Toxicological Sciences 55, 85-96 (2000)
Copyright © 2000 by the Society of Toxicology
Environmental Toxicology |
Effect of Micelle Fatty Acid Composition and 3,4,3',4'-Tetrachlorobiphenyl (TCB) Exposure on Intestinal [14C]-TCB Bioavailability and Biotransformation in Channel Catfish in Situ Preparations
* Department of Veterinary Physiology, Pharmacology and Toxicology, School of Veterinary Medicine, Louisiana State University, South Stadium Drive, Baton Rouge, Louisiana 70803; and
Department of Medicinal Chemistry, University of Florida, Gainesville, Florida 32610
Received September 9, 1999; accepted December 22, 1999
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Polychlorinated biphenyls are transferred in the diet along aquatic food chains. This study investigated the effect of dietary micelle composition and 3,4,3',4'-tetrachlorobiphenyl (TCB) exposure upon the subsequent systemic bioavailability and intestinal metabolism of [14C]-TCB in a catfish in situ intestinal preparation. Initial in vitro experiments examined the solubility of [14C]-TCB in micelles of varying fatty acid composition. Micelles composed of single fatty acids demonstrated greater [14C]-TCB solubility with those fatty acids of longer chain length. Similarly, micelles of the long-chain fatty acid, linoleic acid, solubilized more [14C]-TCB than mixed micelles formulated from equal amounts of myristic (14:0), palmitic (16:0), stearic (18:0), or linoleic (18:2) acids. Systemic bioavailability of [14C]-TCB (60 µM) from an in situ perfused intestinal preparation was 2.2-fold greater when delivered to the intestine in linoleic acid micelles as compared to the mixed micelle preparation. Catfish exposed in vivo to either 0.5 or 5.0 mg TCB/kg feed for 10 days resulted in a 45 to 47% decrease in the subsequent systemic bioavailability of [14C]-TCB in the in situ intestinal preparation. Total intestinal cytochrome P450 content was not significantly affected by TCB preexposure. Immunodetectable CYP1A was found only in the 5.0 mg TCB/kg diet treatment. Corresponding intestinal aryl hydrocarbon hydroxylase (AHH) activities were 2.46 ± 1.16, 2.43 ± 1.58, and 11.35 ± 10.25 pmol/min/mg protein for the control, 0.5, and 5 mg TCB/kg diet groups, respectively. [14C]-TCB in the in situ preparation was metabolized to only a small degree upon a single pass through the intestinal mucosa of the catfish. High variability and low rates of metabolism precluded the association of the magnitude of metabolism with dietary TCB pretreatment. Analysis of tissue sample extracts demonstrated 4 minor peaks, 3 of which were tentatively identified by co-elution with standards as 2-OH-3,4,3',4'-TCB, 4-OH-3,5,3',4'-TCB, and 5-OH-3,4,3',4'-TCB. A fourth remains unidentified. Histological changes in the intestine such as thinning of the submucosa and increased numbers of goblet cells were evident at the 5.0 mg TCB/kg diet dose. These results suggest that TCB intestinal bioavailability may be linked to micelle composition as well as TCB exposure history. Furthermore, single pass intestinal metabolism appears to be a minor contributor to the biotransformational modification of dietary TCB.
Key Words: polychlorinated biphenyls (PCBs); aquatic food chains; lipophilicity of PCBs.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Polychlorinated biphenyls (PCBs), ubiquitous contaminants in the aquatic environment (Fowler, 1990
; Kalmaz and Kalmaz, 1979), exhibit high chemical stability, resistance to degradation, and high lipophilicity (Safe, 1984). The high lipophilicity of PCBs results in the association of these chemicals with all components of the aquatic food chain (Connolly, 1991; James and Kleinow, 1994). PCBs undergo biomagnification through the food chain with higher trophic levels exhibiting greater accumulation than lower trophic levels (Biggs et al, 1980; Mayer et al, 1977; Oliver and Niimi, 1988; Rasmussen et al, 1990; Thomann and Connolly, 1984). The number of trophic steps in the food chain and composition of diet have been correlated to the magnitude of PCB transfer (Rasmussen et al., 1990). Mechanistic models suggest that 90 to 99% of lipophilic contaminants in higher trophic level fish may result from food chain exposure (Thomann, 1989; Thomann et al., 1986).
A variety of physiochemical factors modulate the dietary bioavailability of PCBs. For PCBs within the low to moderate Kow range, an increasing partition coefficient has been shown to result in an increasing rate of uptake in fish (Chiou et al, 1977; Russell et al, 1995). In contrast, a compound Kow above 6 appears to limit bioavailability (Gobas et al., 1988; Opperhuizen and Sijm, 1990). Likewise, halogenation of PCBs has been shown to influence bioavailability with assimilation efficiencies of dietary PCBs inversely proportional to the chlorine content (Bruggeman et al., 1984; Tanabe et al., 1982). In general, compounds with lower molecular weights (Bruggeman et al., 1984; Niimi and Oliver, 1988) and small molecular volumes (Niimi and Oliver, 1988) tend to be more readily absorbed.
Fat in the diet as well as chemical solubility in triglycerides have been shown to facilitate absorption of lipophilic contaminants (Kuksis, 1984). Micelles, formed by bile salt solubilization of dietary fat, are involved in the transport of lipophilic xenobiotics to the intestinal wall (Gobas et al., 1993). Two models, both including lipid as an integral mechanistic component, have been proposed for the gastrointestinal uptake of lipophilic xenobiotics in fish. A fugacity (leaving tendency) based model proposed by Gobas et al. (1993) suggests that the driving force for chemical uptake is generated by digestive processes in the gastrointestinal lumen. Absorption of dietary fat in the form of fatty acids, through reduction of the volume (increasing the relative concentration) and the changing character of the luminal contents, increases the fugacity of lipophilic xenobiotics from the intestinal lumen. This action facilitates the movement of lipophilic contaminants to the intestinal mucosa and ultimately the systemic circulation. A second model proposes that lipophilic xenobiotics are co-assimilated from the gastrointestinal tract in direct association with dietary lipids (Vetter et al., 1985). These models suggest that dietary uptake of xenobiotics against an apparent concentration gradient, as with biomagnification, occurs in the consumer at either the level of the intestine (model 1) or of the tissues of deposition, with the latter occurring largely as a result of lipid incorporation or utilization (model 2).
PCB bioavailability along the food chain can be modulated by biotransformation. In general, higher trophic levels exhibit PCB patterns enriched in highly chlorinated congeners (Boon et al., 1989; Muir et al. 1988). PCBs with unsubstituted adjacent para and meta positions, and congeners with unsubstituted adjacent ortho and meta positions and less than one orthochlorine are more readily biotransformed by oxidative systems such as cytochromes P450 as compared to PCBs with other characteristics (Boon et al., 1992; Walker, 1992). Metabolic activity limits the availability of these lesser chlorinated compounds to higher trophic levels as a result of enhanced elimination by lower organisms. Recent in vitro and in situ studies also suggest that intestinal biotransformation of dietary xenobiotics by the consuming fish can appreciably alter chemical form and systemic bioavailability (Kleinow et al., 1998; Van Veld 1991). Fish intestinal CYP1A activity, inducible by a variety of Ah receptor agonists (James et al., 1997; Kleinow et al., 1998; Van Veld et al., 1987; 1988a,b; 1991), may approach or even exceed concurrent hepatic levels following dietary exposure, when examined on a per mg protein basis (James et al., 1997; Van Veld et al., 1988b, 1991). The relative activity of oxidative pathways in the intestine has been shown to influence the degree of metabolism and composition of absorbed xenobiotics. Similarly, phase II biotransformation pathways in the fish intestinal mucosa have been shown to be operative in the further modification of dietary xenobiotics, with dose and phase I activity as important determinants of phase II contributions (Kleinow et al., 1998).
3,4,3',4'-Tetrachlorobiphenyl (TCB), a coplanar PCB, is abundant in environmental samples and animal tissues (McFarland and Clarke, 1989). The presence of TCB in higher organisms suggests that the compound may be subject to the foregoing dietary processes. While not much is known regarding the fate of TCBs in the fish gastrointestinal tract, TCB has been shown to be an inducer of hepatic CYP1A in fish (Gooch et al, 1989; Lindstrom-Seppa et al, 1994; Monosson and Stegeman, 1991; Otto et al., 1997; Sleiderink and Boon, 1996; White et al., 1997a ; Wirgin et al, 1992 ) and a substrate for fish hepatic CYP1A metabolism (White et al, 1997b). The induction of fish hepatic CYP1A by TCB has been shown to occur in a dose-dependent fashion in the low dosage range, with increases in CYP1A catalytic activity, as well as CYP1A transcription and protein expression both in vitro and in vivo (Gooch et al., 1989; Hahn et al., 1993; Lindstrom-Seppa et al., 1994). In contrast, high TCB concentrations have been reported to elicit an inhibitory effect upon hepatic CYP1A catalytic activity (Hahn et al., 1993; Monosson and Stegeman, 1991; White et al., 1997a).
The following studies examined [14C]-TCB bioavailability from the catfish intestine with alteration of 3 factors: luminal lipid composition, existing TCB body burdens and intestinal biotransformation. The specific objectives of this study were to examine the effect of micelle fatty acid composition upon the solubility of TCB in micelles, the effect of micelle composition on TCB intestinal bioavailability in an in situ preparation, the effect of a 10-day in vivo TCB exposure (0.5 or 5 mg TCB/kg diet) upon the bioavailability and biotransformation of subsequent [14C]-TCB exposures using an in situ intestinal preparation, the effect of various TCB dosages upon CYP1A content and activity in the intestine, and the effect of TCB dietary exposure upon intestinal morphology and integrity.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Chemicals
Radiolabeled (27.3 mCi/mM) [14C]-3,4,3',4'-tetrachlorobiphenyl (TCB) (98% isomer specific) was graciously supplied by H. B. Matthews, NIEHS, Research Triangle Park, NC. Nonradiolabeled TCB was purchased from ChemService Inc., West Chester, PA. The standards for the known hydroxylated TCB metabolites were generously donated by Dr. L. W. Robertson, University of Kentucky, Lexington, KY. Tricane methane sulfonate (MS-222) was obtained from Argent Chemical Company, Redmond, WA. TS-1 tissue solubilizer was procured from Research Products International, Mount Prospect, IL. All other chemicals were obtained from Sigma Chemical Co., St. Louis, MO.
In Vitro TCB Solubility in Micelles
TCB solubility in micelles was examined in vitro in micelles of differing fatty acid composition, using a modification of previously described methods (Hollander and Rim, 1978). The various fatty acids selected were forms commonly found in tissues of catfish held at 20°C as determined by previous assays (Toth et al., 1993). Micelle mixtures (1 ml) were formulated at 40°C, by gentle mixing of 0.9% saline, 10 mM sodium taurocholate (bile salt for fatty acid emulsification), plus 10 mM of either one or a combination of 4 fatty acids (each exhibiting different chain lengths and degrees of saturation): (a) lauric acid 12:0; (b) myristic acid 14:0; (c) linoleic acid 18:2; and (d) a mixture of equal concentrations (2.5 mM) of myristic acid 14:0, palmitic acid 16:0, stearic acid 18:0, and linoleic acid 18:2. Micelle production using these methods was confirmed by optical clarity over time and filtration.
TCB and [14C]-TCB (0.05 µCi) (20 µM total) in a toluene carrier (50 µl) was added on top of 0.9% saline (1 ml) in a conical glass tube. The solvent carrier was slowly evaporated under nitrogen until only a thin surface layer of toluene with the TCB remained. This step removed excess toluene, which would otherwise precipitate the micelle mixture. Warm micelles (1 ml) as formulated above were slowly added below the surface of the toluene, allowing the micelle mixture contact with the underside of the toluene layer by temperature driven convection. TCB was allowed to partition into the micelles as the remaining toluene was evaporated with nitrogen. Following complete evaporation of the toluene layer, the solution was rigorously gassed with nitrogen to remove any remaining toluene and then shaken. Due to the nature of the micelle mixture (micelles in aqueous saline) the hydrophobic TCB not associated with the micelles was partitioned by hydrophobic interactions to the edge of the aqueous phase onto the glass. Relative [14C]-TCB content in micelles of each treatment was measured by liquid scintillation counting of timed samples using 3 replicates (100 µl) from each micelle solution. Total TCB remaining in the micelle solution was expressed as a percentage of the total dose delivered.
In Situ Studies
Animals.
Channel catfish (Ictalurus punctatus) of either sex (1379 ± 319g) were obtained from the Louisiana State University Aquaculture Research Station at Baton Rouge, LA. Fish were kept on a 12-h light/dark photoperiod, in a flow-through system supplied with dechlorinated tap water (pH 8.1 ± 0.2, temperature 19.4 ± 1.4°C, total hardness 27.1 ± 7.0 mg CaCO3/l, alkalinity 114.3 ± 12.9 mg/l). All animals were acclimated to experimental conditions at least 2 weeks prior to use. Animals were fed a custom made, purified, semisynthetic catfish diet (Dyets Inc., Bethlehem, PA) composed of casein 32%, dextrin 29.8%, cellulose 19%, soybean oil 3%, Menhaden oil 3%, gelatin 8%, salt and vitamin mix 5%, and choline chloride 0.17%.
In situ intestinal perfusions.
Catfish of both sexes were used for surgery and the in situ intestinal preparation. The first experiment examined the effect of micelle carrier composition on [14C]-TCB bioavailability in an in situ intestinal preparation of control animals. Following in situ intestinal preparation [14C]-TCB (60 µM) was delivered into the intestinal lumen in a micelle carrier composed of either (1). 2.5 mM monolauroyl-rac-glycerol (a common mono-acyl -glycerol which is an incomplete lipolysis product), 10 mM sodium taurocholate, and the mixture of 2.5 mM myristic acid, 2.5 mM palmitic acid, 2.5 mM stearic acid and 2.5 mM linoleic acid (n = 3), or (2) 2.5 mM monolauroyl-rac-glycerol, 10 mM sodium taurocholate, and 10 mM linoleic acid (n = 5). In a second experiment designed to examine the effect of TCB preexposure upon subsequent [14C]-TCB intestinal bioavailability and metabolism, animals were exposed 10 days prior to the in situ preparation to control diets (n = 3) or diets containing nonradiolabeled TCB at 0.5 mg (n = 6), and 5 mg TCB/kg diet (n = 7). For all these treatments, [14C]-TCB (60 µM) was delivered in the in situ preparation, using the 10 mM linoleic acid micelle.
For the respective treatments of both experiments, control animals were maintained on the semisynthetic diet coated with corn oil (1 ml corn oil/100 g of diet) while for the second experiment TCB was delivered in corn oil applied as a coating on the semisynthetic diet (1 ml corn oil/100 g of diet). Both dietary groups (control and dietary TCB exposure) were maintained on designated experimental diets at 0.5% of fish body weight/day for 10 days prior to the in situ intestinal preparation. Fish were fasted 24 h prior to experimental manipulation.
In situ [14C]-TCB dose preparation.
The micelle solution of defined composition used for intraluminal [14C]-TCB dosing of the intestinal preparation was formulated fresh daily as described in the in vitro section. Micelle compositions containing 60 µM of [14C]-TCB (27.3 mCi/mmol) were formulated to a final volume of 3 ml.
Surgical procedure and dosing.
Surgical preparations were performed as previously described (Kleinow et al., 1998). MS-222 buffered with NaHCO3– was used for anesthesia at induction and maintenance dosages of 106 and 86 mg/L, respectively. The intestine was exteriorized and a ventral loop of proximal intestine of approximately 20 cm in length was selected for the preparation. Vessels perfusing the selected intestinal segment, including a branch of the coeliacomesenteric artery as the afferent (supply) vessel, the corresponding efferent (drainage) vessel, and potential collateral vessels were identified and isolated. The afferent vessel was cannulated using PE-50 tubing filled with saline treated with citrate anticoagulant. Once in place, oxygenated blood containing citrate anticoagulant was pumped into the afferent cannula at a flow rate of 0.1 ml/min. PE-60 tubing was used to cannulate the efferent vessel, and collateral vessels were tied off. Integrity of the perfusion was verified through measurement of efferent blood volumes. Upon completion of the circuit, the intestinal segment was transiently blanched with a small amount of saline containing citrate anticoagulant via the afferent cannula, in order to demarcate the length of intestine perfused by the preparation. Ligatures were placed on the intestine at the borders of the perfused region. Delivery of the micelle solution was performed by inserting the needle of the dosing syringe through the gut wall, securing the preplaced ligature over the needle and injecting the [14C]-TCB micellar dose into a closed intestinal segment. The injected dose was then massaged to allow distribution throughout the segment, and the time was then initiated.
Sample collection.
Throughout the 60-min perfusion, the intestine was moistened with saline, the segment periodically massaged, and a composite blood sample collected. After the 60-min perfusion, the isolated, ligated intestinal segment was drained and the postinfusate collected. Aliquots of the postinfusate were taken for liquid scintillation counting and metabolite analysis. The intestinal segment was then cut open, washed copiously with ice cold saline and blotted dry. The mucosal layer was collected by scraping with a glass slide. Aliquots of weighed mucosal tissue were taken for liquid scintillation counting, metabolite analysis, and as preparations for P450 content and activity. Liver, kidney, and anesthetic water samples were collected and counted for radioactivity, to verify, by the lack of counts, the integrity of the isolated preparation.
Blood, liver, kidney, mucosa, and postinfusate samples (approximately 50 mg) were digested at 50°C in 0.5 ml of TS-1 tissue solubilizer for 24 h, neutralized with 18 µL of glacial acetic acid, and counted in 4.5 ml of scintillant (Ultima Gold, Packard, Downers Grove, IL). Water and preinfusate samples were not digested prior to counting. Corrections for counting efficiency (~95%) and background were utilized for all samples. Preinfusate, postinfusate, intestinal mucosa, and blood sampled for metabolite analysis were snap frozen with liquid N2, wrapped in foil, and kept at –80°C until analysis. Mucosal samples for P450 related analyses were suspended in buffer consisting of 0.25 M sucrose, 0.1 mM EDTA, 0.05 M Tris–HCl, pH 7.4, and 0.1 mM phenylmethyl sulfonyl fluoride (PMSF), and similarly frozen.
Enzyme assays and P450 content.
Mucosal samples were weighed and homogenized in 4 volumes of buffer (0.25 M sucrose, 5 mM EDTA, 0.05 M Tris-HCl, pH 7.4, 0.2 mM PMSF). Microsomal and cytosolic fractions were prepared using the procedure described by James and Little (1983). Total P450 content of microsomes was measured following the method of Estabrook et al. (1972), using a Shimadzu 265 spectrophotometer, as previously described (James et al., 1988). Monooxygenase activity was measured by a fluorescence assay (AHH) of phenolic BaP metabolites (Nebert, 1978).
Western blot analyses.
Microsomal protein fractions (40 µg for intestine, 20 µg for liver), incubated in sample buffer as recommended by BioRad, were resolved in a mini gel format (BioRad) on 4% stacking gel with 8.5% resolving gels, as described by Laemmli (1970). Unstained and prestained molecular weight standards in the range 14,400 to 97,000 (Bio-Rad low molecular weight range) were resolved at the same time as the SDS-treated microsomes. Gentest SupersomesTM expressing rat CYP1A were used to develop a standard curve for quantitation of the antibody response. Electrophoresis was carried out using a 25 mM Tris/192 mM glycine/0.10% SDS buffer at constant voltage of 200V. Western blot transfer of protein to nitrocellulose was performed as described by Towbin et al (1979). The transfer was carried out at 100V in a mini Transblot system (BioRad) using a 25 mM Tris/192 mM glycine/20% v/v methanol/pH 8.3 transfer buffer. The remaining gel was stained with Coomassie blue as an indication of transfer effectiveness.
Immunodetection was carried out using monoclonal antibodies to scup CYP1A (courtesy of Dr. J. J. Stegeman). Transblotted nitrocellulose was rinsed in a 20 mM Tris, 500 mM NaCl, pH 7.5 buffer and nonspecific binding sites blocked with 5%(w/v) dried milk in 20 mM Tris–HCl, pH 7.5, 500 mM NaCl, 0.05% Tween 20 for 45 min. The membrane was washed 4 times with 20 mM Tris, 500 mM NaCl, 0.05% Tween20, pH 7.5 buffer. The primary antibody, diluted 1:10,000 in 5% (w/v) dried milk in 20 mM Tris–HCl, pH 7.5, 500 mM NaCl, 0.05% Tween 20, was incubated with the nitrocellulose for 2 h. The unbound antibodies were washed away in 20 mM Tris–HCl, pH 7.5, 500 mM NaCl, 0.05% Tween 20 buffer and further incubated with a 1:1000 dilution of secondary antibody in blocking agent (rabbit anti-rat antibody conjugated to horseradish peroxidase) for 1 h. After washing with 20 mM Tris–HCl, pH 7.5, 500 mM NaCl, 0.05% Tween 20, the immunoreactive proteins were detected according to the Amersham Western Blotting kit for chemiluminescent detection and the protein bands were visualized by fluorography on Kodak X-OMAT AR films. Fluorograms were subsequently scanned and the protein bands were quantified by scan-analysis densitometry.
Metabolic studies.
Samples of blood and postinfusate (1 ml) were extracted 3 times with (3 ml) heptane:ethanol, 19:1, and the 3 extracts pooled. Intestinal mucosa (0.3 to 0.6 g) was homogenized with 1 ml distilled water and extracted 3 times with (3 ml) heptane:ethanol, 19:1. For each sample, the pooled heptane:ethanol extracts were dried over anhydrous sodium sulfate, evaporated to dryness under nitrogen, and stored at –80°C until HPLC analysis. Before analysis, the residue was dissolved in 0.1 ml 78% methanol:water, the solution filtered (0.45 µm nylon filter) and 50 µl injected onto a pre-equilibrated C18 reverse phase HPLC column. Initial HPLC conditions at a flow rate of 1 ml/min were 78% methanol:22% water (held for 20 min) followed by a gradient to 100% methanol over 5 min (held for 10 min), and then returned over 5 min to 78% methanol. Analysis was accomplished using UV (262 nm) and radiochemical detection (INUS detector). Under these conditions, baseline separation of the known TCB metabolites was achieved. 2-OH,3,3',4,4'-TCB eluted at 17.7 min, 4-OH-3,3',4',5-TCB at 18.2 min, 5-OH-3,3',4,4'-TCB at 18.7 min, 6-OH-3,3',4,4'-TCB at 19.6 min, and 3,3',4,4'-TCB eluted at 22.6 min.
Histological studies.
To assess if any morphological changes were induced in the intestine with TCB exposure catfish (n = 18) were exposed to TCB following the same protocol as used for the in situ preparation. Animals were divided into 3 equal groups: control, 10-day dietary TCB exposure at 0.5 mg/kg diet, and 10-day dietary TCB exposure at 5 mg/kg diet. Following exposure, intestines were harvested, rinsed with ice cold 0.9% saline, and samples of proximal and distal sections collected for histological preparation. Samples were fixed in 10% formalin for at least 24 h, and 4 µm sections stained with hematoxylin-eosin using conventional techniques.
Statistical analysis.
Data analysis was completed using the statistical software SigmaStat (Windows version 1.0, Jandal Corporation, San Rafael, CA). One way ANOVA, Student's t-test, or corresponding nonparametric tests were used to examine for significant differences at p < 0.05. Multiple comparisons (Dunnet`s or Student-Newman-Keuls) were used when ANOVA demonstrated significant differences.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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[14C]-TCB carrying capacity of micelles varied, under in vitro conditions, with the fatty acid acyl side chain composition (Fig. 1
). As the fatty acid chain length increased from C12 to C18, there was a trend towards increased [14C]- TCB solubility. [14C]-TCB mean solubilities were significantly higher (2.6-fold) in linoleic acid (18:2) compared to lauric acid (12:0) micelles (ANOVA, Student Newman-Keuls: p < 0.05), while myristic acid (14:0) micelles showed an intermediate solubility, not significantly different from the other 2 (ANOVA, Student Newman-Keuls: p < 0.05). Mixed micelles composed of equal amounts of myristic acid 14:0, palmitic acid 16:0, stearic acid 18:0, and linoleic acid 18:2 exhibited a 3- to 7.6-fold lower [14C]-TCB solubility when compared to micelles composed of the individual fatty acids.
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The influence of [14C]-TCB micelle solubilization upon systemic bioavailability was examined in the in situ intestinal preparation using the micelle compositions exhibiting the greatest (linoleic acid) and least efficient [14C]-TCB solubilization (mixture). With equivalent [14C]-TCB dose and preparation techniques, linoleic acid micelles facilitated a significantly greater systemic [14C]-TCB bioavailability than the mixed micelle solution (Fig. 2
). The mean systemic bioavailability with linoleic acid micelles (895.9 ± 217.9 pmol [14C]-TCB/ml blood) was approximately 2.2-fold higher than obtained with mixed micelle solution (401.9 ± 334.5 pmol [14C]-TCB/ml blood)(t-test: p < 0.05).
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TCB at both the 0.5 and 5 mg/kg dosages, when administered in the diet for 10 days, significantly decreased subsequent [14C]-TCB systemic bioavailability to the blood of the in situ preparation (Fig. 3
) (ANOVA, Student Newman-Keuls: p < 0.05). When compared to the control animals (896 ± 217.9 pmol [14C]-TCB/ml of blood), TCB at both 0.5 and 5 mg TCB/kg diets decreased blood concentrations of [14C] TCB 47% (475.7 ± 134.4 pmol/ml of blood) and 45% (495.6 ± 160.2 pmol/ml of blood), respectively. There was no significant interdose effect of TCB dietary preexposure upon the intestinal systemic bioavailability of [14C]-TCB.
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[14C]-TCB concentrations in intestinal mucosa, following the in situ perfusion, did not change with the increasing in vivo doses of the TCB preexposure (Fig. 3
). Mucosal [14C]-TCB levels of 16.2 ± 5.6, 15.6 ± 7.9, and 13.9 ± 5.5 nmol/g mucosa were observed for control, 0.5 mg TCB, and 5 mg TCB/kg-diet exposure groups, respectively. Mean postinfusate [14C]-TCB concentrations were 19.6 ± 6.3, 28.4 ± 17.0, and 30.7 ± 9.5 nmol [14C]-TCB/ml for the same treatments (Fig. 3). The effects of TCB preexposure upon [14C]-TCB concentrations were not significant for either the mucosa or postinfusate (ANOVA: p > 0.05). For all treatments, the highest concentrations of [14C]-TCB on a per-unit basis were found in the postinfusate, followed by the mucosa and then the blood. Postinfusate [14C]-TCB concentrations were approximately 1.2, 1.8, and 2.2 times higher than mucosa concentrations in the controls and animals preexposed to 0.5 and 5 mg TCB/kg diets, respectively. Mucosa concentrations compared to blood were 18.1, 28.1, and 32.9 times greater for the same treatments. Consistent over all treatments, the largest contributor to the gradient difference from the intestinal lumen to the blood was that evident between the mucosa and the blood. The gradient differences observed between the lumen and blood were the lowest for controls (21.9x) rising nearly 3-fold for TCB-pretreated animals (59.7 to 61.9x). These results reflect the higher blood and lower postinfusate [14C]-TCB concentrations for the controls and the lower blood and higher postinfusate [14C]-TCB concentrations for TCB-pretreated animals.
Microsomal and cytosolic fractions produced by differential centrifugation of mucosa were examined for [14C]-TCB radioactivity. As compared to the microsomes, lower amounts of radioactivity were associated with the cytosol on a per mg protein basis. The microsomal fractions exhibited similar amounts of [14C]-TCB-derived radioactivity for all treatment groups (0.196 ± 0.039, 0.201 ± 0.60, and 0.236 ± 0.049 nmol [14C]-TCB/mg microsomal protein for control, 0.5mg and 5.0 mg TCB/kg diet treatments, respectively). Control treatment cytosolic radioactivity values (65.6 ± 23.6 pmol [14C]-TCB/mg cytosolic protein) were significantly higher than those for either the 0.5 mg TCB/kg diet (32.4 ± 6.03 pmol [14C]-TCB/mg cytosolic protein) or 5.0 mg TCB/kg diet (41.48 ± 14.57 pmol [14C]-TCB/mg cytosolic protein) treatments (p < 0.05).
Total P450 content, CYP1A content, and aryl hydrocarbon hydroxylase (AHH) activities were measured, to assess the effect of TCB preexposure upon the metabolic capabilities of the catfish intestine. Mean P450 concentrations (between 0.07 and 0.09 nmol/mg protein) were not significantly altered with TCB pretreatment (Fig. 4)(ANOVA: p > 0.05). CYP1A cross reactivity was not detected for either the controls or animals of the 0.5 mg TCB/kg diet treatments. CYP1A levels were variable for the 5.0 mg TCB/kg diet treatment, with values ranging from 0.14 to 24.11 pmol/mg protein (Fig. 5). Composite AHH activities were 2.46 ± 1.16, 2.43 ± 1.58 and 11.35 ± 10.25 pmol/min/mg protein for the control, 0.5 and 5.0 mg TCB/kg diets, respectively (Fig. 6). AHH activities of the 5.0 mg/kg treatment were not significantly greater than controls or the 0.5 mg/kg diet treatments due to the high standard deviation of the data (ANOVA: p > 0.05). Four animals demonstrated large increases (~7-fold) in AHH activities, while 3 animals exhibited levels similar to the controls. AHH activity exhibited a strong correlation (r2 = 0.96) with CYP1A cross reactivity (y = 1.143x + 1.026) (Fig. 7).
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TCB was metabolized to a small degree (0.05 to 1.3%) in the intestinal mucosa of individual catfish in in situ preparations (Table 1
). Analysis of tissue sample extracts by HPLC demonstrated 4 minor peaks which eluted before the parent. Three of these peaks were tentatively identified as 2-OH-3,4,3',4'-TCB, 4-OH-3,5,3'4'-TCB, and 5-OH-3,4,3',4'-TCB by co-elution with standards. The fourth peak eluted much earlier (12.1 min) but remains unidentified.
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TCB elicited few morphological effects in the intestinal tract at the 0.5 mg/kg diet dosage level whereas at 5.0 mg/kg, demonstrable changes were evident when compared to the controls. Among the changes consistently present were a narrowing or thinning of the submucosa with submucosal separation a common feature (Fig. 8
). Goblet cell numbers were also increased along the mucosal folds of TCB-treated catfish.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The solubilization of [14C]-TCB in micelles, when varying the chain length and degree of saturation of the component fatty acids showed a significant 156% increase in [14C]-TCB solubility for micelles containing linoleic acid (18:2) as compared to lauric acid (12:0). An increase in solubilization of TCB was noted with increasing fatty acid chain length (linoleic acid 18:2 > myristic acid 14:0 > lauric acid 12:0). Studies with other compounds including vitamins (Takahashi and Underwood, 1974
) and the PCB Aroclor 1242 (Laher and Barrowman, 1983) have demonstrated increased solubilization in micelles composed of long acyl side chain fatty acids. Laher and Barrowman (1983) demonstrated that Aroclor 1242 solubility in linoleic (18:2) and oleic acid (18:1) micelles was 146 and 85% higher, respectively, compared to octanoic acid (8:0). In this regard, 3,4, 3',4'-TCB behaves in a manner similar to Aroclor 1242. The current studies suggest that 3,4,3',4'-TCB delivery to the intestinal wall and even, perhaps, retention in foodstuff lipid may be dependent upon the fatty acid composition. Stearic factors between the size of the micelle and the compound's molecular volume have been forwarded as a possible explanation for similar findings (Takahashi and Underwood, 1974). Mixed micelles in the current study appear to support a lower 3,4,3',4'-TCB carrying capacity than micelles composed of a single fatty acid type. The reasons for these findings are unknown; however, it appears likely that such a mixed composition influences the determinant stearic factors. Fatty acid composition may be an important consideration for fish, as chain length and even saturation status may change seasonally with temperature and with diet.
In the proximal intestinal segment, as used in our in situ study, the systemic bioavailability of [14C]-TCB molar equivalents (Meq) (composite of [14C]-TCB parent compound and metabolites) was significantly higher (~123%) when delivery was accomplished using linoleic acid micelles as when compared to micelles composed of a fatty acid mixture (myristic, palmitic, stearic, and linoleic acids). These results indicate that the differences in bioavailability of [14C]-TCB Meq in the catfish intestine may be related to the differences in micelle fatty acid composition and the TCB solubility in those micelles. Studies in the killifish indicate that the presence of fat in the diet facilitates absorption of lipophilic contaminants (Van Veld, 1990). Likewise, the solubility of PCB, DDT, and BaP in triglyceride was directly correlated to intestinal absorption efficiency (Van Veld, 1990). The current studies take this concept one step further by suggesting that micelle composition may play a role in the modulation of lipophilic xenobiotic delivery to or through the intestinal wall. One mechanism that may contribute to the apparent differences in [14C]-TCB Meq absorption with differences in micelle composition is the rate- and site-dependent intestinal absorption of fatty acids. Ockner et al. (1972), comparing absorption of linoleic acid micelles with palmitic acid micelles (19.2 mM fatty acid, 9.6 mM glyceryl-1-monooleate, 20 mM sodium taurocholate) in rats, demonstrated a greater uptake of linoleic acid compared to palmitic acid (~37%) in the proximal intestine. In addition, these studies showed that linoleic acid was absorbed primarily by proximal intestine, and palmitic acid by distal intestine. Absorption of triolein in killifish has been shown to occur primarily in the proximal intestine (Honkanen et al., 1985).
Blood [14C]- TCB Meq concentrations were diminished, postinfusate levels were elevated and mucosal concentrations remained about the same for TCB pretreated animals as compared to the controls in the in situ preparations. These trends in tracer distribution suggest that after TCB prexposure at either dose, the subsequent [14C]-TCB Meq dosage was not as effectively absorbed. The systemic bioavailability of [14C]-TCB Meq in the 60-min in situ preparation of control catfish was 2.9% of the total dose delivered in the linoleic acid micelles. Catfish preexposed to 0.5 and 5 mg TCB/kg diet exhibited, under identical in situ exposure conditions, [14C]-TCB Meq bioavailabilities of 1.41 and 1.51%, respectively. In vivo PCB accumulation studies with catfish have demonstrated diminished uptake and rate of uptake following PCB preexposure in the diet (Hansen et al., 1976) or by the sediments (Dabrowska et al., 1996). Similarly, models by Barber et al. (1991) and Clark et al. (1990) have described decreases in PCB uptake with increases in body burden. The in situ character of the current studies indicate that the decreases in [14C]-TCB uptake with TCB preexposure occur on a direct and temporally acute basis at the level of the intestine. Dietary pretreatment with unlabeled TCB effectively altered the [14C]-TCB gradient established by the end of the in situ preparation. The composite postinfusate to blood [14C]-TCB gradients were approximately 21:1 for controls and 59.5 to 61.5:1 for TCB-pretreated animals. These changes with TCB pretreatment appeared to occur by a combined effect of lowering transport to the blood and retention of [14C]-TCB in the postinfusate. These changes in systemic [14C]-TCB uptake were evident in this isolated preparation, although mucosa [14C]-TCB levels were not significantly different. For all treatments, the greatest [14C]-TCB Meq gradients at the end of the perfusion were between the mucosa and the blood (18- to 33-fold) rather than between the postinfusate and the mucosa (1.2- to 2.2-fold). This observation probably reflects the relative lipid content of these compartments.
A variety of mechanisms could decrease the systemic bioavailability of [14C]-TCB Meq following TCB preexposure. Diffusion, one of those mechanisms, is often cited as a major means for the intestinal transfer of uncharged lipophilic xenobiotics in fish (McKim and Nichols, 1994). TCB preexposure may increase TCB tissue loads, which would decrease the diffusion gradient available to [14C]-TCB from the intestinal lumen to the systemic circulation. Unfortunately, unlabeled TCB residues were not measured to directly confirm this hypothesis. While lower blood [14C]-TCB concentrations with TCB pretreatment are consistent with this hypothesis, a couple of pieces of information do not wholly fit. The first is the lack of a clear dose-response relationship for [14C]-TCB at the 0.5 and 5.0 mg TCB/kg dosages. Another confounding feature is the observation that mean mucosal [14C]-TCB concentrations were very similar for all treatments. It would appear logical that the gradient effects would be clearly seen with [14C]-TCB in this intermediate compartment. An interesting observation, which may relate to these discrepancies, is the significantly lower intestinal mucosa cytosolic [14C]-TCB Meq values with TCB pretreatment. This effect may be due to unlabeled TCB occupying or modifying sites on proteins or lipids necessary for transport of [14C]-TCB through the cytosol. In concert with the diffusional process, such a mechanism could impair [14C]-TCB Meq transport to the systemic circulation. A number of cytosolic moieties have been described as transporters for xenobiotics including glutathione S-transferase (Dixit et al., 1982), fatty acid binding proteins (Larsen et al., 1991), nuclear transfer proteins (Tierney et al., 1980) and lipoproteins (Vetter et al., 1985). The lack of a clear relationship of the total mucosal [14C]-TCB counts to the gradient effects seen in other compartments is puzzling. Perhaps it is a reflection of the phenomenon observed with the microsomal membranes whereby [14C]-TCB Meq levels were held constant across treatments. Plasma membranes and other structures removed in the first differential centrifugation accounted for a large part of the total composite mucosal [14C]-TCB Meq and may have demonstrated a similar effect hiding any response to TCB exposure. The mechanism and the significance of such a response will require more investigation.
A number of studies have demonstrated that exposure of animals, including fish, to certain organochlorines alters the distribution of a subsequent tracer dose of the same [14C] organochlorine (Carpenter and Curtis, 1989; 1991; Gilroy et al., 1993; compounds dieldrin and chlordecone). A suggestion has been made that these effects, independent of changes in xenobiotic metabolism or total body lipid content, may be related to lipid composition. Qualitative changes in lipids have been correlated with organochlorine exposure, including changes in fatty acid profiles, alterations in triglycerides and phospholipids (Hansell and Ecobichon, 1974; Ishikawa et al., 1978), and modifications of plasma lipoproteins (Ishikawa et al., 1978). Plasma lipoprotein binding has been shown to facilitate organochlorine transport (including PCBs) in a variety of animal species (including rat, trout, human)(Maliwal and Guthrie, 1982; Plack et al., 1979; Skalsky et al., 1979; Soine et al., 1982; Spindler-Vomachka and Vodicnik, 1984). Given the changes in disposition induced by other organochlorines, the possibility exists that TCB may elicit similar effects altering either the temporary storage or transport by the intestine itself or by the blood.
Other processes that may be influential in xenobiotic bioavailability across the intestine may be inducible by TCB pretreatment. P-glycoprotein, an efflux transporter, has been identified in the catfish intestine (Kleinow et al., 1996). This transporter would reduce bioavailability by the transport of absorbed compound from the intestinal mucosa back into the intestinal lumen. Such an action could account for the observed findings. There currently exist conflicting results in the literature regarding the suitability of PCBs for transport by P-glycoprotein in marine organisms (Galgani et al., 1996; Cornwall et al., 1995). Likewise, there are even contrasting responses relative to the inducibility of P-glycoprotein in the catfish intestine with 3,4,3',4'-TCB dependent upon the assay methodology (Kleinow, unpublished data). Induction of binding proteins by Ah receptor agonists have also been shown to alter the disposition of organochlorines, such as TCDD, in the liver of mice (Poland et al., 1989). The significance of such mechanisms relative to these results can be only speculative at this time.
Since TCB has been shown to be an inducer of CYP1A and its associated activity in the fish liver, pretreatment with TCB may also alter intestinal [14C]-TCB Meq bioavailability by enhancing metabolism and the production of polar metabolites. Metabolism data included herein indicates, however, that [14C]-TCB was very recalcitrant to intestinal metabolism, with only small changes in the metabolite profile from TCB pretreatment. Lack of a correlation between changes in bioavailability and the metabolites formed further suggest that metabolism does not, or at best can only minimally, account for the observed differences.
It is plausible that the decrease in the systemic bioavailability of [14C]-TCB may be related in part to a nonspecific effect, caused by a toxic action of TCB pretreatment on the intestine. The 10-day dietary preexposure in the current study, which provided a TCB dose of approximately 25 µg of TCB/kg body weight/day (5 mg TCB/kg diet at 0.5% body weight), resulted in a thinning of the submucosa and an increase in mucus-secreting goblet cells. An increase in mucus production could contribute to a thickening of the unstirred layer along the intestinal wall, thus increasing the diffusion distance and decreasing the rate of uptake. Similar effects are known to modulate nutritive and xenobiotic absorption in mammals (Kellaway and Marriot, 1975; Smithson et al., 1981). Considerable variation in the gastrointestinal toxicity of PCBs has been reported for fishes. A PCB mixture containing 2-chlorobiphenyl, 2,2'-dichlorobiphenyl, 2,5,2'-trichlorobiphenyl and 2,5,2',5'-tetrachlorobiphenyl (125 µg PCB/kg body wt/day for 28 days) elicited an increase in mucosal exfoliation, cytoplasmic inclusions in the columnar cells and a reduction or absence of the mucosa brush border in some areas of the Chinook salmon intestine (Hawkes et al., 1980). In contrast, channel catfish fed 600 µg PCB (Arochlor 1242)/kg body weight/day for more than a 100-day period exhibited no histopathological changes in the gastrointestinal tract (Hansen et al, 1976). It is unclear whether differences between studies are related to the PCBs used, the dosage, or other factors.
Intestinal AHH activity and CYP1A content were not affected by the 0.5 mg TCB/kg-diet pretreatment and showed induction, but great variability, with the 5.0 mg TCB/kg diet. The consistency of AHH activities at near-control values for the 0.5 mg TCB/kg-diet dose, and the lack of detectable CYP1A, suggest that this dosage may be insufficient to induce intestinal AHH activity when fed at approximately 0.5% of body weight. Conversely, detectable CYP1A content and AHH activities ranging from control levels to more than 12-fold higher with the 5 mg TCB/kg diet dosage indicate that TCB is capable of intestinal induction. Previous studies have demonstrated dose-dependent induction and inhibition of fish hepatic CYP1A catalytic activities by TCB with induction at low and high doses while inhibition occurs only at high dosages (Gooch et al., 1989; Lindstrom-Seppa et al., 1994; White et al., 1997a). The differential response seen in the catfish intestine is most likely differential induction resulting from factors associated with compound ingestion and bioavailability. Direct correlation of AHH activity with CYP1A content for individual animals suggests that the variability was not related to TCB inhibitory effects. Studies determining TCB bioavailability and local TCB concentrations in the intestine following dietary exposure will be required to elucidate the dosimetry and extent of induction and in turn the inhibitory effects of TCB in the catfish intestine.
[14C]-TCB, as administered in the in situ intestinal preparation, was poorly metabolized during the 60-min perfusion. In controls, approximately 0.55% of [14C]-TCB Meq in the mucosa were metabolites while 0.57 and 0.35% of the [14C]-TCB Meq in the 0.5 and 5.0 mg TCB/kg diet treatments were metabolites. Intestinal [14C]-TCB metabolism was highly variable between animals, but in all cases was quantitatively a minor event. Of the small amount of metabolites formed in the mucosa, the predominate form tentatively identified by co-elution with standards for both the controls and TCB-pretreated animals was 2-OH-TCB. 4-OH-TCB was found as a metabolite of [14C]-TCB in only 3 of 10 TCB-pretreated catfish. The catfish intestine exhibited no changes in metabolite profile with dose. In contrast, TCB metabolites in scup (Stenotomus chrysops) bile demonstrated a differing profile with the magnitude of the TCB dosage (White et al., 1997b). At a dosage of 0.1 mg TCB/kg, 5-OH-TCB was the major metabolite (85%) followed by 4-OH-TCB (13%) and 2-OH-TCB and 6-OH-TCB (1% each). Bile of scup treated with 5 mg TCB/kg contained higher amounts of 4-OH-TCB, comparable to the amounts of 5-OH-TCB. White et al. (1997b), in their study with scup, hypothesized that high doses of TCB inhibited CYP1A allowing other CYP to produce greater amounts of 4-OH-TCB through a rearrangement of a 4,5-epoxide. Results of the current study suggest that CYP1A is a minor constituent of the total CYP in the catfish intestine. The foregoing line of reasoning would suggest that the intestine should proportionally produce more 4-OH-TCB rather than less. Further studies are needed to determine if the differences with the current study are organ, species or experimentally related.
These studies have demonstrated (1) that fatty acid composition has an appreciable effect on the micelle-carrying capacity and subsequent intestinal bioavailability of [14C]-TCB, (2) that a previous TCB exposure may reduce the subsequent bioavailability of [14C]-TCB to the systemic circulation (this effect is not related to first pass intestinal metabolism), and (3) that first pass intestinal metabolism of TCB is a minor event with the production of small amounts of metabolites, including 2-OH-3,4,3',4'-TCB, 4-OH-3,5,3',4'-TCB and 5-OH-3,4,3',4'-TCB.
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ACKNOWLEDGMENTS |
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This work was funded by the Superfund Basic Research Program via NIH grant P42 ES-07375. Our special thanks for the technical assistance rendered to M.A.J. and K.M.K. by Sylvia Shreve, RN.
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NOTES |
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1 To whom correspondence should be addressed. Fax: (225) 346–5736. E-mail: kleinow{at}svmmac.vetmed.lsu.edu.
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