Free-ranging Marine Mammals Research Articles

Hepatic microsomal cytochrome P450 enzyme activity in relation to in vitro metabolism/inhibition of polychlorinated biphenyls and testosterone in Baltic grey seal (Halichoerus grypus)

Among other factors, cytochrome P450 (CYP) enzyme activity determines polychlorinated biphenyl (PCB) bioaccumulation, biotransformation, and toxicity in exposed species. We measured the oxidative metabolism in vitro of 12 PCB congeners, representing structural groups based on the number and position of the chlorine atoms, by the hepatic microsomes of one Baltic grey seal (Halichoerus grypus). Microsomal metabolism was observed for several PCBs with vicinal H atoms exclusively in the ortho and meta positions and without any ortho-Cl substituents (CB-15 [4,4'-Cl2] and CB-77 [3,3',4,4'-Cl4]), vicinal meta and para-H atoms (CB-52 [2,2',5,5'-Cl4], and -101 [2,2',4,5,5'-Cl5]) or with both characteristics in combination with either only one ortho-Cl (CB-26 [2,3',5-Cl3], CB-31 [2,4',5-Cl3]) or two ortho-Cl substituents (CB-44 [2,2',3,5'-Cl4]). To allocate PCB biotransformation to specific CYPs, the inhibitive effect of compounds with known CYP-specific inhibition properties was assessed on in vitro PCB metabolism and on regio- and stereospecific testosterone hydroxylase activities. Metabolic inhibition was considered relevant at concentrations < or = 1.0 microM because these inhibitors became decreasingly selective at higher concentrations. At < 1.0 microM, ellipticine (CYPIAI/2 inhibitor) selectively inhibited CB-15, -26, -31, and -77 metabolism, with no significant inhibition of CB-44, -52, and -101 metabolism. Inhibition of CB-52 and -101 metabolism by chloramphenicol (CYP2B inhibitor) started at 1.0 microM and maximized at about 100% at 10 microM. Ketoconazole (CYP3A inhibitor) appeared to selectively inhibit CB-26, -31, and -44 metabolism relative to CB-15, -77, and -52 at concentrations < or = 1.0 microM. Major testosterone metabolites formed in vitro were 2beta-(CYP3A), 6beta- (CYP3A, CYPIA), and 16beta- (CYP2B) hydroxytestosterone and androstenedione (CYP2B, CYP2C11). The CYP forms indicated are associated with the specific metabolism of testosterone in laboratory animals. Inhibition of 2beta- and 6beta-hydroxytestosterone formation at ellipticine and ketoconazole concentrations < or = 1.0 microM suggested that both inhibitors were good substrates of CYP3A-like enzymes in grey seal. Chloramphenicol (model for CYP2B) is apparently not a good inhibitor of CYPI A and CYP3A activities in grey seal because the chemical did not inhibit any metabolic route of testosterone at concentrations from 0.1 to 10 microM. Our findings demonstrated that at least CYP1A- and CYP3A-like enzymes in the liver of grey seals are capable of metabolizing PCBs with ortho-meta and/or meta-para vicinal hydrogens. A CYP2B form might also be involved, but this could not be proven by the results of our experiments. Defining the profiles of CYP enzymes that are responsible for PCB biotransformation is necessary to fully understand the bioaccumulation, toxicokinetics, and risk of PCB exposure in seals and other free-ranging marine mammals.

Vitamin A physiology in the precocious harbour seal (Phoca vitulina): a tissue-based biomarker approach

Vitamin A is a nutrient essential to all mammals for growth and development, as well as for the maintenance of reproductive, endocrine, and immune systems. Environmental contaminant-related disruption of vitamin A has been observed in many wildlife species and can therefore be used as a biomarker of toxic effects. However, the natural processes regulating vitamin A uptake, storage, and distribution among compartments are poorly understood in marine mammals. In this study, 20 young healthy harbour seals (Phoca vitulina) were captured to establish a compartment-based model providing a foundation for a mechanistic understanding of vitamin A physiology and disruption. Vitamin A (retinol, retinyl palmitate, and (or) retinoic acid) was quantified in blood plasma and in biopsy samples of liver, blubber, and skin. Although the highest concentrations of vitamin A were found in liver, blubber represents a more important storage depot, with an estimated 66% of the total retinoid content of the compartments measured. We suggest that vitamin A physiology in the precocious harbour seal has evolved to deal with high vitamin A availability during a short nursing period and to sustain growth during the postweaning fast. Positive correlations in vitamin A concentrations among liver, blubber, and skin support the use of less invasive biopsy sampling of just blubber or skin, which can provide physiologically relevant information in biomarker studies of free-ranging marine mammals.

Evaluation of mitogen-induced responses in marine mammal and human lymphocytes by in-vitro exposure of butyltins and non-ortho coplanar PCBs

The effects of exposure to butyltin compounds (BTs: tributyltin; TBT, dibutyltin; DBT and monobutyltin; MBT) and non-ortho coplanar PCBs (IUPAC 77, 126 and 169) on marine mammals and human lymphocyte were evaluated. Peripheral blood mononuclear cells (PBMCs) isolated from Dall's porpoises (Phocoenoides dalli), bottlenose dolphins (Tursiops truncatus), a California sealion (Zalophus californianus), a larga seal (Phoca largha) and humans (Homo sapiens) were exposed at varying concentrations of BTs and coplanar PCBs. Concanavalin A (Con A)-stimulated mitogenesis found significantly suppressed (P<0.01) when the cells were exposed at 300 nM (89 ng/ml) of TBT and 330 nM of DBT (77 ng/ml), while MBT showed little cytotoxicity at treatment levels of up to 3600 nM (620 ng/ml). BTs concentrations in the liver of Dall's porpoises from Japanese coastal waters ranged between 81–450 ng/g for TBT and 200–1100 ng/g (wet wt.) for DBTs, which is greater than the cytotoxic levels registered in this study. In contrast, non-ortho coplanar PCBs did not suppress cell proliferation at concentrations of up to 30 nM (10 ng/ml). The residue levels of coplanar PCBs in the blubber of Dall's porpoises were 0.12–1.3 ng/g, which were one order of lower than those levels that do cell proliferation. When cells were exposed to a mixture of TBT/DBTand coplanar PCBs, the proliferation was significantly reduced to 33 nM DBT plus 34 nM CB-77 and 33 nM DBT plus 28 nM CB-169 mixtures, respectively. The investigations relating the contaminant-induced immunosuppression in marine mammals have been focused on persistent organochlorines such as PCBs, pesticides and dioxin compounds. However, this study suggested the possibility of BTs could also pose a serious threat to the immune functions in free-ranging marine mammals and humans.

Fine-scale three-dimensional spatial use by diving, lactating female Weddell seals Leptonychotes weddellii

Despite the importance of fine-scale spatial use in understanding an animal's foraging ecology, these data cannot readily be collected for free-ranging marine mammals. We used an acoustic positioning system to quantify, for the first time, the fine-scale 3-dimensional (3D) spatial use of free-ranging Weddell seals swimming under ice. Unlike many other phocid species, lactating Weddell seals spent up to 25% of their time diving. Given the limited foraging range imposed on the seals by the fast ice upon which they breed, this could lead to prey depletion and even inter-specific competition. The seals focused their underwater activity on a relatively small region associated with a steep bottom contour, using the entire water column, with very little time spent at the bottom. This behaviour is consistent with feeding on bentho-pelagic prey such as Pleurogramma antarcticum. The 3D profile of individual dives consisted of the seals making simple, directed dives which gradually converged with the ocean floor. There was some variation from this pattern, usually associated with increased searching time. Such focused foraging activity may result in local prey depletion and intra-specific competition.

Vitamin A physiology and its application as a biomarker of contaminant-related toxicity in marine mammals: a review.

In recent decades, marine mammal populations living in highly polluted areas have experienced incidences of low reproductive success, developmental abnormalities and disease outbreaks. In many of these cases, environmental contaminants were suspected as causal or contributing factors. However, demonstrating a mechanistic link between contaminant exposure and effect in marine mammal populations has proven challenging. Consequently, the development and application of relatively noninvasive biomarkers represents a potentially valuable means of monitoring wildlife populations exposed to elevated levels of contaminants. One touted biomarker is vitamin A (retinol), a "dietary hormone" whose metabolites are required for reproduction, growth, development, immune function, vision and epithelial maintenance. Laboratory studies have shown that many contaminants, including polychlorinated biphenyls (PCBs), polychlorinated dibenzo-para-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs), can disrupt vitamin A physiology and alter the distribution of its essential metabolites. Field studies suggest that complex environmental mixtures of these chemicals can also interfere with vitamin A dynamics in free-ranging marine mammals and other fish-eating wildlife. However, circulatory retinol, which is the least invasive measurement of vitamin A status, appears to have variable responses to contaminant exposure. In addition, "normal" circulatory retinol levels have not yet been described for most wildlife species, and not enough is known about the natural physiological events that can alter these concentrations. Confounding factors must therefore be characterized before retinoids can be used as an effective indicator of adverse health effects in marine mammals exposed to elevated levels of environmental contaminants.

Lipophilic contaminants in marine mammals: review of the results of ten years' work at the Department of Environmental Biology, Siena University (Italy)

Organochlorine contaminants (HCB, DDTs and PCBs) and polycyclic aromatic hydrocarbons (PAHs) were valuated in three Mediterranean cetaceans: the striped dolphin (Stenella coeruleoalba), the bottlenose dolphin (Tursiops truncatus) and the fin whale (Balaenoptera physalus), and in three Argentinean pinnipeds: the southern sea lion (Otaria flavescens), the South American fur seal (Arctocephalus australis) and the subantarctic fur seal (Arctocephalus tropicalis). Two kinds of sample were obtained from the different species of cetaceans and pinnipeds in order to evaluate the toxicological risk to which a species or population is exposed those from stranded specimens and those from free-ranging specimens. In this paper, the use of a non-destructive approach, biopsy sampling, for freeranging marine mammals is recommended.

Marine mammal faeces as a source of DNA

[Extract] Remote collection of skin biopsies from free-ranging marine mammals has been to date, the most effective and noninvasive means of tissue collection for DNA studies (Amos & Hoelzel 1990; Brown et al. 1991; Weinrich et al. 1991). Although it has been shown that DNA extraction from faeces is possible (Hoss et al. 1992), there are no records of DNA extraction from the faeces of marine mammals, perhaps because it is expected their faeces have a shorter life span compared with the faeces of (dry) land mammals. We have been able to extract DNA from the faeces of a free-ranging dugong (Dugong dugon). We present a sequence from this DNA, a region of the mitochondrial DNA which we have used for a genetic population structure study of the dugong. Extraction of DNA from faeces of marine mammals provides an attractive alternative to biopsy sampling. It is less invasive and more economical. The dugong faecal sample was collected from Borroloola (15°8'S 136°7'E) as part of a feeding study (A.R. Preen, pers. comm., 1995). Dugong feeding trails are easily recognizable and their faeces float to the surface of the water. Several intact faeces were collected after floating for an undetermined time (a few hours?) and individually contained in plastic bags. It was about 3h before they were frozen and, although they stayed cold, they were defrosted during a 2 day transit, refrozen, then thawed for DNA extraction.

Developing standards for protecting marine mammals from noise: Lessons from the development of standards for humans

Models for protecting marine mammals from noise have been suggested that are analogous to human noise criteria, specifically (1) weighting functions that model species-specific auditory threshold functions (analogous to A-weighting); (2) threshold models for predicting the proportion of individuals that avoid a noise (analogous to the Schulz model of annoyance); and (3) the equal-energy hypothesis for predicting hearing loss. Models for reducing sleep interference, speech interference, and attention deficits might also be applicable. All these models will be reviewed. Unfortunately, human noise criteria do not apply to a number of effects that could occur in free-ranging marine mammals. Noise could affect nonauditory physiology. Noise could also mimic natural sounds (e.g., seismic impulses that are similar to tail beats), or attract marine mammals into dangerous areas (e.g., attracting killer whales to fishing gear). Given the paucity of research available on noise effects in marine mammals, standards may be difficult to establish, although they are badly needed. At present, management agencies have adopted extremely conservative noise criteria. The experience of regulating noise in human communities suggests that such stringent criteria cannot be enforced consistently. Solutions that have proved practical for human communities will be reviewed.

Assimilation Efficiency of Northern Fur Seals Determined Using Dietary Manganese

We validated the use of dietary manganese (Mn2+) as an inert marker for estimating assimilation efficiency (AE%) of captive northern fur seals (Callorhinus ursinus) for possible application to free-ranging marine mammals. Dietary Mn2+ derived AE% estimates were compared to estimates from CrO, and 51Cr marker techniques. Assimilation efficiency measured using Mn2+ was 90.0 ? 1.2 (SE)% which was not significantly different from estimates using other marker techniques. Excess Mn2+ added to the diet was fully recovered in the feces, indicating that Mn2+ functioned as an unabsorbed marker. The Mn2+ technique might permit measurement of AE% in free-ranging marine mammals. J. WILDL. MANAGE. 54(2):246-251 Animals absorb components of ingested prey with varying efficiencies. Dry matter assimilation efficiency (AE%) varies with digestive tract morphology, food type and quality, seasonality, meal size, time between meals, and the nutritional state of the animal (Golley et al. 1965, Petrusewicz and MacFayden 1970, Moors 1977, Keiver et al. 1984, Ronald et al. 1984, Sibly and Calow 1986). Assimilation efficiency relates food or energy flux through the consumer to the quality or type of diet. Thus, its measurement expresses the quantitative relationships between the energetics and metabolism of an organism and the energy flow through populations or trophic levels (Vogtsberger and Barrett 1973, Ashwell-Erickson and Elsner 1981, Lavigne et al. 1982). Although measuring the AE% of terrestrial animals is generally straightforward using total fecal collection (Kleiber 1975:259) or indigestible marker addition techniques (Arthur 1942, Mautz 1971), applying these methods to some marine mammals is difficult. Assimilation efficiency has been successfully measured by the addition of inassimilable markers in several pinniped species confined in dry pens (Parsons 1977, Miller 1978, Ashwell-Erickson and Elsner 1981, Keiver et al. 1984, Ronald et al. 1984). Most marine mammals, however, are not easily confined for total fecal collections, and markers such as chromic oxide (CrzOQ) or 51Cr may be lost from food into the water if the animal masticates or shreds prey items prior to ingestion. Markers may also be difficult to distribute homogeneously within many potential food species; both of these situations will result in inaccurate AE%

COCCIDIOIDOMYCOSIS IN A CALIFORNIA SEA OTTER (Enhydra lutris)

A weak and emaciated California sea otter (Enhydra lutris) was found stranded on Atascadero Beach in Morro Bay, California. It died three weeks after capture. A diagnosis of coccidioidomycosis was confirmed by histology, serology and culture. This is believed to be the first reported case of this disease from the Morro Bay area of San Luis Obispo County, California as well as the first reported case in a free-ranging marine mammal.