Introduction

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Endothelium, the single cell layer lining blood and lymphatic vessels, provides a physical lining, maintains blood vessel tone, and controls microvascular permeability, platelet clumping, clotting, and lymphocyte traffic (Pearson, 1991). Endothelium initiates angiogenesis essential to vessel growth supporting the development and growth of organisms and the repair of damaged blood vessels (Alberts et al., 1989). Protective functions of endothelium are prominent in the blood-tissue barriers in brain, testis, and retina. Endothelium also may be a site of xenobiotic toxicity. Activation of promutagens in the vasculature is thought to play a role in atherosclerosis (Juchau et al., 1976), and vascular defects and edema are common in developing fish, birds, and mammals exposed to planar halogenated aromatic hydrocarbons (pHAH)¹ (Peterson et al., 1993).

Polynuclear aromatic hydrocarbons (PAH) and pHAH are toxic compounds that induce the expression of several genes, including cytochrome P4501A (CYP1A), via the aryl hydrocarbon receptor (AHR) (Hankinson, 1995). CYP1A can be induced in various cell types in many organs (Goldstein and Linko, 1984; Smolowitz et al., 1991), but endothelium is a site of induction common among organs. In mammals, induction of CYP1A1 has been detected in endothelium of aorta, umbilical vein, heart, kidney, and lung (Dees et al., 1982; Brittebo, 1994; Farin et al., 1994; Thirman et al., 1994; Stegeman et al., 1995). In fish, induction of CYP1A has been seen in endothelium in liver, heart, gill, kidney, gut, gonad, spleen, red muscle, and brain (Miller et al., 1989; Stegeman et al., 1989; Smolowitz et al., 1991; Husoy et al., 1994). Induction in endothelium can be substantial; CYP1A was estimated to account for as much as 25% of the endothelial microsomal protein in the heart of -naphthoflavone (BNF)-treated fish (Stegeman et al., 1989).

The toxicity of AHR agonists is generally AHR-dependent (Poland and Glover, 1980) but may be mediated in part by CYP1A. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) toxicity in fish embryos correlates with CYP1A expression in endothelium, and CYP inhibitors can diminish the effect (Cantrell et al., 1996; Guiney et al., 1997). The toxicological importance of endothelial CYP1A will depend in part on the catalytic capacity and the degree of induction in endothelium. CYP1A is metabolically active in some endothelia. Bovine aorta endothelial cells and microsomes from scup heart, where CYP1A is predominantly endothelial, produce metabolites of benzo[a]pyrene (BP) typical of those formed by CYP1A (Baird et al., 1980; Stegeman et al., 1982). CYP1A and binding of reactive metabolites of heterocyclic amines colocalize in endothelium of mammals and birds (Annas and Brittebo, 1998; Annas et al., 1998), and CYP1A is catalytically active in intact porcine aorta endothelial cells (Stegeman et al., 1995). CYP1A is less active in endothelium than in the liver (e.g., Stegeman et al., 1982), possibly due to a low availability of cytochrome P450 reductase (CPR). CPR mRNA was not detected in rabbit lung endothelium (Overby et al., 1992), and is expressed at low levels in porcine aorta endothelial cells (Stegeman et al., 1995).

In vivo, the patterns of induction of CYP1A in endothelium have been shown to differ with different inducers. In scup, the potent AHR agonist 2,3,7,8-tetrachlorodibenzofuran (TCDF) at 3 µg/kg (0.01 µmol/kg) and the less potent agonist 3,3',4,4'-tetrachlorobiphenyl (TCB) at 1 mg/kg (3 µmol/kg) (both given intraperitoneally) caused similar induction of CYP1A in liver and in sites proximal to large vessels. With TCB, CYP1A induction was seen also in peripheral regions of the organism, but this did not occur with TCDF (Smolowitz et al., 1991). This suggests that CYP1A induction in endothelium of the peripheral vasculature is related to the penetration of inducer into the periphery.

In most organs, endothelium is a minor component, confounding the study of endothelial cell properties in whole organ preparations. We investigated the induction of endothelial CYP1A in extrahepatic organs but particularly in heart and in the capillary network in the rete mirabile of the swimbladder of the eel (Anguilla rostrata). Endothelium represents as much as 8 to 9% of the mass of the teleost heart (Stegeman et al., 1989), whereas the rete mirabile is composed predominantly of endothelial cells (Krogh, 1959). CYP1A was induced in these organs and was metabolically active in microvascular endothelium. Penetration of endothelial CYP1A induction into the capillary bed of the rete mirabile increased with increasing dose of AHR agonists. Our results with this microvascular model suggest that endothelial CYP1A can indicate local levels of inducer and possibly can affect the distribution of AHR agonists.

	Materials and Methods

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Chemicals. Unlabeled BP and BNF were purchased from Aldrich (Milwaukee, WI). [³H]BP (55 Ci/mmol) was obtained from Amersham (Arlington Heights, IL). Unlabeled TCB (congener 77) and [¹⁴C]TCB (ring-labeled; 52.1 mCi/mmol) were purchased from Pathfinder (St. Louis, MO). Soluble tissue dissolver was obtained from NEN Life Science Products (Boston, MA). 7-Ethoxyresorufin was obtained from Molecular Probes (Eugene, OR). MS 222 was the gift of Adria Elskus but can be purchased from Sigma (St. Louis, MO). All other reagents were obtained from Sigma.

Environmental Collections. Female American eels, A. rostrata, were collected from sites near Woods Hole, MA, using traps baited with horseshoe crab meat. Eels were collected from New Bedford Harbor, MA, at the Aerovox site, from Marion Harbor, MA, and from Vineyard Sound, MA. Eels in New Bedford Harbor have been shown to contain concentrations of total polychlorinated biphenyls (PCBs) as high as 700 µg/g whole body dry weight (i.e., approximately 70 mg/kg wet weight) (Lake et al., 1995). Marion Harbor is an enclosed harbor with substantial boat traffic. Vineyard Sound is an open body of water distant from New Bedford Harbor. Fish in the locations away from New Bedford have much lower levels of PCB residues (Elskus et al., 1994). Eels were transported to the laboratory and immediately sacrificed and dissected as described below.

Experimental Exposures. One hundred and twelve female eels collected in Vineyard Sound were purchased from the Marine Biological Laboratory, Woods Hole, MA. Eels were provided a diet of squid once a week. For the first experiment the eels were kept at 20°C. For all other experiments, the eels were kept for at least 3 days at 10°C in flow-through tanks until treated. A previous study in Fundulus heteroclitus showed that, although CYP1A induction occurs more slowly at lower temperatures, the protein turnover is reduced and thus induced protein levels persist for longer periods (Kloepper-Sams and Stegeman, 1992). We reasoned that the lower temperature also would increase retention of CYP1A induction in the eel.

Chemical Distribution Analysis. For analysis of chemical distribution, eels were anesthetized in a solution of MS 222 (6 g/4 liters of sea water) and injected intraperitoneally with corn oil or varying concentrations of [³H]BP (1 and 10 mg/kg) and [¹⁴C]TCB (1 and 10 mg/kg) in corn oil (n = 6). Eight days following injection, each eel was anesthetized in a solution of MS 222 and sacrificed by cervical transection. Portions of freshly dissected organs (0.1-0.25 g each of liver, heart, gill, spleen, gut, kidney, and muscle or 0.03-0.13 g of rete mirabile) were removed, rinsed with water, blotted, and weighed. Bile was collected via syringe, and blood was collected from the caudal vein via syringe. Tissues and bile were placed in 20-ml glass scintillation vials.

Dose-Response Experiments. Eels were anesthetized in a solution of MS 222 and given intraperitoneal injections with corn oil or varying amounts of BNF, BP, or TCB in corn oil. BNF was given at 0.1, 1, 5, 10, and 100 mg/kg (0.4, 4, 20, 40, and 400 µmol/kg), BP at 0.1, 1, and 10 mg/kg (0.4, 4, and 40 µmol/kg), and TCB at 0.1, 1, and 10 mg/kg (0.3, 3, 30, and 60 µmol/kg). There were three to five fish in each dose group. On day 5 (experiment 1) or day 8 (experiments 2-7) eels were anesthetized in aqueous MS 222 and sacrificed by cervical transection. Portions of heart, gill, kidney, and rete mirabile were preserved in 10% neutral buffered formalin. Heart and rete mirabile collected for microsome preparation were frozen in liquid N₂ until use.

Tissue Preparation and Enzyme Assays. Tissues were homogenized in cold buffer (0.15 M KCl, 0.05 M Tris, pH 7.2), and postmitochondrial supernatant (S9 or PMS) and microsomal fractions were prepared as before (Stegeman et al., 1979). Protein content was determined using the bicinchoninic acid method and BSA as a standard. Initial rates of ethoxyresorufin O-deethylase (EROD) activity were measured fluorometrically by the method of Hahn et al. (1993) using a Cytofluor 2300 (Millipore) multiwell plate reader. NADPH-CPR activity was determined as before (Stegeman et al., 1982), with a Shimadzu 210 spectrophotometer.

Western Blotting. Microsomal proteins (80 µg/lane) were resolved using 12% SDS-polyacrylamide gel electrophoresis and electrophoretically transferred to a nylon membrane. The primary antibody was the mouse monoclonal antibody (mAb) 1-12-3 (Park et al., 1986) raised against scup P450E, a CYP1A (Morrison et al., 1995). mAb 1-12-3 is an IgG that is highly specific for CYP1As in fish and CYP1A1 in mammals. The secondary antibody was alkaline phosphatase-linked goat anti-mouse IgG (Bio-Rad, Hercules, CA). The immunoreactive proteins were visualized using enhanced chemiluminescence (Tropix, Bedford, MA). CYP1A content was quantified by densitometric analysis of video images (NIH Image, 1.60b5); values are reported as scup CYP1A equivalents.

Immunohistochemistry. Fixed heart, kidney, gill, and rete mirabile were embedded in paraffin by Pathology Services, Inc. (Cambridge, MA) or by the Laboratory of Aquatic Animal Medicine and Pathology at the MBL (Woods Hole). The staining procedure used was as described by Smolowitz et al. (1991). Tissue sections (5 µm) were mounted on Superfrost Plus slides, and the sections were deparaffinated and hydrated in 1% PBS/BSA, with incubation in 3% H₂O₂ in water to block endogenous peroxidases. Sections were stained using an indirect peroxidase stain [Universal Immunoperoxidase Staining Kit (Murine), Signet Lab, Dedham, MA)] The primary antibody was mAb 1-12-3, and the secondary antibody was a peroxidase-labeled goat antibody to mouse IgG. Staining of CYP1A was scored for intensity of staining (0 = no stain, 1 = mild stain, 2 = moderate stain, 3 = strong stain, 4 = very strong stain) and for occurrence of staining (0 = no cells, 1 = rare cells, 2 = some cells, 3 = many cells, 4 = all cells staining). A stain index was computed by multiplying the intensity and occurrence scores for a scale from 0 through 16. This index has been shown to correlate positively with CYP1A content measured by Western blot.

Statistics. Data were analyzed using SuperAnova for Macintosh (Abacus Concepts, Inc., Berkeley, CA). A one-factor ANOVA combined with comparisons tests or a one-tailed t test were used to analyze data from the environmental collections as indicated in Figures and Tables.

	Results

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Chemical Distribution. Initially, we examined the distribution of [³H]BP and [¹⁴C]TCB following intraperitoneal injection into eels (Fig. 1). Nine organs, bile, and blood were sampled 8 days after a single injection of 1 or 10 mg of BP or TCB/kg. Concentrations of BP and TCB in most of the organs corresponded to the dose given, with approximately 10-fold higher concentrations in organs of eels receiving the greater dose. The patterns of tissue distribution were similar for BP and TCB at each dose. Bile contained the highest concentration of ³H or ¹⁴C, whereas of the organs examined, liver, gut, and kidney contained the greatest concentrations of either BP or TCB label. At the 1 mg/kg dose, concentrations of BP were 2- to 4-fold greater than those of TCB in several organs and in bile, whereas at the high dose, tissue concentrations of the two chemicals were more similar. At both doses, BP and TCB concentrations in gill, heart, and rete mirabile were about 10-fold greater than the concentrations measured in blood, although variability prevented the differences from being significant.

View larger version (25K): [in this window] [in a new window]   Fig. 1.   Tissue distribution of [3H]BP and [14C]TCB in eels. [3H]BP or [14C]TCB were injected intraperitoneally at the doses indicated. A, organs from BP-treated fish; B, organs from TCB-treated fish; C, bile from BP- and TCB-treated fish. Fish were sacrificed 8 days postinjection, and portions of freshly dissected organs were weighed, rinsed with water, blotted, digested with Soluble, minced with scissors, and placed in a 50°C oven until dissolved. Bile was collected via syringe. Blood was collected from the caudal vein via syringe and added to ethanol/Soluble (1:2). H2O2 was added to all vials except those containing gut and muscle. All samples containing Soluble were acidified with HCl. Scintiverse was added to the vials, and the samples were allowed to quench overnight before counting. Data are means ± S.D. of three to six individuals. For A and B: a, significantly greater than 1 mg/kg dose (P < .05, Student's t test); b, significantly greater than blood (P < .05, Dunnett's). Concentrations in bile were significantly greater than those in blood.

Microsomal CYP1A Induction in Endothelium-Rich Organs. Microsomal preparations of heart and rete mirabile from animals treated with the different inducers were analyzed for CYP1A content and EROD activity and compared with results in liver (Table 1). In both heart and rete mirabile, CYP1A protein content and EROD activity showed dose-dependent increases in eels treated with BP (Fig. 2, Table 1). Comparing the content of CYP1A in tissues from animals given different doses of BP showed that the degree of induction in heart and rete mirabile was as great or greater than that in liver of these fish (Table 1). Comparing responses in animals given BP, BNF, or TCB at the same dose of 10 mg/kg (30-40 µmol/kg) showed that the extent of CYP1A induction in heart was less in fish given BNF than in those given BP, whereas TCB produced even less induction of CYP1A and EROD (Table 1). Likewise, induction of microsomal CYP1A and EROD activity in rete mirabile was less in animals given BNF, and particularly in those given TCB, than in the eels given BP. The lesser induction by TCB also was evident in liver (Table 1).

View larger version (34K): [in this window] [in a new window]   Fig. 2.   Immunoblot of CYP1A in rete mirabile microsomes from eels treated with BNF, BP, and TCB. Eels were sacrificed 8 days postinjection, and rete mirabile microsomal fractions were electrophoresed, transferred to nylon, immunoblotted with mAb 1-12-3 against scup CYP1A, and visualized using enhanced chemiluminescence. The treatment with BNF, BP, or TCB and the doses represented in the various lanes are: Lane 1, scup CYP1A standard; lane 2, control; lane 3, 1 mg/kg BP; lane 4, 0.01 mg/kg BP; lane 5, 10 mg/kg BP; lane 6, 10 mg/kg TCB; lane 7, 10 mg/kg BNF.

Immunohistochemistry. Immunohistochemical analyses were performed to evaluate the dose and inducer dependence of CYP1A induction in endothelium of heart and rete mirabile, and selected other organs. An initial examination of H&E-stained sections showed no cellular necrosis in the organs from fish given any dose of inducer.

View larger version (46K): [in this window] [in a new window]   Fig. 3.   Immunohistochemical analysis of CYP1A protein in endothelium of organs from eels treated with BNF, BP, and TCB. A, BNF-treated eels (N ranged from 3 to 10). B, BP-treated eels (N ranged from 4 to 8). C, TCB-treated eels (N ranged from 3 to 15). Eels were injected intraperitoneally with BNF, BP, or TCB at the doses indicated on the figure and sacrificed 8 days postinjection. Tissues were dissected, embedded, sectioned, and stained immunohistochemically for CYP1A as described by Smolowitz et al. (1991). Staining of CYP1A was scored for intensity of staining (0 to 4) and for occurrence of staining (0 to 4), and a stain index was computed by multiplying the intensity and occurrence scores for a scale from 0 through 16. Data from experiment 1 were not significantly different from the other experiments; therefore, all data were combined. *Significantly different from control. aSignificantly different from control and 0.1 mg/kg. bSignificantly different from control, 0.1 and 1 mg/kg. cSignificantly different from 1 mg/kg. dSignificantly different from 5 mg/kg. Significance was determined as P < .05 (Tukey-Kramer).

CYP1A Immunostaining in Rete Mirabile. Sections illustrating CYP1A staining by mAb 1-12-3 in rete mirabile are shown in Fig. 4. The rete mirabile structure has prerete arteries leading to afferent collecting vessels (heart pole) that diverge into the capillary bed of the rete. These capillaries anastomose at the site of gas secretion into the swimbladder (bladder pole) and then diverge into efferent capillaries that return blood to the body. Rete mirabile from control fish had variable staining (mild to strong) in prerete arteries only (Table 2). All treated fish showed strong to very strong staining in these prerete arteries. However, the extent to which CYP1A staining penetrated further into the body of the rete mirabile differed with inducer and dose.

View larger version (120K): [in this window] [in a new window]   Fig. 4.   CYP1A staining in rete mirabile of eels treated with BNF. Eels were injected intraperitoneally with BNF and sacrificed 8 days postinjection. Dose (and sections shown) are: A, control (heart pole); B, 0.1 mg/kg (heart pole); C, 1 mg/kg (heart pole); D, 5 mg/kg (heart pole); E, 5 mg/kg (bladder pole). The immunostaining procedure used is essentially as described by Smolowitz et al. (1991).

Endothelial CYP1A Induction in Environmentally Exposed Eels. The above results indicate that the magnitude and penetration of CYP1A induction in vessels of the heart and rete mirabile are associated with the magnitude of exposure to AHR agonists. To determine whether induced levels of CYP1A occur in endothelium of eels exposed to CYP1A inducers in the environment, we examined CYP1A expression in heart and rete mirabile of eels from New Bedford Harbor and from two less contaminated sites, Marion Harbor and Vineyard Sound. Microsomal CYP1A content and activity were elevated significantly in heart of eels from New Bedford Harbor (Table 3). The relative levels of cardiac microsomal EROD matched the relative levels of CYP1A content in liver microsomes of these same animals (Schlezinger and Stegeman, 2000). Immunohistochemical analysis also was performed with heart, and the CYP1A stain index in cardiac endothelium corresponded to the relative levels of cardiac microsomal CYP1A content in eels from the three sites, with a strong increase in eels from New Bedford Harbor. Likewise, microsomal preparations of rete from New Bedford Harbor eels had elevated EROD rates and CYP1A content, compared with the samples from Marion Harbor or Vineyard Sound.

	Discussion

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Most vertebrate organs have multiple cell types in which CYP1A can be induced, however, endothelium is a common site of strong induction of CYP1A in virtually all organs of vertebrates. Yet, endothelial cells usually represent a very small proportion of organ mass, which greatly confounds the study of endothelial CYP1A. Here we investigated the dose and inducer dependence of endothelial CYP1A induction in several organs of eel, focusing on heart and the rete mirabile in the swimbladder, a countercurrent microvascular structure that increases the partial pressure of oxygen to aid its secretion into the bladder (Krogh, 1959). Fish heart has been used as a model for CYP1A induction in endothelial cells, because in some fish endothelium represents as much as 8 to 9% of the mass of the heart (Stegeman et al., 1989). That proportion is less than in the rete mirabile, which is composed predominantly of capillary endothelium (Krogh, 1959), and which, in the eel, is relatively large and accessible. Thus, we deduced that the rete mirabile may be a unique site in which to study regulation and function of CYP1A in endothelium.

BP elicited a dose-dependent induction of microsomal CYP1A protein and EROD activity in rete mirabile and in heart, and BP, BNF, and TCB all elicited dose-dependent induction of CYP1A immunostaining in rete mirabile and heart endothelium. Eels from New Bedford Harbor, an environment highly contaminated by AHR agonists, also had induced CYP1A content and/or activity in the rete mirabile and the heart. In both experimentally and environmentally induced eels, the patterns of response observed in rete mirabile paralleled those observed in heart and liver, indicating that a similar AHR signal transduction pathway is operating in the microvascular endothelium of the rete.

Immunohistochemical staining of CYP1A induced in some endothelial cells of the heart or rete mirabile was as strong or stronger than that in hepatocytes (Fig. 3), indicating that CYP1A is as highly induced in those endothelial cells as in hepatocytes. Despite that, the content of CYP1A detected in rete or heart microsomes was much less than that in liver microsomes. In liver, the majority of cells showed strong induction, whereas in heart or rete mirabile, a smaller percentage of the cells showed strong staining.

Given the cellular localization of CYP1A, the induction of EROD activity indicates that the induced CYP1A is catalytically active in microvascular endothelium of the rete, and in endothelium of the heart. Other studies have shown that CYP1A is catalytically active in endothelial cells in culture (Stegeman et al., 1995). As with CYP1A content, EROD rates in heart and rete mirabile microsomes from induced eels were substantially less than in microsomes from liver, even of control eels (Table 1). However, the heart and rete mirabile had estimated turnover numbers for EROD (EROD per unit of CYP1A equivalent) that were similar to or greater than the value for liver. Other studies in fish heart and in mammalian endothelial cells in culture have indicated that CYP1A has a lower catalytic capacity in the endothelium than in the liver, suggested to result from a content of CPR in endothelial cells that is not sufficient to support the full catalytic capacity of induced levels of CYP1A in endothelium (Stegeman et al., 1982, 1995; Overby et al., 1992). The observation that EROD turnover in heart and rete mirabile of eel is more like that in liver suggests that the eel may differ from other species in this regard. Consistent with that, the rates of CPR in rete microsomes were as high as the CPR rates often seen in liver of fish, a novel finding for an endothelium-rich organ.

It is possible that different CYP1As are expressed in endothelium and liver of eel. Single CYP1As have been identified in most fish examined (Morrison et al., 1998). Recently, however, Itakura and colleagues identified two CYP1A genes that are expressed in a congeneric eel species, A. japonica (Ogino et al., 1999); there could be a divergence of CYP1As also in A. rostrata. mAb 1-12-3, which we used in immunoassays, recognizes an epitope conserved among all fish CYP1As sequenced thus far, and that epitope is likely to occur in multiple CYP1As in eels. Thus, whether eel endothelium expresses a distinct isoform remains to be determined.

As with induction of hepatic CYP1A in these same eels (Schlezinger and Stegeman, 2000), BP, BNF, and TCB differed in their ability to induce microsomal CYP1A protein and EROD and CYP1A staining. Induction by BNF peaked at intermediate doses, and at comparable doses TCB elicited a lesser response than BNF or BP. The dose-dependent penetration of CYP1A staining into the rete also was much less with TCB. The slighter response to TCB in rete and heart suggests that this pHAH is not as efficacious as PAH in endothelium. The basis for this difference is not understood. In the rete mirabile, the difference is not likely due to a lower tissue dose of TCB, because at 10 mg/kg the concentrations of BP and TCB in rete differed little. Thus, it appears that some inducer-specific factors are involved in the degree to which CYP1A is induced along a vessel. TCB but not BP has been shown to suppress CYP1A via oxidative inactivation following uncoupling (J. J. Schlezinger, W. D. J. Struntz, and J. J. Stegeman, submitted). TCB does inactivate CYP1A in eel liver microsomes (Schlezinger et al., 2000), but whether a similar process occurs in endothelium is not known.

Several of our results are consistent with the hypothesis that endothelial CYP1A can indicate and possibly influence the pharmacokinetics of AHR agonists. The distribution of BP and TCB showed that rete mirabile, an organ composed predominantly of endothelial cells, can retain AHR agonists. The accumulation of AHR agonists from the blood and the correspondence between greater penetration of CYP1A induction and greater dose indicate that endothelial cells remove inducers from the circulation. Similar to the dose-dependent penetration seen in the rete mirabile, in a study of human placenta, endothelial cells from placenta of smokers had elevated aryl hydrocarbon hydroxylase activity, whereas endothelial cells from the umbilical vein did not (Manchester et al., 1984). The maternal portion of placenta from smokers also has significantly higher levels of CYP1A than the fetal portion (B. P. Slezak, J. Fisher, M. Alashari, J. Schlezinger, J. J. Stegeman, and J. R. Olson, in preparation). In gill, there was a dose-dependent transition of CYP1A immunostaining from staining only in endothelial cells to staining as well in epithelial cells adjacent to the endothelium, suggesting that at the higher doses inducer had crossed the endothelium.

Together, the results above provide support for the suggestion that endothelial CYP1A participates in reducing the penetration of AHR agonists to underlying tissues and into distal regions. Thus, the absence of CYP1A staining in efferent vessels of the rete mirabile of animals treated with any inducer could result if the inducers did not penetrate into those efferent structures. Yet, the possibility that there may be some difference in the responsiveness of afferent and efferent capillary endothelium cannot be excluded. Determining the extent of uptake into endothelial cells, and the clearance of CYP1A inducers in a pass through the vascular bed, will be important in resolving this issue.

Substrate binding by CYP1A could contribute to the role of endothelium as a protective cell layer in blood-tissue barriers and perhaps throughout the body. Contribution to that barrier function could occur whether or not CYP1A induced in endothelium is catalytically efficient with a particular substrate. CYP1A2, which binds but does not oxidize TCDD and other pHAH (Poland et al., 1989a,b), is responsible for sequestration and accumulation of TCDD in rat liver (Diliberto, 1997). TCB is tightly bound but is only slowly metabolized by fish CYP1A (White et al., 1997), whereas BP and BNF bind to fish (and mammalian) CYP1A1 but are rapidly metabolized. Thus, some substrates might be effectively "bound" by the CYP1A, and thereby sequestered in the endothelium. In systems where CYP1A in the endothelium is catalytically inefficient, CYP1A might then slowly metabolize PAH as well as pHAH, perhaps with a slow elimination of the compounds from the cells and potentially a slower rate of formation of reactive metabolites. Eel may provide a contrasting system for study, one in which endothelial CYP1A appears to be more active.

Overt toxicity and apoptosis caused by TCDD in lake trout embryos (Guiney et al., 1997) and in medaka embryos (Cantrell et al., 1996, 1998) correlate with CYP1A expression in endothelium, and CYP inhibitors can diminish the effect (Cantrell et al., 1996). Planar halogenated aromatic hydrocarbon exposure results in edema and cardiac dysmorphogenesis in fish embryos (Wisk and Cooper, 1990; Spitsbergen et al., 1991; Walker et al., 1991) and in other vertebrates (Brunstrom and Andersson, 1988). One potential route by which CYP1A might participate in such effects is alteration of arachidonic acid metabolism. PAH treatment of canine coronary and rabbit pulmonary arteries leads to dose-dependent increases in arachidonic acid-induced endothelium-dependent relaxations (Pinto et al., 1986), which can be reversed by general P450 inhibitors (Pinto et al., 1986) or the CYP1A-preferred substrate, 7-ethoxyresorufin (Fulton et al., 1992). Toxicity also could involve production of reactive oxygen species in endothelium; CYP1A binds but slowly metabolizes planar halogenated aromatic hydrocarbon (e.g., TCB), resulting in uncoupling and the release of reactive oxygen species (Schlezinger et al., 1999). Porcine aorta endothelial cells respond to TCB treatment with increased CYP1A1 content and activity as well as reactive oxygen species production and lipid peroxidation (Toborek et al., 1995). N-Acetylcysteine, an antioxidant, can prevent apoptosis of vascular cells, protecting against TCDD toxicity in fish embryos (Cantrell et al., 1996, 1998). Whether CYP1A is uncoupled by TCB or other substrates under conditions present in the endothelium must be determined.

In summary, in the eel model, CYP1A is induced by PAH and pHAH in a dose-dependent manner in microvascular endothelium, where it is metabolically active. The patterns of induction were inducer-specific in endothelium, as they were in liver (Schlezinger and Stegeman, 2000), with BNF and BP having an apparently greater potency than TCB. Penetration of CYP1A induction into the vascular bed of the rete mirabile is consistent with the idea that endothelial CYP1A can indicate local concentrations of inducers and possibly remove AHR agonists from the circulation, thereby potentially reducing their penetration peripherally. Endothelial CYP1A may both enhance and ameliorate the toxicity of AHR agonists by holding them in a sensitive cell type but also by slowing their spread to underlying tissues and distal areas. Rete mirabile in the eel swimbladder is a potential model for the study of CYP1A expression in endothelium in vivo and its role in the toxicity of AHR agonists.