Simon C. Courtenay

A comparison of the dose and time response of CYP1A1 mRNA induction in chemically treated Atlantic tomcod from two populations

Abstract Quantification of cytochrome P4501A1 (CYP1A1) mRNA levels in environmentally exposed Atlantic tomcod (Microgadus tomcod) has revealed significantly induced gene expression in fish from contaminated locales including the Hudson River, New York, and the Miramichi River, New Brunswick. In order to calibrate this response, determine its sensitivity and dose-responsiveness, levels of hepatic CYP1A1 mRNA were quantified in depurated Atlantic tomcod intraperitoneally (i.p.) injected with various concentrations of: β-naphthoflavone (β-NF), the PAH benzo[a]pyrene (B[a]P), the non-ortho coplanar PCB congener-3,3′,4,4′- tetrachlorobiphenyl (IUPAC: PCB-77), and the dioxin 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD). Additionally, the rates of CYP1A1 mRNA induction and disappearance were quantified in depurated Atlantic tomcod i.p. injected with single doses of these chemicals and sacrificed at times ranging up to 72 days. Levels of CYP1A1 mRNA were dose-responsive for all four chemicals with maximum induction ranging from 50- to 460-fold and first significant induction being observed in the low mg per kg fish (wet weight) range for β-NF and B[a]P, μg/kg range for PCB-77 and ng/kg range for 2,3,7,8-TCDD. However, while tomcod from the Miramichi River responded to both PAHs and halogenated aromatic hydrocarbons (HAHs), Hudson River tomcod responded only to PAHs indicating population level differences in CYP1A1 mRNA inducibility in tomcod. Furthermore, differences in the responsiveness to PAHs and HAHs suggest that more than one molecular mechanism mediates CYP1A1 transcription in Atlantic tomcod. Kinetic profiles of CYP1A1 mRNA induction differed greatly between tomcod treated with HAHs and PAHs. Initial induction occurred within hours of treatment with PAHs and peaked after 1–3 days, compared to initial induction 4–7 days after treatment with HAHs, and maximum induction not occurring for up to 72 days after exposure. Quantification of halogenated aromatic hydrocarbons (HAH) in the livers of tomcod caught in the Hudson and Miramichi Rivers confirmed exposure and accumulation of known CYP1A1 inducing chemicals including 2,3,7,8-TCDD at concentrations as high as 1.5 μg/kg lipid (554 ng/kg w.w.) and PCB-77 at concentrations as high as 108 μg/kg lipid (15 μg/kg w.w.). These results suggest that hepatic CYP1A1 mRNA concentration can be a useful bioindicator of exposure to some aromatic hydrocarbon compounds in the aquatic environment and that profiles of gene induction and disappearance may help identify environmental inducers provided that gene responsiveness is also evaluated under controlled laboratory conditions.

Effects of prior exposure history on cytochrome P4501A mRNA induction by PCB congener 77 in atlantic Tomcod

Abstract Levels of P450IA mRNA in tomcod collected from the Miramichi River, Canda and the Hudson River, New York were measured by slot blot analyses after the fish had been depurated and i.p. injected with Aroclor 1254, PCB congener 77, or PCB congener 105. Elevated expression of P450IA mRNA was observed in congener 77-treated Miramichi tomcod, whereas no induction was seen in the Hudson River fish. Tomcod from both rivers were induced by injection with beta-naphthoflavone. These results suggest that the prior exposure history of the Hudson River tomcod may have affected their non-inducibility by treatment with PCB congener 77.

Induction and clearance of cytochrome P4501A mRNA in Atlantic tomcod caged in bleached kraft mill effluent in the Miramichi River

Abstract Levels of cytochrome P4501A mRNA were measured in Atlantic tomcod caged in the effluents of a bleached kraft pulp and paper mill on the Miramichi River, NB, Canada and two sites downstream, and compared to levels in fish caged upstream of the mill and in unexposed laboratory-maintained tomcod. Additionally, the rate of clearance of CYP1A mRNA in tomcod exposed at the mill and transferred to clean water was compared to that observed in tomcod treated in the laboratory with a single dose of 2,3,7,8-TCDD (0.5 μg/kg). Levels of CYP1A mRNA in tomcod caged upriver of the mill were low and comparable to those observed in untreated laboratory control fish. Levels of CYP1A mRNA were elevated up to 11-fold in tomcod caged at the mill site and a gradient in levels of gene expression was seen at the two downstream sites. Tomcod i.p. injected with a single dose of 2,3,7,8-TCDD exhibited a profile of prolonged CYP1A mRNA induction of at least 25 days. Levels of CYP1A mRNA in tomcod caged at the mill site and transferred to clean water remained significantly induced for at least 3 days post-transfer. In combination, these results suggest that CYP1A mRNA was induced in caged tomcod by constituents of the mills effluents and that the rates of clearance of CYP1A mRNA may provide additional information concerning the identity of environmental inducers.

Histopathological evaluation of Atlantic tomcod (Microgadus tomcod) collected at estuarine sites receiving pulp and paper mill effluent

Abstract Histopathological examination of liver, pancreas, spleen and gonads was conducted in Atlantic tomcod ( Microgadus tomcod ) collected during September–November 1994 from estuarine sites receiving pulp and paper mill effluents in the Restigouche Estuary, the Miramichi Estuary and Pictou Harbour, and reference sites in the Kouchibouguac and Margaree Estuaries. This study was part of a larger investigation into the environmental health of estuaries and the coastal environment of the southern Gulf of St. Lawrence. Density and surface area of pigmented macrophage aggregates (PMA), and presence or absence of inflammatory, preneoplastic and neoplastic changes were evaluated in spleen and liver. Stages of maturity of the gonads were determined. In the Miramichi Estuary only, 28% of the fish had extensive multifocal granulomatous lesions centered on PMA in the spleen, likely caused by an infectious agent. In these affected fish, the density of PMA was slightly increased, and the surface area of PMA was markedly increased (10×) compared to normal fish from the same site. Both the density and the surface area of PMA were lower in spleens of fish from the Restigouche Estuary than at other sites. Condition factor and stage of maturity of the gonads were higher at this site. In the Restigouche Estuary only, more than 90% of the fish had zones of vacuolation in the pancreas. In the liver, density of PMA was generally low and did not differ significantly among sites. No preneoplastic or neoplastic lesions were observed. Further studies are needed to identify the causes and pathogenesis of the two lesions that were observed only at contaminated sites: the multifocal granulomatous lesions in the spleen and the zones of vacuolation in the pancreas. A larger sample of older fish should be examined to evaluate prevalence of preneoplastic and neoplastic changes. Histopathological markers have successfully identified potential effects of pulp and paper effluents on fish health, but the observed lesions cannot, at this stage, be attributed specifically to toxic exposure.

Chemical–environment interactions affecting the risk of impacts on aquatic organisms: A review with a Canadian perspective — interactions affecting vulnerability

Environmental change can increase the vulnerability of aquatic species to toxic chemicals by challenging an organism’s aptitude to respond to chemicals or to repair toxic injury or by modifying animal behaviours like migration or predation. On the other hand, xenobiotics may affect the capacity of aquatic species to adapt to environmental challenges that come with change (e.g., pathogens, temperature). Across Canada we have identified a number of circumstances where chemicals and environmental variability have likely worked together to affect vulnerability of aquatic organisms. For example in the Maritimes, exposure to municipal wastewater or bleached kraft pulp mill effluent altered immune function in bivalves and increased their risk of developing haemocytic neoplasia, a disease known to cause high mortality. Northwest Atlantic cod stocks have experienced large-scale changes in environment and exhibit marked seasonal cycles in energy reserves. The risk associated with subsequent redistribution of persis...

Seasonal use of estuaries by winter flounder in the Southern Gulf of St. Lawrence

Abstract The seasonal distribution of winter flounder Pleuronectes americanus in the southern Gulf of St. Lawrence was examined by analyzing data from research surveys conducted from 1986 to 1994 in the southern Gulf and from beach seining, trawling, and sampling of commercial fisheries in the Miramichi Estuary. During June–August, most winter flounder were found in coastal waters (<40 m deep) and within a temperature range of 3–16°C. Abundance in coastal waters declined sharply between September and late November–early December. Winter flounder were not captured in deep offshore waters during winter; rather, large numbers moved into the Miramichi Estuary during October and November and remained until spring. The brackish water under the ice of the Miramichi Estuary was warmer (–1 to 0°C) than the full salt water under the ice of the southern Gulf (below –1.5°C) and could provide a thermal refuge for winter flounder as well as protection from ice contact during winter storms. Adult winter flounder left th...

Is hepatic cytochrome P4501A1 expression predictive of hepatic burdens of dioxins, furans, and PCBs in Atlantic tomcod from the Hudson River estuary?

Hepatic cytochrome P4501A1 (CYP1A1) expression in fishes is frequently used to evaluate bioavailable aromatic hydrocarbon contamination of aquatic ecosystems. In controlled laboratory experiments, CYP1A1 expression in naïve fishes is usually dose-responsive to aromatic hydrocarbons and in field studies levels of gene expression in natural populations often correspond with known levels of sediment-borne contaminants. We quantified CYP1A1 mRNA levels in juvenile Atlantic tomcod Microgadus tomcod from 42 sites in the Hudson River estuary to evaluate the correspondence between hepatic CYP1A1 expression and hepatic concentrations of persistent halogenated aromatic hydrocarbons and to determine the utility of CYP1A1 expression as a biomarker in evaluating the microgeographic distribution of bioavailable contaminants within a large aquatic ecosystem. We found significant spatial heterogeneity in CYP1A1 mRNA levels among collection sites with levels of gene expression differing in some cases by 23-34 folds. CYP1A1 mRNA expression was highest in tomcod from the Newark Bay complex and lowest in tomcod from the most upriver collection sites in the main stem of the Hudson River. Although levels of PCDDs, PCDFs, and PCBs expressed as TCDD TEQs and CYP1A1 mRNA were highest in tomcod from the Newark Bay complex, there was no relationship between hepatic halogenated aromatic hydrocarbon levels and hepatic CYP1A1 mRNA in tomcod from sites in the main stem of the Hudson River. These results suggest that levels of CYP1A1 expression in fish from sites highly polluted with mixtures of halogenated aromatic hydrocarbons and other xenobiotics may not always be reflective of levels of bioavailable aromatic hydrocarbon contaminants. Based on these results and earlier controlled laboratory experiments, we hypothesize that elevated levels of CYP1A1 expression in tomcod from the Hudson River may be due primarily to PAHs or other contaminants not measured in this study.

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.

Water Renewal Estimates for Aquaculture Developments in the Richibucto Estuary, Canada

Water renewal in semi-enclosed coastal areas is crucial for the supply of oxygen and seston and for the removal of organic loadings from finfish or shellfish aquaculture sites. Water renewal depends on hydrodynamic processes and can have a complex spatial distribution due to irregular topographic features. This study describes some physical oceanography observations gathered in the Richibucto estuary, New Brunswick, Canada, and provides an estimate of the spatial distribution of water renewal in the North Arm, a location in the estuary where the largest American oyster (Crassostrea virginica) aquaculture operation in eastern Canada is located. The estuary changes from a well mixed estuary to a partially stratified estuary depending on runoff conditions. Tides are mixed but mainly diurnal due to the nearby presence of the second M2 amphidromic point in the Gulf of St. Lawrence. Tidal amplitudes vary from 0.3 to 0.6 m and show a slight increase some 35 km upstream. Currents in the main channel can reach over 0.60 m s−1 during ebb and 0.3 m s−1 during flood, with a slack water period of approximately 8 h. Low frequency sea level fluctuations have a range of 0.5 m at the mouth and are coherent within the estuary. Hydrodynamic and advection-dispersion models are used to calculate the spatial distribution of the local renewal time (LRT) in the North Arm for high and low freshwater discharge conditions, using the dissolved tracer method. Results show that the LRT varies from less than 5 d at the downstream end of the North Arm to over 100 d further upstream. When averaged over the entire North Arm, the integral renewal time (IRT) is estimated to vary only from 8 to 21 d depending on the season. The LRT and IRT estimates are major improvements over conventional renewal estimates using tidal prism methods.

Utility of morphological abnormalities during early‐life development of the estuarine mummichog, Fundulus heteroclitus, as an indicator of estrogenic and antiestrogenic endocrine disruption

To evaluate the use of morphological abnormalities for standard testing of endocrine-disrupting substances (EDS), we tested the hypothesis that developmental abnormalities are a sensitive indicator of exposure to waterborne estrogenic and antiestrogenic EDS during embryonic, larval, and juvenile stages in the common estuarine killifish, the mummichog (Pisces: Cyprinodontidae). Static exposures with daily renewal were carried out with 10 to 10,000 ng/L of the estrogen agonist 17alpha-ethynylestradiol (EE2) or antagonist ZM189,154 (ZM) for the first 25 or 60 d of life. Incidence of skeletal abnormalities (scoliosis, lordosis, head, craniofacial, jaw, fin) and soft tissue abnormality (anal swelling) were significantly increased by EE2 but only at high concentrations (1,000 or 10,000 ng/L). Sixty-day exposure produced more severe abnormalities than 25-d exposure and in a higher proportion of fish. Within the longer exposure, 10,000 ng/L EE2 produced more abnormal fish than 1,000 ng/L (100% vs 51.6%) and more abnormalities per abnormal fish (5.73 vs 1.47). Fish reared to 12 months in clean water after exposure for 60 d to 10,000 ng/L EE2 survived at a lower rate than controls, retained abnormalities with the exception of anal swelling and, like fish exposed to other concentrations of EE2 and ZM, showed increased weight at length at 6 and 12 months. Sixty-day exposure to ZM increased the incidence of scoliosis (1,000 ng/L) but decreased the overall incidences of abnormal fish and lordosis (10 and 10,000 ng/L). No impacts of EE2 or ZM were observed before hatch, and clearing and staining of larvae demonstrated that expression of vertebral abnormalities coincided temporally with ossification. We conclude that morphological abnormalities in mummichogs are not a sensitive indicator of exposure to estrogenic or antiestrogenic waterborne EDSs at environmentally relevant concentrations.