Assessment of zebrafish embryo photomotor response sensitivity and phase-specific patterns following acute- and long-duration exposure to neurotoxic chemicals and chemical weapon precursors.

Zebrafish are an attractive model for chemical screening due to their adaptability to high-throughput platforms and ability to display complex phenotypes in response to chemical exposure. The photomotor response (PMR) is an established and reproducible phenotype of the zebrafish embryo, observed 24 h post-fertilization in response to a predefined sequence of light stimuli. In an effort to evaluate the sensitivity and effectiveness of the zebrafish embryo PMR assay for toxicity screening, we analyzed chemicals known to cause both neurological effects and developmental abnormalities, following both short (1 h) and long (16 h+) duration exposures. These include chemicals that inhibit aerobic respiration (eg, cyanide), acetyl cholinesterase inhibitors (organophosphates pesticides) and several chemical weapon precursor compounds with variable toxicity profiles and poorly understood mechanisms of toxicity. We observed notable concentration-responsive, phase-specific effects in the PMR after exposure to chemicals with a known mechanism of action. Chemicals with a more general toxicity profile (toxic chemical weapon precursors) appeared to reduce all phases of the PMR without a notable phase-specific effect. Overall, 10 of 20 chemicals evaluated elicited an effect on the PMR response and eight of those 10 chemicals were picked up in both the short- and long-duration assays. In addition, the patterns of response uniquely differentiated chemical weapon precursor effects from those elicited by inhibitors of aerobic respiration and organophosphates. By providing a rapid screening test for neurobehavioral effects, the zebrafish PMR test could help identify potential mechanisms of action and target compounds for more detailed follow-on toxicological evaluations. Approved for public release: distribution unlimited.

Cryopreserved hepatocytes from rainbow trout (Oncorhynchus mykiss): a validation study to support their application in bioaccumulation assessment.

Determination of biotransformation rates of xenobiotics in freshly isolated trout hepatocytes has been demonstrated to significantly improve the performance of bioaccumulation assessment models. In order to promote this in vitro approach, trout hepatocytes need to be cryopreserved to facilitate their availability while ensuring their metabolic competency. In the present study, we obtained basal level metabolic enzyme activities for cytochrome P450 (CYP) 1A, CYP3A, glutathione-S-transferase, and uridine 5'-diphospho-glucuronosyltransferase from trout hepatocytes cryopreserved for various periods of time up to three months and compared their values with those obtained from freshly isolated hepatocytes. Similarly, we compared intrinsic clearance (CL(int)) values determined in cryopreserved trout hepatocytes to those determined in freshly isolated hepatocytes for reference compounds molinate, michler's ketone, 4-nonylphenol, 2,4-ditert-butylphenol, benzo(a)pyrene, and pyrene. Our results show that cryopreserved trout hepatocytes maintained greater than 75% of their basal level enzyme activities and greater than 72% of xenobiotic biotransformation capabilities, regardless of the length of cryostorage. As a result, bioconcentration factors of the reference compounds were adequately predicted based on the CL(int) values. We simulated the condition for shipping cryopreserved trout hepatocytes and demonstrated that 24 h dry ice storage did not negatively affect the rates of xenobiotic biotransformation. We conclude that cryopreserved trout hepatocytes are suitable for biotransformation rate determination of xenobiotics in vitro, and therefore, are an acceptable alternative to freshly isolated trout hepatocytes in the application in bioaccumulation assessment.