UC Berkeley

DISSERTATION ABSTRACT

The adverse outcome pathway (AOP) approach combines patterns of molecular events across levels of biological organization and complex endpoints such as reproduction, behavior, and toxicity, and provides regulatory decision criteria for Environmental Protection Agency (EPA). Useful empirical data for AOPs is derived from key indicators of toxicant exposure such as conserved transcriptional factors, nuclear receptors and their targets. One approach to establish changes in toxicant response is gene expression profiling, or ‘omics’ tools, which can lead to improved mechanistic models of toxicity in closely related species (fathead minnow and zebrafish), and divergent species (fish and crustaceans). Gene expression patterns have been used to characterize toxicant-dependent signaling for crustaceans exposed to metals, flame retardants, and narcotic toxicants. Gene expression studies can identify new assay development needs, and generate new hypotheses about conserved molecular pathways leading to direct evidence from knockout models.

In the first study, we focus on conserved nuclear receptors in the model crustacean Daphnia magna exposed to endocrine disrupting chemicals (EDCs). The Daphnia reproductive X-Y axis model involves key orthologous genes which drive metabolism and endocrine-toxicity. Previous work in Daphnia hypothesized a unified mechanism of endocrine disruption that leads to inappropriate production of male offspring through effects on a sex-differentiation gene (dsx-1) in the methyl farnesoate (MF) pathway. We carried out a microarray gene expression study with eight different EDCs in female Daphnia and found two distinct patterns of transcriptional response induced by male-inducing chemicals. Pyriproxifen and methoxychlor produced similar gene expression patterns overall and in key endocrine response genes, while methyl farnesoate and arochlor exposure shared a distinct response pattern. In particular, there were inverse responses at the ecdysteroid receptor (EcR), a PAS-binding protein that regulates key steroid biosynthesis metabolism between the two sets. We also noted differences in key molting genes (CYP450s) from the ecdysteroid pathway. Together our study demonstrates a dsx-1 dependent mechanism for male-differentiation (dsx-1) in the ovary, and a dsx-1 independent EDC mechanism for altered reproductive mode.

In the next study, we assessed Daphnia magna exposure to polycyclic aromatic hydrocarbons (PAHs) and pyrethroids, which may lead to neurotoxicity. Since PAH exposure activates the conserved AHR pathway in vertebrates, we tested the hypothesis that PAH exposure in Daphnia would induce AHR-related gene expression endpoints (cytochrome P450s, oxidative stress, metabolism). Despite the lack of a clear orthologous AhR gene, we observed induction of antioxidant pathways and CYP450-related oxidoreductase enzymes that suggest similar crustacean and vertebrate mechanisms of PAH toxicity.

To link molecular endpoints to an ecological phenotype, we used an automated video-tracking system to assess Daphnia neuro-behavioral endpoints including swimming speed and tank location preference. Altered Daphnia magna swim behaviors were upward (top of tank) and downward (bottom of the tank), similar to phototactic behaviors recognized in zebrafish anxiety-related research. Pyrethroids reduced swim speed and induced a bottom swimming phenotype at doses well below acute toxicity. Swim responses differed within the PAH chemical class. Daphnia behaviors in light and dark environments are dependent upon circadian clock mechanisms, which may be sensitive to toxicant exposure. Thus, we hypothesized that altered circadian clock (CLK) mechanisms may be a nexus between toxicant exposure and altered behavior. We observed that exposure to phenanthrene (PHE), pyrene, and cyfluthrin influenced cryptochrome-2 expression, an upstream regulator of Daphnia circadian clock genes. We also identified a broad range of functional genes involved in PHE-altered neurological pathways, involving dopamine and the Mbln12 gene involved in photo-receptor cell growth. The behavioral and molecular studies suggest that these chemicals disrupt neurologic function which results in altered behavioral outcomes.

In the final study, we used a zebrafish developmental and behavioral chemical toxicity screening assay to assess the toxicity of a candidate biofuel (2,5-DMF) and related furan compounds. Because of structural similarity to aromatic hydrocarbons, we hypothesized that 2,5-DMF targets the AhR signaling pathway and produces characteristic zebrafish developmental toxicity. AhR agonists such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and beta-napthoflavone (BNF) cause characteristic pericardial edema and Cyp1a1 activation in zebrafish. However, we found that 2,5-DMF toxicity, as well as some polyaromatic hydrocarbons, did not produce morphological defects. Non-AhR mechanisms that include hypoxia and neuro-behavioral effects were observed. We assessed AHR-dependent mechanisms using morphological endpoints, AHR-XRE cell-based reporter assays, and in silico AhR-LBD binding models. Zebrafish were also assessed for embryonic and larval photomotor responses. We evaluated several furan derivatives and found that 2-ethyl furan and 2,3-dihydrofuran had the least biological effect in our assays. The highest aquatic risk to developing fish was from 2,5-dimethyl furan, and 2-methyl furan. Overall, we determined that zebrafish exposure to biofuel candidates did not induce AhR mediated toxicity, but produced significant behavioral toxicity from non-AhR mechanisms.