* Woods Hole Oceanographic Institution, Biology Department, Woods Hole, Massachusetts 02543; Ocean Alliance, Lincoln, MA 01773;
Marine Biological Laboratory, Woods Hole, MA 02543
Received November 7, 2003; accepted February 29, 2004
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ABSTRACT |
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Key Words: marine mammal; CYP1A1; skin; ß-naphthoflavone; sperm whale; endothelium; dose-response.
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INTRODUCTION |
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High concentrations of organochlorine pollutants deleteriously affect the endocrine, reproductive, immune, and nervous systems of laboratory animals and elicit adverse responses such as skin and liver damage, thymic atrophy, weight loss, and neurobehavioral problems (Geyer et al., 1984). Contaminant tissue burdens equal to or above levels found harmful in laboratory animals have been reported in several cetaceans and pinnipeds, including beluga whales (Dephinapterus Leucas) of the Saint Lawrence Estuary, long-finned pilot whales (Globicephala melas) from the Faroe Islands, killer whales (Orcinus Orca) from the North Pacific, and animals involved in recent mass stranding events (Colborn and Smolen, 1996
; Kannan et al., 2000
; Kuehl et al., 1991
; Martineau et al., 1987
; Ross et al., 2000
). While a direct link between contaminant burden and cetacean epizootics or mass stranding has not been established, some of the highest PCB concentrations found in wildlife have been reported in these animals (Aguilar and Borrell, 1994
; Kannan et al., 1993
). However, the concentrations of chemicals present in marine mammal tissues can provide only a partial insight as to the actual toxicity to the animal.
Linking biological effects with exposure to organochlorines and other pollutants is particularly challenging in marine mammals because of their legal status as protected species, the complex logistics involved in studying them in their natural habitat, the impracticality of laboratory studies, and the complex ethical issues involved. To our knowledge, the only in vivo exposure experiments involving organic contaminants reported in the literature for cetaceans are those of Geraci and St Aubin (1982) in the late 1960 s, when three bottlenose dolphins (Tursiops truncatus) and one Risso's dolphin (Grampus griseus) were exposed topically to crude oil or orally to machine oil. Topical exposure resulted in transient cell damage in the epidermis, while the extensive hepatic and pancreatic fibrosis observed after oral exposure were attributed to trematode parasites.
To date, the effects of chemicals in cetaceans have been inferred largely from correlations between high body burdens and pathologies and by extrapolation from dose-response relationships for both toxicities and molecular effects in other species. Molecular effects correlated with toxicity include the induction of cytochrome P450 enzymes (CYP) and particularly of CYP1A1 by chemicals such as polycyclic aromatic hydrocarbons, polychlorinated biphenyls, dioxins, and furans via the aryl hydrocarbon receptor (AHR) signaling pathway (Poland and Knutson, 1982). CYP1A1 induction is widely used as a biomarker of exposure and molecular effects in animal species (Stegeman et al., 1992
). Among the few studies of cytochrome P450 s in cetaceans, several have examined the metabolism of foreign chemicals in hepatic microsomes or cell cultures (Boon et al., 1998
; Goksøyr et al., 1986
; Murk et al., 1994
; White et al., 1994
, 2000
), and a CYP1A1 has been identified in several species (Teramitsu et al., 2000
). Correlations between non-ortho and mono-ortho PCB burdens in blubber and hepatic CYP1A1 content and activity were observed in beluga whales (White et al., 1994
). Such correlations generally support the use of CYP1A1 induction as a biomarker of exposure to AHR agonists in cetaceans, but data to directly demonstrate the concentration dependence of induction is critically absent (Angell et al., 2004
). We employed skin biopsy slices to show directly a link between chemical exposure and CYP1A1 induction in cetacean tissues. The use of skin biopsy for measuring CYP1A1 activity in marine mammals has been advocated as a valid nondestructive method since the early 1990 s (Fossi et al., 1992
, 2003
). We treated sperm whale skin biopsy sections with various concentrations (0600 µM) of ß-naphthoflavone (BNF), a prototypical CYP1A1 inducer. Levels of CYP1A1 induction in the endothelium (a prominent site of induction in vertebrates), in smooth muscle cells, and in fibroblasts were then determined using immunohistochemical staining.
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MATERIALS AND METHODS |
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Biopsy treatment. Immediately after collection, we manually cut two thin (about 2-mm thick) slices spanning the epidermis and dermis from each of 50 biopsies. We incubated one of the two slices (treated slice) for 24 h in cell culture media with BNF. Incubation was carried at the ambient temperature of the air-conditioned pilothouse. Temperature logs indicate the ambient temperature in the pilothouse to be maintained at about 32°C when air conditioned. Treatment groups were 0, 0.6, 6, 60, or 600 µM BNF prepared in dimethylsulfoxide (DMSO) as carrier, with ten animals per treatment group. The 0 µM BNF corresponded to DMSO alone and allowed us to test for carrier effect. For each biopsy, we incubated the other slice (untreated slice) for 24 h in media alone. After the 24-h incubation in media, untreated and treated slices were placed in 10% neutral buffered formalin until embedding in paraffin to ensure protein preservation.
Media and chemicals. DMEM (Dulbecco's Modified Eagle's Medium, Sigma, St. Louis, MO) medium was prepared with 5.96 g HEPES free acid (N-[2-Hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]; 4-[2-Hydroxyethyl]piperazine-1-[2-ethanesulfonic acid],Sigma), 2.2 g NaHCO3 (sodium bicarbonate, Sigma) and 0.58 g NaCl (sodium chloride, Fisher Scientific, Pittsburgh, PA) per liter. Medium was adjusted to pH 7.5, filter-sterilized, and refrigerated before use. BNF was purchased from Aldrich (Milwaukee, WI), and DMSO from Fisher Scientific. BNF was chosen for its low toxicity to humans, which allowed for safety protocols compatible with our fieldwork.
Immunohistochemical (IHC) analysis. Biopsy slices were prepared for immunohistochemical staining of cytochrome P4501A1. Slices fixed in 10% neutral buffered formalin were embedded in paraffin. Serial microtome sections (5 µm thick) were obtained from within the 0.2-mm outer layers and then stained using a peroxidase anti-peroxidase detection system (Signet Laboratories, Dedham, MA) with either a monoclonal antibody against scup CYP1A (MAb 1-12-3, 0.3 µg/ml) or a purified mouse myeloma protein nonspecific antibody (MOPC31, 0.3 µg/ml, Sigma, St. Louis MO USA), as previously described (Smolowitz et al., 1991). MAb 1-12-3 is highly specific for CYP1A1 in mammals (Drahushuk et al., 1998
), and the epitope recognized is a CYP1A1 specific epitope (unpublished data). CYP1A1 staining was evaluated under light microscopy after incubation with amino-9-ethylcarbazole as chromogenic substrate (AEC, Signet Laboratories) and counterstaining with Mayer's hematoxylin (Sigma). For each section, CYP1A1 staining scores (scale of 015) were determined as the products of the staining occurrence (scale of 03) and the staining intensity (scale of 05) in each cell type. A staining occurrence of 0 corresponds to no staining, while a staining occurrence of 3 corresponds to staining in all cells. The staining intensity represents the average intensity observed for each cell type throughout a section. A staining intensity of 0 corresponds to an absence of staining or to a staining equal to that observed with the control MOPC antibody. A staining intensity of 5 corresponds to a very strong staining equal to that observed in a highly CYP1A-induced liver section of scup treated with 3,3',4,4' tetrachlorobiphenyl (TCB). Serial liver sections of this TCB-induced scup were used as controls for staining intensity among IHC runs. IHC staining scores have been shown to reflect accurately the content of CYP1A1 protein measured by immunoblotting techniques (Woodin et al., 1997
). A treatment-specific staining score was determined for each animal as the difference between the staining scores of the treated (with DMSO or BNF) and untreated (media alone) biopsy sections. For each treatment group, two biopsies were randomly selected for hematoxylin and eosin staining of both treated and untreated sections (H and E, Richard Allen Scientific, Kalamazoo, MI) according to standard protocols (Allen, 1992
). We evaluated tissue integrity (nuclear stain intensity, nucleus shape, eosinophilia) in all sections using IHC and hematoxylin- and eosin-treated slides.
Statistical analyses. Differences among treatment-specific CYP1A staining scores for endothelial and smooth muscle cells were statistically analyzed by one-way ANOVA using Fisher's Protected LSD test for equal sample size (n = 10) using the SuperANOVA (Abacus Concepts). One untreated biopsy sample in the DMSO group could not be scored for fibroblasts due to the faintness of the counterstain. The differences among treatment-specific CYP1A1 staining scores for fibroblasts were therefore statistically analyzed by one-way ANOVA using the Tukey HSD Compromise test for unequal sample size (n = 9 for DMSO group, and n = 10 for all other groups) using the SuperANOVA (Abacus Concepts). The = 0.05 level was considered significant.
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RESULTS |
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DISCUSSION |
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To specifically address this issue, we adapted a tissue slice protocol for use with cetacean skin biopsies. In contrast with in vitro exposure studies that rely on cell culture, the normal tissue architecture (including cell heterogeneity and cellcell interactions) is maintained in this protocol. We collected skin biopsies from 50 sperm whales in a minimally invasive manner and exposed biopsy sections to 0, 0.6, 6, 60, or 600 µM BNF, a prototypical CYP1A1 inducer. We selected this wide range (0.6 to 600 µM BNF) of BNF concentrations to increase the likelihood of detecting changes in CYP1A1 induction. 600 µM BNF was selected because it neared the highest concentration of BNF that could conveniently be prepared in DMSO. We used 0.6 µM BNF as the lowest concentration based on a previous study on porcine endothelial cells (Stegeman et al., 1995). The use of tissue slices for studies of cytochrome P450 activities and inducibility by chemicals such as BNF, TCDD, and Aroclor® 1254 (commercial PCB mixture) has been validated by comparisons with in vivo experiments (Drahushuk et al., 1996
; Lake et al., 1993
). In mammals, precision-cut tissue slices and outer layers of generally thicker manually cut slices have been shown to retain viability and metabolic capacity for at least 24 h (Parrish et al., 1995
). Similarly, we did not observe any apparent alteration of the dermal cells in all biopsy sections after 24 h, treated or untreated.
The faint staining observed in all untreated slices (91% of these slides had a staining score below 5) probably reflects environmental exposure of the sperm whales to CYP1A1 inducers; such environmental induction has been suggested in biopsies from numerous cetacean species (Angell et al., 2004). DMSO has been reported to have a protective action on CYP enzymes in vitro that may be due to the scavenging of hydroxyl radicals, while`its effects in vivo are unclear with both increased and decreased monooxygenation rates having been reported (Glockner and Muller, 1995
). However, the average DMSO treatment-specific scores for all three cell types examined were not statistically different from zero, indicating the suitability of this compound as a carrier in our experiments. In BNF-treated slices, statistically significant induction of CYP1A1 was detected in endothelial cells, smooth muscle cells, and fibroblasts, and at all four concentrations tested (Table 1, Fig. 2). The results showed a concentration-dependent relationship for cetacean CYP1A1 inducibility in endothelial and smooth muscle cells, with three statistically different levels of CYP1A1 induction observed in each cell type (Fig. 3). In rat liver slices, CYP1A1 induction has been detected at the protein, mRNA, and enzyme activity levels after incubation with 25 µM BNF for 24 h (Lupp et al., 2001
; Muller et al., 1996
), and a concentration-dependent induction was also detected enzymatically after both 48 h and 72 h incubation with 050 µM BNF (Lake et al., 1993
). Therefore, our observation of CYP1A1 protein induction in sperm whale skin biopsies occurred at concentrations comparable to those known to produce induction and to show a concentration-dependent effect in rat liver slices. For endothelial cells, smooth muscle cells, and fibroblasts, the 0.6 µM BNF treatment group resulted in the lowest observed effect level in our study, but this could be an overestimation since lesser concentrations were not tested.
Endothelial cells are in immediate contact with blood-borne xenobiotics and may play an important toxicological role in their transfer and metabolism. Both in vitro and in vivo studies have shown CYP1A1 to be catalytically active and inducible in endothelium of terrestrial mammals (Hennig et al., 2002; Stegeman et al., 1995
). Our results confirm that CYP1A1 is also inducible in cetacean endothelium. Chlorinated dioxins and coplanar PCBs can generate oxidative stress and an inflammatory response in mammalian endothelial cells after being activated by CYP1A1 in vitro (Hennig et al., 2002
). Organochlorines that are not rapidly metabolized by CYP1A1 also may produce oxidative stress or radical-induced damage, resulting from uncoupling of CYP1A1 (Schlezinger et al., 1999
). While contaminant-induced toxic effects in endothelial cells are still to be characterized in cetaceans, our findings underline the importance of examining the endothelial tissue when assessing exposure to and potential effects of environmental contamination in these animals.
CYP1A1 mRNA expression and inducibility have been reported in smooth muscle cells of laboratory animals and humans (Kerzee and Ramos, 2001; Zhao et al., 1998
). However, some studies suggest the existence of a labile repressor preventing a basal transcriptional activation in vascular smooth muscle cells of adult rodents (Giachelli et al., 1991
; Kerzee and Ramos, 2001
). The sperm whales sampled for our study are of unknown age but likely included immature and mature animals. While our results demonstrate CYP1A1 protein was inducible by BNF in cetacean smooth muscle cells, CYP1A1 basal expression could not be assessed, since environmental exposure in our untreated samples cannot be ruled out. In fibroblasts, we did not detect a statistically significant concentration effect, possibly because of a relatively high variability of response in this cell type, or because the range of BNF concentrations used caused maximum induction or was otherwise inadequate to reveal a concentration effect. In terrestrial mammals, CYP1A1 expression and inducibility in fibroblasts appear to vary widely in both primary cultures and cell lines (Gradin et al., 1999
; Kim et al., 1997
). Based on our findings, CYP1A1 is inducible in dermal smooth muscle cells and fibroblasts, and the toxicological significance of induction in these two cell types in cetaceans needs to be established.
In human and rodents, skin CYP1A1 is known to play a significant role in xenobiotic metabolism: it is inducible after topical or systemic treatment by BNF and other AHR agonists, and activity levels can reach 27% of that of the liver, the major organ of xenobiotic metabolism (Ahmad et al., 1996). In aquatic mammals, few studies have examined how changes in skin tissue may relate to overall systemic effects of environmental chemicals. Significant correlations between certain planar PCB blubber burdens and hepatic CYP1A1 content and activity have been observed in beluga whales (White et al., 1994
). A recent study on captive river otter (Lontra canadensis) chronically fed crude oil demonstrated a dose-dependent induction of dermal endothelial CYP1A1 (Ben-David et al., 2001
). That study illustrates the validity of using skin tissues for contaminant exposure through the oral route, generally the most important source of exposure in marine mammals. However, there still remains a need for further research investigating and modeling the relationships among contaminant concentrations, toxicokinetics in blubber and whole body, dermal CYP1A1 expression and biological effects in marine mammals.
This report provides the first direct demonstration that an AHR agonist can induce CYP1A1 in cetacean tissue in a concentration-dependent manner. In the case of endangered or protected species, studies using incubation or culture of skin biopsies may indeed be the sole avenue for investigating responses to contaminant exposure in live tissues. The protocol presented here could be adapted to investigate experimental exposure to specific chemicals or chemical mixtures, at selected concentrations and incubation times and with specific biological endpoints of interest. It also could be modified in order to preserve exposed tissue sections for enzyme activity or mRNA analyses. In future studies, the use of precision-cut slices would ensure standardization of slice dimensions and enhance viability in culture (by creating thinner slices) and, therefore, could provide opportunities for detailed metabolic studies.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed at Woods Hole Oceanographic Institution, Biology Department, 45 Water Street, Woods Hole, MA 02543. Fax: 508-457-2134. E-mail: cgodard{at}whoi.edu.
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