Alaska: projects/activities

Persistent organic pollution is a global problem. This fact is especially apparent in the Arctic where pesticides currently used in distant environments accumulate, in some cases to higher levels than those observed in the source region. This pollution threatens the well-being of the aboriginal inhabitants of these regions. Most of the traditionally harvested animals in the Arctic are long-lived and from the higher trophic levels of the food chain, thereby providing an opportunity for considerable bioaccumulation and biomagnification of persistent contaminants. This has prompted a growing concern by the Alaska Inupiat that pollutants in the environment might be contributing to their unique morbidity and mortality rates, especially of their children. Our studies are currently focused on two specific organic pollutants found in the Arctic environment; 1}hexachlorobenzene (HCB), a byproduct during manufacture of several different chlorinated compounds and consistently detected in the Arctic and, 2} dichlorodiphenyl dichloroethylene (p,p’-DDE), a chlorinated environmental breakdown product measured in the Arctic population at significantly higher concentrations than the parent pesticide, DDT. We hypothesize that mammalian embryonic cell exposure to these chemicals, individually or as mixtures at environmentally relevant concentrations and ratios, will alter the cell cycle and/or cause death by apoptosis, rather than by necrosis. We also predict synergistic cytotoxicity of the chemical mixture because of an accumulation of deleterious effects at different cellular target sites by each chemical. We further hypothesize that while some chemicals target non-genetic cellular components (such as a cell membrane or cytosolic component), other chemical effects will occur primarily at the genetic level, directly or indirectly. Our experiments have been designed as a set of sensitive cellular and molecular assays to compare levels and types of cytotoxic and genotoxic activity of the above chemicals (individual and mixture), at environmentally relevant concentrations, upon embryonic cells in culture. Our experimental evidence thus far is that these chemicals, separately or as a mixture at concentrations and molar ratios relevant to that measured in the Arctic environment, do have cytotoxic and/or genotoxic effects that could result in profound consequences to exposed tissues of a developing embryo or fetus. We have further experimental evidence that exposure to both chemicals at environmentally relevant concentrations is more toxic to the cell than the sum of effects by exposure to the individual chemicals. Experimental results indicate this is due to different cellular target sites for each chemical (Appendix A: Preliminary Results).

I. Objectives: I.1. To determine the normal range of values (natural variability due to time of year, age, gender) for basic nutritional and health parameters (blubber characteristics, essential and non-essential elements, structure of basic tissues) in the bowhead whale. a. Blubber thickness (depth and girth), chemical composition (lipids, water, calories), and tissue structure (light microscopy and special stains) will be assessed. b. Essential and non-essential elements (heavy metals) will be measured in liver and kidney. c. Tissue structure (light microscopy) characteristics obviously related to nutritional status in liver (glycogen, lipid and lipofuscin stores), pancreas (zymogen granules), and intestine (mucosal microvilli) and any evidence of inactivity/atrophy will be examined. d. Documentation of "normal" structure of basic tissues and evaluation for evidence of disease will also be conducted. I.2. Using data from Objective 1 to identify the parameters most important in assaying the health status of other mysticetes residing in the Bering Sea or Western Arctic that are harvested or stranded. I.3. Using data from Objective 1 to help determine the role of the bowhead whale as an indicator of ecosystem health and development of an optimized protocol for assessing mysticete health for the Bering Sea and Western Arctic, and other regions.

The first part of the present study evaluated tissue concentrations of twelve essential and non-essential elements in four arctic marine mammal species important as subsistence resources to indigenous Alaskans. Species sampled included: bowhead whales, beluga whales, ringed seals, and polar bears. Concentrations of As, Cd, Co, Cu, Pb, Mg, Mn, Hg, Mo, Se, Ag, and Zn, were analyzed in liver, kidney, muscle, blubber, and epidermis (the latter in cetaceans only). Elements that were identified as having tissue concentrations, which in domesticated species would have been considered higher than normal and/or even toxic, were Cd, Hg, Ag, and Se. However, the concentrations of these elements were consistent with previous reports for arctic marine mammals. Remaining elements were at concentrations within normal ranges for domesticated species, although Cu was found frequently at concentrations that would be considered marginal or deficient in terrestrial domesticated animals. Across-species comparisons revealed that Cd was highest in kidney, followed by liver in all four species. Its concentrations were frequently correlated with Cu, Zn, Hg, and Se. Cadmium accumulated with age in bowhead and beluga whales, especially in liver and kidney. The relationships between Cd and Hg, and between Cd and Se were believed to be due to mutual accretion with age, although direct interactions could not be ruled out, especially with respect to Cd and Se. Associations between Cd and Cu, and Cd and Zn were potentially attributable to mutual binding with the inducible protein, metallothionein. This assumption was supported by the observation that Cd:Zn ratios in liver and kidney displayed a significant linear relationship to age and that this ratio either increased slightly (in kidney and liver of bowheads) or remained constant (in kidney and liver of belugas) with age. In general, Se was highest in liver and kidney of all four species, where it was frequently at concentrations that would have been deemed elevated or toxic for domesticated species, although within ranges previously reported for arctic marine mammals. Selenium increased with age indices, and was highly correlated with Hg, and often with Cd as well. Mercury also increased with age, and liver contained the highest tissue concentration in the cetacean and pinniped species. The pattern of Se accumulation in polar bears differed, with highest concentrations found in kidney, which suggested that this tissue may be the primary site for Hg detoxification in this species, as is the case for terrestrial mammals. Compared to the other three species, bowhead whales had very low Hg concentrations in all tissues. The highly significant linear relationship between Hg and Se noted in various tissues (particularly liver) of all four species was presumed due to binding of these two elements to each other following demethylation of MHg. This assumption was supported by the observations that while Se and Hg both accumulated with age, the fraction of total Hg that was composed of MHg decreased with age. The quantity that represented the difference between total Hg measured directly and calculated total Hg [i.e., SHg = Hg(II) + MHg], also increased with age in beluga liver. This connoted that a portion of the total Hg present was in an organic form other than MHg, and that this form accumulated with age. Alternatively, this portion, which was apparently not measured by either the Hg(II) or MHg procedures, may have been lost during extraction. Species in this study had mean hepatic Hg:Se molar ratios that were below unity. This implies that Hg concentrations may have been below some threshold level, after which subsequent accumulation proceeds in a 1:1 molar ratio fashion with Se. Alternatively, it might suggest that a 1:1 Hg:Se molar ratio is not a prerequisite for protection from Hg toxcosis among marine mammals, because none of the animals in the present study exhibited lesions typically associated with Hg toxicosis. In beluga liver, concentrations of Ag were elevated when compared to domesticated species. The only element that showed a significant linear association to Ag was Cu—a relationship that was observed in all four species. This suggested that Ag and Cu may be associated through a common ligand, possibly metallothionein. The association between Ag and Se in beluga liver was less strong than that between Hg and Se; moreover, Ag did not increase with age. These findings indicate that Ag probably does not compete with Hg for Se binding, and therefore is unlikely to substantially inhibit detoxification of Hg in beluga whales. In the second portion of this research, tissues from bowhead whales, beluga whales and ringed seals were examined at both the gross and light microscopic level. The purpose of this evaluation was three-fold: to describe the normal histologic appearance of tissues; to perform a routine histologic survey of tissues that would contribute to a general health assessment, and; to scrutinize tissues for lesions that might support a diagnosis of toxicosis caused by Cd, Hg, Ag, or Se. Tissues examined were chosen on the basis of their propensity to be targets for toxicologic injury from the specified elements (with the exception of brain) and included, but were not limited to, the tissues analyzed chemically. Special stains were used to identify particular pigments or tissue components. Overall, the bowhead whales evaluated appeared healthy and had low parasite burdens. The most common lesion, which was observed in all bowheads, was a non-inflammatory chronic renal periglomerular and interstitial fibrosis. This lesion was not typical of Cd-induced nephropathy, and it did not appear to be associated with renal Cd burdens. Nevertheless, thresholds of Cd-induced renal injury are not known for cetacean species, and more whales need to be examined histologically in conjunction with analysis of tissue Cd residues. Acute myodegeneration was observed in cardiac and/or skeletal muscle of a few bowheads, and was presumed to reflect a hunting-induced exertional myopathy. The beluga whales examined were generally in good body condition and appeared healthy grossly, but they had much higher parasite burdens than bowhead whales. In particular, prevalence in belugas of pulmonary nematodiasis was high, being especially common among whales obtained from Pt. Hope compared to those from Pt. Lay. Grossly, firm, caseous nodules were associated with lungworms, while histologically, the associated pulmonary changes ranged from mild chronic inflammation and focal granuloma formation to catarrhal granulomatous and eosinophilic verminous bronchopneumonia. Another change observed in some belugas and believed to be associated with lungworm infection, was multifocal pulmonary arterial medial hypertrophy and degeneration. Beluga whales harvested at Pt. Lay (summer) frequently showed evidence of hepatic and pancreatic atrophy, while whales taken at Pt. Hope (spring) did not. This was believed to result from anorexia during migration—a supposition corroborated by the lack of stomach contents among Pt. Lay whales. Another prominent histologic finding among belugas was hepatic telangectasia, which occurred with significantly greater frequency and severity in Pt. Hope belugas than in those from Pt. Lay. The etiology and significance of this lesion could be not be ascertained, although it was not believed to be associated with any of the elements analyzed in this study. Mild thickening of Bowman’s capsule was seen frequently in belugas. However, this lesion was not typical of Hg or Cd-induced nephropathies, and did not appear correlated with kidney concentrations of these metals. This lesion was believed to be a normal consequence of aging in belugas, although a metal etiology for it could not be excluded irrefutably. In general, ringed seals were in good body condition and appeared healthy on gross examination. Among seals evaluated histologically, the most common finding was a mild, chronic, focal or periportal hepatitis, with focal hepatocellular necrosis sometimes apparent. Although a metal etiology for this lesion could not be definitively ruled out, in the absence of other lesions that would support a diagnosis of metal toxicosis, an infectious etiology was considered more credible. Two out of sixteen seals had embryologic remnants (an epidermoid cyst and an ultimobranchial cyst)—lesions that are usually considered incidental. While no toxic (metal or otherwise) etiology could be ascertained for these lesions, the incidence of retained embryologic remnants seemed high. A number of xenobiotics are known to be endocrine-disruptors, and the potential for such an etiology among these seals should be examined further. Lipofuscin deposition was ubiquitous among all three species examined histologically. Lipofuscin was most prevalent in hepatocytes, but also commonly was observed in various other tissue and cell types, especially in cardiac and skeletal myocytes, and in uriniferous tubular epithelial cells. The third portion of this study employed autometallographic (AMG) development of light microscopic tissue sections to amplify and localize deposition of inorganic Hg in liver and kidney of beluga and bowhead whales. No staining occurred among bowhead tissues, confirming the extremely low concentration of Hg determined through chemical analyses. In beluga kidney sections, AMG granules were seen throughout the uriniferous tubular epithelium, showing that Hg deposits throughout the nephric tubule, and not solely in the proximal tubular epithelium. In liver tissue, AMG granules were deposited primarily in periportal regions among whales with lower hepatic Hg burdens. In addition to periportal deposition, AMG granules were observed in pericentral and mid-zonal regions in the belugas sampled that had higher liver Hg concentrations (generally older animals). Granules were densely concentrated in stellate macrophages, especially near portal triads. Granules also were distributed in hepatocellular cytoplasm, generally concentrated toward the bile cannalicular domain of the cell. Granules were discrete, potentially indicating that Hg was confined within lysosomes. These observations suggested that inorganic Hg deposits initially in periportal regions of young animals, with subsequent accumulation occurring pericentrally, and finally, midzonally as the whales age. Computer-assisted densitometric analysis was used for semi-quantitative evaluation of AMG staining intensities. These AMG staining intensities were well correlated with concentrations of Hg determined via chemical analysis. Areas with AMG-staining were identified and compared with location of lipofuscin in the same field, visualized with fluorescent microscopy. While AMG granules and lipofuscin deposits sometimes were co-localized, they more often were not. In addition, abundant lipofuscin deposition was seen in livers of younger belugas with little to no Hg-catalyzed AMG staining. Also, lipofuscin concentrated predominantly in pericentral regions. These observations suggested that in the healthy marine mammals of this study, marked hepatic lipofuscin deposition most often occurred independently of Hg accumulation. Consequently, hepatic lipofuscin is likely to be a poor indicator of Hg-induced damage in belugas. The abundant lipofuscin deposition in livers of marine mammals was interpreted as most likely denoting a heightened exposure to oxidative stress that is probably inherent to a marine mammalian existence. These oxidative stressors may include a diet high in polyunsaturated fatty acids (PUFAs), alternating hypoxia and abundant oxygenation, and periodic bouts of anorexia associated with migration.