I. Summary

When considering the entirety of evolution, human-mediated aquatic pollution is a relatively new and extreme pressure on species survival due to the persistence of organic pollutants, such as dioxin-like compounds (DLCs) from industrialism. DLCs include certain kinds of polychlorinated biphenyls (PCBs), pollutants that continue to persist, despite their ban in the 1970s by the U.S. Environmental Protection Agency (EPA). Atlantic killifish (Fundulus heteroclitus) are one of the most extensively used animal models for studying organismal adaptations to polluted environments due to their resistance to DLCs. However, the mechanism underlying its ability to survive in polluted waters is still not fully understood. The purpose of this study is to investigate the physiological adaptations that allow the Atlantic killifish to survive in the PCB-polluted waters of New Bedford Harbor (NBH), MA. Due to old textile manufacturing waste disposal, NBH is believed to possess lethal concentrations of PCB for all aquatic species. Nevertheless, the Atlantic killifish is found to survive within the harbor’s PCB contaminated water. We hypothesize the Atlantic killifish will begin to develop physiological resistance to PCB126-mediated toxicity after translocation from the non-PCB-polluted waters of Scorton Creek (SC), MA to PCB126-polluted waters in the laboratory. The findings from this study will provide broad insights into different biological mechanisms that  help organisms adapt to polluted environments.  

In aim 1, a PCB-sensitive sample of killifish from SC will be translocated to PCB126-polluted waters, and its hepatic metabolism and histology will be compared to a non-translocated PCB-sensitive SC sample of killifish and a PCB-resistant NBH sample to determine whether its metabolism adapts similarly to the changes in its environment that has been previously observed in NBH killifish. 

Aim 2 will measure PCB resistance between killifish embryos that have been exposed to an anoxic environment and killifish embryos that have not undergone exposure to an anoxic environment for both SC killifish and NBH killifish. Previous research suggests that nitric oxide synthase (NOS) grants increased PCB resistance, so NOS RNA levels will be measured and compared between the killifish exposed and not exposed to, anoxic environments.

The purpose of aim 3 will be to measure expression levels of the toxin-metabolizing enzymes CYP1A1 and CYP1B1 in liver tissues of the PCB126-translocated samples; previous research has demonstrated reduced metabolic activity as a biological hallmark of the PCB-resistance in this species. Data will be gathered through immunohistochemistry techniques and then measured with a valid subjective stain scoring method. 

The final aim seeks to identify whether prolonged exposure to PCBs reduces induction of CYP1A1 and CYP1B1 via epigenetic changes. Previous studies have failed to show changes in DNA methylation but propose investigating alternative epigenetic events such as histone acetylation. Histone acetylation will be measured using chromatin immunoprecipitation quantitative PCR (ChIP-qPCR); the results will be compared to the SC and NBH samples. 

II. Background 

When considering the entirety of species evolution, human-mediated water pollution is a relatively recently developed pressure on species for survival. Unfortunately, many byproducts and chemicals involved with mass production find their way into an abundant amount of bodies of water. Numerous chemicals possess immense potential to consequently harm populations of aquatic species in various known and unknown ways. Typically, it is assumed that aquatic species are not capable of adapting under such dramatic human-altering environments. Nevertheless, populations of the Atlantic killifish (Fundulus heteroclitus) have been known to reside in some of the most polluted aquatic environments that would otherwise be expected to extinct any aquatic populations. The Atlantic killifish’s unique ability to survive these harsh environments provides potential insight into how species cope with environmental pollution. Therefore, the Atlantic killifish is one of the most extensively studied model organisms for observing organismal adaptations to human-mediated polluted environments. 

PCBs are a widespread water pollutant known to be found in numerous aquatic locations, and PCB toxicity has been shown to have many adverse effects on species. In general, these effects include a variety of cancers, and negative impacts to the immune, reproductive, nervous, and endocrine systems. Further, exposure to PCBs has been demonstrated to elicit a negative correlation with Atlantic killifish populations2. The ability of the killifish to reside, while possibly exhibiting physiological fitness costs can be attributed to the metabolic pathway PCBs act on. 

PCB126 is a dioxin-like PCB congener known to act as a ligand involved in the induction of the aryl hydrocarbon receptor (AHR) pathway. The DLC-dependent activation of the AHR transcription factors (TFs) leads to induction of the cytochrome P450 enzymes-CYP1A1 and CYP1B1- responsible for breaking down toxic compounds in the cell. Interestingly, Atlantic killifish embryos and larvae from NBH displayed reduced sensitivity to dioxin-like compounds DLCs via reduced induction of these cytochrome enzymes1,3. This observed contrast in Atlantic killifish exposed to toxic pollution provides evidence towards adaptations aiding these killifish populations to survive. However, research is limited in establishing a clear, specific mechanistic adaptation that allows the Atlantic killifish to reside in PCB126-polluted waters. In addition, it is currently unclear whether there are other confounding ecological variables that may be contributing to the observed resistance to PCBs in the Atlantic killifish.  The purpose of this study is to investigate the physiological adaptations that allow the Atlantic killifish to survive in this human-mediated extreme environment. It is currently unknown whether Atlantic killifish will develop complete physiological resistance to PCB126 over an adult lifetime. However, epigenetic modifications are believed to show evidence in many species within a lifetime. Thus, we hypothesize that within the Atlantic killifish’s (Fundulus heteroclitus) adult lifetime, the species will begin to develop signs of physiological resistance to PCB126-mediated toxicity after translocation from SC to PCB126-polluted waters in the laboratory.

III. Significance 

The Atlantic killifish serves as a valuable model for predicting how species will adapt to increasing levels of pollution. By studying the adaptive changes in the killifish, we may gain a deeper understanding of biological mechanisms that allow species to survive under various environmental challenges. Due to their persistence in the environment, PCBs have contaminated the food chain, with ingestion of fish caught from contaminated sites being a major exposure pathway,. Thus, the research conducted is relevant to organisms beyond the Atlantic killifish. The biological mechanisms elucidated from this research will serve to predict the effect of Atlantic killifish resistance and its physiological compromises on surrounding ecosystems. The altered metabolic pathways and associated pathologies may also provide predictive insights into how humans and other organisms will respond to similar environmental stressors. 

1. Specific Aims

Aim 1:  Fish from NBH have been observed to show hepatic pathology resembling non-alcoholic fatty liver disease (NAFLD), which could possibly be due to the metabolic adaptations previously observed in NBH killifish that allow it to survive in the PCB126 polluted environment. Therefore, it is hypothesized that translocated killifish from SC will show similar levels of metabolites and have similar NAFLD liver pathology to NBH killifish as a result of their adapted metabolism. In addition, it is hypothesized that non-translocated killifish from SC will show statistically significant differences in concentrations of hepatic metabolites compared to the translocated sample of SC killifish and to the NBH sample of killifish.  

Aim 2: The purpose of this aim is to determine whether exposure to anoxic environments during embryonic phases increases survival rates for killifish and whether increased expression of nitric oxide synthase (NOS) is linked to survival rate increase. Previous research has shown that nitric oxide-the product of NOS-has a protective effect against PCBs and is controlled by anoxia signals.1 Nitric oxide and hypoxia signal pathways may interact with the AHR pathway and regulate CYP1A1 activity through inhibition of CYP1A1 promoter activity.9 Embryonic killifish from SC and NBH will be subjected to anoxia environments, and their survival rates will be compared to those of embryos subjected to  oxygenated environments in both PCB and non-PCB environments; NOS expression levels will then be measured and compared. We expect NBH killifish exposed to anoxia environment will have higher NOS levels and survival rates than embryos who are not subjected to anoxia. We also expect NBH killifish exposed to anoxia environment but not PCBs will have higher NOS levels than control killifish, but lower survival rates, due to the metabolic costs of NOS. Finally, we expect that SC killifish will have similar trends in, but overall lower, NOS levels compared to NBH killifish, as well as decreased survival rates when exposed to PCBs compared to NBH killifish. 

Aim 3: The purpose of this aim is to determine  whether there are reduced CYP1A1 and CYP1B1 enzymatic expression levels in liver tissues of the Atlantic killifish following exposure to PCB-polluted waters. Levels of enzyme expression will be compared between a SC killifish baseline, an experimental PCB126-polluted killifish condition, and an NBH killifish positive control group. Data will be gathered through immunohistochemistry techniques and then measured with a valid subjective stain scoring method. In response to dangerous effects of CYP1A1 and CYP1B1 enzymes producing toxic metabolites, and the hyperactivity of the AHR pathway, which is involved in the facilitation of CYP1A1 and CYP1B1 expression, previous literature has displayed PCB126-mediated AHR pathway suppression3. Therefore, we expect an overall reduction of CYP1A1 and CYP1B1 expression as a protective adaptation following PCB126 exposure, thus showing evidence of PCB126 physiological resistance.

Aim 4: The final aim will be conducted in parallel to aim 3, with the goal of identifying the epigenetic alterations behind the reduced induction of CYP1A1 and CYP1B1 after exposure to PCB-polluted waters. 

Since histone deacetylation has been suggested as a possible epigenetic alteration but has never been measured , acetylation of the activating marker H3K27ac will be analyzed with chromatin immunoprecipitation quantitative PCR (ChIP-qPCR). We hypothesize PCB exposure will reduce H3K27ac in as early as the first screening at six months.

1. Design and Methods 

Aim 1: Hepatic metabolism and anatomy of killifish following translocation into PCB-polluted water.  

Rationale: A previous study compared PCB-resistant Atlantic Killifish that are native to PCB laden waters in New-Bedford Harbor (NBH), Massachusetts to PCB-sensitive Atlantic Killifish that are native to the freshwaters of Scorton Creek (SC), Sandwich, Massachusetts, and found that there were differences in hepatic metabolites between the two populations of fish.6 Many of these hepatic metabolites are associated with one-carbon metabolism, an important metabolic pathway for DNA methylation, amino acid metabolism, and nucleic acid synthesis.6 These results suggest that PCB-resistant fish living in NBH and PCB.Sensitive fish living in SC have physiologically adapted to their respective environments over time by altering their hepatic metabolism.6 Even though NBH Killifish have clearly been able to successfully adapt to living in high concentrations of PCBs, there is evidence that there is a higher metabolic cost for them to maintain physiological homeostasis since many killifish from NBH show hepatic pathology resembling non-alcoholic fatty liver disease (NAFLD), which is typically asymptomatic.6 It is unknown, however, whether the differences in hepatic metabolism between NBH and SC fish and the hepatic pathology observed in NBH fish are specifically due to PCBs, or whether there are other confounding ecological variables that could be contributing to the observed differences.6 Therefore, this aim will in part attempt to determine whether PCBs are the singular variable that causes the observed differences in metabolism between NBH and SC fish. We will place SC killifish in a tank containing the same concentration of PCB126 typically found in NBH. In addition, this aim will attempt to determine whether the adaptations in hepatic metabolism observed in NBH fish induce hepatic pathology resembling NAFLD in fish that are translocated from SC fresh waters. This will serve to provide data that either supports or refutes a causal link between the observed alterations in metabolism and the hepatic pathology observed in NBH fish.    

Method:  100 fresh water killifish from SC will be caught and placed into a fish tank containing water from SC. The first step of the protocol will be to collect blood from the caudal artery of each SC fish. Liver function tests will be carried out by analyzing the serum concentrations of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP).  High levels of these enzymes would indicate the presence of hepatic illness. Any fish from this fresh water sample that has high liver enzymes will be removed from the study since this liver damage would not be the result of PCB126. The remaining killifish will then be transferred to a tank with water containing PCB126. After six months, each fish will be euthanized, and the livers from each fish will be removed and immediately weighed. Then, a small piece of liver from each fish will be homogenized (the rest of the liver will be temporarily stored in liquid nitrogen), and hepatic metabolites will be extracted and analyzed using targeted metabolomics.6 The rest of the liver will be removed from liquid nitrogen storage and will be cyrosectioned into 50 µm sections, followed by Oil Red-Oil staining, which stains for neutral lipids.6 These same methods (excluding liver function tests) will then be applied to another sample of 100 killifish inhabiting PCB126-polluted waters in NBH and to another sample of 100 killifish from SC. Each sample will be collected and placed in a tank containing water from NBH and SC, respectively. Data will then be collected for the SC sample (negative control) and the NBH sample (positive control). The hepatic metabolites from the translocated sample of killifish that originated from SC will be compared to the metabolites of killifish from the NBH sample and the non-translocated SC sample by using a One-way ANOVA. Additionally, the histological sections of hepatic tissue that were previously stained from the translocated sample of killifish from  SC, the sample from NBH, and the non-translocated sample from SC, will then be observed under the microscope and examined for the presence of lipid accumulation in hepatocytes.6 

Predictions: It is hypothesized that killifish from NBH and fish that are translocated from clean waters to PCB126-polluted waters will show mild signs of hepatic pathology resembling NAFLD and will have similar concentrations of hepatic metabolites. Additionally, it is hypothesized that killifish from SC that were not translocated will not show signs of hepatic pathology resembling NAFLD and will show statistically significant differences in concentrations of hepatic metabolites compared to the translocated sample of SC killifish and to the NBH sample of killifish. 

Aim 2: Anoxia as Nitric Oxide Synthase promoting signal inducing increased PCB resistance 

Rationale: Nitric oxide synthase (NOS) increases PCB tolerance levels of killifish.1 NOS levels may be influenced by embryonic exposure to oxygen poor environments;Zebrafish embryos which have been exposed to oxygen poor environments have shown increased tolerance to 2,3, 7,8-Tetrachlorodibenzo-p-dioxin (TCDD) which has a similar chemical structure to PCBs. NOS levels have also been suggested to have an effect on the AHR pathway and the CYP1A1 enzyme. This is significant as it indicates a mechanism that is influenced by embryonic exposure to oxygen poor environments that increases tolerance to toxins. Since TCDD has a similar structure to PCBs, it is possible that a similar mechanism could lead to both increased TCDD and PCB tolerance. Past research has also demonstrated that killifish embryos exposed to an anoxia environment, are able to enter a dormant phase (diaphase) with unique energy usage patterns allowing for survival and recovery even after months in an anoxia environment. Killifish embryos’ unique adaptation to survive in anoxia environments may also promote NOS production increasing PCB tolerance. NBH embryos may also have increased tolerance levels compared to SC embryos due to environmental conditions favoring phenotypes that favor the ability to synthesize high levels of NOS. Increased NOS production may be unfavorable under non-toxic environments due to increased metabolic costs of NOS production. Killifish survival rates will be measured for NBH and SC embryo populations who are exposed to PCB and/or anoxia conditions, where NOS levels will then be measured, with a control for each population exposed to neither. 

Method: Killifish from SC and NBH will be collected and taken into a lab setting, where embryo collection will occur after initiating a breeding colony.  Embryos who will be exposed to anoxia environment will be exposed for varying amounts of time (5, 10, 20, 50 days) (method described in Podrabsky et al., 2012). Those not exposed to anoxia environment will be in a neutral environment. After anoxia exposure, embryos will be given a rest period of 24 hours, after which they will be exposed to PCB126-waters which will vary in PCB126 concentrations (0.0125-4 ng/ml) for  varying time periods (Every 12 hours through 96 hours).10 Embryos not exposed will maintain in neutral environment. After PCB exposure (or, for non-exposure groups, after an amount of time similar to how long the exposure would’ve taken has passed) embryos will be sacrificed for RNA collection (methods described in Whitehead et al., 2010). Embryos not exposed to either PCB or anoxia environments are the controls for the aim. A Two-Way ANOVA will be conducted for survival rates for each population. A simple regression will be run to determine effect of anoxia on NOS levels. A Two-Way ANOVA will be conducted to determine NOS on survival. A Two-way ANOVA will be used to measure between populations. 

Predictions: Since NOS is believed to increase tolerance, for NOS levels to increase due to anoxia, we predict that NOS levels will have a positive correlation with anoxia duration, and thus a positive correlation with survival rates. We also predict that NBH embryos will have a higher tolerance than SC embryos.

Aim 3: PCB126-induced reduction of CYP1A1 & CYP1B1 enzymatic expression levels in the liver

Rationale: Previous studies have established that Atlantic killifish (Fundulus heteroclitus) are able to survive in habitats occupied by PCBs in lethal concentrations. These PCBs are known to activate the AHR pathway and ultimately induce expression of CYP1A mRNA3. CYP1A encodes for CYP1A1 protein, known to be involved in degradation of harmful chemicals. However, research has demonstrated that suppression of the AHR pathway seems to be a mechanism of surviving PCB-polluted waters3. These seemingly surprising results can be explained by the fact that, although CYP1 enzymes are involved in detoxification, it has also been established that they are possibly involved in generating toxic metabolites that can facilitate factors of increased toxic effects. Therefore, researchers believe AHR pathway suppression is necessary to prevent its hyperactivity, which would ultimately lead to cell death3. Suppression of the AHR pathway may indicate that there will be an observed and measurable reduction in the downstream target of the AHR genes: CYP1A1. Furthermore, previous research in Atlantic killifish has identified reduction in another enzyme relevant to the CYP1 family known as CYP1B1 in fish exposed to PCB12612. Thus, the goal of this aim will be to measure the expression of CYP1A1 & CYP1B1 enzymatic expression in response to AHR pathway suppression via PCB126. 

Method: Data will be collected from adult Atlantic killifish from SC following translocation to PCB126-polluted waters in the lab (experimental group). Baseline enzymatic expression of both CYP1A1 and CYP1B1 will be measured upon initially retrieving SC killifish to serve as a negative control comparison. Baseline enzymatic expression of both CYP1A1 and CYP1B1 will also be measured in NBH fish to serve as a positive control comparison. Since Atlantic killifish reach maturity around 2 years old and are known to typically live to 4 years old, within the PCB126-polluted experimental group, CYP1A1 and CYP1B1 expression will be measured every 6 months for 2 years after SC translocation to PCB126-polluted lab waters. To quantify the expression levels of CYP1A1 and CYP1B1 enzymes, immunohistochemistry techniques will be applied to collected liver tissues. Scoring techniques that have shown construct validity from previous research will be used. Sections of liver tissues will be scored in two categories, first on occurrence of cells, then second on intensity of stain, following a staining index from. Each time data is collected, the mean of each final staining index from the experimental condition will be calculated, and data will be analyzed using a one-way ANOVA statistical comparison between SC baseline killifish (negative control), PCB126-polluted killifish (experimental condition), and NBH killifish (positive control). 

Predictions: Since we expect PCB126 exposure to have suppressive effects on the AHR pathway, we expect an overall significant reduction of both CYP1A1 and CYP1B1 enzymatic expression in liver tissues over the Atlantic killifish adult lifetime.

Aim 4: Epigenetic mechanisms behind reduced sensitivity of the AHR pathway

Rationale: Studies have shown reduced signaling in the aryl hydrocarbon receptor signaling pathway via reduced induction of cytochrome P4501A (CYP1A1); however, the mechanism of this decreased activation has not been fully elucidated. Insight into the link between epigenetic alterations and reduced CYP1A1 expression was established in 2002, when researchers characterized the CYP1A1 response of three generations of laboratory-raised and offspring of Elizabeth River killifish to exposure of CYP1A1-inducing chemicals. The variances in CYP1A1 induction between the first-generation embryos, and second and third generation fish, indicated non-genetic mechanism involvement in CYP1A1 downregulation. It was later hypothesized that non-induction of CYP1A1 stems from changes in DNA methylation in its promoter region upon exposure to PCBs; however, no methylated cytosines were observed between contaminated and reference-site fish7. The epigenetic alterations due to PCB exposure are clearly yet to be established. Non-differential DNA methylations prompts this aim to study other epigenetic alterations; histone deacetylation was suggested to play a possible role in reduced induction of CYP1A17. This would be worth investigating as histone acetylation is associated with gene expression. CYP1B1 histone acetylation will also be measured, as the expression level of this enzyme will be measured in aim 3. 

Method: Data will be collected from Atlantic killifish living in SC (negative control), Atlantic killifish living in NBH (positive control), and Atlantic killifish following translocation to PCB126-polluted waters in the lab (experimental group). Acetylation of lysine 27 on histone 3 (H3K27ac) is an activating histone modification, making it an attractive candidate epigenetic marker to study. Every six months a sample of the translocated fish will be tested for CYP1A1 and CYP1B1 H3K27ac. A chromatin immunoprecipitation assay (ChIP) will be used to quantify H3K27ac from hepatic DNA samples. Histone-DNA complexes will be immuno-precipitated using H3K27ac-specific antibodies. H3K27ac will then be quantified using quantitative PCR (qPCR). An input sample will be used to determine the starting amount of chromatin and will be used to calculate % IP16. For each condition-fish from SC, fish from NBH, and translocated fish-, the % IP will be calculated based on the relative levels of amplicon produced in the qPCR. As a negative control, a set of gene targets that are unbound by acetylated histones will be used to ensure the specificity of the antibodies used. A no-template control will also be run to ensure there is no contamination16. The mean %IPs of acetylated CYP1 enzymes will be compared between the baseline condition (SC) and the translocated killifish, followed by a one-way ANOVA to calculate significance following translocation. 

Predictions: It is hypothesized that killifish exposed to PCB-polluted waters will exhibit reduced acetylation CYP1 enzyme H3K27ac; this would provide an explanation for the reduced induction previously reported 3,12. It is possible that there will be no significant decreases in H3K27ac following translocation. Other epigenetic alterations, such as methylation of lysine 4 on histone 3 (H3K4me1) are also activating markers15. It is possible other genes in the AHR pathway are causing reduced induction of CYP1 enzymes; an alternative approach is to conduct high-throughput screening, in which global histone modifications are measured using ChIP-seq and then correlated to PCB exposure. 

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