Environmental Toxicology Research Interests and Experience
Dalhousie University, Halifax, NS, Canada
My interdisciplinary environmental toxicology research program encompasses studies of multixenobiotic resistance in aquatic organisms at the level of the ecosystem, population, and organism, down to the cellular and molecular realms. This work combines aquatic environmental toxicology and ecology by integrating field research studying intertidal biodiversity at gradients from polluted coastal sites with cell biology studies of the metabolism and excretion of environmental contaminants in pollutant-tolerant organisms. These results are further applied to address the water management issues of pollution abatement and conservation education. Although the diversity of intertidal species decreases dramatically with increased proximity to a pollution source such as a pulp mill, a small number of hardy species are found adjacent to pollution sources which tolerate chronic exposure to hazardous environmental contaminants. My interest is to identify these hardy species through field studies then in the laboratory elucidate the underlying biochemical and molecular mechanisms that contribute to their multixenobiotic resistance phenotype. My studies focus on one multixenobiotic resistance strategy analogous to multidrug resistance (MDR) conferring by several ATP-binding cassette (ABC) transporters that facilitate the cellular export of a wide variety of environmental toxicants. Elucidating the function and regulation of xenobiotic transporters is an important issue in human health because defects in ABC transporters are responsible for several human genetic diseases and ABC transporters facilitate the cellular efflux of certain anti-cancer drugs in human tumor cells thus conferring MDR and confounding chemotherapy.
Multixenobiotic Resistance in Marine Species
Understanding the cellular mechanisms that permit animals to tolerate extreme chemical challenges such as exposure to anthropogenic organic contaminants, natural product toxins, or chemotherapeutics, has been of long-term interest to me. The traditional detoxification paradigm describes the biotransformation of chemicals by phase I (e.g. cytochrome P450 1A) and phase II (e.g. glutathione-S-transferase) drug metabolizing enzymes to more easily excretable hydrophilic forms. That the excretion of these metabolites is an energy dependent process facilitated by multixenobiotic (i.e. multidrug) resistance transport proteins such as the ATP-binding cassette (ABC) transporters is currently under appreciated. Insufficient attention has been focused on the influence of efflux (as well as uptake) transporters on biodisposition, bioavailability, and toxicity of anthropogenic organic contaminants.
My goal is to elucidate the role ABC transporters play in the multixenobiotic resistance phenotypes observed in some wild populations of fish which are highly exposed to putative ABC substrates, such as organic pollutants and algal toxins, in their aquatic environment. Furthermore, I aim to characterize the complementary roles that ABC transporters and detoxification enzymes play in conferring resistance: ABCB1 transports parent compounds and/or metabolites of phase I detoxification enzymes, such as cytochrome P450 3A, while ABCC2 and ABCG2 exports many phase II metabolites including glutathione conjugates (Figure 1).
Figure 1. Schematic model of the role of ABC transporters on xenobiotic disposition in polarized hepatocytes in relation to relevant solute carrier (SLC) family uptake transporters, and phase I and II metabolizing enzymes. Amphipathic xenobiotics may enter the cell by diffusion, cation uptake is facilitated by basolateral (sinusoidal) SLC transporters members of the organic cation transporter family (OCTs), while anion uptake is facilitation by other SLC members including those of the organic anion transporter (OATs) family and the organic anion transporting polypeptide (OATPs) family. Unmodified amphipathic xenobiotics and phase I (predominantly cytochrome P450) metabolites are exported by the canalicular ABCB1 (formerly P-glycoprotein [P-gp]). Organic anions and their phase II conjugates are exported with some overlapping substrate specificity but varying affinity by the ABCC family (formerly MPR family), including the basolateral ABCC1 and canalicular ABCC2 (predominantly glutathione conjugates, lesser extent sulfate and glucoronate), basolateral ABCC3 (preferentially glucoronate and sulfate conjugates), and canalicular ABCG2 (predominantly sulfate and glutamylate conjugates.) ABCB11 can excrete at low-affinity certain ABCB and ABCC family xenobiotic substrates. Hepatic uptake of endogenous bile salts is facilitated by SLC transporters, predominantly the sodium-taurocholate transporting polypeptide (NTCP), and to a lesser extent, in a sodium-independent manner by OATPs. Monovalent bile salts are predominantly actively excreted by canalicular ABCB11 and to a lesser extent by canalicular ABCG2 and basolateral ABCC3 which also transport divalent bile salts at low-affinity as does canalicular ABCC2. (Bard & Paetzold, Aquatic Toxicology in preparation.)
Current Environmental Toxicology Research Projects
In vivo P-glycoprotein Transport Activity
Lack of amenable experimental in vivo models from wild vertebrate populations has hindered the progression of xenobiotic transporter research in the environmental realm. To help remedy this deficiency, I developed the first protocol for a rapid in vivo assay to simultaneously evaluate xenobiotic transport activity mediated by ATP-binding cassette (ABC) transporters in multiple organs of fish (Bard et al., submitted). ABCs, such as P-glycoprotein (P-gp/ABCB1) and the glutathione conjugate transporter MRP2 (ABCC2), confer multidrug resistance. These data provide the first evidence that P-gp plays a major role in xenobiotic transport in ovary and blood-brain barrier in fish. Using this assay, I determined that benzo[a]pyrene nor its cytochrome P4501A (CYP1A) metabolites are P-gp substrates but its transport may be mediated by MRP2. This novel system will be a useful screening tool to identify environmental relevant contaminants and marine natural products that are ABC substrates in vivo, and to compare ABC transporter activity in multiple organs between populations of fish living in different chemical environments. Contaminants of interest we are testing include: ivermectin and emamectin benzoate, fish aquaculture pesticides (anti-sea louse); ethinyl estradiol, a major chemical contaminant in municipal sewage originating from contraceptive pill; artificial musks (toiletry perfumes) found in municipal sewage shown to be chemosensitizers that inhibit P-gp transport activity.
In collaboration with neuroscientist and ethologist (behavioural expert) Dr. Simon Gadbois (Neuroscience & Psychology, Dalhousie University), we have developed a suite of novel behavioural toxicology assays to assess the neuroprotective activity of P-gp at the blood-brain barrier in killifish (Fundulus heteroclitus). We provided the first in vivo demonstration that in fish, P-gp activity protects against neuropathology and mortality from the aquaculture antiparasitic drug ivermectin (Bard and Gadbois, 2007). This assay provides us with a tool to compare the behavioural effects of potential environmental neurotoxins between fish with intact P-gp activity versus fish exposed to environmental chemosensitizers (chemicals that inhibit P-gp activity). We plan to use this assay to assess behaviour in wild populations of chemically resistant killifish from the Sydney Tar Pondso that subtle effects of environmental toxins can be measured. We were assisted in conducting the labour intensive observations by undergraduate researchers and honours thesis students Keren Menashe, Paola Santarelli, Kim Bourque, Beth Campbell, and half a dozen undergraduate volunteers.
Multixenobiotic Resistance Conferred by P-glycoprotein Transporters
The role of ABC transporters in conferring cellular chemical resistance and its interaction with traditional drug metabolizing enzymes has been underappreciated in the environmental toxicology field. To address this deficiency, I characterized the role P-gps play in conferring a multixenobiotic resistance phenotype in natural populations of fish chronically exposed to anthropogenic contaminants (Bard et al., 2002a, Bard et al., 2002b). Polychlorinated biphenyl-resistant fish and oil exposed fish both expressed elevated levels of P-gp compared to reference fish. I investigated whether P-gp might be involved in induction of the detoxification enzyme CYP1A. Although hepatic CYP1A and P-gp were both elevated in oil exposed fish, there was no induction of P-gp in fish exposed to the CYP1A inducers ß-naphthoflavone or 2,3,7,8-tetrachloro-dibenzofuran. These data suggest that P-gp expression is not regulated by the aryl hydrocarbon receptor pathway.
Chemical Resistance in Sydney Tar Pond Fish
A chemically resistance population of estuary killifish (Fundulus heteroclitus) have been discovered in the highly polluted Sydney Tar Ponds in Nova Scotia. The Tar Ponds were used as a disposal site for waste from the former Sydney steel mill and are contaminated with high levels of polycyclic aromatic hydrocarbons, polychlorinated biphenyls, and raw sewage. We are working to elucidate the cellular defense mechanisms responsible for multixenobiotic resistance in this chemically tolerant fish population. We are investigating the role of a suite of detoxification genes including ABC transporters (B11/P-gp, C2/MRP, G2/BCRP) and phase I and II drug metabolizing enzymes in conferring chemical resistance in these fish by qPCR mRNA assessment and protein expression. The functional role of multidrug resistance proteins in chemical resistance will be assessed using a novel in vivo transport assay to evaluate P-glycoprotein activity simultaneously in multiple compartments (ovary, brain, liver, bile, kidney, gut, blood). This work is currently being conducted by my MSc student Christine Paetzold and in collaboration with protein biochemist Dr. Neil Ross at the National Research Council, Halifax, NS, analytical chemist Dr. Jocelyne Hellou, Department of Fisheries and Oceans, Halifax, NS and community ecologist Prof. Martha Jones, Cape Breton University, NS. Featured on CKDU radio November, 2004.
ABC Transporters in Antarctic Fish
In collaboration with the Italian Antarctic Research Program, University of Siena (Prof. Ilaria Corsi) and University of Ancona (Prof. Francesco Regoli), we are investigating the role of ABC transporters in conferring chemical resistance in the Antarctic emerald rockcod (Trematomus bernacchii) exposed to multiple anthropogenic contaminants. We are examining differential protein and mRNA expression of ABC transporters (B1/P-gp, C family/MRPs, G2/BCRP) and phase I and II drug metabolizing enzymes (CYP1A1, GST, etc.) in fish exposed in vivo to different combinations of polycyclic aromatic hydrocarbons and heavy metals. This work is currently being conducted by my PhD student Sara Zucchi who is co-supervised by Prof. Corsi with funding from the Italia Programma Nazionale Ricerche in Antartide.
P-glycoprotein in Cholangiocellular Carcinomas
In fish with contaminant–associated liver tumours, P-gp (ABCB1) was highly expressed in bile canaliculi of non-tumourous liver surrounding cholangiocellular carcinomas, but was not detected within tumours. Tissue adjacent to human tumours is rarely examined for P-gp and this unexpected finding provides insight into the physiological function and clinical significance of hepatic multidrug resistance. These data suggest that elevated hepatic P-gp was induced by cellular stress caused by impaired bile elimination due to unorganized proliferative biliary structures (Bard et al., submitted).
Natural Product Toxicogenomics
Saxitoxin is a potent Paralytic Shellfish Toxin generated by certain algal species during bloom conditions and can causes toxicity and morbidity in marine vertebrate species, including commercial fish. We are investigating the effects of saxitoxin on the differential hepatic gene expression in farmed Atlantic salmon (Salmo salar). To examine saxitoxin detoxification pathways in the liver, we used quantitative PCR to assess expression of a suite of genes coding for xenobiotic metabolizing enzymes (e.g. cytochrome P450 1A, glutathione-S-transferase) and multidrug resistance transport proteins ( e.g. P-glycoprotein, MRP). To investigate what other genes are affected by saxitoxin treatment, hepatic expression of 4000 different amplicons was assessed using a microarray generated by our collaborators at the National Research Council, Halifax, NS. Using this technology, we identified 4 genes differently regulated that had not previously been associated with either detoxification or a general stress response (Bard et al., 2006). In collaboration with the National Research Council, we are initiating a metabolomic study to fingerprint the hepatic metabolites generated during saxitoxin exposure to shed light on the chemical defense strategies these fish employ to tolerate the effects of natural product toxins produced during algal blooms.
British Columbia Pulp Mill Effluent and Intertidal Diversity
Coastal Scene Investigation (CSI) [formerly Project S.O.S.: Save Our Shores, founded 1985]
My interest in research developed as a teenager when I queried why once abundant intertidal species such as sea urchins and abalone had become scarce along the coast of British Columbia. I founded a youth marine environmental group to promote proper beach etiquette (not removing animals from the shore for souvenirs) and to publicize the detrimental environmental effects of overharvesting and marine pollution. I wanted to evaluate whether pollution from local pulp and paper mills contributed to the problem. At the time, it was difficult to determine the effect of the mills as no baseline data existed as to biological diversity in the areas before the mills were in place. In response to this inadequacy, for my Stanford undergraduate honors thesis, I secured several (10) foundation and Stanford University grants to conduct a 4-year independent research and public service project. I trained >200 volunteers from coastal communities to aid me to conduct rocky intertidal biodiversity quadrat surveys at 28 sites surrounding 5 coastal pulp mills. I developed a biological index to rate pollution exposure at sites based on the hardiness of resident rocky intertidal species (Bard, 1998). This index has been useful to pinpoint depleted sites for pollution abatement and identify pristine highly diverse sites for candidate marine protected area designation. In the early 1990s, I documented depressed rocky intertidal diversity up to 20 km downstream from pulp mill effluent outflows. These data helped to focus public attention on the issue.
The government responded to this public pressure by strictly enforcing pollution abatement laws and creating the Environmental Effects Monitoring (EEM) Program through Environment Canada. EEM led to improvements in effluent quality at mills so that dioxin levels, for example, are now below detection in intertidal invertebrates surrounding many of these mills. Unfortunately no baseline data was collected as the associated environmental monitoring program began AFTER the effluent processing changes were completed, making it difficult to quantify environmental improvements attributable to EEM.
In 2004 & 2005 with funding from NSERC Discovery grant, we resurveyed three pulp mill regions: Howe Sound, Powell River and Prince Rupert. In all three regions, intertidal species richness decreases significantly as distance to a pulp mill decreases. Compared to the intertidal baseline study conducted in 1990-1993 (Bard 1998) in each region, mean species richness has increased over the past decade coincident with decreases in biochemical oxygen demand, total suspended solids, and adsorbable organic halide levels in pulp mill effluent (Bard et al., 2007 submitted). Depleted sites impacted by historical contamination that are in need of remediation were identified. The lowest diversity sites were negatively impacted by physical disturbance from accumulated wood debris. We advocate the adoption of rocky intertidal monitoring for Pacific region pulp mills with marine outfalls as a complement to the current EEM program. We are recommending to Environment Canada that in addition to monitoring current effluent discharge, that the EEM mandate include concrete steps for remediation of historical contamination by the pulp and paper industry and suppliers. Over 200 field volunteers have assisted with this research which has produced two MSc theses (F. de Raedemaecker, W. Willems) and three BSc Honours theses (R. Wickramanayake, A. Baccardax, S. Truesdell) which are currently being prepared as manuscripts for submission to peer-review journals.
To enable community groups to conduct their own biodiversity quadrat studies, I wrote a marine exploration guide with simple instructions in field methods (Bard, 1990). For volunteers to see the impact of their contribution to our research results, I have developed an extensive marine environmental science educational and research website. My education platform and the Quadrat Study Program were adopted by the Georgia Strait Alliance (GSA) in 1990 to establish Straitkeepers, the first community volunteer based long-term intertidal monitoring program in B.C. that annually surveys 33 additional sites with the aid of scientists, local student, and community members with funding from Department of Fisheries and Oceans (DFO) and Environment Canada. In 2007, the California State Environmental Protection Agency and Water Quality Board invited me to serve as an advisor to help create an intertidal monitoring program along their coastline as they plan to use the aforementioned biological index (Bard, 1998) as a sensitive measure of coastal ecosystem health.
DNA Barcoding of British Columbia Intertidal Species
Our long-term marine ecotoxicological monitoring program along BC pulp mill pollution gradients depends on accurate species identification. Our efforts are hampered by the lack of authoritative field guide of intertidal species identification for coastal BC. We are undertaking a program to sequence a 650 base pair portion of the mitochondrial cytochrome c oxidase I (COI) gene to DNA barcode all rocky intertidal macroscopic species. This project is being conducted in collaboration with Paul Hebert (UGuelph) and Gary Saunders (UNB) with the support of the Consortium of the Barcode of Life and the global Census of Marine Life. This project is the first effort to barcode a discrete ecosystem in Canada and will include 27 broad taxonomic groups and over 250 species represented as egg cases, juvenile or adults in the habitat. A number of the organisms we observe appear to be novel and the DNA barcode would allow us to “name” this species. In addition, we are unable to easily identify the many cryptic juvenile specimens and egg cases found in the intertidal zone – which serves as a nursery for many subtidal species. DNA barcoding technology would facilitate species identification and permit application beyond the initial ecotoxicological aims of this project. We are currently developing a freely available illustrated online searchable field guide of intertidal species. DNA barcode database would allow trophic level studies between subtidal and intertidal species using gut analysis. This tool would allow us to detect the increased range of southern species or the invasion of alien species at even the egg case or juvenile stage. Accurately measuring species diversity will help in identification of candidate marine protected areas for highly diverse regions (work we are undertaking with World Wildlife Fund). Several undergraduate and post-graduate researchers have assisted in conducting this work.
Halifax Harbour Intertidal Ecotoxicology with Application to Marine Management
Halifax Regional Municipality currently discharges raw sewage into the receiving marine environment. Due to the foul conditions, few researchers have chosen to conduct work in Halifax Harbour. In anticipation of future primary sewage treatment (anticipated 2008) and in order to provide baseline biological data in which to permit concrete assessment of remediation efforts, from 2005-06, we evaluated the health of Halifax Harbour intertidal system by evaluating intertidal diversity and community ecology, incidence of imposex and intersex in marine snails, and immune function in mussels. We observed reduced biomass and diversity of the Fucoids and Ascophyllum macro-algae in the harbour (Bard and Coray, 2007 in preparation). We demonstrated the extirpation of community structuring predatory whelks (Nucella lapillus) as a result of endocrine disruption from historical contamination from tributyl tin (Coray and Bard, 2007), resulting in imposex and mortality, and subsequent dominance of harbour intertidal communities by mussels. These mussels suffer immunomodulation and high rates of fungal infections (Coray et al., 2007 submitted). We plan to continue this work post-pollution abatement to evaluate the effectiveness of primary treatment and determine whether more advanced treatment is warranted. Work was conducted by my MSc student Camille Coray & 2 Honours theses E. Ring & C. Kendall.
Research has been featured on CBC radio, Chronicle-Herald, The Coast.
Role of Integrated Management and Science in Predicting and Reducing Land Based Impacts on Nova Scotia Coastal Ecosystem Health
To examine the relationships between land use patterns and the biodiversity of receiving marine and coastal environments in Nova Scotia, we are undertaking an interdisciplinary study using a suite of indicators of coastal ecosystem health in collaboration with the Atlantic Centre for Global Change and Ecosystem Research at Acadia University (CRC Chair & Prof. John Roff). We are also evaluating the effectiveness of current integrated coastal management practices and pinpointing the deficiencies in translating science to informed environmental policy, funded by Social Sciences and Humanities Research Council (Mercer Clarke, Roff, and Bard 2007 in preparation). This work is being conducted by my PhD candidate Colleen Mercer Clarke who is co-supervised by Dr. Roff.
Aquatic scientists, engineers, and environmental managers, have long been frustrated by the difficulties of effectively track the dispersion of contaminants in the aquatic environment. I have undertaken an interdisciplinary collaboration with Prof. Chris Taggart & Prof. Barry Ruddick (Oceanography, Dalhousie) to apply novel particle tracer-technology that they have developed, to resolve environmental toxicology problems such as documenting pollution plumes. Development of cost effective and efficient aquatic tracer systems can be applied to numerous environmental science issues including effluent discharge, outfall plumes, contaminant dispersal, produced water from ocean drilling activities, a whole range of aquaculture issues, and the many source-sink and dispersion problems tied to erosion, bedform transport, flow and diversion, particulates, flocs, and biological propagules. Such technologies have significant potential to trace the dispersion of fluidized particulates in a manner that is complementary to conventional technologies and ideal for improving and validating conventional methods, including numerical flow and dispersion models. The use of these tracers to track contaminants will provide valuable data to government environmental managers to make informed policy decisions on a large range of issues from wastewater to fisheries. To further this cross-disciplinary work, we organized a regional workshop sponsored by NSERC-Atlantic in 2006. In response to the high interest and support from the initial conference, we are developing an interdisciplinary national network on aquatic tracer technology.
Catastrophic Modeling of Toxic Legacy in British Columbian Waters
In 1975 near Powell River, BC, 4 tanks containing 400 tonnes of liquid chlorine were lost at sea during a storm and have never been located. In collaboration with colleagues in Mathematics and Chemical Engineering, an atmospheric spreading model, geographic information system (GIS), and risk assessment were developed to model the human and environmental health effects of a catastrophic release of chlorine were the tank to rupture during an earthquake or via corrosion (Bard et al., 2004.) We are currently in discussion with the Canadian Navy and a civil submarine technologist to plan an oceanographic expedition for the search, environmental assessment, and safe recovery of any remaining chlorine. Featured in Globe and Mail Nov. 30, 2004.