Induction of Cetacean Cytochrome P4501A1 by ß-Naphthoflavone Exposure of Skin Biopsy Slices

Céline A. J. Godard*,{dagger},1, Roxanna M. Smolowitz{ddagger}, Joanna Y. Wilson*, Roger S. Payne{dagger} and John J. Stegeman*

* Woods Hole Oceanographic Institution, Biology Department, Woods Hole, Massachusetts 02543; {dagger} Ocean Alliance, Lincoln, MA 01773; {ddagger} Marine Biological Laboratory, Woods Hole, MA 02543

Received November 7, 2003; accepted February 29, 2004


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Marine mammals can accumulate environmental contaminants in their blubber at concentrations harmful to laboratory animals. Induction of the cytochrome P450 1A1 (CYP1A1) enzyme is widely used as a biomarker of exposure and molecular effects in animal species, yet the validity of this biomarker has not been established in marine mammals. In vivo studies are generally precluded in these protected species, but skin biopsies (epidermis and dermis) can be collected in a minimally invasive way. We developed an in vitro assay using skin biopsy slices to examine CYP1A1 protein induction in marine mammals in response to chemical exposure. Skin biopsies from sperm whale (Physeter macrocephalus) were exposed for 24 h to ß-naphthoflavone (BNF), a prototypical CYP1A1 inducer, and CYP1A1 induction was detected by immunohistochemical staining in endothelial cells, smooth muscle cells, and fibroblasts. Biopsy slices were exposed to a range of BNF concentrations (0.6–600 µM), and a significant concentration-effect relationship was observed in both endothelial and smooth muscle cells (p = 0.05). This is the first study using skin biopsy slices to examine exposure of cetacean tissue to a CYP1A1 inducer. It demonstrates a causal relationship between chemical exposure and CYP1A1 induction and therefore validates the use of CYP1A1 expression in skin biopsies as a biomarker in cetaceans. Our protocol can be adapted to the investigation of chemicals, mixtures, concentrations, incubation times, or biological endpoints of choice. This should prove particularly relevant for these and other protected species that cannot be studied in the laboratory.

Key Words: marine mammal; CYP1A1; skin; ß-naphthoflavone; sperm whale; endothelium; dose-response.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The oceans are the final sink for many toxicants, and there is growing concern for the health of marine mammals, because they are known to accumulate high levels of polyhalogenated aromatic hydrocarbons, pesticides, and other lipophilic contaminants in their blubber (Aguilar and Borrell, 1994Go; Colborn and Smolen, 1996Go; Kannan et al., 1993Go; Martineau et al., 1987Go; Ross et al., 2000Go; Tanabe et al., 1981Go). Marine mammals can bioaccumulate and biomagnify these lipophilic marine contaminants due to a high position in marine food chains, large fatty tissue reserves, and longer life spans than many other marine organisms (Boon et al., 1992Go).

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., 1984Go). 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, 1996Go; Kannan et al., 2000Go; Kuehl et al., 1991Go; Martineau et al., 1987Go; Ross et al., 2000Go). 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, 1994Go; Kannan et al., 1993Go). 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)Go 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, 1982Go). CYP1A1 induction is widely used as a biomarker of exposure and molecular effects in animal species (Stegeman et al., 1992Go). 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., 1998Go; Goksøyr et al., 1986Go; Murk et al., 1994Go; White et al., 1994Go, 2000Go), and a CYP1A1 has been identified in several species (Teramitsu et al., 2000Go). 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., 1994Go). 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., 2004Go). 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., 1992Go, 2003Go). We treated sperm whale skin biopsy sections with various concentrations (0–600 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Biopsy collection. Sperm whale biopsies were obtained in the Sea of Cortez, Mexico in the summer and fall of 1999 (August–October) between 24°41.6' N and 28°31.0' N latitude and 110°02.0' W and 112°41.7' W longitude. The biopsy cruise was part of the Voyage of the Odyssey, a 5-year research program headed by Ocean Alliance and designed to gather baseline data on the levels and effects of synthetic contaminants in the marine environment worldwide, using sperm whales as an indicator species. The skin biopsies were collected using a 150-lb draw weight compound crossbow (Barnett RC-150). The skin biopsies collected included epidermis and dermis layers. Biopsy arrows with 40 mm by 7 mm internal diameter tips were designed and fabricated by Finn Larsen of the Danish Institute for Fisheries Research, Charlottenlund, Denmark. Samples were obtained under U.S. National Marine Fisheries Service permit No. 1004 to Ocean Alliance, and Mexico Secretaria de Medio Ambiente Recursos Naturales y Pesca permit No. 4903 to Dr. Jorge Urban Ramirez of the Universidad Autonoma de Baja California Sur, Mexico.

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., 1991Go). MAb 1-12-3 is highly specific for CYP1A1 in mammals (Drahushuk et al., 1998Go), 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 0–15) were determined as the products of the staining occurrence (scale of 0–3) and the staining intensity (scale of 0–5) 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., 1997Go). 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, 1992Go). 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 {alpha} = 0.05 level was considered significant.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Biopsy Collection and Treatment of Tissue Slices
Biopsies from fifty sperm whales were treated with BNF in DMSO or with DMSO alone for 24 h, with ten animals per treatment group (0, 0.6, 6, 60, or 600 µM BNF). We examined all biopsy sections for tissue integrity. Some samples exhibited perinuclear vacuolation in the epithelium and partial cell junction loss between epithelium and dermis, but no apparent alteration was observed in the dermal cells, the focus of our IHC study. Immunohistochemical analysis carried out to evaluate the expression of CYP1A1 in skin biopsy sections showed CYP1A1 staining in three different cell types: the endothelial cells comprising the lining of all blood vessels including capillaries, the smooth muscle cells present in the larger blood vessels, and fibroblasts. In all fifty sperm whales studied, no staining was observed in the antibody-control slides (incubated with the nonspecific antibody MOPC) prepared for each untreated and treated biopsy section, indicating that the staining observed in sections incubated with the monoclonal antibody to CYP1A1 is specific. The specificity of the CYP1A1 staining is illustrated in Figure 1.



View larger version (115K):
[in this window]
[in a new window]
 
FIG. 1. Specificity of the staining for CYP1A1. Serial sections of sperm whale PM99-371 skin biopsy slice treated with 600 µM BNF for 24 h were stained with specific (MAb 1-12-3) or nonspecific (MOPC31) antibodies. Magnification is 600x. Panel A shows blood vessels (v) after staining with nonspecific MOPC31 antibody, and Panel B shows same vessels after CYP1A1 specific staining (staining is red).

 
Inducibility of Cetacean CYP1A1
Table 1 presents the average CYP1A1 IHC staining scores for the untreated and treated biopsy sections, along with the average treatment-specific staining scores (i.e., the difference between untreated and either BNF- or DMSO-treated sections). Faint staining can be observed in all untreated sections (with 91% of the staining scores below 5), probably reflecting environmental exposure of the whales to CYP1A1 inducers. Average staining scores for endothelial cell, smooth muscle cell, and fibroblast staining in all untreated biopsies were 2.5, 2.3, and 2.9, respectively (standard deviation STD = 1.4, 1.3, and 2.0; range = 1–6, 1–4.5, and 0.5–9). For all cell types, the average BNF-specific staining scores were significantly different from the DMSO-specific average staining scores, consistent with induction of CYP1A1 in sperm whale endothelial cells, smooth muscle cells, and fibroblasts. This is illustrated in Figure 2, which shows CYP1A1 staining in untreated and treated biopsies from three animals (PM99-346, PM99-336, and PM99-352). Untreated sections showed only a faint staining (average staining score of 2.2, STD of 1.6) while there was an increase in staining intensity with BNF treatment in all three cell types. For the three animals illustrated in Figure 2, the treatment-specific staining scores for endothelial cells, smooth muscle cells, and fibroblasts were, respectively, 2.5, 2.5, and 3 in the DMSO group; 7, 7, and 7 in the 0.6 mM BNF group; and 13.5, 10.5, and 13 in the 600 µM BNF group. It is important to note that the staining intensity for any particular section reflects the average intensity observed for all vessels in that biopsy section and that at times both low and high intensity staining vessels were observed within the biopsies treated with BNF.


View this table:
[in this window]
[in a new window]
 
TABLE 1 Cytochrome P450 1A1 Expression in Sperm Whale Endothelial Cells, Smooth Muscle Cells, and Fibroblasts

 


View larger version (113K):
[in this window]
[in a new window]
 
FIG. 2. CYP1A1 expression in untreated and BNF-treated sperm whale biopsy slices. Biopsy slices shown are from three whales: animal PM99-346 (DMSO group), animal PM99-336 (0.6 µM BNF group), and animal PM99-352 (600 µM BNF group). Biopsy sections were immunohistochemically stained with MAb 1-12-3. CYP1A1-specific staining is red. Magnification is 600x. Symbols are: v for blood vessel, c for capillary, r for red blood cell, and f for fibroblast. Inset boxes showing fibroblasts are from a different area of the slide in each case.

 
Concentration-Effect in Cetacean CYP1A1 Inducibility
As illustrated in Figure 3, statistical analyses showed a concentration-dependent relationship for cetacean CYP1A1 inducibility in endothelial and smooth muscle cells. In fibroblasts, all treatment groups had CYP1A1 scores statistically different from the control (DMSO) group, but staining did not statistically differ between doses of BNF. In both endothelial and smooth muscle cells, there were three statistically different levels of CYP1A1 staining. Treatment groups were assigned randomly, but interestingly, we observed higher CYP1A1 expression in endothelium and smooth muscle cells from untreated biopsy sections than in the matched sections treated with DMSO alone. However, these higher CYP1A1 expression scores were not statistically different from the CYP1A1 expression scores observed in untreated biopsies matched to the BNF treatment groups. To further ensure that the higher CYP1A1 expressions in the untreated biopsy sections matched with DMSO-treated sections did not bias our results, we also calculated treatment-specific staining scores in two other ways. Thus, treatment-specific staining scores were calculated by subtracting either the mean staining score of all untreated sections, or the mean staining score of the untreated biopsies matched to the DMSO treatment group, from the BNF-treated biopsy staining score for each animal (data not shown). Regardless of the statistical method used, we detected significant concentration-dependent increases in CYP1A1 staining in response to BNF exposure in endothelium and smooth muscle cells.



View larger version (19K):
[in this window]
[in a new window]
 
FIG. 3. CYP1A1 is inducible in multiple cell types of cetacean skin. Levels of CYP1A1 expression due to BNF treatment are given as treatment-specific staining scores. Treatment-specific staining scores were determined for each animal as the difference between the staining scores of the untreated and treated biopsy sections. n = 10 animals for each treatment group. Within each panel, scores with different letters are statistically different at {alpha} = 0.05. Panel A: endothelial cells. Panel B: smooth muscle cells. Panel C: fibroblasts.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A higher overall incidence of mass mortalities, epizootics, pathologies, and reproductive failures has been reported in marine mammal populations in the last 30 years (Colborn and Smolen, 1996Go). It has been hypothesized that this elevated incidence may be related to the mass production of organochlorines that started in the 1940 s and the subsequent effects of long-term chronic exposure in the first generation of animals exposed, and/or of developmental and early postnatal exposure in their offspring (Colborn and Smolen, 1996Go). Numerous studies have established that marine mammals can accumulate high levels of organochlorines and other lipophilic contaminants (Martineau et al., 1987Go; Ross et al., 2000Go) but studies reporting on the molecular or physiological effects of these compounds are scarce. Minimally invasive methods for biomarkers of exposure and effect are critically needed to investigate the impact of pollution on marine mammals (Fossi et al., 1992Go). CYP1A1 induction is widely used in animal species as a biomarker of exposure to AHR agonists. Because the induction reflects a change in gene or protein expression levels or enzymatic activity, it is also considered a biomarker of molecular effects. Correlations between CYP activity and levels of some organochlorines (PCBs, dichlorodiphenyltrichloroethanes) in skin biopsies of common dolphins (Delphinus delphis) have been reported (Fossi et al., 2003Go), indicating that CYP1A1 induction may be a valid biomarker of exposure in these animals. However, the validity of this biomarker (i.e. demonstrating that it responds in a dose-dependent manner to toxicant exposure) remains to be directly established in cetacean species.

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 cell–cell 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., 1995Go). 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., 1996Go; Lake et al., 1993Go). 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., 1995Go). 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., 2004Go). 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, 1995Go). 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., 2001Go; Muller et al., 1996Go), and a concentration-dependent induction was also detected enzymatically after both 48 h and 72 h incubation with 0–50 µM BNF (Lake et al., 1993Go). 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., 2002Go; Stegeman et al., 1995Go). 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., 2002Go). 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., 1999Go). 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, 2001Go; Zhao et al., 1998Go). 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., 1991Go; Kerzee and Ramos, 2001Go). 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., 1999Go; Kim et al., 1997Go). 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., 1996Go). 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., 1994Go). 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., 2001Go). 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.


    ACKNOWLEDGMENTS
 
We thank Dr. Jorge Urban Ramirez of the Universidad Autonoma de Baja California Sur, Mexico for use of his permit No. 4903 from the Mexico Secretaria de Medio Ambiente Recursos Naturales y Pesca. We thank the crew of the Ocean Alliance R/V Odyssey for providing a logistical platform as well as skilled assistance in approaching sperm whales and collecting skin biopsies. We thank Dr. Finn Larsen of the Danish Institute for Fisheries Research, Charlottenlund, Denmark, for his custom-made biopsy arrows. We thank Rebecca Clark from Ocean Alliance for technical assistance. We thank Bruce Woodin, Carolyn Angell, and Dr. Michael Moore from the Woods Hole Oceanographic Institution, Woods Hole, MA, for providing valuable feedback in immunohistochemistry staining techniques. We thank Dr. Victoria Starczak from the Woods Hole Oceanographic Institution for assistance with statistical analyses. Funding for Ocean Alliance was graciously provided by the Pacific Life Foundation, the World Wildlife Fund, and the Summit Foundation. This study was also supported in part by NIEHS grant P42-ES-04696 and by NOAA Sea Grant Number NA86RG0075 R/B-162. Contribution 10980 from the Woods Hole Oceanographic Institution.


    NOTES
 

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.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Aguilar, A., and Borrell, A. (1994). Reproductive transfer and variation of body load of organochlorine pollutants with age in Fin whales (Balaenoptera physalus). Arch. Environ.l Contam. Toxicol. 27, 546–554.

Ahmad, N., Agarwal, R., and Mukhtar, H. (1996). Cytochrome P-450-dependent drug metabolism in skin. Clin. Dermatol. 14, 407–415.[CrossRef][ISI][Medline]

Allen, T. (1992). Hematoxylin and eosin. In Laboratory Methods in Histotechnology (E. Prophet, B. Mills, J. Arrington, and L. Sobin, Eds.), pp. 53–58. American Registry of Pathology, Washington, DC.

Angell, C., Wilson, J., Moore, M., and Stegeman, J. (2004). Cytochrome P450 1A1 expression in cetacean integument: Implications for detecting contaminant exposure and effects. Mar. Mamm. Sci. (in press).

Ben-David, M., Kondratyuk, T., Woodin, B., Snyder, P., and Stegeman, J. (2001). Induction of cytochrome P450 IAI expression in captive river otters fed Prudhoe Bay crude oil: Evaluation by immunohistochemistry and quantitative RT-PCR. Biomarkers 6, 218–235.[CrossRef][ISI]

Boon, J., Sleiderink, H., Helle, M., Dekker, M., van Shanke, A., Roex, E., Hillebrand, M., Klamer, H., Govers, B., Pastor, D., et al. (1998). The use of microsomal in vitro assay to study phase I biotransformation of chlorobornanes (Toxaphene) in marine mammals and birds. Possible consequences of biotransformation for bioaccumulation and genotoxicity. Comp. Biochem. Physiol. CToxicol. Pharmacol. 121, 385–403.

Boon, J. P., Van Arnheim, E., Jansen, S., Kannan, N., Petrick, G., Schulz, D., Duinker, J. C., Reijnders, P. J., and Goksøyr, A. (1992). The toxicokinetics of PCBs in marine mammals with special reference to possible interactions of individual congeners with the cytochrome P450-dependent monooxygenase system: An overview. In Persistent Pollutants in Marine Ecosystems (C. H. Walker and D. R. Livingstone, Eds.), pp. 119–159. SETAC Special Publication Series, Pergamon Press, Oxford.

Colborn, T., and Smolen, M. J. (1996). Epidemiological analysis of persistent organochlorine contaminants in cetaceans. Rev. Environ. Contam. Toxicol. 146, 91–172.[ISI][Medline]

Drahushuk, A., McGarrigle, B., Larsen, K., Stegeman, J., and Olson, J. (1998). Detection of CYP1A1 protein in human liver and induction by TCDD in precision-cut liver slices incubated in dynamic organ culture. Carcinogenesis 19, 1361–1368.[Abstract]

Drahushuk, A., McGarrigle, B., Tai, H., Kitareewan, S., Goldstein, J., and Olson, J. (1996). Validation of precision-cut liver slices in dynamic organ culture as an in vitro model for studying CYP1A1 and CYP1A2 induction. Toxicol. Appl. Pharmacol. 140, 393–403.[CrossRef][ISI][Medline]

Fossi, M. C., Marsili, L., Giovanni, N., Natoli, A., Politi, E., and Panigada, S. (2003). The use of a non-lethal tool for evaluating toxicological hazard of organochlorine contaminants in Mediterranean cetaceans: New data 10 years after the first paper published in MPB. Mar. Pollut. Bull. 46, 972–982.[CrossRef][ISI][Medline]

Fossi, M. C., Marsili, L., Leonzio, C., Notarbartolo Di Sciara, G., Zanardelli, M., and Focardi, S. (1992). The use of non-destructive biomarker in Mediterranean cetaceans: Prelimiray data on MFO activity in skin biopsy. Mar. Pollut. Bull. 24, 459–461.[CrossRef][ISI]

Geraci, J. and St. Aubin, D. (1982). Study of the effects of oil on cetaceans. US Department of the Interior, Bureau of Land Management. Washington, D.C. Contract No AA-551-CT9-29.

Geyer, H., Freitag, D., and Korte, F. (1984). Polychlorinated biphenyls (PCBs) in the marine environment, particularly in the Mediterranean. Ecotoxicol. Environ. Saf. 8, 129–151.[ISI][Medline]

Giachelli, C., Majesky, M., and Schwartz, S. (1991). Developmentally regulated cytochrome P-4501A1 expression in cultured rat vascular smooth muscle cells. J. Biol. Chem. 266, 3981–3986.[Abstract/Free Full Text]

Glockner, R., and Muller, D. (1995). Ethoxycoumarin O-deethylation (ECOD) activity in rat liver slices exposed to ß-naphthoflavone (BNF) in vitro. Exp. Toxicol. Pathol. 47, 319–324.[ISI][Medline]

Goksøyr, A., Solbakken, J. E., Tarlebø, J., and Klungsøyr, J. (1986). Initial characterization of the hepatic microsomal cytochrome P-450 system of the piked whale (minke) Balaenoptera acutorostrata. Mar. Environ. Res. 19, 185–203.[CrossRef][ISI]

Gradin, K., Toftgard, R., Poellinger, L., and Berghard, A. (1999). Repression of dioxin signal transduction in fibroblasts. Identification of a putative repressor associated with Arnt. J. Biol.l Chem. 274, 13511–13518.[Abstract/Free Full Text]

Hennig, B., Meerarani, P., Slim, R., Toborek, M., Daugherty, A., Silverstone, A., and Robertson, L. (2002). Proinflammatory properties of coplanar PCBs: In vitro and in vivo evidence. Toxicol. Appl. Pharmacol. 181, 174–183.[CrossRef][ISI][Medline]

Kannan, K., Blankenship, A., Jones, P., and Giesy, J. (2000). Toxicity reference values for the toxic effects of polychlorinated biphenyls to aquatic mammals. Hum. Ecol.l Risk Assess. 6, 181–201.

Kannan, K., Tanabe, S., Borrell, A., Aguilar, A., Focardi, S., and Tatsukawa, R. (1993). Isomer-specific analysis and toxic evaluation of polychlorinated biphenyls in striped dolphins affected by an epizootic in the Western Mediterranean Sea. Arch. Environ. Contam. Toxicol. 25, 227–233.[ISI][Medline]

Kerzee, J., and Ramos, K. (2001). Constitutive and inducible expression of Cyp1a1 and Cyp1b1 in vascular smooth muscle cells. Role of the AhR bHlH/PAS transcription factor. Circ. Res. 89, 573–582.[Abstract/Free Full Text]

Kim, P., DeBoni, U., and Wells, P. (1997). Peroxidase-dependent bioactivation and oxidation of DNA and protein in benzo[a]pyrene-initiated micronucleus formation. Free Radic. Biol. Med. 23, 579–596.[CrossRef][ISI][Medline]

Kuehl, D., Haebler, R., and Potter, C. (1991). Chemical residues in dolphins from the U.S. Atlantic coast including Atlantic bottlenose obtained during the 1987/88 mass mortality. Chemosphere 22, 1071–1084.[CrossRef][ISI]

Lake, B., Beamand, J., Japenga, A., Renwick, A., Davies, S., and Price, R. (1993). Induction of cytochrome P450-dependent enzyme activities in cultured rat liver slices. Food Chem. Toxicol. 31, 377–386.[CrossRef][ISI][Medline]

Lupp, A., Danz, M., and Muller, D. (2001). Morphology and cytochrome P450 isoforms expression in precision-cut rat liver slices. Toxicology 161, 53–66.[CrossRef][ISI][Medline]

Martineau, D., Béland, P., Desjardins, C., and Lagacé, A. (1987). Levels of organochlorine chemicals in tissues of beluga whales (Delphinapterus leucas) from the St. Lawrence Estuary, Québec, Canada. Arch. Environ. Contamin. Toxicol. 16, 137–147.[ISI]

Muller, D., Glockner, R., and Rost, M. (1996). Monooxygenation, cytochrome P4501A1 and P4501A1-mRNA in rat liver slices exposed to beta- naphthoflavone and dexamethasone in vitro. Exp. Toxicol. Pathol. 48, 433–438.[ISI][Medline]

Murk, A., Morse, D., Boon, J., and Brouwer, A. (1994). In vitro metabolism of 3,3',4,4'-tetrachlorobiphenyl in relation to ethoxyresorufin-O-deethylase activity in liver microsomes of some wildlife species and rat. Eur. J. Pharmacol. 270, 253–261.[Medline]

Parrish, A., Gandolfi, A., and Brendel, K. (1995). Precision-cut tissue slices: Applications in pharmacology and toxicology. Life Sci. 57, 1887–1901.[CrossRef][ISI][Medline]

Poland, A., and Knutson, J. (1982). 2,3,7,8-Tetrachlorodibenzo-p-dioxin and related halogenated aromatic hyrocarbons: Examination of the mechanism of toxicity. Ann. Rev. Pharmacol. Toxicol. 22, 517–554.[CrossRef][ISI][Medline]

Ross, P., Ellis, G., Ikonomou, M., Barett-Lennard, L., and Addison, R. (2000). High PCB concentrations in free-ranging Pacific killer whales, Orcinus orca: Effects of age, sex and dietary preference. Mar. Pollut. Bull. 40, 504–525.[CrossRef]

Schlezinger, J., White, R., and Stegeman, J. (1999). Oxidative inactivation of cytochrome P450 1A stimulated by 3,3',4,4'-tetrachlorobiphenyl: Production of reactive oxygen by vertebrate CYP1As. Mol. Pharmacol. 56, 588–597.[Abstract/Free Full Text]

Smolowitz, R., Hahn, M., and Stegeman, J. (1991). Immunohistochemical localization of cytochrome P-450IA1 induced by 3,3',4,4'-tetrachlorobiphenyl and by 2,3,7,8-tetrachlorodibenzoa-furan in liver and extrahepatic tissues of the teleost Stenotomus chrysops (scup). Drug Metab. Dispos. 19, 113–123.[Abstract]

Stegeman, J., Brouwer, M., Di Gulio, R., Forlin, L., Fowler, B., Sanders, B., and Van Veld, P. (1992). Molecular responses to environmental contamination: Enzyme and protein systems as indicators of chemical exposure and effects. In Biomarkers. Biochemical, Physiological, and Histological Markers of Anthropogenic Stress (R. Huggett, R. Kimerle, P. Mehrle, and H. Bergman, Eds.), pp. 235–335. Lewis Publishers. SETAC Special Publications Series, Boca Raton, FL.

Stegeman, J. J., Hahn, M. E., Weisbrod, R., Woodin, B. R., Joy, J. S., Najibi, S., and Cohen, R. A. (1995). Induction of cytochrome P4501A1 by aryl hydrocarbon receptor agonists in porcine aorta endothelial cells in culture and cytochrome P4501A1 activity in intact cells. Mol. Pharmacol. 47, 296–306.[Abstract]

Tanabe, S., Tanaka, H., Maruyama, K., and Tatsukawa, R. (1981). Ecology and bioaccumulation of Stenella coeruleoalba: Elimination of chlorinated hydrocarbons from mother striped dolphins through parturition and lactation. In Studies on the Levels of Organochlorine Compounds and Heavy Metals in Marine Organisms (T. Fujiyama, Ed.), pp. 115–121. University of Ryuskyus, Okinawa.

Teramitsu, I., Yamamoto, Y., Chiba, I., Iwata, H., Tanabe, S., Fujise, Y., Kazusaka, A., Akahori, F., and Fujita, S. (2000). Identification of novel cytochrome P450 1A genes from five marine mammal species. Aquat. Toxicol. 51, 145–153.[CrossRef][ISI][Medline]

White, R., Hahn, M. E., Lockhart, W. L., and Stegeman, J. J. (1994). Catalytic and immunochemical characterization of hepatic microsomal cytochromes P450 in beluga whale (Delphinapterus leucas). Toxicol. Appl. Pharmacol. 126, 45–57.[CrossRef][ISI][Medline]

White, R., Shea, D., Schlezinger, J., Hahn, M., and Stegeman, J. (2000). In vitro metabolism of polychlorinated biphenyl congeners by beluga whale (Delphinapterus leucas) and pilot whale (Globicephala melas) and relationship to cytochrome P450 expression. Compar. Biochem. Physiol. C Toxicol. Pharmacol. 126, 267–284.

Woodin, B., Smolowitz, R., and Stegeman, J. (1997). Induction of cytochrome P4501A in the intertidal fish Anoplarchus purpurescens by Prudhoe Bay Crude Oil and environmental induction in fish from Prince William Sound. Environ. Sci. Technol. 31, 1198–1205.[CrossRef][ISI]

Zhao, W., Ramos, K. S., and Parrish, A. R. (1998). Constitutive and inducible expression of cytochrome P4501A1 and P4501B1 in human vascular endothelium and smooth muscle cells. In Vitro Cell. Dev. Biol. 34, 671–673.[ISI]





This Article
Abstract
FREE Full Text (PDF)
All Versions of this Article:
80/2/268    most recent
kfh124v1
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Search for citing articles in:
ISI Web of Science (2)
Disclaimer
Request Permissions
Google Scholar
Articles by Godard, C. A. J.
Articles by Stegeman, J. J.
PubMed
PubMed Citation
Articles by Godard, C. A. J.
Articles by Stegeman, J. J.