Superfund Research Program
Developmental Effects of Superfund Hydrocarbon Mixtures in Fundulus heteroclitus
Project Leader: Richard T. Di Giulio
Co-Investigators: David E. Hinton, Marjorie F. Oleksiak (University of Miami)
Grant Number: P42ES010356
Funding Period: 2000-2011
Final Progress Reports
A key component of this project is a population of killifish (Fundulus heteroclitus) that inhabits an estuary in the Elizabeth River, Virginia, a tributary of the James River just below the Chesapeake Bay. The study site in this river is adjacent to the Atlantic Wood Superfund site and is highly polluted by a complex mixture of chemicals, dominated by polycyclic aromatic hydrocarbons (PAHs) and various nitrogen-, sulfur- and oxygen-substituted PAHs.
While exhibiting elevated rates of liver cancer due to high PAH exposures, the population of killifish inhabiting this site are resistant to other toxic effects of the contaminated sediments compared to killifish from nearby unpolluted sites, and this resistant phenotype is heritable. The most sensitive effects Dr. Di Giulio’s research team has observed in developing killifish embryos are cardiovascular effects including edema in the yolk sac and deformities of the heart.
The key questions addressed by this study are: (1) What mechanisms underlie the adaptations to this pollution scenario exhibited by Elizabeth River killifish? (2) In adapting to this pollution scenario, have these fish paid a price, i.e., are there fitness costs associated with the adaptations that allow killifish to inhabit and reproduce in this harsh environment? (3) What mechanisms underlie the effects of this chemical mixture on cardiovascular development?
In this past year, the project investigators have continued work on question 1, but have focused on question 3. In addressing mechanisms of adaptation, they employed differential display to explore differences between populations. This method aids in identification of differences in patterns of gene expression that are not hypothesis-driven, in contrast to the researchers earlier work. This work identified a number of differentially expressed genes, based upon population and sex differences, or both. Of particular interest were genes associated with anaerobic metabolism, which were consistent with the observed fitness cost of increased sensitivity to hypoxia in the chemically-resistant Elizabeth River population. This work, together with published gene sequences is enabling the research team to now develop a killifish microarray that will have utility in this and other projects.
The discovery of the marked sensitivity of cardiovascular development to Elizabeth River sediment extracts has led the researchers into explorations of this phenomenon that has ramifications for both ecological and human health (question 3). Results to date suggest important interaction between AHR agonists and CYP1A inhibitors that are both plentiful at this site. The researcher’s initial observation came from dose response studies with varying dilutions of sediment extracts and employed a novel assay for measuring CYP1A enzyme activity, a marker for AHR agonism, in living embryos without harm to these organisms. The reserachers observed an inverted U-shaped curve where activity increased up to a point and then fell, ultimately to control values. At the break point where activity began falling coincided with the appearance of deformities that then exhibited a positive dose-response. Subsequent studies with model agonists (such as β-naphthoflavone and BaP, the latter abundant in the extract) and antagonists (such as α- naphthoflavone and flouranthene, the latter abundant in the extract) tracked the results with extracts; that is, co-exposure to inhibitors at non-toxic concentrations greatly synergized (100 to 1000-fold) the cardiovascular toxicity of agonists and inhibited CYP1A activity. Interestingly, this effect of inhibitors is the opposite of their effect in combination with chlorinated AHR agonists such as dioxin (TCDD) and PCB 126.
Dr. Di Giulio’s team’s most recent work employs the zebrafish model that allows them to use the morpholino gene knock-down methodology; this approach is more specific than the chemical inhibitor approach they have used with killifish. They initially observed the same synergy between AHR agonists and CYP1A inhibitors in zebrafish as they previously observed with killifish. This same pattern of synergy was observed when CYP1A morpholinos were used rather than chemical inhibitors. Conversely, morpholinos against AHR1 provided protection, supporting a role for AHR-mediated events in the cardiovascular toxicity of these mixtures. Current work is expanding the zebrafish-morpholino approach to elucidate mechanistic relationships among AHR agonism, CYP1A inhibition, oxidative stress, and cardiovascular development.
These studies have several important ramifications, including: (1) Multi-generational exposures of vertebrate populations to Superfund chemicals can apparently alter the evolution of exposed populations. (2) An important component of altered evolution is the occurrence of fitness costs such as reduced tolerance to hypoxia and to UV radiation in chemically-adapted organisms. (3) Cardiovascular deformities are a very sensitive endpoint with these PAH mixtures, and also represent a human health outcome for which there is great concern. (4) PAHS with different molecular effects are markedly synergistic with respect to cardiovascular development and raise concerns for current additive models employed to estimate risks from exposures to PAH mixtures. Collectively, the researcher’s mechanistic studies with the killifish and zebrafish models have important implications for both human and ecological health.