Detection of endocrine disrupting chemicals in Danio rerio and Daphnia pulex: Step-one, behavioral screen.

Anthropogenic surface and ground water contamination by chemicals is a global problem, and there is an urgent need to develop tools to identify and elucidate biological effects. Contaminants of emerging concern (CECs) are not typically monitored or regulated and those with known or suspected endocrine disrupting potential have been termed endocrine disrupting chemicals (EDCs). Many CECs are known to be neurotoxic (e.g., insecticides) and many are incompletely characterized. Behavioral responses can identify chemicals with neuroactive properties, which can be relevant to EDC mechanisms (e.g., neuroendocrine disturbances). Two freshwater species, Daphnia pulex and Danio rerio, were evaluated for swimming behavior alterations resulting from 24-hr exposure to 9 CECs: triclosan, triclocarban, chlorpyrifos, dieldrin, 4-nonylphenol, bisphenol-A, atrazine, metformin, and estrone. This is the first step in the development of a bioassay for detecting estrogenic and/or anti-androgenic activity with the goal to evaluate complex mixtures of uncharacterized contaminants in water samples. The second step, described in a subsequent report, examines transcriptome alterations following chemical exposure. Significant differences in the swimming behavior response and sensitivity were found across chemicals within a species and across species for a given chemical in this unique optical bioassay system. In the concentration ranges studied, significant behavioral alterations were detected for 6 of 9 CECs for D. pulex and 4 of 9 CECs for D. rerio. These results underscore the utility of this bioassay to identify behavioral effects of sublethal concentrations of CECs before exploration of transcriptomic alterations for EDC detection.

Cisplatin-induced hair cell loss in zebrafish neuromasts is accompanied by protein nitration and Lmo4 degradation.

Generation of reactive oxygen species, a critical factor in cisplatin-induced ototoxicity, leads to the formation of peroxynitrite, which in turn results in the nitration of susceptible proteins. Previous studies indicated that LMO4, a transcriptional regulator, is the most abundantly nitrated cochlear protein after cisplatin treatment and that LMO4 nitration facilitates ototoxicity in rodents. However, the role of this mechanism in regulating cisplatin-induced hair cell loss in non-mammalian models is unknown. As the mechanosensory hair cells in the neuromasts of zebrafish share many features with mammalian inner ear and is a good model for studying ototoxicity, we hypothesized that cisplatin treatment induces protein nitration and Lmo4 degradation in zebrafish hair cells, thereby facilitating hair cell loss. Immunostaining with anti-parvalbumin revealed a significant decrease in the number of hair cells in the neuromast of cisplatin treated larvae. In addition, cisplatin treatment induced a significant decrease in the expression of Lmo4 protein and a significant increase in nitrotyrosine levels, in the hair cells. The cisplatin-induced changes in Lmo4 and nitrotyrosine levels strongly correlated with hair cell loss, implying a potential link. Furthermore, a significant increase in the expression of activated Caspase-3 in zebrafish hair cells, post cisplatin treatment, suggested that cisplatin-induced decrease in Lmo4 levels is accompanied by apoptosis. These findings suggest that nitrative stress and Lmo4 degradation are important factors in cisplatin-induced hair cell loss in zebrafish neuromasts and that zebrafish could be used as a model to screen the otoprotective efficacy of compounds that inhibit protein nitration.

Developmental exposure to Pb2+ induces transgenerational changes to zebrafish brain transcriptome.

Lead (Pb2+) is a major public health hazard for urban children, with profound and well-characterized developmental and behavioral implications across the lifespan. The ability of early Pb2+ exposure to induce epigenetic changes is well-established, suggesting that Pb2+-induced neurobehavioral deficits may be heritable across generations. Understanding the long-term and multigenerational repercussions of lead exposure is crucial for clarifying both the genotypic alterations behind these behavioral outcomes and the potential mechanism of heritability. To study this, zebrafish (Danio rerio) embryos (<2 h post fertilization; EK strain) were exposed for 24 h to waterborne Pb2+ at a concentration of 10 µM. This exposed F0 generation was raised to adulthood and spawned to produce the F1 generation, which was subsequently spawned to produce the F2 generation. Previous avoidance conditioning studies determined that a 10 µM Pb2+ dose resulted in learning impairments persisting through the F2 generation. RNA was extracted from control- and 10 µM Pb2+-lineage F2 brains, (n = 10 for each group), sequenced, and transcript expression was quantified utilizing Quant-Seq. 648 genes were differentially expressed in the brains of F2 lead-lineage fish versus F2 control-lineage fish. Pathway analysis revealed altered genes in processes including synaptic function and plasticity, neurogenesis, endocrine homeostasis, and epigenetic modification, all of which are implicated in lead-induced neurobehavioral deficits and/or their inheritance. These data will inform future investigations to elucidate the mechanism of adult-onset and transgenerational health effects of developmental lead exposure.