Heart rate assays in wild-type zebrafish embryos have been limited to analysis of one embryo per video/imaging field. Here we present for the first time a platform for high-throughput derivation of heart rate from multiple zebrafish (Danio rerio) embryos per imaging field, which is capable of quickly processing thousands of videos and ideal for multi-well platforms with multiple fish/well. This approach relies on use of 2-day post fertilization wild-type embryos, and uses only bright-field imaging, circumventing requirement for anesthesia or restraint, costly software/hardware, or fluorescently-labeled animals. Our original scripts (1) locate the heart and record pixel intensity fluctuations generated by each cardiac cycle using a robust image processing routine, and (2) process intensity data to derive heart rate. To demonstrate assay utility, we exposed embryos to the drugs epinephrine and clonidine, which increased or decreased heart rate, respectively. Exposure to organic extracts of air pollution-derived particulate matter, including diesel or biodiesel exhausts, or wood smoke, all complex environmental mixtures, decreased heart rate to varying degrees. Comparison against an established lower-throughput method indicated robust assay fidelity. As all code and executable files are publicly available, this approach may expedite cardiotoxicity screening of compounds as diverse as small molecule drugs and complex chemical mixtures.

Urinary mutagenicity reflects systemic exposure to complex mixtures of genotoxic/ carcinogenic agents and is linked to tumor development. Coal combustion emissions (CCE) and diesel engine exhaust (DEE) are associated with cancers of the lung and other sites, but their influence on urinary mutagenicity is unclear. We investigated associations between exposure to CCE or DEE and urinary mutagenicity. In two separate cross-sectional studies of nonsmokers, organic extracts of urine were evaluated for mutagenicity levels using strain YG1041 in the Salmonella (Ames) mutagenicity assay. First, we compared levels among 10 female bituminous (smoky) coal users from Laibin, Xuanwei, China, and 10 female anthracite (smokeless) coal users. We estimated exposure-response relationships using indoor air concentrations of two

Exposure to fine particulate matter (PM) air pollution causes adverse cardiopulmonary outcomes. Yet, the limited capacity to readily identify contributing PM sources and associated PM constituents in any given ambient air shed impedes risk assessment efforts. The health effects of PM have been attributed in part to its capacity to elicit irritant responses. A variety of chemicals trigger irritant behavior responses in zebrafish that can be easily measured. The purposes of this study were to examine the utility of zebrafish locomotor responses in the toxicity assessment of fine PM and its chemical fractions and uncover mechanisms of action. Locomotor responses were recorded in 6-day-old zebrafish exposed for 60 min in the dark at 26 °C to the extractable organic matter of a compressor-generated diesel exhaust PM (C-DEP) and 4 of its fractions (F1-F4) containing varying chemical classes of increasing polarity. The role of the transient receptor potential (TRP) cation channel TRPA1, a chemical sensor in mammals and zebrafish, in locomotor responses to C-DEP, was also examined. Acrolein, an environmental irritant and known activator of TRPA1, and all extracts induced concentration-dependent locomotor responses whose potencies ranked as follows: polar F3 > weakly polar F2 > C-DEP > highly polar F4 > nonpolar F1, indicating that polar and weakly polar fractions that included nitro- and oxy-polyaromatic hydrocarbons (PAHs), drove C-DEP responses. Irritant potencies in fish positively correlated with mutagenic potencies of the same extracts in strains of Salmonella sensitive to nitro- and oxy-PAHs, further implicating these chemical classes in the zebrafish responses to C-DEP. Pharmacologic inhibition of TRPA1 blocked locomotor responses to acrolein and the extracts. Taken together, these data indicate that the zebrafish locomotor assay may help expedite toxicity screening of fine PM sources, identify causal chemical classes, and uncover plausible biological mechanisms.

Background: Wildland firefighters conducting prescribed burns are exposed to a complex mixture of pollutants, requiring an integrated measure of exposure. Objective: We used urinary mutagenicity to assess if systemic exposure to mutagens are higher in firefighters after working at prescribed burns versus after non-burn work days. Other biomarkers of exposure and oxidative stress markers were also measured. Methods: Using a repeated measures study design, we collected urine before, immediately after, and the morning after a work shift on prescribed burn and non-burn work days from 12 healthy subjects. Urines were analyzed for malondialdehyde (MDA), 8-isoprostane, 1-hydroxypyrene (OH-Pyrene), and mutagenicity in Salmonella YG1041 +S9. Particulate matter (PM2.5) and carbon monoxide (CO) measurements were collected by personal monitoring. Light-absorbing carbon (LAC) of PM2.5 was measured as a surrogate for black carbon exposure. Linear mixed-effect models were used to assess cross-work shift (pre- to post-work shift) changes in urinary biomarkers. Results: No significant differences occurred in creatinine-adjusted urinary mutagenicity across the work shift between burn days (48 samples) and non-burn day (21 samples). Firefighters lighting fires had a non-significant, 1.6-fold increase in urinary mutagenicity for burn- versus non-burn day exposures. Positive associations were found between cross-work shift (pre- to post-) changes in creatinine-adjusted urinary mutagenicity and MDA (p = 0.0010), OH-Pyrene (p = 0.0001), and mass absorption efficiency, which is the LAC/PM2.5 ratio (p = 0.2245), respectively. No significant effect of day type or work task on cross-work shift (pre- to post-) changes in MDA or 8-isoprostane was observed. Conclusion: Urinary mutagenicity may serve as a suitable measure of occupational smoke exposures among wildland firefighters, especially among those lighting fires for prescribed burns.

Maternal behavior (MB) is a complex response to infant cues, orchestrated by postpartum neurophysiology. While mesolimbic dopamine contributes to MB, little is known about real-time dopamine fluctuations during the postpartum period. Thus, we used fast-scan cyclic voltammetry (FSCV) to measure individual dopamine transients in the nucleus accumbens of early postpartum rats, and compared them to dopamine transients in virgins and in postpartum females exposed to cocaine during pregnancy, which is known to disrupt MB. We hypothesized that dopamine transients are normally enhanced postpartum, and support MB. In anesthetized rats, electrically-evoked dopamine release was larger and clearance was faster in postpartum females than in virgins, and gestational cocaine exposure blocked the change in clearance. In awake rats, control mothers showed more dopamine transients than cocaine-exposed mothers during MB. Salient pup-produced stimuli may contribute to differences in maternal phasic dopamine by evoking dopamine transients; supporting the feasibility of this hypothesis, urine composition (glucose, ketones and leukocytes) differed between unexposed and cocaine-exposed infants. These data, resulting from the novel application of FSCV to models of MB, support the hypothesis that phasic dopamine signaling is enhanced postpartum. Future studies with additional controls can delineate which aspects of gestational cocaine reduce dopamine clearance and transient frequency.