characteristics and transport processes.

Cellular RNA is chemically modified by exposure to air pollution mixtures.

RNAs are more susceptible to modifications than DNA, and chemical modifications in RNA have an effect on their structure and function. This study aimed to characterize chemical effects on total RNA in human A549 lung cells after exposure to elevated levels of major secondary air pollutants commonly found in urban locations, including ozone (O3), acrolein (ACR) and methacrolein (MACR). Enzyme-linked immunosorbent assays (ELISA) were used to measure levels of interleukin (IL)-8 in the growth media and 8-oxoguanine (8OG) levels in total cellular RNA, and lactate dehydrogenase (LDH) in the growth media was measured by a coupled enzymatic assay. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to measure levels of microRNA 10b (miR-10b). The study found that 1-h exposure to all tested pollutant mixtures consistently caused significant increases in the levels of 8OG in total RNA. In the case of 4 ppm O3 exposures, measured levels of IL-8, LDH and miR-10b each showed consistent trends between two independent trials, but varied among these three targets. After 1-h exposures to an ACR+MACR mixture, measured levels of IL-8, LDH and miR-10b showed variable results. For mixtures of O3+ACR+MACR, IL-8 measurements showed no change; miR-10b and LDH showed variable results. The results indicate that short-term high-concentration exposures to air pollution can cause RNA chemical modifications. Chemical modifications in RNAs could represent more consistent markers of cellular stress relative to other inflammation markers, such as IL-8 and LDH, and provide a new biomarker endpoint for mechanistic studies in toxicity of air pollution exposure.

From the Field to the Laboratory: Air Pollutant-Induced Genomic Effects in Lung Cells.

Current in vitro studies do not typically assess cellular impacts in relation to real-world atmospheric mixtures of gases. In this study, we set out to examine the feasibility of measuring biological responses at the level of gene expression in human lung cells upon direct exposures to air in the field. This study describes the successful deployment of lung cells in the heavily industrialized Houston Ship Channel. By examining messenger RNA (mRNA) levels from exposed lung cells, we identified changes in genes that play a role as inflammatory responders in the cell. The results show anticipated responses from negative and positive controls, confirming the integrity of the experimental protocol and the successful deployment of the in vitro instrument. Furthermore, exposures to ambient conditions displayed robust changes in gene expression. These results demonstrate a methodology that can produce gas-phase toxicity data in the field.

Application of chemical vapor generation systems to deliver constant gas concentrations for in vitro exposure to volatile organic compounds.

Exposure to volatile organic compounds from outdoor air pollution is a major public health concern; however, there is scant information about the health effects induced by inhalation exposure to photochemical transformed products of primary emissions. In this study, we present a stable and reproducible exposure method to deliver ppm-ppb levels of gaseous standards in a humidified air stream for in vitro cell exposure through a direct air-liquid interface. Gaseous species were generated from a diffusion vial, and coupled to a gas-phase in vitro exposure system. Acrolein and methacrolein, which are major first-generation photochemical transformation products of 1,3-butadiene and isoprene, respectively, were selected as model compounds. A series of vapor concentrations (0.23-2.37 ppmv for acrolein and 0.68-10.7 ppmv for methacrolein) were investigated to characterize the exposure dose-response relationships. Temperature and the inner diameter of the diffusion vials are key parameters to control the evaporation rates and diffusion rates for the delivery of target vapor concentrations. Our findings suggest that this exposure method can be used for testing a wide range of atmospheric volatile organic compounds, and permits both single compound and multiple compound sources to generate mixtures in air. The relative standard deviations (%RSD) of output concentrations were within 10% during the 4-hour exposure time. The comparative exposure-response data allow us to prioritize numerous hazardous gas phase air pollutants. These identified pollutants can be further incorporated into air quality simulation models to better characterize the environmental health risks arising from inhalation of the photochemical transformed products.

Epigenetic events determine tissue-specific toxicity of inhalational exposure to the genotoxic chemical 1,3-butadiene in male C57BL/6J mice.

1,3-Butadiene (BD), a widely used industrial chemical and a ubiquitous environmental pollutant, is a known human carcinogen. Although genotoxicity is an established mechanism of the tumorigenicity of BD, epigenetic effects have also been observed in livers of mice exposed to the chemical. To better characterize the diverse molecular mechanisms of BD tumorigenicity, we evaluated genotoxic and epigenotoxic effects of BD exposure in mouse tissues that are target (lung and liver) and non-target (kidney) for BD-induced tumors. We hypothesized that epigenetic alterations may explain, at least in part, the tissue-specific differences in BD tumorigenicity in mice. We evaluated the level of N-7-(2,3,4-trihydroxybut-1-yl)guanine adducts and 1,4-bis-(guan-7-yl)-2,3-butanediol crosslinks, DNA methylation, and histone modifications in male C57BL/6 mice exposed to filtered air or 425 ppm of BD by inhalation (6 h/day, 5 days/week) for 2 weeks. Although DNA damage was observed in all three tissues of BD-exposed mice, variation in epigenetic effects clearly existed between the kidneys, liver, and lungs. Epigenetic alterations indicative of genomic instability, including demethylation of repetitive DNA sequences and alterations in histone-lysine acetylation, were evident in the liver and lung tissues of BD-exposed mice. Changes in DNA methylation were insignificant in the kidneys of treated mice, whereas marks of condensed heterochromatin and transcriptional silencing (histone-lysine trimethylation) were increased. These modifications may represent a potential mechanistic explanation for the lack of tumorigenesis in the kidney. Our results indicate that differential tissue susceptibility to chemical-induced tumorigenesis may be attributed to tissue-specific epigenetic alterations.

The Gillings Sampler--an electrostatic air sampler as an alternative method for aerosol in vitro exposure studies.

There is growing interest in studying the toxicity and health risk of exposure to multi-pollutant mixtures found in ambient air, and the U.S. Environmental Protection Agency (EPA) is moving towards setting standards for these types of mixtures. Additionally, the Health Effects Institute's strategic plan aims to develop and apply next-generation multi-pollutant approaches to understanding the health effects of air pollutants. There's increasing concern that conventional in vitro exposure methods are not adequate to meet EPA's strategic plan to demonstrate a direct link between air pollution and health effects. To meet the demand for new in vitro technology that better represents direct air-to-cell inhalation exposures, a new system that exposes cells at the air-liquid interface was developed. This new system, named the Gillings Sampler, is a modified two-stage electrostatic precipitator that provides a viable environment for cultured cells. Polystyrene latex spheres were used to determine deposition efficiencies (38-45%), while microscopy and imaging techniques were used to confirm uniform particle deposition. Negative control A549 cell exposures indicated the sampler can be operated for up to 4h without inducing any significant toxic effects on cells, as measured by lactate dehydrogenase (LDH) and interleukin-8 (IL-8). A novel positive aerosol control exposure method, consisting of a p-tolualdehyde (TOLALD) impregnated mineral oil aerosol (MOA), was developed to test this system. Exposures to the toxic MOA at a 1 ng/cm(2) dose of TOLALD yielded a reproducible 1.4 and 2-fold increase in LDH and IL-8 mRNA levels over controls. This new system is intended to be used as an alternative research tool for aerosol in vitro exposure studies. While further testing and optimization is still required to produce a "commercially ready" system, it serves as a stepping-stone in the development of cost-effective in vitro technology that can be made accessible to researchers in the near future.

Cumulative risk: toxicity and interactions of physical and chemical stressors.

Recent efforts to update cumulative risk assessment procedures to incorporate nonchemical stressors ranging from physical to psychosocial reflect increased interest in consideration of the totality of variables affecting human health and the growing desire to develop community-based risk assessment methods. A key roadblock is the uncertainty as to how nonchemical stressors behave in relationship to chemical stressors. Physical stressors offer a reasonable starting place for measuring the effects of nonchemical stressors and their modulation of chemical effects (and vice versa), as they clearly differ from chemical stressors; and "doses" of many physical stressors are more easily quantifiable than those of psychosocial stressors. There is a commonly held belief that virtually nothing is known about the impact of nonchemical stressors on chemically mediated toxicity or the joint impact of coexposure to chemical and nonchemical stressors. Although this is generally true, there are several instances where a substantial body of evidence exists. A workshop titled "Cumulative Risk: Toxicity and Interactions of Physical and Chemical Stressors" held at the 2013 Society of Toxicology Annual Meeting provided a forum for discussion of research addressing the toxicity of physical stressors and what is known about their interactions with chemical stressors, both in terms of exposure and effects. Physical stressors including sunlight, heat, radiation, infectious disease, and noise were discussed in reference to identifying pathways of interaction with chemical stressors, data gaps, and suggestions for future incorporation into cumulative risk assessments.

Photochemically altered air pollution mixtures and contractile parameters in isolated murine hearts before and after ischemia.

BACKGROUND: The cardiopulmonary effects of the individual criteria air pollutants have been well investigated, but little is known about the cardiopulmonary effects of inhaled multipollutant mixtures that more realistically represent environmental exposures. OBJECTIVES: We assessed the cardiopulmonary effects of exposure to photochemically altered particle-free multipollutant mixtures. METHODS: We exposed mice to filtered air (FA), multipollutant mixtures, or ozone (O3) for 4 hr in a photochemical reaction chamber. Eight hours after exposure, we assessed cardiac responses using a Langendorff preparation in a protocol consisting of 20 min of global ischemia followed by 2 hr of reperfusion. Cardiac function was assessed by measuring the index of left-ventricular developed pressure (LVDP) and contractility (dP/dt) before ischemia. On reperfusion after ischemia, recovery of postischemic LVDP and size of infarct were examined. We used bronchoalveolar lavage (BAL) cell counts to assess lung inflammation. RESULTS: Exposure to the multipollutant mixtures decreased LVDP, baseline rate of left ventricular contraction (dP/dtmaximum), and baseline rate of left ventricular relaxation (dP/dtminimum) compared with exposure to FA. Exposure to O3 also decreased heart rate and dP/dtminimum. Time to ischemic contracture was prolonged in the multipollutant-mixture group relative to that in the FA group. Mice in the multipollutant-mixture group had better recovery of postischemic LVDP and smaller infarct size. Exposure to multipollutant mixtures and to O3 exposure increased numbers of macrophages in the BAL fluid. CONCLUSIONS: Exposure to photochemically altered urban air pollution appears to affect cardiac mechanics in isolated perfused hearts. Inhalation of acute multipollutant mixtures decreases LVDP and cardiac contractility in isolated non-ischemic murine hearts, prolongs ischemic contracture, increases postischemic recovery of LVDP, and reduces infarct size.

Hazard assessment of United Arab Emirates (UAE) incense smoke.

Incense burning inside the home, a common practice in Arabian Gulf countries, has been recognized as a potentially modifiable source of indoor air pollution. To better understand potential adverse effects of incense burning in exposed individuals, we conducted a hazard assessment of incense smoke exposure. The goals of this study were first to characterize the particles and gases emitted from Arabian incense over time when burned, and secondly to examine in vitro human lung cells responses to incense smoke. Two types of incense (from the United Arab Emirates) were burned in a specially designed indoor environmental chamber (22 m(3)) to simulate the smoke concentration in a typical living room and the chamber air was analyzed. Both particulate (PM) concentrations and sizes were measured, as were gases carbon monoxide (CO), sulfur dioxide (SO2), oxides of nitrogen (NOx), formaldehyde (HCHO), and carbonyls. During the burn, peak concentrations were recorded for PM (1.42 mg/m(3)), CO (122 pm), NOx (0.3 ppm), and HCHO (85 ppb) along with pentanal (71.9 µg/m(3)), glyoxal (84.8 µg/m(3)), and several other carbonyls. Particle sizes ranged from 20 to 300 nm with count median diameters ranging from 65 to 92 nm depending on time post burn-out. PM, CO, and NOx time-weighted averages exceeded current government regulation values and emissions seen previously from environmental tobacco smoke. Charcoal emissions were the main contributor to both the high CO and NOx concentrations. A significant cell inflammatory response was observed in response to smoke components formed from incense burning. Our hazard evaluation suggests that incense burning contributes to indoor air pollution and could be harmful to human health.