Monitoring redox stress in human airway epithelial cells exposed to woodsmoke at an air-liquid interface.

Wildland fires contribute significantly to the ambient air pollution burden worldwide, causing a range of adverse health effects in exposed populations. The toxicity of woodsmoke, a complex mixture of gases, volatile organic compounds, and particulate matter, is commonly studied in vitro using isolated exposures of conventionally cultured lung cells to either resuspended particulate matter or organic solvent extracts of smoke, leading to incomplete toxicity evaluations. This study aimed to improve our understanding of the effects of woodsmoke inhalation by building an advanced in vitro exposure system that emulates human exposure of the airway epithelium. We report the development and characterization of an innovative system that permits live-cell monitoring of the intracellular redox status of differentiated primary human bronchial epithelial cells cultured at an air-liquid interface (pHBEC-ALI) as they are exposed to unfractionated woodsmoke generated in a tube furnace in real time. pHBEC-ALI exposed to freshly generated woodsmoke showed oxidative changes that were dose-dependent and reversible, and not attributable to carbon monoxide exposure. These findings show the utility of this novel system for studying the molecular initiating events underlying woodsmoke-induced toxicity in a physiologically relevant in vitro model, and its potential to provide biological plausibility for risk assessment and public health measures.

A new cell culture exposure system for studying the toxicity of volatile chemicals at the air-liquid interface.

A cell culture exposure system (CCES) was developed to expose cells established at an air-liquid interface (ALI) to volatile chemicals. We characterized the CCES by exposing indigo dye-impregnated filter inserts inside culture wells to 125 ppb ozone (O3) for 1 h at flow rates of 5 and 25 mL/min/well; the reaction of O3 with an indigo dye produces a fluorescent product. A 5-fold increase in fluorescence at 25 mL/min/well versus 5 mL/min/well was observed, suggesting higher flows were more effective. We then exposed primary human bronchial epithelial cells (HBECs) to 0.3 ppm acrolein for 2 h at 3, 5, and 25 mL/min/well and compared our results against well-established in vitro exposure chambers at the U.S. EPA's Human Studies Facility (HSF Chambers). We measured transcript changes of heme oxygenase-1 (HMOX1) and interleukin-8 (IL-8), as well as lactate dehydrogenase (LDH) release, at 0, 1, and 24 h post-exposure. Comparing responses from HSF Chambers to the CCES, differences were only observed at 1 h post-exposure for HMOX1. Here, the HSF Chamber produced a ∼6-fold increase while the CCES at 3 and 5 mL/min/well produced a ∼1.7-fold increase. Operating the CCES at 25 mL/min/well produced a ∼4.5-fold increase; slightly lower than the HSF Chamber. Our biological results, supported by our comparison against the HSF Chambers, agree with our fluorescence results, suggesting that higher flows through the CCES are more effective at delivering volatile chemicals to cells. This new CCES will be deployed to screen the toxicity of volatile chemicals in EPA's chemical inventories.

A Device that Allows Rodents to Behaviorally Thermoregulate when Housed in Vivariums.

Laboratories and vivariums typically are maintained at ambient temperatures of 20 to 24 °C, leading to cold stress in mice. When mice are inactive and sleeping during the light phase, their zone of thermoneutrality associated with a basal metabolic rate is 30 to 32 °C. If given a choice, mice will use thermoregulatory behavior to seek out thermoneutral temperatures during the light phase. The cold stress of a vivarium can be problematic to researchers requiring an animal model that is not stressed metabolically. However, it may not be practical or economically feasible to maintain an animal vivarium at thermoneutral temperatures. One problem with raising the ambient temperature of a vivarium is that personnel wearing protective equipment will be subject to considerable heat stress. Here we present plans for the construction and operation of a device that allows mice to access a heated floor that is maintained at an approximate thermoneutral temperatures (30 to 32 °C). The device is made of inexpensive, readily available materials and uses a disposable hand warmer as a heat source. One hand warmer provides a thermoneutral environment for approximately 12 h. This device is easily adapted to a standard mouse or rat cage and requires only brief daily maintenance to change the heating pad. With this device in a standard cage, mice can select a warmer environment associated with thermoneutral conditions during the light phase and cooler ambient temperatures during the dark phase.

Rat tail skin temperature monitored noninvasively by radiotelemetry: characterization by examination of vasomotor responses to thermomodulatory agents.

INTRODUCTION: Measurement of tail skin temperature (T(sk)) of the rat can provide important information on mechanisms pertaining to physiology, pharmacology, and toxicology. T(sk) is largely under control of peripheral vascular tone, which is also sensitive to most experimental manipulations such as handling and restraint. Hence, it is extremely difficult to acquire long-term measurements of T(sk) that are free of artifacts from experimental manipulation. The purpose of this study is to demonstrate the utility of a radiotelemetric probe to provide continuous, long-term measurements of T(sk) in undisturbed rats. METHODS: A telemetry probe is placed on the base of the tail and secured with a protective guard to prevent the rat from chewing on the probe. T(sk) is continuously monitored with standard radiotelemetric software and computer technology. Core temperature (T(c)) is monitored in duplicate sets of rats at the same time but the current system does not allow for simultaneous measurement of T(sk) and T(c) from the same animal. Rats were subjected to a variety of experimental manipulations to demonstrate the utility of the probe. RESULTS/DISCUSSION: A marked increase in T(sk) was seen during the transition from light to dark phase, reflecting an increase in heat loss to lower T(c); a decrease in T(sk) during the development of endotoxin-induced fever, reflecting a reduction in heat loss to facilitate an elevation in T(c); an increase in T(sk) following exposure to the organophosphate insecticide chlorpyrifos, reflecting an increase in heat loss to facilitate a hypothermia response; and a direct effect of increasing ambient temperature on T(sk). The probe is relatively inexpensive and is used with no surgery and provides long-term measurement (e.g., >24 h) of T(sk) in unrestrained rats.