Top predators and monitoring methods - Polar Bear - Project title:
	Immune response and Chlorinated Organic Pollution in Polar Bears.
Co-ordinating institution
	Norwegian Polar Institute
Final report
	A preliminary report was delivered: Derocher, A.; Larsen, H.J.; Skåre, J.U. and Wiig, Ø.:  Immune response and Chlorinated Organic Pollution in Polar Bears.  The work on polar bear toxification proceeded with funding from several other sources after the delivery of this report to the Transport and Effects Programme.  See list of scientific publications below for references.
Summary and results
	This project represents a truly unique research venture between scientists in many diverse disciplines from both Norway and Canada. Results from this study will provide definitive answers on the likelihood, and possible mechanisms, by which persistent organic pollutants, POPs, can affect the immune function in polar bears. Field research for this study in both Svalbard (high pollution) and Hudson Bay, Canada (lower pollution) were completed in autumn 1998 and 1999, respectively. All proposed field components of the study were successfully completed. The research includes studying the relationship between immune globulin levels and PCB exposure in polar bears, the effect of high PCB exposure on the ability of the bears to produce antibodies to various vaccines, the relationship between PCB exposure and the lymphocyte proliferation in polar bears and the effect of in vitro exposure of lymphocytes to PCB congeners. The experimental and laboratory work is completed. The study suffered a major setback when a laboratory fire at The Norwegian School of Veterinary Science, Oslo resulted in a seven-month delay in finalizing OC results. Data analysis of the very complex result matrix (including data on OC levels, immune parameters, sex hormones, other hormones, ecology and polar bear biology) is on-going. Due to the complexity of the data matrix the quality assurance of the final results is considered of outmost importance. Thus, results will be referred in this report only to the extent that we consider appropriate with regard to quality assurance. An inter-laboratory calibration of analytical methods for analysis of organochlorines, OCs, in plasma has been carried out between the National Wildlife Research Centre, Hull, Canada and the Norwegian Veterinary Institute laboratory. Excellent agreement was found. The project has resulted in the development of a good methodology for assessment of white blood cell activities under field condition. This is important for studies of cell biology, physiology and immunology in polar bears. It further represents the basic methodology for measuring cell-mediated immunity to microbes, for testing of how environmental pollutants, infections, nutritional factors, starvation and stress may influence the lymphocyte function. Significant lower lymphocyte responses to lipopolysaccharide from E. coli and Mycobacteria were found in polar bears with high PCB-156 concentration in the blood. In vitro exposure to selected PCB congeners of mitogen induced lymphocyte cultures was found to suppress the lymphocyte response, with the strongest reduction in cultures from polar bears having the highest plasma concentration of PCBs. The present study confirmed our previous finding of a significant inverse correlation between IgG and PCBs, both sum PCB and specific congeners (PCB 99, 118 and 194). The vaccination study revealed significantly higher plasma concentrations of PCBs 118, 156, 180 and 194 in polar bears from Svalbard as compared to Canada together with significantly higher antibody titres to influenza virus, reo virus and herpes virus in Canada as compared to Svalbard. These results together with the fact that there was inverse correlation between IgG and PCB 99, 118 and 194 in the same material, demonstrate that the effect of PCBs on the IgG concentration is associated with decreased ability to produce antibodies following immunization. Impaired antibody production following immunization and decreased specific lymphocyte response in polar bears with high PCB exposure indicated decreased resistance to infections. This is supported by the findings that polar bears with high plasma PCB have been more exposed to the Pasteurella bacteria, one of the most common microbes in the environment, that attack the respiratory system. Thus, the study has demonstrated that high PCB exposures of the polar bears impair lymphocyte function, IgG concentration and the ability to produce antibodies following immunization of the bears with certain microbes. By analysing the present combined effects of PCBs on the immune system it is reasonable to assume that PCB is associated with decreased resistance to infections of the polar bears. Furthermore, preliminary results have revealed a negative correlation between individual PCBs and testosterone concentrations and between sum PCB and testosterone, indicating that PCB exposure in polar bears is associated with a decrease in male testosterone concentrations. In the arena of Arctic ecotoxicology, this study represents a major new initiative in experimental design to rigorously test the effects of POPs on wildlife. With the information from this study, new methods of monitoring and impact assessment will be possible. Given the delay in the analysis of PCB data, this report should be considered a general summary of the results to date. Statistic treatment of the combined result matrix is on-going. Scientific papers are under preparation and will be prepared within the next year.
Scientific publications
	The Transport and Effects Programme was a co-sponsor for a bigger research initiative on contaminants in Polar Bears. The other main funding sources, apart from internal resources provided by the participants, are the Toxic Substances Research Initiative (Canada) and the Norwegian Research Council.   The list of publications below contains what has been published unntil 2004. Publications in peer-reviewed journals: Andersen, M, Lie, E, Belikov, SE, Bernhoft, A, Boltunov, AN, Derocher, AE, Garner, GW, Skaare, JU and Wiig, Ø (2001): Geographic variation of selected PCB congeners in polar bear (Ursus maritimus) from Svalbard east to Chukchi Sea. Polar Biol 2001, 24, 231-238. Braathen M, Haave M, Olsen GH, Derocher AE, Skaare JU, Ropstad E, Wiig Ø, Sormo EG, Jenssen BM. PCB contamination in relation to levels of thyroid hormones and progesterone, and migration patterns in polar bears (Ursus maritimus). Organohalogen Compounds, 2000, 49, 438-441.  Braathen M; Derocher AE; Wiig Ø; Sørmo EG; Lie E; Skaare JU; Jenssen BM (2004): Relationships between PCBs and thyroid hormones and retinol in female and male polar bears. Environ Health Perspect 2004. Vol 112: 8: 826-833 Haave M, Ropstad E, Derocher AE, Lie E, Dahl E, Wiig Ø, Skaare JU, Jensen BM.(2003): Polychlorinated Biphenyls and reproductive hormones in female polar bears at Svalbard. Environ Health Persp, 2003, 4 (volume 111): 431-436. Henriksen EO, Wiig Ø, Skaare JU, Gabrielsen GW, Derocher AE. (2001): Monitoring PCB in polar bears: lesson Learned from Svalbard. J Environ Monitoring; 3: 1-7  Lie E, Derocher AE, Wiig Ø, Skaare JU. Polychlorinated biphenyls in mother/offspring pairs of polar bears (Ursus maritimus) at Svalbard. Organohalogen Compounds 2000. 49, 457-460.       Lie E, Bernhoft A, Riget F, Belikov SE, Boltunov AN, Derocher AE, Garner GW, Wiig Ø, Skaare JU. Geographical distribution of organochlorine pesticides (OCPs) in polar bears (Ursus maritimus) in the Norwegian and Russian Arctic. Sci Total Environ 2003, 306:159-170.     Lie E, Larsen HJS, Larsen S, Johansen GM, Derocher AE, Lunn NJ, Norstrom RJ, Wiig Ø, Skaare JU. Does high organochlorine (OC) exposure impair the resistance to infection in polar bears (Ursus maritimus)? Part I: Effect of OCs on the humoral immunity. J Toxicol Environ Health 2004; Part A, 67:555-582.            Lie E, Larsen HJS, Larsen S, Johansen GM, Derocher AE, Lunn NJ, Norstrom RJ, Wiig Ø, Skaare JU. Does high organochlorine (OC) exposure impair the resistance to infection in polar bears (Ursus maritimus)? Part II: Effect of OCs on mitogen and antigen induced lymphocyte proliferation. J Toxicology & Environmental Health Olsen GH, Mauritzen M, Derocher AE, Sørmo ES, Skaare JU, Wiig Ø, Jenssen, BM. Space-use strategy determines PCB burdens in female polar bears. Environ Sci Technol. 2003, 37: 4919-4924.  Oskam, I.C., Derocher, A.E., Lie, E., Skaare, J.U., Wiig, Ø., Larsen, S., Dahl, E. and Ropstad, E. (2001) Organochlorines effect the major androgenic hormone, testosterone, in male polar bears (Ursus maritimus). Pharmacol. Toxicol. 88, suppl.1, 23.                   Oskam IC, Ropstad E, Dahl E, Lie E, Derocher AE, Wiig Ø, Larsen S, Wiger R, Skaare JU. Organochlorines affect the major androgenic hormone, testosterone, in male polar bears (Ursus maritimus) at Svalbard. J. Toxicology & Environmental Health 2003; part A, 66/2119-2139.                Oskam IC, Ropstad E, Lie E, Derocher AE, Wiig Ø, Dahl E, Larsen S, Skaare JU. . Organochlorines affect the steroid hormone cortisol in polar bears (Ursus maritimus) at Svalbard, Norway. J. Toxicology & Environmental Health 2004; part A, 67/12: 959-977.  Skaare JU, Bernhoft A, Derocher A, Gabrielsen GW, Goksøyr A, Henriksen E, Larsen HJ, Lie E, Wiig Ø (2000) Organochlorines in top predators at Svalbard – occurrence, levels and effects.  Toxicol. L. 112-113:103-109.   Skaare, J.U., Larsen, H.J., Lie, E., Ropstad, E., Bernhoft, A., Derocher, A.E., Lunn, N.J., Norstrom, R. and Wiig, Ø. (2001b) Relationships between high loads of organic contaminants and health effects in artic mammals-Immune response and chlorinated environmental pollutants in polar bear. Pharmacol. Toxicol. 88, suppl.1, 14.  Skaare JU, Bernhoft A,Wiig Ø, Norum KR, Haug E, Eide DM, Derocher AE. Relationships between plasma levels and organochlorines, retinol and thyroid hormones from polarbears (Ursus maritimus) at Svaldbard. Toxicol. Environm. Health 2001; Part A, 62:101 - 115 ; samt: J Toxicol Environ Health Pt A 2001;62,(4): 227-241. Skaare, J.U., Larsen, H.J.S., Lie, E., Bernhoft, A., Derocher, A.E., Norstrom, R, Ropstad, E, Wiig, Ø. (2002) Ecological risk assessment of persistent organic pollutants in the arctic. Toxicology.   Skaare JU, Larsen HJ, Lie E, Bernhoft A, Derocher AE, Norstrom R, Lunn N, Ropstad E, Wiig Ø. Polar bear case study. Organohalogen Compounds 2003, 61: 299-302. PhD thesises: Elisabeth Lie (2004): Organochlorine contaminants in polar bears (Ursus maritimus); Geographical trends and possible immunotoxic effects. Thesis Dr. Scient., Norwegian College of Veterinary Medicine/Norges Veterinærhøgskole, Oslo, 26.08.04 Irma Oskam (2004): Effects of organochlorine contaminants on mammalian reproductive and endocrine systems (studies on functional changes in steroid hormones and reproductive parameters in mice, goats and polar bears). Thesis Dr. Scient., Norwegian College of Veterinary Medicine/Norges Veterinærhøgskole, Oslo, 27.10.04.   Cand scient thesises Marte Haave (2001): Variation in plasma progesterone and estradiol of female polar bears (Ursus maritimus) in relation to reproductive status and concentrations of polychlorinated biphenyls (PCBs). Cand.Scient. thesis in ecotoxicology. NTNU, Trondheim. Marte Braathen (2001): Endocrine disruption in polar bears- does PCB alter retinal thyroid hormone levels? Cand.Scient. thesis in ecotoxicology. NTNU, Trondheim. Gro Harlaug Olsen (2001): Space-use strategies affect PCB levels in female polar bears (Ursus maritimus) in the Norwegian arctic. Cand.Scient. thesis in ecology/marine biology. NTNU, Trondheim. Several abstracts and posters have been published as well.
Original project description
	Background Polar bears are the top predator in the Arctic marine ecosystem and rely largely on seal blubber as their main energy source. Due to their position in the food web and their diet, polar bears are exposed to high levels of environmental pollution. The impacts of pollution are likely to be expressed through impacts on the physiology of polar bears operating at the cellular level which are ultimately integrated into population level effects. Immune function is known to be effected by persistent organic pollutants. Reduced immune function increases the risk of a population to be decreased by an epizootic. Further, if toxic chemicals interrupt the normal hormonal balance, the result may be altered growth rates, behaviour and reduced reproductive rates. Polar bears have low rates of reproduction that they compensate for by having high adult survival rates. If either reproductive or survival rates of polar bears are reduced by the impacts of pollution, the population can enter into a negative growth rate. Once a polar bear population is in a declining state, recovery will take decades and only once the factor causing the decline is removed. Reduced immune function in polar bears represents a major threat to the population. A disease epizootic could result in large (20-50%) changes in population size within a period of months depending on the disease and degree of impairment of the immune system. Changes in hormone levels affecting other physiological factors could result in much more subtle declines in the population which may go undetected for many years. Understanding the cellular level threats to polar bears allow assessment of population level threats. The working hypothesis for this proposal is that understanding immune system and hormone function in polar bears with high pollution levels, compared to polar bears with lower pollution levels, will provide insight into the effects of pollution. Further, the methods used will provide a monitoring method for assessing population status over time. This project represents a strong element of competence building with respect to polar bear immune system and wildlife immune toxicology in general. We will develop both reagents and methods to measure polar bear immune function. Immune pathological studies of PCB effects on the polar bear immune system, immune response ability, and infection resistance are important bases for development of sound monitoring methods. Such methods could involve measurements of polar bear immune globulins (IgG, IgM, and IgA) in blood, which constitute the combined antibodies against for example bacteria, viruses, and parasites. Such methods are usually based on "polyclonal" reagents of restricted amount and constitution. We will develop "monoclonal" reagents and thereby have unlimited access to reagents. Monoclonal antibodies will be made available for national and international institutions for use in monitoring programs. The monitoring of organochlorine pollutants since the 1970’s has detected a global pattern of long-range transport especially by air from industrialised areas in Europe, Asia and North America. Long-range transport has resulted in accumulation of these compounds in the Arctic environment. PCBs (polychlorinated biphenyls) are considered one of the most serious environmental pollutants and have been found in high concentrations in polar bears (Norstrom et al. 1988, Norheim et al. 1992, Bernhoft et al. 1997, Norstrom et al. 1998). Polar bears in the east Greenland, Barents and Kara seas areas have much higher PCB levels than bears from other areas (Norstrom et al. 1998). In the Svalbard area, high levels of PCBs have also been found in arctic fox (Wang-Andersen et al. 1993), glaucous gull (Gabrielsen et al. 1995, Mehlum and Daelemans, 1995) and seals (Muir et al. 1992, Daelemans et al. 1993). The main food of polar bears are seals and they often only eat the subcutaneous fat of the seals that are rich in organochlorines. Therefore, PCBs bio-accumulate in polar bears. Extreme concentrations of highly chlorinated PCBs accumulate with age, especially in male polar bears (Bernhoft et al. 1997). Females, however, transfer PCBs to their offspring through milk. The highest chlorinated PCBs are not transported with the milk since they are accumulated more effectively in the subcutaneous fat depots in the females. Levels of most other organochlorines are higher in the fat depots of offspring than in their mother (Bernhoft et al. 1997). Differential metabolisation of low chlorinated PCBs and handling of high chlorinated organochlorines is the explanation for differential distribution of PCBs in different species (Daelemans et al. 1993). The polar bear seems to have a better ability to metabolise several organochlorines than other marine mammals (Norstrom et al. 1994). This probably protects the bears against toxic effects from the pollutants. The question is whether some organochlorines, especially PCBs, causes cellular dysfunction which effects the resistance against diseases. Some experiments on lab animals and from human beings show that PCBs have severe effects on the immune system. This information is largely from laboratory studies, often with PCB doses which induce cell damage and in subtoxic doses (Tryphonas 1994, Luster and Rosenthal 1993, Holsapple et al. 1991). The results from these studies can be summarised: PCBs reduce the ability to produce antibodies after vaccination, PCB reduce the ability to kill microbes due to dysfunction in the white blood cells (phagocytes, natural killer cells), High PCB levels give high infection susceptibility to several micro-organisms, PCBs have effects on the concentrations of immune globulin in blood, PCBs have effect on cell mediated immunity. Results from laboratory animals and humans are necessarily not applicable to polar bears with a quite different metabolism. Further, laboratory experiments can not simulate the real environment in which the polar bear get exposed for these pollutants. During our NFR project «Klorerte organiske miljøgifter i isbjørn på Svalbard. Forekomst nivåer og effekter», we rinsed immune globulin G (IgG) from polar bears, produced antibodies for these, and established methods for quantifying immune globulin in polar bears. In addition, we measured IgG in blood samples collected in the period 1990-1996 and compared the levels with the levels of PCBs in the same individuals. IgG was found to be negatively correlated with PCBs (sum of 22 congeners). IgG is the sum of all antibodies of this type immune globulin in polar bear blood. This indicates that the PCB burden in polar bears has a much higher negative effect on the immune system than we had expected. It is therefore very important to characterise the effects of organochlorines on the immune system of polar bears in detail. Documentation of immune suppressive effects and the implications this has on the resistance to infection and animal health of polar bears in the Svalbard area. We will produce antibodies for IgM and IgA (the main immune bodies of circulation and mucous systems). During the project, we started lymphocyte proliferation and cryo-preservation of white blood cells for immune function testing. Both in laboratory and in wild animals persistent organic pollutants influence the thyroid hormones and retinoid homeostatis (Boily et al., 1994; Brouwer & van den Berg, 1986; Jenssen et al., 1995; Henriksen et al., 1998). The thyroid hormones and retinols are important for regulation of cell differentiation, development and growth, reproduction and immune system function. Persistent organic pollutants can have large consequences for other physiological functions in organisms which ultimately result in changes at the population level. It is therefore important to understand how persistent pollutants influence thyroid hormone function because they play an important role in the physiological adaptations of polar bears to the extreme Arctic conditions. In addition, thyroid hormones and retinols can be used as non-destructive effect biomarkers for exposure to pollutants. Such biomarkers are reliable indicators for monitoring of higher trophic level vertebrates. Similar to top predator species (see Jenssen et al., 1995; Haugen, 1996) there are indications in polar bears that thyroid hormones and plasma retinol concentrations are negatively correlated with PCBs (Skaare et al., 1994). The mechanistic understanding of how persistent organic pollutants affect thyroid hormone and retinol homeostasis is partially understood. It is thought that the pollutant metabolites interfere with the Ah-receptor (Brouwer & van den Berg, 1986). It is essential that several thyroid hormone parameters are measured from polar bears (TT4, FT4, TT3, FT3, rT3) as well as thyroid stimulating hormone (TSH). Aims The aim of the project is to clarify the linkages between PCB levels in polar bears and their immune system capability. By using specific immune function tests and induction of protective antibodies, we will obtain information on how PCBs effect the infection resistance of polar bears. The project therefore represents a clear extension of existing knowledge on polar bear ecotoxicology and will contribute new knowledge in this field for ongoing monitoring programs. Methods Two main experiments will be performed. The first will include bears from Svalbard, the Barents Sea and western Russia, where blood samples are taken only once. This experiment will describe the effect of PCBs on different functions of white blood cells in polar bears. The experiment also represents the completion of work on establishment of lymphocyte proliferation tests, cryo-preserving of lymphocytes for function tests and phagocytosis testing. Concerning this, we will establish an operational cell culture laboratory in the field and measure general immune response of lymphocytes. In addition, we will examine how the individual immune competence of lymphocytes might be affected after in vitro exposure to PCB congeners. The responsive capability of lymphocytes is measured by lymphocyte proliferation testing after in vitro stimulation with different mitogens (Phytohemagglutinin, Concanavalin A, Poke Weed mitogen, PPD, Lipopolysakkarid). To test the effect of in vitro exposure of PCBs on the general response level of lymphocytes, different levels of selected PCBs will be added to lymphocyte cultures. PCB congeners with strong effects on immune globulin will be used (NFR- 110750/720). In this connection, we will use both rinsed lymphocytes and a full blood technique (Larsen 1979). The methods are not described for polar bears but we have years of experience with such methods on livestock, fur-bearing animals, dogs, fowl, and salmon. The initial experiments with polar bear lymphocytes show good response with cultured lymphocytes. This is a part of method development. If the full blood technique functions well for measurements of suppression of lymphocyte proliferation at a cellular level, because of immune-suppressive factors in plasma, laboratory equipment needed for field experiments can be lowered significantly. This part of the project includes cryo-preservation of separated lymphocytes in order to test the possibility for in vitro function tests for different phagocytosis activity. The comparison of lymphocyte proliferation from fresh and cryo-preserved lymphocyte cultures will give important additional information. This main experiment will also include hematology, hormones (cortisol, oestradiol, testosterone) in addition to measuring levels and immune globulin and antibodies against environmental microbes. The second main experiment is an international project which includes polar bears from Canada with low PCB levels in order to compare them with the Svalbard bears. This work represents a vaccination model with recapture 4-6 weeks after immunisation. The experiment is designed to measure specific immune response in bears from areas with different PCB exposure. The bears are immunised with different herpes-, reo-, and influenza viruses, tetanus toxoid and keyhole limpet hemocyanin (KLH). Antibodies are established and protective antibodies like virus neutralising antibodies, virus hemagglutinations inhibition antibodies and toxin neutralising antibodies. In vitro lymphocyte stimulation will also be performed with mitogens specific antigens. The measurement of antibody production after immunisation will detect possible immune suppressive effects on the immune system in the period up to recapture. This also represents a means of measuring a combined in vivo effect of PCBs on all parts of the immune system which are involved in the production of antibodies (phagocytose, antigenpresentation, T-lymphocyte function, cytokinproduction, B-lymphocyte stimulation and differentiation in addition to the antibody production of the plasma cells). By neutralising virus infections in cell cultures, inhibition of virus hemaglutination and toxin neutralisation, we will measure the resistance factor directly. Therefore, the effect of PCBs on the infection resistance can be measured without being dependent on infection experiments or doing registration in relation to disease outbreaks. By comparing measurements of antibody response against viruses, toxoids, and KLH with the results from lymphocyte responses, we will see the effect of PCBs on the combined immune system. In addition, the component of lymphocyte functions which are most affected can be determined. The study will also include in vitro exposure of lymphocytes to PCB congeners, so that we can register the effect of PCBs directly on the proliferation response to the lymphocytes from polar bears wit varying PCB exposure. This main experiment will also include haematology, hormone analyses and the measurement of immune globulin and antibodies against environmental microbes. Earlier results from this project show that the immune function is affected by the hormone status of the animal. Steroid hormones like testosterone, progesterone and cortisol were significantly related to several immune parameters. Earlier investigations also show that the level of thyroid hormones are related to the environmental exposure to PCB in polar bears. The analysis of hormones gives knowledge on the physiological status of animals. This might give important additional information for the evaluation of their immune function. The following hormones will be investigated in the present project: testosterone oestradiol, progesterone, cortisol and thyroxin. Goals The main goal of the project is to assess the immunological impact of high PCB exposure in polar bears in Svalbard. Subgoal 1: To define the relationship between PCB exposure and lymphocyte proliferation ability in polar bears. Subgoal 2: Develop methods for cryo-preservation of lymphocytes for in vitro testing of phagocyte function and lymphocyte responses. Subgoal 3: Study the relation between immunoglobuline levels and PCB exposure in polar bears. Subgoal 4: Detect if high PCB exposure decreases the ability to produce antibodies in polar bears. Subgoal 5: Determine if PCB exposure affects the infection resistance of polar bears. Subgoal 6: Understand the impact of PCBs on reproductive hormones, thyroid hormones, and retinol. Subgoal 7: Assess the threat that PCBs pose to the immune system and hormone homeostasis in polar bears. Subgoal 8: Develop methods and non-destructive biomarkers for monitoring the effects of toxic chemicals on polar bears. Ethics The immunising will be conducted with dead vaccines and will have no clinical effects on the polar bears. All capture and handling of polar bears will be conducted by the Norwegian Polar Institute in accordance with internationally accepted standard methods. The project has been approved by the Norwegian Experimental Animal Committee. Time Plan Phase 1: Svalbard: ending winter 1998 Field work with sampling in Svalbard: summer-autumn 1998 Immunising of polar bears 1, August 1998 Recapture of vaccinated polar bears, September 1998 Measuring of neutralised antibodies from herpes and influenza viruses Measuring of hemagglutination inhibition antibodies against influenza virus Measuring of antibodies against tetanus toxin Lymphocyte stimulation with mitogen and antigens In vitro exposure of lymphocytes with PCB congeners Lymphocyte proliferation in full blood and lymphocyte culturing technique development Cryo-preservation of lymphocyte cultures Lymphocyte proliferation in cryo-preserved cultures Phagocytosis and respiratory burst in cryo-preserved cultures using flow cytometric analysis methods Measuring of immune globulin Hematology Measuring of antibodies with environmental microbes hormone analyses (sex steroids and thyroid hormones) PCB measurements in far samples and blood plasma Phase 2 Part 1: Churchill, Manitoba, Canada: ending winter 1999: Field work with sampling in Svalbard: summer-autumn 1998 Immunising of polar bears 1, August 1998 Recapture of vaccinated polar bears, September 1998 Measuring of neutralised antibodies from herpes and influenza viruses Measuring of hemagglutination inhibition antibodies against influenza virus Measuring of antibodies against tetanus toxin Lymphocyte stimulation with mitogen and antigens In vitro exposure of lymphocytes with PCB congeners Lymphocyte proliferation in full blood and lymphocyte culturing technique development Cryo-preservation of lymphocyte cultures Lymphocyte proliferation in cryo-preserved cultures Phagocytosis and respiratory burst in cryo-preserved cultures using flow cytometric analysis methods Measuring of immune globulin Haematology Measuring of antibodies with environmental microbes Hormone analyses (sex steroids and thyroid hormones) PCB measurements in fat samples and blood plasma. Part 2: Svalbard area, ending winter 1999: Field work with sampling in Svalbard: summer-autumn 1998 Immunising of polar bears 1, August 1998 Recapture of vaccinated polar bears, September 1998 Measuring of neutralised antibodies from herpes and influenza viruses Measuring of hemagglutination inhibition antibodies against influenza virus Measuring of antibodies against tetanus toxin Lymphocyte stimulation with mitogen and antigens In vitro exposure of lymphocytes with PCB congeners Lymphocyte proliferation in full blood and lymphocyte culturing technique development Cryopreservation of lymphocyte cultures Lymphocyte proliferation in cryopreserved cultures Phagocytosis and respiratory burst in cryopreserved cultures using flow cytometric analysis methods Measuring of immune globulin Haematology Measuring of antibodies with environmental microbes Hormone analyses (sex steroids and thyroid hormones) PCB measurements in fat samples and blood plasma Production of monoclonal antibodies for polar bears Publication of Results The primary distribution of results from this project will be in publications in international peer reviewed scientific journals. Presentations at international and national meeting and conferences will provide additional distribution of the information. Popular scientific article and popular media will be used to provide the information to the public. Budget Total budget for 1998: NOK 736 000,-. NOK 346 000,- is applied for from the Effects Programme, while NOK 390 000,- is covered by the Norwegian Polar Institute. Total budget for 1999: NOK 1 037 000,-. NOK 637 000,- is applied for from the Effects Programme, while NOK 400 000,- is covered by the Norwegian Polar Institute. References Bernhoft, A., Wiig, Ø. & Skaare, J.U.: Organochlorines in polar bears (Ursus maritimus) at Svalbard. Environ. Poll., 95, No. 2, pp. 159-175, 1997. Boily, M. H., Champoux, L., Bourbonnais, D. H., DesGranges, J. L., Rodrigue, J. & Spear, P. A.: ß-carotene and retinoids in eggs of great blue herons (Ardea herodias) in relation to St Lawrence River contamination. Ecotoxicology, 3, pp. 271-286, 1994. Brouwer, A. & van den Berg, K. J.: Binding of a metabolite of 3,4,3',4'-tetreachlorobiphenyl to transthyretin reduces serum vitamin A transport by inhibiting the formation of the protein complex, carrying both retinol and thyroxin. Toxicol. Appl. Pharmacol, 85, pp. 301-312, 1986. Daelemans, F.F., Mehlum, F., Lydersen, C. & Schepens, P.J.C. Mono-ortho and non-ortho substituted PCBs in Arctic ringed seal (Phoca hispida) from the Svalbard area: Analyses and determination of their toxic threat. Chemosphere, 27, 429-437, 1993. Gabrielsen, G.W., Skaare, J.U., Polder, A. & Bakken, V.: Chlorinated hydrocarbons in glaucous gulls (Larus hyberboreus) in the southern part of Svalbard. Sci. Tot. Environ., 160/161, 337-346, 1995. Haugen, O.: The effects of polychlorinated biphenyls (PCBs) on plasma vitamin A concentrations in free ranging grey seal (Halichoerus grypus) pups at Froan. Cand. scient., Department of Zoology, Norwegian University of Science and Technology (NTNU), 1996. Henriksen, E. O., Gabrielsen, G. W., Skaare, J. U., Skjegstad, N. & Jenssen, B. M.: Relationships between PCB levels, hepatic EROD activity and plasma retinol in glaucous gulls, Larus hyperboreus. Marine Environ. Research, 46, pp. 45-49, 1998. Holsapple, M.P., Snyder, N.K., Wood, S.C., Morris, D.L.: A review of 2, 3, 7, 8- tetrachlordibenzo-p-dioxin-induced changes in immunocompetence: 1991 update. Toxicology, 69, 219-255, 1991. Jenssen, B. M., Skaare, J. U., Woldstad, S., Nastad, A. T., Haugen, O., Kløven, B. & Sørmo, E. G.: Biomarkers in blood to assess effects of PCBs in free-living grey seal pups. In Whales, seals, fish and man. ed. A. S. Blix, L. Walløe, & Ø. Ulltang (Eds.). Elsvier Science Publishers B.V., Amsterdam, pp. 607-615, 1995. Larsen, H.J.: A whole blood method for measuring mitogen-induced transformation of sheep lymphocytes. Research in Veterinary Science, 1979, 27, 334-338. Luster, M.I., Rosenthal, G.J.: Chemical agents and the immune response. Environ. Health Perspect., 100, 219-236, 1993. Mehlum, F. & Daelemans, F.F.: PCBs in Arctic seabirds from the Svalbard region. Sci. Tot. Environ., 160/161, 441-446, 1995. Muir, D.C.G., Wagemann, R., Hargrave, B.T., Thomas, D.J., Peacall, D.B. & Norstrom, R.J.: Arctic marine ecosystem contamination. Sci. Tot. Environ., 122, 75-134, 1992. Norheim, G., Skaare, J.U. & Wiig, Ø.: Some heavy metals, essential, elements, and chlorinated hydrocarbons in polar bear (Ursus maritimus) at Svalbard. Environ. Pollut., 77, 51-57, 1992. Norstrom, R.J. & Muir, D.C.G.: Chlorinated hydrocarbon contaminants in arctic marine mammals. Sci. Tot. Environ., 154, 107-128, 1994. Norstrom, R.J., Simon, M., Muir, D.C.G. & Schweinsberg, R.E.: Organochlorine contaminants in arctic marine food chains: Identification, geographical distribution, and temporal trends in polar bears. Environ. Sci. Technol., 22, 1063-1071, 1988. Norstrom, R.J., Malone, B., Born, E.W., Garner, G.W., Olpinski, S., Ramsay, M.A., Scliebe, S., Stirling, I., Taylor, M.K. & Wiig, Ø.: Chlorinated hydrocarbon contaminants in polar bears from Eastern Russia, North America, Greenland and Svalbard: Biomonitoring of hemispheric pollution. Arch. Envir. Cont. Toxicol., 35, No. 2, pp. 354-367, 1998. Tanabe, S., Iwata, H. & Tatsukawa, R.: Global contamination by persistent organochlorines and their ecotoxicological impact on marine mammals. Sci. Tot. Environ., 154, 163-177, 1994. Tryphonas, H.: Immunotoxicity of polychlorinated biphenyls: Present status and future considerations. Exp. Clin. Immunogenet. 11, 149-162, 1994. Wang-Andersen, G., Skaare, J.U., Prestrud, P. & Steinnes, E.: Levels and congener pattern of PCBs in arctic fox, Alopex lagopus, in Svalbard. Environ. Pollut., 82, 269-275, 1993.