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<p>Chronic exposure to toxicant mixtures is a serious threat to environmental and
human health. It is especially important to understand the effects of these exposures
for contaminants, such as polycyclic aromatic hydrocarbons (PAHs), which are toxic,
ubiquitous, and increasingly prevalent. Furthermore, estuarine systems are of particular
concern, as they are highly impacted by a wide variety of pollutants; fish there are
often exposed to some of the highest levels of contaminants of any vertebrate populations,
along with other stressors such as fluctuations in water level, dissolved oxygen,
and temperature. A population of <italic>Fundulus heteroclitus</italic> (the Atlantic
killifish or mummichog, hereafter referred to as killifish) inhabits a Superfund site
heavily contaminated with a mixture of PAHs from former creosote operations; they
have developed resistance to the acute toxicity and teratogenic effects caused by
the mixture of PAHs in sediment from the site. The primary goal of this dissertation
was to better understand the mechanism(s) by which Elizabeth River killifish resist
the developmental toxicity of a complex mixture of PAHs and to investigate the tradeoffs
associated with this resistance. Because the aryl hydrocarbon receptor (AHR) pathway
plays an important role in mediating the effects of PAHs, one major hypothesis of
my work was that suppression of the AHR response plays an important role in the resistance
of Elizabeth River killifish. For this reason, investigation of the activation of
the AHR pathway, as measured by CYP induction, is a unifying thread throughout the
work. Another major hypothesis of this work is that adaptation to PAHs has secondary
consequences for Elizabeth River killifish, such as altering their response to other
xenobiotics. To investigate these hypotheses, a series of experiments were carried
out in PAH-adapted killifish from the Elizabeth River and in reference fish. The
morpholino gene knockdown technique was modified for use in killifish; we demonstrated
that CYP1A knockdown exacerbates PAH-driven cardiac teratogenesis and AHR2 (but not
AHR1) knockdown rescues PAH-driven cardiac teratogenesis. Using acute toxicity tests
of larval killifish, we showed that Elizabeth River killifish are less sensitive than
reference larvae to chlorpyrifos, permethrin, and carbaryl. These results demonstrated
that the adaptation was able to protect from multiple xenobiotics, not just PAHs.
Using the in ovo ethoxyresorufin-o-deethylase (EROD) assay and a subjective cardiac
deformity screen, we showed that the adaptation was spread throughout the killifish
subpopulations of the Elizabeth River estuary. However, the adaptive response varied
greatly among the subpopulations, which showed that AHR pathway suppression was not
required for some level of protection from PAH toxicity. Finally, using the quantitative
real-time PCR, the EROD assay, and cardiac deformity screening, we demonstrated that
the adaptation was heritable for two generations of fish reared in clean laboratory
conditions. The findings in this dissertation will help to reveal how mixtures of
PAHs exert their toxic action in un-adapted organisms. Furthermore, these studies
will hopefully demonstrate how chronic exposure to PAH mixtures can affect organisms
at the population and even evolutionary level. Perhaps most importantly, they will
help us to better predict the consequences and tradeoffs for organisms and populations
persisting in PAH-contaminated environments.</p>
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