Hans Jørgen S. Larsen

Possible immunotoxic effects of organochlorines in polar bears (Ursus maritimus) at Svalbard.

Associations between immunoglobulin G (IgG) levels and the organochlorine contaminants (OCs) polychlorinated biphenyls (PCBs), chlordanes, 1,1-dichloro-2,2-bis(4-chlorophenyl) ethylene (DDE), hexachlorobenzene (HCB), and hexachlorocyclohexanes (HCHs) in blood plasma from polar bears caught at Svalbard were determined. The blood samples were collected from free-living polar bears of different age and sex between 1991 and 1994. The IgC concentration increased with age and was significantly higher in males than in females. IgG was negatively correlated with sigmaPCB level and with three individual PCB congeners, IUPAC numbers 99, 194, and 206. HCB was also negatively correlated with IgG. The significant negative OC correlation with IgG levels may indicate an immunotoxic effect.

Does high organochlorine (OC) exposure impair the resistance to infection in polar bears (Ursus maritimus)? Part I: Effect of OCs on the humoral immunity.

This study was undertaken to assess if high levels of organochlorines (OCs) are associated with decreased ability to produce antibodies in free-ranging polar bears (Ursus maritimus) and thus affect the humoral immunity. In 1998 and 1999, 26 and 30 polar bears from Svalbard, Norway, and Churchill, Canada, respectively, were recaptured 32–40 d following immunization with inactivated influenza virus, reovirus, and herpes virus and tetanus toxoid. Blood was sampled at immunization and at recapture for determination of plasma levels of polychlorinated biphenyls (PCBs) and organochlorine pesticides (OCPs), serum immunoglobulin G (IgG) concentrations, and specific antibodies against influenza virus, reovirus, and herpes virus, tetanus toxoid, and Mannheimia haemolytica. The OCs alone contributed with up to 7% to the variations in the immunological parameters. The combination of ∑PCBs (sum of 12 individual PCB congeners), ∑OCPs (sum of 6 OCPs), and biological factors accounted for 40–60% of the variation in the immunological parameters. Negative associations were found between ∑PCBs and serum immunoglobulin G (IgG) levels and between ∑PCBs and increased antibody titers against influenza virus and reovirus following immunization. In contrast, a positive association was registered between ∑PCBs and increased antibodies against tetanus toxoid. ∑OCPs also contributed significantly to the variations in the immunological responses. OCs did not have the same impact on the antibody production against M. haemolytica. The present study demonstrated that high OC levels may impair the polar bears ability to produce antibodies and thus may produce impaired resistance to infections.

Does high organochlorine (OC) exposure impair the resistance to infection in polar bears (Ursus maritimus)? Part II: Possible effect of OCs on mitogen- and antigen-induced lymphocyte proliferation.

Previous studies have reported alarmingly high levels of organochlorines (OCs), particularly polychlorinated biphenyls (PCBs), in free-ranging polar bears (Ursus maritimus). In this study plasma concentration of PCBs ranged from 14.8 to 200 ng/g wet weight. The aim of the study was to investigate associations between OCs and lymphocyte proliferation after in vitro stimulation with different mitogens and antigens. In 1998 and 1999, 26 and 30 free-ranging polar bears from Svalbard and Churchill, Canada, respectively, were recaptured 32–40 d following immunization with inactivated tetanus toxoid and hemocyanin from keyhole limpets (KLH) to sensitize lymphocytes. At recapture, blood was sampled for determination of plasma levels of PCBs and organochlorine pesticides (OCPs) and lymphocyte proliferation after in vitro stimulation with specific mitogens—phytohemagglutinin (PHA), pokeweed mitogen (PWM), concanavalin A (Con A), lipopolysaccharide (LPS), and purified protein derivative of Mycobacterium aviumsubsp. paratuberculosis (PPD)—and antigens: tetanus toxoid and KLH. The combinations of ΣPCBs (sum of 12 individual PCB congeners), ΣOCPs (sum of 6 OCPs), and their interactions contributed up to 15% of the variations in the lymphocyte responses. By using multiple regression analyses, followed by classical mathematic function analyses, thresholds for immunomodulation were estimated. Depending on the lymphocyte proliferation response studied, the estimated thresholds for significant immunomodulation were within the concentration ranges 32–89 ng/g wet weight (ww) and 7.8–14 ng/g ww for ΣPCBs and ΣOCPs, respectively. Thus, this study demonstrated that OC exposure significantly influences specific lymphocyte proliferation responses and part of the cell-mediated immunity, which also is associated with impaired ability to produce antibodies (Lie et al., 2004). The authors thank the Norwegian Research Council (NFR, numbers 125693/720 and 140730/720), the Norwegian Ministry of Environment Transport and Effect Program, and the Toxic Substances Research Initiative in Canada for funding this study. The authors thank Tine Borgen for technical assistance in the lymphocyte proliferation test.

Organochlorines in top predators at Svalbard : occurrence, levels and effects

Alarmingly high polychlorinated biphenyl (PCB) levels have been found in the top predators such as glaucous gull (Larus hyperboreus) and polar bear (Ursus maritimus) at Svalbard [Gabrielsen, G.W., Skaare, J.U., Polder, A., Bakken, V., 1995. Chlorinated hydrocarbons in glaucous gull (Larus hyperboreus). Sci. Total Environ. 160/161, 337-346; Bernhoft, A., Skaare, J.U., Wiig, O., 1997. Organochlorines in polar bears (Ursus maritimus) at Svalbard. Environ. Pollut. 95, 159-175; Henriksen, E.O., Gabrielsen, G.W., Trudeau, S., Wolkers, H., Sagerup, K., Skaare, J.U., 1999. Organochlorines and possible biochemical effects in glaucous gull (Larus hyperboreus) from Bear Island, the Barents Sea. Arch. Environ. Contam. Toxicol. (in press). ]. Studies of the possible toxic effects, particularly on the immune system and reproduction, of the very high PCB levels in these species are currently being investigated. Data obtained in the field (f.i. reproductive success in polar bears and intestinal nematodes in glaucous gulls), as well as levels of various biochemical and physiological parameters (f.i. thyroid hormones, retinol, EROD activity, CYP1A, IgG), have been coupled with the PCB levels [Skaare, J.U., Wiig, O., Bernhoft, A., 1994. Klorerte organiske miljogifter; Nivâer og effekter i isbjorn. Norwegian Polar Institute Reportseries no. 86, 1-23 (in Norwegian); Bernhoft, A., Skaare, J.U., Wiig, O., 1997. Organochlorines in polar bears (Ursus maritimus) at Svalbard. Environ. Pollut. 95, 159-175; Bernhoft, A., Skaare, J.U., Wiig, O., Derocher, A.E., Larsen, H.J., 2000. Possible immunotoxic effects of organochlorines in polar bears (Ursus maritimus) at Svalbard (in press); Henriksen, E.O., Gabrielsen, G.W., Skaare, J.U., Skjegstad, N., Jensen, B.M., 1998a. Relationship between PCB levels, hepatic EROD activity and plasma retinol in glaucous gull, Larus hyperboreus. Marine Environ. Res. 46, 45-49; Henriksen, E.O., Gabrielsen, G.W., Trudeau, S., Wolkers, H., Sagerup, K., Skaare, J.U. , 1999. Organochlorines and possible biochemical effects in glaucous gull (Larus hyperboreus) from Bear Island, the Barents Sea. Arch. Environ. Contam. Toxicol. (in press); Sagerup, K., Gabrielsen, G.W., Skorping, A., Skaare, J.U., 1998. Association between PCB concentrations and intestinal nematodes in glaucou gulls, Larus hyperboreus, from Bear Island. Organohalogen compounds 39, 449-451; Skaare, J.U., Wiig, O., Bernhoft, A., 1994. Klorerte organiske miljogifter; Nivâer og effekter i isbjorn. Norwegian Polar Institute Reportseries no. 86, 1-23. (in Norwegian)].

Ecological risk assessment of persistent organic pollutants in the arctic

Ecological risk assessment of persistent organic chemicals in marine mammals in the Arctic would comprise assessments of both exposure and effect and risk characterization. Results from a comprehensive collaborative study between Norwegian and Canadian researchers, comprising epizoological (ecological) studies (reproductive rate, offspring survival), experimental studies on the immune system function, monitoring studies (e.g. physiological assays of thyroid hormones, retinol, IgG and testosterone) and registration of biological data (e.g. sex, age, reproductive status, nutritional status) were coupled with PCB exposure data. Assessment of the results indicates that population status and health of polar bears with very high PCB levels may be at risk.

Endocrine Disruption Induced by Organochlorines (OCs): Field Studies And Experimental Models

Long-range transport of persistent organic compounds by air and ocean currents from industrialized areas resulted in high levels of these pollutants in food webs in the Svalbard area. With the aim to test if organochlorine (OC) exposure in free-living polar bears from Svalbard affected their plasma steroid hormone concentrations, it was found that polychlorinated biphenyls (PCBs) were associated with increased progesterone levels in females. The sum of pesticides (∑pesticides) and ∑PCBs contributed significantly negative to the variation of the plasma testosterone in males, and the overall contribution of the OCs to the plasma cortisol variation was negative. A second objective was to study the effects of selected OCs (i.e., PCB 153 and PCB 126) on animal health as a consequence of effects on endocrine-regulated functions such as reproduction and immunity in a goat model focusing on long-term and low-level exposure during the periods of fetal development and in the neonatal period. Additionally, acute exposure was studied in adult mice. The results indicated that exposure to low doses of PCB 153 in utero and in the suckling period influenced reproductive functions and both PCB 153 and PCB 126 exerted immunomodulatory effects on the offspring, whereas acute exposure of adult mice had minor effects on male reproductive function.

Combined effects of selected Penicillium mycotoxins on in vitro proliferation of porcine lymphocytes

The in vitro effect of combinations of the Penicillium mycotoxins citrinin (CIT), cyclopiazonic acid (CPA), ochratoxin A (OTA), patulin (PAT), penicillic acid (PIA) and roquefortine C (RQC) on mitogen induced lymphocyte proliferation was determined using purified lymphocytes from six piglets. Dose–response curves for each mycotoxin and mycotoxin combinations were generated. The combined effects of toxin pairs based on IC20 were illustrated in isobole diagrams and statistically calculated. OTA and CIT elicited a synergistic effect. Four toxin pairs elicited additive effects, four pairs less–than–additive effects and six pairs independent effects. Thus, the majority of toxin pairs tested produced lower combined effects than an additive effect. The results indicate that the sum effect of all toxins is less than that from the summation of concentrations of the individual compounds, adjusted for differences in potencies.

EFFECTS OF PERINATAL EXPOSURE TO LOW DOSES OF PCB 153 AND PCB 126 ON LYMPHOCYTE PROLIFERATION AND HEMATOLOGY IN GOAT KIDS

Pregnant does (10 goats/group) were dosed orally with either PCB 153 or PCB 126 dissolved in corn oil or only corn oil (control group) from day 60 of gestation until delivery. Effects on in vitro mitogen-induced lymphocyte proliferation and blood cell counts in their goat kids exposed to low levels of PCB 153 and PCB 126 during gestation and lactation were assessed. The concentrations of PCB 153 and PCB 126 in adipose tissue in the goat kids 9 mo postpartum were 5800 ng/g (fat weight) and 0.49 ng/g (fat weight), respectively. Kids exposed to PCB 153 had a significantly higher number of white blood cells, neutrophils, and lymphocytes at 2 wk of age compared to controls. In the kids exposed to PCB 126 there was a significantly lower concentration of monocytes at 2, 4, and 8 wk of age. The mean lymphocyte response to phytohemagglutinin (PHA) and to concanavalin A (Con A) was significant lower in the PCB 153 compared to the control group at wk 2, 4, and 8 postnatally. The results of the present study support previous reports on immunotoxic effects of PCB exposure in animals. However, this is the first report to demonstrate immunotoxicity in animals by using low doses of PCB 153. The difference in results between PCB 126 and PCB 153 treatment groups may strengthen the hypothesis that PCBs mediate immunotoxic effects through both AhR-dependent and independent mechanisms.

The effects of the Penicillium mycotoxins citrinin, cyclopiazonic acid, ochratoxin A, patulin, penicillic acid, and roquefortine C on in vitro proliferation of porcine lymphocytes

The in vitro effect of each of the Penicillium mycotoxins citrinin (CIT), cyclopiazonic acid (CPA), ochratoxin A (OTA), patulin (PAT), penicillic acid (PIA) and roquefortine C (RQC) on mitogen induced lymphocyte proliferation was determined using purified lymphocytes from 6 piglets. Dose response curves for each mycotoxin were generated and the concentrations producing 50% inhibition of cell proliferation (IC50) were estimated. OTA and PAT were the most potent toxins with IC50 of 1.3 and 1.2 µmol/l, respectively (0.52 and 0.18 mg/l, respectively). Based on molar concentrations, OTA was 15, 30, 40, and 65 times more potent as an inhibitor than PIA, CIT, CPA and RQC, respectively.

Immunotoxicity of Polychlorinated Biphenyls (PCB) in Free-Ranging Gray Seal Pups with Special Emphasis on Dioxin-Like Congeners

Increased pinniped and dolphin mortality rates have led to speculations that persistent pollutants, in particular polychlorinated biphenyls (PCB), are immunomodulatory, making individuals susceptible to infections. The aim of the present study was to investigate effects that PCB may exert on peripheral blood mitogen-induced lymphocyte proliferation responses in free-ranging gray seal (Halichoerus grypus) pups from the polluted Baltic Sea and from the cleaner open waters of the Atlantic Ocean. T-cell mitogen (phytohemagglutinin [PHA] and concanavalin A [Con A]) and T-/B-cell mitogen (pokeweed mitogen [PWM]) responses were significantly lower in the more PCB-contaminated Baltic gray seal pups than in reference pups from the Atlantic. Best regression analysis revealed that dioxin-like mono-ortho PCB accounted for the immunosuppressive responses. In the Baltic seals, the immunosuppressive effects were limited to the more persistent dioxin-like mono-ortho PCB. In the Atlantic seals a positive correlation existed between PCB concentrations and mitogen-induced lymphocyte proliferation responses. Although direct cause–effect relationships are difficult to demonstrate in free-ranging animals, our results are consistent with the weight of evidence available that PCB, and dioxin-like PCB in particular, modulate lymphocyte function in free-ranging marine mammals. Thus, exposure to PCB most likely result in increased susceptibility to infections or reduced host resistance in pinnipeds.