R. Christopher Chambers

Mechanistic Basis of Resistance to PCBs in Atlantic Tomcod from the Hudson River

Chronic pollution of the Hudson River, New York, results in rapid evolution of resistance to the pollutants. The mechanistic basis of resistance of vertebrate populations to contaminants, including Atlantic tomcod from the Hudson River (HR) to polychlorinated biphenyls (PCBs), is unknown. HR tomcod exhibited variants in the aryl hydrocarbon receptor 2 (AHR2) that were nearly absent elsewhere. In ligand-binding assays, AHR2-1 protein (common in the HR) was impaired as compared to widespread AHR2-2 in binding TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) and in driving expression in reporter gene assays in AHR-deficient cells treated with TCDD or PCB126. We identified a six-base deletion in AHR2 as the basis of resistance and suggest that the HR population has undergone rapid evolution, probably due to contaminant exposure. This mechanistic basis of resistance in a vertebrate population provides evidence of evolutionary change due to selective pressure at a single locus.

Evidence of spatially extensive resistance to PCBs in an anadromous fish of the Hudson River.

Populations of organisms that are chronically exposed to high levels of chemical contaminants may not suffer the same sublethal or lethal effects as naive populations, a phenomenon called resistance. Atlantic tomcod (Microgadus tomcod) from the Hudson River, New York, are exposed to high concentrations of polycyclic aromatic hydrocarbons (PAHs) and bioaccumulate polychlorinated biphenyls (PCBs), polychlorinated dibenzo-p-dioxins (PCDDs), and polychlorinated dibenzofurans (PCDFs). They have developed resistance to PCBs and PCDDs but not to PAHs. Resistance is largely heritable and manifests at early-life-stage toxic end points and in inducibility of cytochrome P4501A (CYP1A) mRNA expression. Because CYP1A induction is activated by the aryl hydrocarbon receptor (AHR) pathway, as are most toxic responses to these compounds, we sought to determine the geographic extent of resistance to CYP1A mRNA induction by PCBs in the Hudson River tomcod population. Samples of young-of-the-year tomcod were collected from seven locales in the Hudson River, extending from the Battery at river mile 1 (RM 1) to RM 90, and from the Miramichi River, New Brunswick, Canada. Laboratory-reared offspring of tomcod adults from Newark Bay, in the western portion of the Hudson River estuary, were also used in this study. Fish were partially depurated in clean water and intraperitoneally injected with 10 ppm coplanar PCB-77, 10 ppm benzo[a]pyrene (BaP), or corn oil vehicle, and levels of CYP1A mRNA were determined. CYP1A was significantly inducible by treatment with BaP in tomcod from the Miramichi River, from laboratory-spawned offspring of Newark Bay origin, and from all Hudson River sites spanning 90 miles of river. In contrast, only tomcod from the Miramichi River displayed significantly induced CYP1A mRNA expression when treated with PCB-77. Our results suggest that the population of tomcod from throughout the Hudson River estuary has developed resistance to CYP1A inducibility and probably other toxicities mediated by the AHR pathway. Tomcod from the Hudson River may represent the most geographically expansive population of vertebrates with resistance to chemical pollutants that has been characterized.

Characterization of the aryl hydrocarbon receptor repressor and a comparison of its expression in Atlantic tomcod from resistant and sensitive populations

Atlantic tomcod from the Hudson River, USA, are resistant to cytochrome P4501A1 (CYP1A1) mRNA induction and early life stage toxicities induced by coplanar polychlorinated biphenyls (PCBs) or tetrachlorodibenzo-p-dioxins but not polycyclic aromatic hydrocarbons. We sought to determine if basal expression or inducibility of aryl hydrocarbon receptor repressor (AHRR) mRNA is higher in tomcod from the resistant Hudson River population than in those from sensitive populations. Tomcod AHRR cDNA was characterized and its expression quantified in different tissues and life stages of tomcod from the Hudson River, Miramichi River, Canada (sensitive), and among environmentally exposed tomcod from these two sources and the St. Lawrence River, Canada. Phylogenetic analysis revealed that tomcod AHRR falls within the clade of other vertebrate aryl hydrocarbon receptors (AHRs) but is most closely related to the four previously identified AHRR genes. Induction of AHRR mRNA was observed in all tissues of PCB77-treated juvenile tomcod of Miramichi River descent, and expression differed among tissues and was significantly related to levels of CYPIAI mRNA expression. Aryl hydrocarbon receptor repressor mRNA was similarly inducible in F2 embryos of Miramichi and Hudson River descent by benzo[a]pyrene but less by PCB77 in Hudson River offspring. A significant, positive correlation was observed between CYP1A1 mRNA and AHRR mRNA concentrations in environmentally exposed tomcod from the three rivers. We conclude that differences in basal expression or inducibility of AHRR mRNA are not the mechanistic basis of resistance but that levels of AHRR often mirror those of CYP1A1, suggesting that a common AHR pathway-related mechanism may modulate expression of both genes.

Toxic effects of PCB126 and TCDD on shortnose sturgeon and Atlantic sturgeon

Exposure to chemical contaminants is often invoked to explain recruitment failures to populations of sturgeon worldwide, but there is little empirical evidence to support the idea that young sturgeon are sensitive at environmentally relevant concentrations. The authors used shortnose sturgeon (Acipenser brevirostum) and Atlantic sturgeon (Acipenser oxyrinchus) as models to investigate the sensitivities of sturgeon to early-life-stage toxicities from embryonic exposures to graded doses of polychlorinated biphenyl 126 (PCB126) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Survival to hatching of shortnose sturgeon decreased with increasing dose, although the duration of the embryonic period was not significantly altered by exposure in either species. Morphometric features of larvae of both species were affected by dose, including shortening of the body, reduction in head size, reduction in quantity of yolk reserves, and reduction in eye size. Eye development in both species was delayed with increasing dose for both chemicals. The persistence of larvae in a food-free environment decreased inversely with dose in both species, with sharp declines occurring at PCB126 and TCDD doses of ≥1 ppb and ≥0.1 ppb, respectively. Dose-responsive early-life-stage toxicities reported here are among the more sensitive found in fish and occurred at burdens similar to those found in situ in a sympatric bottom-dwelling bony fish in the Hudson River Estuary. The present study is among the first demonstrating the sensitivity of any sturgeon to the hallmark early-life-stage toxicities induced by aryl hydrocarbon receptor agonists.

Detecting critical periods in larval flatfish populations

Abstract We evaluate the time-course of deaths and evidence of periods of increased mortality (i.e., critical periods) in laboratory populations of larval flatfish. First, we make the distinction between age-at-death and abundance-at-time data for fish larvae, the latter being typical in studies of natural populations. Next, we describe an experimental investigation of age- and temperature-dependent mortality in larval winter flounder, Pseudopleuronectes americanus . The survivorship curves of these populations differed significantly in both the magnitude and time-course of mortality among the four water temperatures evaluated (7, 10, 13, and 16°C). Mortality was highest in the cooler temperatures and concentrated in the third quarter of larval life, largely concurrent with settlement of surviving members of the cohort. Among the statistical methods for analysing survival data, the proportional-hazards model with time-varying covariates proved best at capturing the patterns of age-specific mortalities. We conclude that fair appraisals of recruitment hypotheses which are predicated on periods of high, age-specific mortality that vary with environmental conditions (e.g., Hjorts critical period hypothesis) will require: (1) data that are based on age, not time; (2) data that are of higher temporal resolution than commonly available at present and (3) analytical methods that are sensitive to irregularities in survivorship curves. We suggest four research approaches for evaluating critical periods in nature.

Characterization and expression of cytochrome P4501A in Atlantic sturgeon and shortnose sturgeon experimentally exposed to coplanar PCB 126 and TCDD

The AHR pathway activates transcription of CYP1A and mediates most toxic responses from exposure to halogenated aromatic hydrocarbon contaminants such as PCBs and PCDD/Fs. Therefore, expression of CYP1A is predictive of most higher level toxic responses from these chemicals. To date, no study had developed an assay to quantify CYP1A expression in any sturgeon species. We addressed this deficiency by partially characterizing CYP1A in Atlantic sturgeon (Acipenser oxyrinchus oxyrinchus) and shortnose sturgeon (Acipenser brevirostrum) and then used derived sturgeon sequences to develop reverse transcriptase (RT)-PCR assays to quantify CYP1A mRNA expression in TCDD and PCB126 treated early life-stages of both species. Phylogenetic analysis of CYP1A, CYP1B, CYP1C and CYP3A deduced amino acid sequences from other fishes and sturgeons revealed that our putative Atlantic sturgeon and shortnose sturgeon CYP1A sequences most closely clustered with previously derived CYP1A sequences. We then used semi-quantitative and real-time RT-PCR to measure CYP1A mRNA levels in newly hatched Atlantic sturgeon and shortnose sturgeon larvae that were exposed to graded doses of waterborne PCB126 (0.01-1000 parts per billion (ppb)) and TCDD (0.001-10 ppb). We initially observed significant induction of CYP1A mRNA compared to vehicle control at the lowest doses of PCB126 and TCDD used, 0.01 ppb and 0.001 ppb, respectively. Significant induction was observed at all doses of both chemicals although lower expression was seen at the highest doses. We also compared CYP1A expression among tissues of i.p. injected shortnose sturgeon and found significant inducibility in heart, intestine, and liver, but not in blood, gill, or pectoral fin clips. For the first time, our results indicate that young life-stages of sturgeons are sensitive to AHR ligands at environmentally relevant concentrations, however, it is yet to be determined if induction of CYP1A can be used as a biomarker in environmental biomonitoring.

Characterization of AHR2 and CYP1A expression in Atlantic sturgeon and shortnose sturgeon treated with coplanar PCBs and TCDD

Atlantic sturgeon and shortnose sturgeon co-occur in many estuaries along the Atlantic Coast of North America. Both species are protected under the U.S. Endangered Species Act and internationally on the IUCN Red list and by CITES. Early life-stages of both sturgeons may be exposed to persistent aromatic hydrocarbon contaminants such as PCBs and PCDD/Fs which are at high levels in the sediments of impacted spawning rivers. Our objective was to compare the PCBs and TCDD sensitivities of both species with those of other fishes and to determine if environmental concentrations of these contaminants approach those that induce toxicity to their young life-stages under controlled laboratory conditions. Because our previous studies suggested that young life-stages of North American sturgeons are among the more sensitive of fishes to coplanar PCB and TCDD-induced toxicities, we were interested in identifying the molecular bases of this vulnerability. It is known that activation of the aryl hydrocarbon receptor 2 (AHR2) in fishes mediates most toxicities to these contaminants and transcriptional activation of xenobiotic metabolizing enzymes such as cytochrome P4501A (CYP1A). Previous studies demonstrated that structural and functional variations in AHRs are the bases for differing sensitivities of several vertebrate taxa to aromatic hydrocarbons. Therefore, in this study we characterized AHR2 and its expression in both sturgeons as an initial step in understanding the mechanistic bases of their sensitivities to these contaminants. We also used CYP1A expression as an endpoint to develop Toxicity Equivalency Factors (TEFs) for these sturgeons. We found that critical amino acid residues in the ligand binding domain of AHR2 in both sturgeons were identical to those of the aromatic hydrocarbon-sensitive white sturgeon, and differed from the less sensitive lake sturgeon. AHR2 expression was induced by TCDD (up to 6-fold) and by three of four tested coplanar PCB congeners (3-5-fold) in Atlantic sturgeon, but less so in shortnose sturgeon. We found that expression of AHR2 and CYP1A mRNA significantly covaried after exposure to TCDD and PCB77, PCB81, PCB126, but not PCB169 in both sturgeons. We also determined TEFs for the four coplanar PCBs in shortnose sturgeon based on comparison of CYP1A mRNA expression across all doses. Surprisingly, the TEFs for all four coplanar PCBs in shortnose sturgeon were much higher (6.4-162 times) than previously adopted for fishes by the WHO.

A Dramatic Difference in Global Gene Expression between TCDD-Treated Atlantic Tomcod Larvae from the Resistant Hudson River and a Nearby Sensitive Population

Abstract Atlantic tomcod in the Hudson River Estuary bioaccumulate high hepatic burdens of environmental toxicants. Previously, we demonstrated that Hudson River tomcod developed resistance to TCDD and PCB toxicity probably through strong natural selection during their early life-stages for a variant of the Aryl Hydrocarbon Receptor2 (AHR2). Here, we evaluated the genomic consequences of the resistant genotype by comparing global gene expression in larval tomcod from the Hudson River with expression in larvae from a nearby sensitive population (Shinnecock Bay). We developed an annotated draft tomcod genome to explore the effects of multigenerational exposure to toxicants and a functionally impaired AHR2 on the transcriptome. We used the tomcod genome as a reference in RNA-Seq to compare global gene expression in tomcod larvae from the Hudson River and Shinnecock Bay after experimental exposure of larvae to graded doses of TCDD. We found dramatic differences between offspring from the two populations in the number of genes that were differentially expressed at all doses (0.01, 0.1, and 1 ppb) and even in the vehicle controls. At the two lowest TCDD doses, 250 and 1,141 genes were differentially expressed in Shinnecock Bay larvae compared with 14 and 12, respectively, in Hudson River larvae. At the highest dose (1.0 ppb), 934 genes were differentially expressed in Shinnecock Bay larvae and 173 in Hudson River larvae, but only 28 (16%) of affected genes were shared among both populations. Given the large difference between the two populations in the number and identity of differentially expressed genes, it is likely that the AHR2 pathway interacts directly or indirectly with many genes beyond those known in the AHR2 battery and that other regulatory systems may also respond to TCDD exposure. The effects of chronic multi-generational exposure to environmental toxicants on the genome of Hudson River tomcod are much greater than previously expected.