Grantee Research Project Results
Final Report: Determination of the Relative Sensitivity of the Northern Elephant Seal andOther California Pinnipeds to the Toxic Effects of Polychlorinated Biphenyls
EPA Grant Number: R823415Title: Determination of the Relative Sensitivity of the Northern Elephant Seal andOther California Pinnipeds to the Toxic Effects of Polychlorinated Biphenyls
Investigators: Tjeerdema, Ronald S. , Olsen, Hugh
Institution: University of California - Santa Cruz
EPA Project Officer: Hahn, Intaek
Project Period: August 1, 1995 through September 1, 1998
Project Amount: $294,218
RFA: Exploratory Research - Environmental Biology (1995) RFA Text | Recipients Lists
Research Category: Biology/Life Sciences , Human Health , Aquatic Ecosystems
Objective:
Description - The co-planar polychlorinated biphenyls (PCBs), polychlorodibenzo-p-dioxins (PCDDs), and polychlorodibenzofurans (PCDFs) are globally distributed environmental organochlorine contaminants of concern due to their persistence and potent toxicity. Toxic effects in laboratory species include immunosuppression, teratogenicity, carcinogenicity, reproductive toxicity, neurotoxicity, and disturbances in energy metabolism, which produce a characteristic wasting syndrome (1,2). These compounds are bioconcentrated, biomagnified, and residues have been found in liver, adipose tissue, blood, and milk of seals (pinnipeds) and other marine mammals (3). Toxic effects of these compounds are initiated through binding to a cytosolic Aryl Hydrocarbon (Ah) receptor (1,4). In pinnipeds these compounds have been linked to reproductive failure (5,6), immune system dysfunction (7), and may disturb thermoregulation through changes in T3 and T4 thyroid hormones (8). Concentrations of organochlorine residues in the milk of lactating pinnipeds sampled along the coast of California exceeded those from seals residing in the Arctic, the Antarctic, and Australia (9). In California, researchers at the Marine Mammal Center (San Francisco), and the University of California have documented a skin disease in elephant seals (10). This condition is of unknown etiology, but has characteristics in common with epithelial hyperkeratinization produced by PCDDs, PCDFs, and co-planar PCBs (10). Pinniped populations along the coast of California are experiencing toxicity from unknown sources, e.g. transitional cell carcinoma in California Sea Lions (11), and maybe experiencing toxic effects related to exposure to these compounds. Dose response data for pinniped species are unavailable for most toxic endpoints. This area of research has been limited by the protected status of pinniped species, which precludes in vivo toxicity testing. Furthermore, there is a continuing need for relevant and well-characterized clinical test procedures for assessing subtle toxic endpoints impacting disease and longeviety in wildlife populations.This project developed toxicity dose response data for the California Sea Lion, Zalophus californianus, California Harbor Seal, Phoca vitulina, and Northern Elephant Seal, Mirounga angustirostris. Model compounds used in this study were the most effective Ah receptor agonists of the series of co-planar PCB congeners (3,3', 4,4'-tetrachlorobiphenyl) and PCDD congeners (2,3,7,8-tetrachlorodibenzo-p-dioxin or TCDD). Original study design included determination of relationships between body burdens of co-planar PCBs, serum levels of T3 and T4 thyroid hormones, and lipoprotein lipase activity in large populations of Northern Elephant Seals, matched for period of fasting, age, and sex. Individual variability and collection of sufficient numbers of samples from wild populations proved to be problematic. This study was unable to correlate lipoprotein lipase activity with serum T3 and T4 thyroid hormone levels or PCB residues for these reasons. Experimental approach was revised in two ways. First, use of ex vivo tissue culture methods allowed individuals to serve as their own control in dose response studies, and proved an effective way to deal with individual variability within wild populations. This approach also decreased the numbers of samples needed for analysis. Second, ex vivo culture of white blood cells was included in the study based on ready availability of blood samples and applicability (discussed below). Adipose tissue was selected for study because of its key importance for survival of pinniped species. This tissue serves as insulation, as a source of metabolic energy for thermoregulation, as the key source of metabolic energy during normal periods of fasting associated with life cycle, and as the source of energy transferred to offspring through lactation and nursing. Adipose tissue is a principle target tissue for wasting syndrome caused by co-planar PCBs, PCDDs and PCDFs. White blood cells were selected for study based on the presence of glucose transport systems sensitive to these agents, the accessibility of these cells for sampling, their critical role in immune function, and documented immunotoxic effects of these compounds to these cells. Four biochemical end points were examined in adipose tissue and white blood cells. These included: 1) activation of Ah receptor/tyrosine kinase associated signal transduction systems, 2) inhibition of glucose transport through the facilitative glucose transporter system (GLUTs), 3) inhibition of lipoprotein lipase, and 4) Ah receptor mediated lipid peroxidation. Bioassays compared pinniped responses with responses in the guinea pig, rat, and mouse run as controls in these studies.
Overall Project Objectives - To determine susceptibility of the Northern Elephant Seal, Mirounga angustirostris, California Harbor Seal, Phoca vitulina, and California Sea Lion, Zalophus californianus, to the toxic effects of co-planar PCBs, and PCDDs on energy metabolism and adipose tissue function.
Summary/Accomplishments (Outputs/Outcomes):
Specific Aim I- Evaluation of species sensitivity of the Northern Elephant Seal, California Harbor Seal, and California Sea Lion based on activation of tyrosine kinase activity as the initial cellular biochemical response to 3,3', 4,4'-tetrachlorobiphenyl, and 2,3,7,8-tetrachlorodibenzo-p-dioxin. Cytosolic supernatants (100,000-x g) were prepared from ex vivo experiments with control (1,4-dioxane vehicle treated) and PCB/TCDD treated adipose tissues, then incubated with 33P g-ATP and protein phosphorylation examined and quantitated using SDS-PAGE and autoradiography techniques as previously described (13). Both adipose tissue and white blood cells from all three pinniped species exhibited a dose dependent change in tyrosine kinase activity in response to the model co-planar PCB, 3,3', 4,4'-tetrachlorobiphenyl and model dioxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin. Response to Ah receptor blockers, and PCB congeners were consistent with the hypothesis that changes in tyrosine phosphorylation in all species are Ah receptor mediated. Analysis of SDS-PAGE gels found two main bands of approximately 130 and 35 kDa, which contained tyrosine residues whose phosphorylation was altered in response to treatment with test agent. Dose response measured as change in phosphorylation of the 130 and 35 kDa bands exhibited several characteristics. All three pinniped species tested, responded to the model co-planar PCB and Dioxin. In some individuals, tissues treated ex vivo with test agent concentrations greater than 10-11 M final tissue concentration exhibited a negative response, i.e. a decrease in phosphorylation of the 130 and 35 kDa bands with increasing dose. Threshold for response varied between individuals but responses were seen in some individuals at estimated final tissue concentrations of approximately 10-12 M. These results are comparable with sensitivity of guinea pig, rat, and mouse tissues run in parallel as controls in these studies. The identity of kinases involved in the phosphorylation of the 130 and 35 kDa cytosolic protein bands identified by SDS-PAGE were investigated using immunoprecipitation techniques and commercially available antibodies against Raf and MAP kinases. Results for all three pinniped species suggest that activation of the growth factor/Raf/MAP kinase-signaling pathway is a common response to exposure to these compounds.Specific Aim II- Evaluation of species sensitivity of the Northern Elephant Seal, California Harbor Seal, and California Sea Lion to inhibition of glucose transport in adipose tissue and white blood cells. Using techniques previously described (14-15), adipose tissue and white blood cells were treated with the model co-planar PCB, 3,3', 4,4'-tetrachlorobiphenyl or the model dioxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin. Rates of glucose transport were determined by incubation of tissues with 3H-methyl-glucose as previously described (16,17). A dose response for inhibition of glucose transport in adherent white blood cells including monocytes and macrophages were observed for all three pinniped species. Individual variation in responses were seen, however some individuals showed statistically significant decreases in glucose transport of treated cells at doses as low as 10-12 molar for adherent white blood cells in selected pinniped species. Changes in glucose transport after treatment with combinations of Ah receptor blockers and dioxin or PCB were consistent with the hypothesis that inhibition of glucose transport is Ah receptor mediated in adherent white blood cells and were consistent with responses in laboratory species. In contrast, non-adherent lymphocytes isolated from seal blood samples exhibited a positive dose-response relationship for glucose transport with increasing dose of model compound. Cell type appears to be an important determinate in response to these agents. Inhibition of glucose transport in adipose tissue incubated ex vivo with model compounds was less consistent than results with adherent white blood cells. Glucose transport in adipose tissue of the Northern Elephant seal, was statistically less sensitive to inhibition of glucose transport than adherent white blood cells.
Specific Aim III- Evaluation of species sensitivity of the Northern Elephant Seal, California Harbor Seal and California Sea Lion to inhibition of lipoprotein lipase activity in adipose tissue. Evaluation of lipoprotein lipase activity in ex vivo treated tissues using enzyme assays (18) yielded variable results. Methods for quantitation of total LPL protein levels in samples using antibodies and Western blotting techniques have been developed. Analyses of archived samples are in progress.
Specific Aim IV- Establish and evaluate lipid peroxidation as a toxic response to PCBs, and PCDDs in adipose tissue. Adipose tissue samples from the California Sea Lion were treated ex vivo with the model co-planar PCB, 3,3',4,4'-tetrachlorobiphenyl or the model dioxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin. Tissue homogenates were prepared and lipid peroxidation quantified using the method of Buege and Aust (17). Tissues exhibited a dose dependent increase in lipid peroxidation with significant increases in levels of lipid peroxides at doses as low as 10-10M of 2,3,7,8-tetrachlorodibenzo-p-dioxin. Lipid peroxidation appears to be a valid endpoint for toxicity from PCBs and dioxins in adipose tissue. Results in this study are consistent with observations of others using ex vivo treated adipose tissue from the guinea pig (18). Further work in other pinniped species is in progress.
Conclusions:
Dose response data for tyrosine kinase induction in ex vivo treated tissues of the Northern Elephant Seal, Mirounga angustirostris, California Harbor Seal, Phoca vitulina, and California Sea Lion, Zalophus californianus, are consistent with these species having a functional Ah receptor system. This system responds in a similar fashion to laboratory species used as controls and appears to have binding affinities for co-planar PCBs and dioxins that are not greatly different from rodent species tested. Responses subsequent to the presumed binding to the Ah receptor show differences in dose response. Results of ex vivo studies of inhibition of glucose transport found adherent white blood cells were more sensitive to the effects of model compounds than adipose tissue. Taken together, these results suggest that pinniped adipose tissue may not be the most sensitive target tissue for toxicity. White blood cells have proven to be an accessible and responsive tissue for analysis and clinical tests based on this observation could prove useful. The documentation of increased lipid peroxidation in response to these compounds has implications for other toxic effects produced through the presumed generation of free radicals. Further work in pinnipeds is needed to resolve these questions and to identify the most sensitive toxic responses to these compounds. Ex vivo tissue culture proved to be a valuable approach for assessing species and tissue sensitivity.Journal Articles:
No journal articles submitted with this report: View all 2 publications for this projectSupplemental Keywords:
Dioxin, TCDD, PCB, Pinniped, Northern Elephant Seal, Mirounga angustirostris, California Harbor Seal, Phoca vitulina, California Sea Lion, Zalophus californianus Protein Phosphorylation, Lipid Peroxidation, Glucose Transport, Lipoprotein Lipase, Raf, MAP kinase, ex vivo, Adipose Tissue, White Blood Cells., RFA, Health, Scientific Discipline, Toxics, Geographic Area, Ecology, Environmental Chemistry, Endocrine Disruptors - Environmental Exposure & Risk, HAPS, pesticides, State, Chemistry, endocrine disruptors, Risk Assessments, Children's Health, Endocrine Disruptors - Human Health, Biology, neurotoxic, dioxin, Lindane (all isomers), endocrine disrupting chemicals, northern elephant seal, Chlordane, Dieldrin, metabolic activation, PCB, DDT, Toxaphene (chlorinated camphene), Chlordane (technical mixture and metabolites), Toxaphene, pinnipeds, immune systems, teratogen mixtures, California (CA), cancer riskProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.