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Department of Medicinal Chemistry (M.O.J., Z.T.), University of Florida-Gainesville; The Whitney Laboratory (M.O.J., A.H.A., K.M.), University of Florida-St. Augustine; and Department of Veterinary Physiology, Pharmacology, and Toxicology (K.M.K.), Louisiana State University
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Abstract |
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Abstract
Introduction
Results
Discussion
References
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These studies demonstrated that intestinal mucosa of the channel
catfish contained activities comparable with liver for several phase 2 xenobiotic-metabolizing enzymes, and showed that CYP1A-dependent monooxygenase activities were inducible in intestine but not liver by
dietary exposure to low concentrations of the Ah agonist,
-naphthoflavone (BNF). The diets administered were
laboratory-prepared, semisynthetic pellets of known composition,
commercial chow, or chow supplemented with BNF at 10 or 100 mg BNF/kg
chow. Very low intestinal benzo(a)pyrene hydroxylase [aryl
hydrocarbon hydroxylase (AHH)] and ethoxyresorufin O-deethylase (EROD) activities were found in catfish fed
the semisynthetic diet. Intestinal EROD and AHH activities were
elevated by the commercial chow diet and further induced by
supplementation with 10, but not 100, mg BNF/kg diet. In
vitro studies showed that catfish EROD and AHH activities were
sensitive to inhibition by BNF, with mean IC50 values of
0.078 and 2.2 µM, respectively. Thus, residues of BNF retained in
intestinal mucosa may have masked monooxygenase induction in catfish
fed the 100 mg BNF/kg diet. Microsomal UDP-glucuronosyltransferase and
cytosolic PAPS-sulfotransferase activities with
3-hydroxybenzo(a)pyrene as substrate were largely unaffected by the diets studied, and intestinal activities were similar
to hepatic activities. Glutathione S-transferase activity was slightly induced in intestinal, but not hepatic cytosol of catfish
treated with BNF at the 10 mg/kg diet level relative to chow controls.
Epoxide hydrolase activity with styrene oxide as substrate was not
affected by diet in intestinal microsomes.
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Introduction |
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Abstract
Introduction
Results
Discussion
References
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The fate of organic xenobiotics that are present in foodstuff is determined in large part by interactions in the GI1 tract (1). Xenobiotics may be absorbed or excreted from the GI tract as parent compound, or may be wholly or partially biotransformed in the GI tract before systemic absorption or elimination in feces. Diets of aquatic species that inhabit polluted environments are likely to contain chemical pollutants that will undergo initial processing in the stomach and intestine. Intestinal biotransformation of environmental chemicals, which alters their lipid and water solubility, is likely to affect the intestinal bioavailability of the chemical. As part of a study of the bioavailability of low, environmentally relevant concentrations of polycyclic aromatic hydrocarbons and their phase 1 and phase 2 metabolites in carcinogen-sensitive and carcinogen-resistant aquatic species, we have investigated the ability of isolated perfused catfish intestine to metabolize BaP and BaP metabolites (2-4).
Studies in mammals, reviewed by Kaminsky and Fasco (5), and a few marine fish species [including spot (6, 7), toadfish (8), and mummichog (9)] have shown that CYP1A-dependent monooxygenase activities, such as EROD and AHH, are readily induced in the intestine by dietary exposure to Ah agonists. Studies with a freshwater fish (the trout) showed that dietary BNF at concentrations of >50 ppm (50 mg BNF/kg diet/day fed for 7 days) induced hepatic EROD and CYP1A, compared with treatment for 7 days with a diet containing <25 ppm BNF that did not generally induce EROD activity in the liver (10). In the trout study, intestinal monooxygenase activities were not measured (10). There have been no previous studies of the drug-metabolizing capacity of catfish intestinal preparations, although catfish liver is known to contain at least two constitutive forms of CYP that cross-react with antibodies to trout liver constitutive forms (11). Treatment of catfish with BNF (10 mg/kg ip) resulted in induction of hepatic EROD activity and a hepatic CYP that cross-reacted with a CYP1A-specific monoclonal antibody (12). Treatment of mice and rats with Ah agonists also results in the induction of activities of several UGT and GST isozymes (13), but induction of these phase 2 enzymes is more variable in the liver or intestine of fish exposed to Ah agonists (reviewed in ref. 14).
Objectives of the present study were to examine the effects of the composition of the diet, and the presence in the diet of a known Ah receptor agonist inducing agent, BNF, on intestinal and hepatic enzymes in the channel catfish that are important in the biotransformation of polycyclic aromatic hydrocarbons. BNF was selected because it is an extensively studied and relatively nontoxic Ah agonist that is known to induce intestinal and hepatic CYP1A in marine fish species (9, 14). The parameters studied in intestinal and hepatic subcellular fractions of the channel catfish were total P450, EROD, AHH, EH, GST, and UGT and ST with 3-, 7-, and 9-hydroxy-BaP as substrates. Results showed that intestinal EROD and AHH activities in catfish were very low in control fish and very sensitive to the composition of the diet and the presence in the diet of low levels of the CYP1A inducer, BNF. Hepatic monooxygenase activities were not affected by the low levels of dietary BNF used in these studies. EH and phase 2-conjugating enzyme activities in intestinal mucosal fractions were similar to those found in the liver, and these activities were relatively insensitive to the composition of the diet.
Materials and Methods
Animals and Treatments.
Catfish used in these studies were obtained from the Louisiana State
University Ben Hur aquaculture research facility at Baton Rouge, LA.
Male and female catfish, body weight of 450-1200 g, were used in these
studies. Catfish were maintained in well water (pH 7.1) at a
temperature of 24°C, and were fed morning and evening a total daily
ration of 30 g of food/kg of body weight. Groups of 5-12 catfish
were fed one of the following five diets for at least 2 weeks before
being killed. One diet was a semisynthetic purified feed that was
formulated according to guidelines established for warm water fish by
the National Research Council (15) and stored at 
20°C until use.
This diet contained the following major components: casein, 32%;
dextrin, 30%; cellulose flour, 17%; gelatin, 8%; and fatty acids,
6%. The fatty acid composition was 95% saturated (14:0, 16:0, and
18:0) and 5% unsaturated (2% 22:6n-3, 1% 18:3n-3, 1% 18:2n-6, and
1% 20:5). Fatty acid components were selected based on the fatty acid
composition of the fat of catfish acclimated to 24°C (16). Minor
components of the purified diet were: vitamins, ICN vitamin diet
fortification, 1% of diet; minerals, U.S.P. XIV salt mixture, 4% of
diet and 2% carboxymethylcellulose (a binder). Components of the diet
were obtained from ICN Biomedicals, (Irvine, CA). A second diet was
commercially available Silvercup fish chow (Murray Elevators, Murray,
UT) used for normal maintenance of the fish and stored at 4°C. A
third diet was Silvercup chow supplemented with BNF (10 mg BNF/kg
diet). This diet was fed to the catfish for 2-3 weeks, then the
catfish were fed chow without BNF for 1 day before being killed. The
fourth diet was Silvercup chow with 100 mg BNF/kg diet. The fourth diet
was fed to catfish for 2-3 weeks and followed by chow without BNF for
1-3 days before being killed. The fifth diet was Silvercup chow to
which corn oil vehicle was added. The BNF-treated chow was prepared
freshly each day as follows. For the 10 mg BNF/kg chow diet, 0.1 ml of a solution of BNF in corn oil (10 mg/ml) was dripped onto 100 g
chow so that the chow was evenly coated with the solution. After waiting for 5 min to allow absorption of the corn oil solution, the
chow was fed to the fish. The same procedure was used for the 100 mg
BNF/kg chow diet, except that more of the corn oil solution (1 ml/100
g) was added. Vehicle control diets had corn oil (0.1 ml/100 g diet)
added before being fed to the fish.
Chemicals.
Radiolabeled BaP (G-3H), specific activity 60 Ci/mmol, was
obtained from Amersham International (Chicago, IL) and purified by
alumina chromatography before use (2). Unlabeled BaP (3-, 7-, and
9-hydroxy-BaP), glucuronide, and sulfate conjugates of 3-, 7-, and
9-hydroxy-BaP and other BaP metabolite standards were obtained from the
NCI Chemical Carcinogen Repository through Chemsyn (Lenexa, KS).
Purities of BaP and 3-, 7-, and 9-hydroxy-BaP used as substrates were
examined by C18 reversed-phase HPLC with UV (280 nm) and
fluorescent (excitation 375 nm, emission 435 nm) detection as described
previously (2), and found to be >99%. Ethoxyresorufin was prepared
from ethyl iodide and resorufin as described previously (17) and
purified by TLC on silica gel plates. UDPGA and adenosine
3
-phosphate-5-phosphosulfate were purchased from Sigma Chemical Co.
(St. Louis, MO). All other reagents and solvents used were of the
highest available quality and were purchased from Sigma or Fisher
Scientific (Orlando, FL).
Microsome and Cytosol Preparation.
Catfish were immobilized in ice water and killed by severing the spinal
cord. Livers were removed, rinsed in ice-cold buffer 1 [composed of
1.15% KCl, 0.05 M potassium phosphate (pH 7.4), 0.2 mM PMSF],
weighed, and homogenized in 4 volumes of buffer 1. Intestines were
severed from the stomach and rinsed thoroughly with ice-cold buffer 2 [0.25 M sucrose, 5 mM EDTA, 0.05 M Tris-Cl (pH 7.4), 0.2 mM PMSF] to
remove contents. The proximal section (first one-third of the
intestinal length) was cut off, opened, and mucosal cells removed by
scraping with a scalpel into 10 ml of buffer 2. Mucosal cells were
sedimented at 2000 g, weighed, and homogenized in 4 volumes
of buffer 2. Washed microsomal and cytosolic fractions were prepared
from the homogenates of liver and intestine using the procedure
described by James and Little (18). Intestinal monooxygenase assays
were done on the day the fish were killed. Aliquots of each subcellular
fraction were flushed with nitrogen and stored at 80°C until used
in assays.
Assays. Phase 1. The total P450 content of microsomes was measured by the method of Estabrook et al. (19) in a Shimadzu 265 or Perkin-Elmer Lambda 4B spectrophotometer, as described by James et al. (20).
EROD activity was measured in intestinal microsomes by the method of Prough et al. (21) and in liver microsomes by the method of Pohl and Fouts (22). For intestinal microsomes, an ethoxyresorufin substrate concentration of 2.5 µM was used, and the formation of resorufin in the presence of 1 mg microsomal protein was monitored at excitation 550 nm, emission 585 nm over a period of 30 min in a fluorescence spectrophotometer cuvette containing 0.1 M HEPES-NaOH buffer (pH 7.6) and 2 mM NADPH in a total volume of 2.5 ml. For hepatic microsomes, five substrate concentrations in the range 0.2-3 µM were used, and tubes containing 1% bovine serum albumin, 0.1 M HEPES (pH 7.6), 0.1 mg microsomal protein/ml, and 2 mM NADPH were incubated for 5 min before stopping the reaction with methanol, centrifuging to precipitate protein, and reading the fluorescence of the protein-free supernatant at excitation 550 nm, emission 585 nm. In studies of BNF inhibition of EROD activity, varying concentrations of BNF (0.001-1 µM final concentration) were added from the acetone solution; and the solvent was removed before adding ethoxyresorufin (2.5 µM) and the other assay components. BaP monooxygenase activity was measured by one of two assay methods: a fluorescence assay (AHH) of phenolic BaP metabolites (23) and a radiochemical HPLC assay of all BaP metabolites (18). The radiochemical HPLC assay was used to determine the metabolite profile and validate the use of the more sensitive fluorescence assay for most studies of intestinal activity. For studies of apparent KM values, BaP concentrations ranging from 0.5 to 10 µM were used. Tubes contained 0.1 M HEPES-NaOH buffer (pH 7.6), BaP (0.5-10 µM, with 10 µCi [3H]BaP for assay of all metabolites), and intestinal or hepatic microsomes [0.15-0.5 mg protein and 2 mM NADPH (added last) in a volume of 1 ml]. In studies of the effect of BNF on AHH activity, varying concentrations of BNF (0.01-10 µM) were added to the assay tubes from acetone solution and the solvent removed before adding BaP (10 µM), and the other assay components. After incubation for 5 min (liver) or 15 min (intestine), the assay was stopped by adding 1 ml ice-cold acetone. For the fluorescence assay, tubes were extracted with 3 × 3 ml heptane, the pooled heptane extracts were backextracted into 3 ml of 1 N NaOH, and the fluorescence of the NaOH measured at excitation 392 nm, emission 513 nm. Products were quantitated as 3-hydroxy-BaP using a standard curve developed with authentic 3-hydroxy-BaP. For the radiochemical assay, tubes were extracted with 3 × 3 ml ethyl acetate, and the pooled organic phases were dried over anhydrous sodium sulfate and evaporated under nitrogen. Residues were reconstituted in 0.15 ml of 55% methanol and analyzed by HPLC exactly as described previously (18). EH activity was measured with [14C]styrene oxide as substrate, as described previously (24).Phase 2. UGT was assayed by a modification of the method of Singh and Wiebel (25). Tubes contained 3-, 7-, or 9-hydroxy-BaP, added in methanol solution such that the final concentration of substrate would be 1 µM. In some studies, varying concentrations of BNF (to give final concentrations of 0.1-250 µM) were added to tubes from acetone solution at the same time as the substrate. Tube contents were evaporated to dryness under nitrogen to remove the methanol and acetone solvents that inhibit UGT. After removal of solvent, 0.1 M Tris-Cl (pH 7.6), 5 mM MgCl2, 0.02-0.05 mg microsomal protein solubilized with 1 mg Lubrol/mg microsomal protein and water were added to a volume of 0.45 ml, and tubes were placed in a bath at 35°C. The reaction was started by adding UDPGA (200 µM) in 0.05 ml and terminated after 10 (liver) or 15 (intestine) min by addition of cold methanol (2 ml). Methanol was added to blanks before UDPGA. Tubes were centrifuged to precipitate protein, and 2 ml of the supernatant was added to 0.5 ml of 1N NaOH. After mixing, the fluorescence was measured at excitation 300, emission 421 (BaP-3-glucuronide); excitation, 295, emission 408 (BaP-7-glucuronide); and excitation 295, emission 415 (BaP-9-glucuronide). Under these alkaline conditions, the hydroxy-BaP substrate was in the phenolate form, and the substrate fluorescence did not interfere with the glucuronide conjugate fluorescence, because the fluorescence maxima for the BaP phenolates were excitation 285, emission 510 (9-hydroxy-BaP); excitation 285, emission 586 (7-hydroxy-BaP); or excitation 390, emission 545 nm (3-hydroxy-BaP). A calibration curve was prepared for each glucuronide standard to convert fluorescence reading to pmoles of product formed. Fluorescence of a 3 µg/ml solution of quinine sulfate was routinely measured at each wavelength pair as a standard.
ST was measured using the same principle employed in assaying UGT (see previous paragraph) to separate the fluorescence of the sulfate conjugate from that of the phenolic BaP substrate under alkaline conditions. Hydroxy-BaP substrates in methanol solution were added to assay tubes, so that the final concentrations would be 1 µM, and the methanol evaporated under nitrogen. In some cases, BNF (0.1-250 µM) was added from acetone solution and the acetone removed under nitrogen. Tris-Cl (0.1 M; pH 7.0), cytosol (0.01 mg protein), and water were added up to 0.45 ml; and tubes were placed in a water bath at 35°C. The reaction was started by the addition of 20 µM PAPS (saturating) in 0.05 ml and stopped after 5 min (liver) or 10 min (intestine) with methanol, 2 ml. Methanol was added to blanks before PAPS. Tubes were centrifuged, and 2 ml of supernatant was added to 0.5 ml of 1 N NaOH. Fluorescence of the sulfate conjugate was measured at excitation 294, emission 408 (BaP-7-sulfate); excitation 285, emission 410 (BaP-9-sulfate); or excitation 294, emission 415 (for BaP-3-sulfate). Authentic BaP-sulfate conjugates were used to develop standard curves for converting fluorescence into pmoles of product formed. GST activity was assayed with CDNB as substrate as described previously (24). The CDNB concentrations used ranged from 0.5 to 5 mM, the GSH concentration was 1 mM, and 0.01 mg cytosolic protein was added to assay cuvettes containing 0.1 M HEPES-NaOH buffer (pH 7.6). In some cases, BNF (1-250 µM) was included in the assay mixture with 1 mM CDNB and 1 mM GSH.| |
Results |
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Abstract
Introduction
Results
Discussion
References
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Catfish. Some of the characteristics of the fish used in these studies are presented in table 1. Liver and intestinal mucosa/body weight ratios and protein yields for microsomes and cytosols were similar for fish maintained on purified diet, chow, chow with corn oil vehicle, and chow with 10 mg BNF/kg. Chow with 100 mg BNF/kg resulted in fish with slightly smaller liver/body weight ratios, and markedly lower intestinal mucosa/body weight ratios, thus suggesting a possible toxic reaction in the intestine. Although we did not monitor food intake or weight gain per fish, because the fish were housed in groups, we observed that fish on all feeding regimens quickly ate the food ration of 3% body weight/day and seemed in good health. Fish of a range of beginning sizes were used; in the size range studied, there was no apparent effect of size on the enzyme activities measured. For the induction studies, larger fish were needed to obtain enough intestinal mucosa to complete all assays on each fish.
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Monooxygenase Activities. Monooxygenase activity with BaP was very low in the intestine and approached the limits of detection with the radiochemical HPLC assay at BaP concentrations below 2 µM. The profile of BaP metabolites formed by both liver and intestine microsomes from a single catfish maintained on the purified diet (table 2) showed that 3-hydroxy-BaP was a major metabolite. Other catfish maintained on purified diet showed similar profiles, and the other major identified metabolites (benzo-ring phenols and dihydrodiols) were similar to those previously demonstrated with other fish species (20, 26, 27). With catfish liver and intestinal microsomes, 32.6 ± 2.8% and 25.6 ± 3.1% of the BaP metabolites formed, respectively, comigrated with 3-hydroxy-BaP (mean ± SD, N = 3 for each tissue). Because 3-hydroxy-BaP was a major metabolite, the more sensitive and rapid fluorescence assay was used for further studies, including all induction studies. Apparent KM values for BaP were 1-2 µM in the intestine and 0.4-0.7 µM in the liver, and did not change with diet (table 3; data not shown). Composition of the diet was an important determinant of monooxygenase activity in the intestine (fig. 1). In intestinal microsomes, total P450 content was unchanged by altering the diet (fig. 1A), but EROD activity and the AHH Vmax were significantly increased by changing from the purified diet to the commercial fish chow (fig. 1, B and C). There were no differences in intestinal microsomal P450 contents, EROD, or AHH activities of catfish-fed chow or chow with corn oil vehicle; therefore, the results from these fish were combined as chow controls (table 3, fig. 1). Further increases in intestinal AHH and EROD activities were achieved by incorporating a low concentration of BNF (10 mg/kg chow) in the diet. Fish fed 100 mg BNF/kg chow exhibited a decrease in EROD activity relative to fish fed 10 mg/kg BNF. Even in induced fish, EROD activities in intestine were too low to measure KM values accurately with the limited amount of intestinal microsomes available. Composition of the diet did not affect hepatic monooxygenase activities or P450 contents (fig. 1). Hepatic AHH activities were higher than intestinal AHH activities in control fish, but similar in fish receiving BNF in the diet. Hepatic EROD activities were much higher than intestinal EROD activities in all groups studied (fig. 1). Hepatic apparent KM values for ethoxyresorufin ranged from 0.3 to 3 µM (data not shown), but did not exhibit significant differences with diet.
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EH and Conjugating Activities. Intestinal EH activity was unaffected by the composition of the diet (table 4). We did find, however, that EH activities in hepatic microsomes from catfish fed 100 mg BNF/kg diet were markedly higher than those found in hepatic microsomes from fish maintained on other diets (table 4).
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In Vitro Effects of BNF. Because BNF was known to affect xenobiotic-metabolizing enzymes directly, we also determined the effect of adding BNF to assay tubes before measuring GST, 9-hydroxy-BaP-ST, 9-hydroxy-BaP-UGT, and AHH activities in the appropriate intestinal subcellular fraction from control (chow-fed) catfish. Because EROD activity was so low in intestinal microsomes, the effect of BNF on EROD activity was studied in hepatic microsomes from control catfish. BNF did not inhibit GST activity over a range from 0.1 to 250 µM. BNF did inhibit the other activities, and the results are shown in figs. 2 and 3. EROD and AHH were both very sensitive to inhibition by BNF, but there were substrate-selective differences in the potency of BNF as a monooxygenase inhibitor. The IC50 for EROD was 0.078 ± 0.022 µM and the IC50 for AHH was 2.2 ± 0.09 µM (fig. 2). UGT and ST activities with 9-hydroxy-BaP were somewhat sensitive to inhibition by BNF and showed similar inhibition profiles. For both ST and UGT, there was ~15% inhibition of activity at 5 µM BNF. IC50 values were 48.0 ± 3.0 µM (ST) and 46.9 ± 15.1 µM (UGT) under the assay conditions used (fig. 3).
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Discussion |
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Abstract
Introduction
Results
Discussion
References
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Several studies have demonstrated a marked effect of diet and
exposure to Ah receptor agonists on intestinal monooxygenase activities
in rodents (5, 28-30), but there are few comparative studies in fish.
Van Veld et al. (6) showed that oral administration of BaP
(16 mg/kg food) to a marine fish (the spot) resulted in increased
biotransformation of BaP in the intestine, but not the liver.
Similarly, incorporation of 3MC (10 mg/kg food) in the diet of the spot
resulted in 18-fold induction of intestinal CYP1A expression, 36-fold
induction of intestinal EROD, and 17-fold induction of intestinal AHH
activity, whereas starvation reduced AHH and EROD activities to
undetectable levels (7). The spot fed 3MC (10 mg/kg food) did not show
hepatic induction of CYP1A, AHH, or EROD. The present study showed that
the composition of the diet is an important determinant of intestinal
monooxygenase activity in a freshwater fish
the channel catfish.
Intestinal, but not hepatic, EROD and AHH activities were higher in
catfish maintained on a commercial fish chow than in catfish maintained on a purified diet, and were further induced by low dietary
concentrations of BNF (fig. 1). It is not known what components of the
commercial chow induce EROD and AHH activity, and presumably CYP1A.
Possible inducers are cereal or other plant-derived natural products
that are weak Ah agonists, such as indole-3-carbinol and bioflavonoids (28, 31, 32).
Although CYP1A-dependent activities, EROD and AHH, were induced by the chow diet and by BNF, there was no increase in total intestinal cytochrome P450 content. This lack of effect on total P450 may be explained by considering that the CYP1A content of uninduced intestinal microsomes was so low that, even after induction, CYP1A was too small a fraction of the total intestinal microsomal P450 content to affect total content. Another possibility is that whereas CYP1A was upregulated, one or more constitutive isozymes were downregulated by dietary BNF. Studies with control and 3MC-fed spot described previously are consistent with either of these interpretations, in that an 18-fold induction of the specific CYP1A (P450E) content of intestinal microsomes was reported, but the total P450 content of intestinal microsomes was <2-fold higher in 3MC-fed spot vs. controls (7).
It was of interest that, in the present study, intestinal AHH activity was induced to near hepatic levels by chow or 10 mg/kg BNF, whereas even induced activities of EROD were well below hepatic activities. The extent of induction of AHH activity by 10 mg/kg BNF, relative to chow or purified diet controls, was higher than the extent of induction of EROD (table 3 and fig. 1). These results were in contrast with results in which a marine fish (the spot) were fed 3MC-containing diets, wherein the extent of intestinal induction of AHH and EROD were similar (7). In another marine fish (the mummichog), high dietary concentrations of BNF (250 mg/kg food) resulted in a 10-fold induction of intestinal EROD and a 5.6-fold induction of hepatic EROD (9). In catfish, the high BNF diet (100 mg/kg diet) did not induce either AHH or EROD. Thus, mummichog respond differently to high dietary BNF, compared with the catfish used in these studies. Catfish used in the present studies had much lower uninduced EROD activities (3.6 ± 2.3 pmol/min/mg protein for chow controls) than the control spot (56 ± 7 pmol/min/mg protein) or mummichog (65 ± 34 pmol/min/mg protein), as well as lower AHH activities (23 ± 15 pmol/min/mg protein for catfish chow controls vs. 42 ± 5 pmol/min/mg for control spot). The lower induction of EROD observed in catfish in this study, relative to these previous studies with small marine fish (the spot and mummichog), may be due to differences in the clearance of residues of BNF from catfish intestine that may relate to the lower basal EROD and AHH activities. Previous studies have shown that residual BNF is capable of inhibiting both EROD and AHH activities (10, 17, 33, 34). The present study showed that catfish EROD was exquisitely sensitive to BNF, with an IC50 of <0.1 µM. Because the low intestinal EROD activity in catfish necessitated the use of large amounts of microsomal protein in assays (1 mg/2.5 ml assay volume), it is possible that assay tubes contained enough residual BNF from intestinal microsomes to inhibit EROD. AHH activity was also very sensitive to BNF inhibition, although higher concentrations of BNF were needed for inhibition of AHH than EROD activity (fig. 2). Thus, low residual concentrations of BNF in the washed intestinal microsomes would have less effect on AHH activity than EROD activity. The substrate selectivity of BNF effects on AHH and EROD is consistent with the interpretation that, in catfish intestine, more than one form of P450 is responsible for BaP metabolism (some of which are less sensitive to inhibition by BNF than others), whereas only one form, presumably CYP1A, is responsible for EROD activity. This form is very sensitive to the presence of traces of BNF. Trout CYP1A was also found to be very sensitive to BNF inhibition, with a Ki for EROD of 7.6 ± 1.1 nM (34).
We found high variability in the intestinal EROD and AHH activities of the group of catfish given 100 mg BNF/kg diet, with 2 of the 4 having EROD and AHH activities similar to those of the 10 mg BNF/kg diet group, and two having activities in the range found for chow controls. As discussed previously, it was shown that BNF was a potent inhibitor of EROD and AHH in catfish intestine, and residues of BNF persisting into the washed intestinal microsomes would inhibit EROD and AHH. Another factor that might influence CYP1A-dependent activities, as well as other activities in catfish fed 100 mg BNF/kg diet, was the effect of the high-dose BNF diet on intestinal mucosal cell weight. Catfish fed chow containing 100 mg BNF/kg had significantly lower intestinal cell/body weight ratios, compared with catfish fed 10 mg BNF/kg diet (table 1). Although the cause of this effect of BNF on catfish intestinal cells is unknown, it may indicate some cellular toxicity that could impact xenobiotic metabolism. In a study of the effect of dietary administration of BNF to brown bullhead (a fish species related to the catfish), it was shown that bullhead fed 500 mg BNF/kg diet for 90 days, followed by 150 days of untreated chow, were dramatically smaller (length and weight) than control bullhead not exposed to BNF (35). The authors noted that all of the fish in the study seemed to eat their chow diets eagerly, although the exact amounts consumed by each group of fish were not recorded (35). If the BNF and control fish did consume similar amounts of food, the observed effect on size would be consistent with an effect on intestinal function, perhaps with retardation of food absorption.
EH activity with styrene oxide as substrate was similar in intestinal and hepatic microsomes from control catfish. The low-dose BNF diet did not induce EH activity, whereas the diet containing 100 mg BNF/kg resulted in a 2-fold increase in hepatic, but not intestinal, EH activity. The elevated hepatic microsomal EH activity may be the result of a direct effect of BNF on EH activity or could be due to elevated EH content of hepatic microsomes from the high-dose BNF group as a result of induction or interindividual variability. BNF is known to be a direct stimulant of microsomal EH activity in human lymphocytes (36). It is very unlikely, however, that enough BNF was present in the hepatic, but not intestinal, microsomes from the treated catfish to cause a 2-fold increase in activity in hepatic microsomes only, thus suggesting that EH enzyme content was higher in the livers of the group fed the high BNF diet. Previous studies with the marine fish species sheepshead and scup have revealed large individual differences in EH activities of hepatic microsomes (24, 37). The Ah agonist, 3MC, is a weak inducer of microsomal EH activity in rats (38). No other studies with fish have shown induction of EH activity by Ah agonists (20, 39-41). Further studies would be needed to determine the reasons for the elevated EH activity in the high-dose BNF diet group; but, the most likely explanation is that these fish were high normal EH content individuals.
There have been very few studies of intestinal phase 2 enzymes in fish or of the effects of inducing agents on these activities. GST was found in the intestine, as well as in the liver of several marine fish, with 1,2-dichloro-4-nitrobenzene, BaP-4,5-oxide, styrene oxide, and octene-1,2-oxide as substrates (24). There are conflicting reports in the literature on the inducibility of GST in the liver or intestine of marine or freshwater fish exposed to Ah agonists. The extent of induction of GST in fish, if induction occurs, is rarely more than 2- to 3-fold, whereas the extent of induction of CYP1A-dependent activities (discussed previously) is often >5-fold. Some studies have shown no effect of Ah agonists on hepatic GST activity (20, 39, 42, 43), whereas others have shown slight induction, especially if the fish were sampled >10 days after the dose of the inducer (41, 43). Intestinal and hepatic GST in mummichog sampled from sites heavily polluted with creosote was higher than in mummichog taken from a reference site (9). Mummichog treated with dietary BNF (250 mg/kg food) for 2 weeks had 2.5-fold higher GST in intestine, but no induction of GST in the liver, compared with mummichog maintained on the same diet without BNF (9). In the present study, there was a small induction of GST activity in intestinal cytosol of catfish exposed to 10 mg BNF/kg diet for 2-3 weeks, relative to control chow-fed fish, but in cytosols from catfish given 100 mg BNF/kg diet; there was no further increase in GST activity. Because the in vitro addition of BNF to assay tubes did not affect catfish GST activity, a possible explanation of the lack of induction in the 100 mg BNF/kg diet group was a general toxic effect on the intestine (as discussed previously). These studies also showed that fish fed the purified diet had higher GST activity than chow-fed control fish. Recent studies have shown that plant polyphenols, which may be present in the commercial chow, inhibit GST in rodents (44). Further studies would be needed to determine the cause of the dietary effects on GST activity.
This study showed that ST and UGT activities with BaP phenols were high in the catfish intestine, suggesting that low concentrations of hydroxylated polycyclic aromatic hydrocarbon would be readily conjugated in catfish intestine. UGT activities with other phenolic and alcoholic substrates have been studied in liver and extrahepatic organs of several fish species, as reviewed by George (45). Although induction of UGT activity with phenolic substrates has been observed in the liver and kidney of some marine fish after treatment with BNF, Aroclors, or 3MC, the extent of induction is usually <2-fold (41, 46, 47). UGT activities with 3-, 7-, and 9-hydroxy-BaP in channel catfish intestine were not induced by treatment with BNF in the present study, and in fish receiving the higher dose activity with 7- and 9-hydroxy-BaP was lower than in fish fed other diets. In vitro studies showed that BNF could inhibit UGT activity with 1 µM 9-hydroxy-BaP as substrate, although the IC50 for BNF inhibition was 46.9 µM (fig. 3B). Significant inhibition of UGT activity (13 ± 6%) was found by the in vitro addition of 5 µM BNF (fig. 3B). There are several possible explanations of the lower UGT activity in intestinal microsomes from the high-dose fish. Residues of BNF retained in intestinal cells after administration of the 100 mg BNF/kg diet could directly inhibit activity. There may be intestinal cell toxicity, as discussed previously, or the lower activities in the higher dose group may reflect individual variability in this activity for unknown reasons. ST activity is not generally inducible by Ah agonists and, as expected, we did not find induction of ST activity. ST activity with 9-hydroxy-BaP, but not 3- or 7-hydroxy-BaP, was lower in intestinal cytosol from the 100 mg BNF/kg chow group, compared with the 10 mg BNF/kg chow group. Others have shown that ST activities with acetaminophen or minoxidil as substrates were inhibited by plant bioflavonoids (48). BNF was found to be an inhibitor of catfish intestinal 9-hydroxy-BaP-ST activity with IC50 of 48 ± 3 µM, and significant inhibition (16 ± 4%) was observed at 5 µM BNF (fig. 2). If residues of BNF were present in the intestinal mucosa, the amount of intestinal cytosol used in assays (10 µg protein/0.5 ml assay volume) was unlikely to have contained enough BNF to cause the extent of inhibition of 9-hydroxy-BaP-ST activity observed (table 5). The lower 9-hydroxy-BaP-ST activity found in the 100 mg BNF/kg chow group, compared with the 10 mg BNF/kg chow group, may be a reflection of large interindividual variations unrelated to diet or due to general intestinal toxicity resulting from exposure to the 100 mg BNF/kg diet.
In summary, intestinal EROD and AHH activities of channel catfish were very sensitive, and GST activity was moderately sensitive to the composition of the diet and the presence of the Ah agonist, BNF. Total P450 content, ST, UGT, and EH activities were less sensitive to or were unaffected by the composition of the diet. Hepatic xenobiotic-metabolizing activities were unaffected by the composition of the diet or the presence of low concentrations of dietary BNF. The catfish intestine possesses high activities of many xenobiotic-metabolizing enzymes and is clearly important in the first-pass biotransformation of low levels of xenobiotics.
| |
Acknowledgments |
|---|
The BaP metabolites used in these studies were obtained from the NCI Chemical Carcinogen Repository. The authors gratefully acknowledge the assistance of Heidi Feistner, Chris Frost, and Sean Boyle in the catfish treatment studies.
| |
Footnotes |
|---|
Received August 2, 1996; accepted December 12, 1996.
This work was supported by the U.S. Public Health Service, National Institutes of Health Grant ES 05781 (to M.O.J., K.M.K.), and by a National Science Foundation undergraduate fellowship to the Whitney Lab. A preliminary account of this work was presented at the 10th Conference on Microsomes and Drug Oxidations, Toronto, Canada, 1994.
2 M. O. James et al., manuscript in preparation.
3 Z. Tong and M. O. James, manuscript in preparation.
Send reprint requests to: Dr. Margaret O. James, Department of Medicinal Chemistry, P.O. Box 100485, College of Pharmacy, University of Florida, Gainesville, FL 32610-0485.
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Abbreviations |
|---|
Abbreviations used are:
GI, gastrointestinal;
BaP, benzo(a)pyrene;
CYP, cytochrome P450;
EROD, ethoxyresorufin
O-deethylase;
AHH, aryl hydrocarbon hydroxylase;
BNF, -naphthoflavone;
UGT, UDP-glucuronosyltransferase;
GST, glutathione
S-transferase;
P450, cytochrome P450;
EH, epoxide hydrolase;
ST, PAPS-sulfotransferase;
NCI, National Cancer Institute;
UDPGA, UDP-glucuronic acid;
PMSF, phenylmethylsulfonylfluoride;
PAPS, 3-phosphoadenosine-5-phosphosulfate;
CDNB, 1-chloro-2,4-dinitrobenzene;
GSH, glutathione;
IC50, inhibitory concentration of 50%;
3MC, 3-methylcholanthrene.
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References |
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Abstract
Introduction
Results
Discussion
References
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) was 0.078 ± 0.022 µM. The IC50 for BNF inhibition of intestinal
microsomal AHH activity (
) was 2.20 ± 0.09 µM.
) was
46.9 ± 15.1 µM.