Disposition and Metabolism of 2,3,7,8-Tetrachlorodibenzofuran by Rainbow Trout (Oncorhynchus mykiss)

TOXICOLOGY AND APPLIED PHARMACOLOGY ARTICLE NO.

139, 418–429 (1996)

0183

Disposition and Metabolism of 2,3,7,8-Tetrachlorodibenzofuran by Rainbow Trout (Oncorhynchus mykiss)1,2 A. RUTH STEWARD, ROBERT MASLANKA, JYOSTNA PANGREKAR,3 SUBODH KUMAR,

AND

HARISH C. SIKKA4

Environmental Toxicology and Chemistry Laboratory, Great Lakes Center for Environmental Research and Education, State University of New York College at Buffalo, Buffalo, New York 14222 Received November 9, 1995; accepted April 25, 1996

Disposition and Metabolism of 2,3,7,8-Tetrachlorodibenzofuran by Rainbow Trout (Oncorhynchus mykiss). STEWARD, A. R., MASLANKA, R., PANGREKAR, J., KUMAR, S., AND SIKKA, H. C. (1996). Toxicol. Appl. Pharmacol. 139, 418–429. The disposition and metabolism of 2,3,7,8-tetrachlorodibenzofuran (TCDF) was investigated in rainbow trout (Oncorhynchus mykiss) in order to better understand the metabolic and physiological factors that modulate the fate of this extremely toxic compound in rainbow trout compared to other species. The fish were dosed orally with [3H]TCDF (1 mg/kg); fish were terminated at 1–19 days for the determination of whole body half-life or at 0.3–28 days for determination of tissue distribution. Unassimilated TCDF (51.5% of the dose) was eliminated with a half-life of 0.84 days. The assimilated body burden of TCDF equivalents decreased with a half-life of 14.8 days (determined between 3 and 19 days). Trout muscle showed a relatively high capacity to accumulate and retain (unmetabolized) TCDF, accounting, at 3 days, for 32% of the body burden of TCDF equivalents (half-life in muscle, 15.2 days). Trout liver, on the other hand, showed a relatively low capacity to accumulate and metabolize TCDF. At 3 days, the concentrations of TCDF equivalents in liver and bile were, respectively, 0.37 ng/g liver (0.88% of the body burden) and 4.8 ng/ml bile. The data suggest that the relatively high affinity of lipid-rich trout muscle for TCDF limits the ability of the liver to accumulate and metabolize TCDF. The major TCDF metabolites found in trout liver and bile were, respectively, 4-OH-TCDF and TCDF-4-O-glucuronide. q 1996 Academic Press, Inc.

Polychlorinated dibenzofurans (PCDFs)5 such as 2,3,7,8tetrachlorodibenzofuran (TCDF), like their analogs the polychlorinated dibenzodioxins (PCDDs), together designated 1

Supported by New York Sea Grant (Grant No. R/CTP-4). Presented in part at the 6th International Symposium on Responses of Marine Organisms to Pollutants, April 24–26, 1991, Woods Hole, MA, and at the 11th International Symposium on Chlorinated Dioxins and Related Compounds, September 23–27, 1991, Research Triangle Park, NC. 3 Present address: Molecular Oncology Research, Children’s Hospital of Pittsburgh, Pittsburgh, PA 15213. 4 To whom correspondence should be addressed. Fax: (716) 878-5400. 5 Abbreviations used: CI, confidence interval; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDF, 2,3,7,8-tetrachlorodibenzofuran; 4-OH-TCDF, 42

TOX 7876

/

6h0f$$$421

hydroxy-2,3,7,8-TCDF; 3-MeO-TCDF, 3-methoxy-2,4,7,8-TCDF; PCDF, polychlorinated dibenzofuran; PCDD, polychlorinated dibenzo-p-dioxin; PCDX, PCDFs and PCDDs; SAL, saccharic acid-1,4-lactone.

418

0041-008X/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

AID

PCDXs, are highly toxic manufacturing by-products and pyrolysis products of chlorinated aromatic compounds as well as chlorination products resulting from the bleaching of paper pulp with chlorine. These compounds are widely dispersed in the environment and have been found in the fish and sediments of the Great Lakes, the Hudson River and New York Bight, the Baltic Sea, and in other industrialized and heavily populated areas (Fletcher and McKay, 1993; Whittle et al., 1992; van der Weiden et al., 1993; Stalling et al., 1985; Safe, 1990). Although the major environmental reservoirs of PCDXs are sediments, TCDF and other congeners are taken up and biomagnified by the food web (Cook et al., 1991; Opperhuizen and Sijm, 1990). The distribution and persistence of these highly lipophilic compounds within the ecosystem depend on a number of factors, including the bioavailability and extent of bioaccumulation of specific congeners by various organisms. Predicting the ultimate hazard of these persistent and ubiquitous environmental toxicants to humans (and to environmental organisms at high trophic levels) requires a knowledge of the metabolic fate and disposition of the various congeners at lower levels of the food web and particularly in fish species and other organisms eaten by humans. Although the metabolic fate, disposition, and toxicity of certain congeners, particularly 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and TCDF, have been extensively investigated in mammals, including humans, studies in fish species have been relatively limited (Van der Berg et al., 1994). The studies that have been done indicate that TCDF, which is prominently present in the biota of PCDX-contaminated environments such as Lake Ontario (Whittle et al., 1992), is metabolized and eliminated much more slowly by fish than by mammals (Van der Berg et al., 1994). Half-lives of 40– 77 days were reported by Muir et al. (1992) for TCDF in rainbow trout. A half-life of 135 days was reported for TCDF

07-10-96 08:10:56

toxas

AP: Tox

DISPOSITION AND METABOLISM OF TCDF BY TROUT

in carp (Kuehl et al., 1987). In contrast, the half-life of this compound in rats and mice is reported to range from 1 to 4 days (Birnbaum et al., 1980; Decad et al., 1981). Thus, TCDF is an interesting and important model compound for an investigation of the physiological parameters underlying species differences in metabolic fate and disposition. Rainbow trout was selected as one species for the present investigation of the kinetics of tissue distribution, metabolism, and elimination of TCDF because it is an important economic species that is reported to eliminate TCDF only very slowly (Muir et al., 1992). This investigation has included analysis of bile, liver, and muscle for TCDF and its metabolites and the identification of the major metabolites in bile and liver. The overall goal of the investigation was to assess physiological parameters of trout (and possibly other fish species) that determine the metabolic fate and disposition of TCDF in the respective species. The same parameters are a critical aspect of the assessment of the hazard of eating these fish captured from a contaminated environment. Portions of this work have been reported in abstract form (Maslanka et al., 1992; Steward et al., 1992). Parallel studies on the metabolic fate and disposition of TCDF in channel catfish are reported in a companion paper (Steward et al., 1996). MATERIALS AND METHODS 3

Chemicals. [ H]TCDF (sp act 22.2 or 45 Ci/mmol; obtained, respectively, from ChemSyn Scientific, Lenexa, KS, or as a gift from Dr. Stephen Safe of Texas A & M University, College Station, TX) was purified by reversed-phase high-performance liquid chromatography (HPLC) on a Zorbax ODS column eluted at 1.5 ml/min with a 30-min gradient from 70 to 100% methanol. Fractions were collected and TCDF was monitored at 306 nm and by radioassay of small aliquots of each collected fraction. TCDF eluted at 34 min under these conditions. Appropriate fractions were combined and the solvent was removed under nitrogen. The [3H]TCDF was dissolved in acetone and the purity was determined to be greater than 96% by chromatography on a clean column. [3H]TCDF was diluted with unlabeled TCDF (from ChemSyn) to the indicated specific activities. The following authentic methylated derivatives of TCDF metabolites were a gift of Dr. Leo T. Burka of the National Institute of Environmental Health Sciences (Research Triangle Park, NC): 1-methoxy2,3,7,8-tetrachlorodibenzofuran (1-MeO-TCDF), 4-MeO-2,3,7,8-TCDF (4-MeO-TCDF), 3-MeO-2,4,7,8-TCDF (3-MeO-TCDF), 3-hydroxy-2,4,7,8TCDF (3-OH-TCDF), 2-MeO-3,7,8-trichlorodibenzofuran(2-MeO-TriCDF), and 2,2*-di-MeO-4,4*5,5*-tetrachlorobiphenyl[2(MeO)2-TCB] (see Burka and Overstreet, 1989). To minimize potential photodegradation of these chemicals, all work was done under subdued yellow light or under fluorescent lamps protected by ARM-A-Lite Filter Ray shields (Thermoplastic Processes, Stirling, NJ). Biochemicals were obtained from Sigma (St. Louis, MO). Animals. Rainbow trout were obtained from Whispering Pines Fish Farm (Holland, NY). The fish were held in the laboratory in flowing charcoal-filtered dechlorinated tap water at 15 { 17C under a 12-hr/12hr light/dark photoperiod. The fish were fed daily with Ziegler Trout Chow (Ziegler Brothers, Gardner, PA) at a rate of 1.5% of body weight per day. The fish were acclimated to the laboratory for at least 2 weeks prior to dosing. Tissue distribution and half-life. For half-life determination, trout (49 { 8 g) were dosed orally (by No. 4 gelatin capsule, Lilly Co.) with

AID

TOX 7876

/

6h0f$$$422

07-10-96 08:10:56

419

[3H]TCDF (sp act 22.2 Ci/mmol) (per 50 g fish, 50 ng TCDF in 0.65 ml acetone). For the tissue distribution studies, trout (96 { 6 g) received [3H]TCDF of sp act 35.0 Ci/mmol (100 ng/100 g fish). To facilitate the retention of the capsules, the fish were chilled to 37C for 30 min before dosing. After dosing, the flowing, dechlorinated water was gradually warmed to 157C and subsequently maintained at that temperature. The fish were fed daily except from 24 hr before treatment until 24 hr after treatment. At each time point for the half-life study (1, 3, 5, 7, 9, 11, 13, 15, 17, and 19 days), four to six fish were terminated by a sharp blow to the head and frozen at 0207C until analyzed. For determination of tissue distribution, three or four fish were terminated at each time point (8 hr and 1, 3, 7, 14, and 28 days). The trout were partially anesthetized by chilling to 57C, the tail was excised, and the blood was collected into heparinized glass tubes. Each fish was then terminated by a sharp blow to the head. Three 25-ml aliquots of blood from each fish were each solubilized in 1 ml of a 2:1 solution of isopropyl alcohol/Soluene 350 (Packard) overnight. Each solubilized blood sample was then decolorized with 500 ml of a 30% hydrogen peroxide solution, mixed with 10 ml of HionicFluor scintillation fluid (Packard) and (after standing overnight) quantified by liquid scintillation counting. Bile was obtained from the gallbladder and immediately placed on ice (in the dark). Three 25-ml aliquots of bile were solubilized, decolorized, and analyzed in the same manner as the blood. The remainder of the bile was then frozen at 0807C pending further analysis. Liver, kidney, stomach, intestines, abdominal fat, and muscle fillets were dissected, weighed, and stored at 0207C pending analysis. Tissues (and whole fish cut into pieces) were thawed and then homogenized in 9 vol of 0.02 M Tris buffer (pH 7.4) in a Waring blender; triplicate aliquots (typically 100 ml) of each homogenate were then solubilized in 500 ml of Soluene 350 overnight at room temperature, mixed with 10 ml of HionicFluor scintillation fluid, and assayed by scintillation counting, with quench correction by external standard. Background was subtracted. Analysis of TCDF-derived radioactivity in liver and muscle. Liver homogenates were extracted three times with 3 vol of ethyl acetate. The combined extracts were dried with Na2SO4 and concentrated under nitrogen. The resulting liver residues were dissolved in ether and then transferred to a Wheaton microapparatus for methylation with diazomethane (produced from N-methyl-N-nitroso-N-nitroguanidine). The ether was then evaporated and the residues were dissolved in methanol for analysis by HPLC. Bile analysis. To determine conjugated derivatives of TCDF, bile was analyzed by enzymatic hydrolysis with glucurase (bovine b-glucuronidase from Sigma) or with arylsulfatase (type VIII; Sigma) as previously described (Steward et al., 1995), with the following modifications: Aliquots of the bile (diluted with 0.2 M sodium acetate buffer) were incubated with hydrolytic enzymes following a preextraction (at pH 6.5) (three times) with 2 vol of ethyl acetate, followed by exhaustive evaporation of the residual organic solvent. Other aliquots of bile, prepared and incubated in parallel with the first aliquots, were not preextracted with ethyl acetate. As previously described, two controls were used, containing and not containing saccharic acid-1,4-lactone (SAL). The samples (adjusted to pH 5.5) were incubated, with shaking, for 4 hr at 287C. The pH was then adjusted with 0.5 M K2PO4 to pH 6.5 for the final extraction (three times) with 2 vol of ethyl acetate. Each extract was dried over Na2SO4 and evaporated under nitrogen. The resulting residues were methylated by being dissolved in ether containing diazomethane generated in a Wheaton microapparatus. Following evaporation of the ether, each residue was dissolved in methanol (80–100 ml) for analysis by HPLC. HPLC analysis. In general, aliquots of the methylated extracts of bile, liver, or muscle were mixed with UV-detectable amounts of authentic standards [2-(MeO)2-TCB, 1-MeO-TCDF, 3-MeO-TCDF, 4-MeO-TCDF, 2MeO-TriCDF, 3-MeO-TriCDF, and TCDF] and injected onto a Zorbax

toxas

AP: Tox

420

STEWARD ET AL.

ODS column (4.6 1 250 mm), eluted for 25 min with 1.5 ml/min of 80% methanol, followed by a 20-min gradient to 90% methanol. This method was routinely used for bile analyses to quantify both the major metabolite and the unidentified polar metabolites (see Fig. 7). Inasmuch as 3-MeOTCDF and 4-MeO-TCDF were not separated by this method, a confirmatory analysis used the same column, eluted for 25 min with 1.5 ml/min of 75% acetonitrile, followed by a 20-min gradient to 90% acetonitrile. In all cases, fractions were collected every 20 sec or 1 min, mixed with scintillation fluid (Scintiverse E from Fisher), and assayed by liquid scintillation counting. Peaks were identified by comparing retention times of the radioactive peaks with those of the authentic standards (detected at 300 nm) using a lag time between the detector and the fraction collector determined to be 20 sec. The identity of each authentic standard in the UV chromatogram was confirmed spectrally using a Hewlett–Packard diode-array detector. An alternative method used to confirm the identity of the major metabolite found in liver was as follows: An aliquot of unmethylated liver extract was cleaned up by chromatography on a Zorbax ODS column eluted at 1.5 ml/ min with 80% methanol. The fractions eluting between 4 and 6 min (which contained the largest amount of [3H]TCDF-derived radioactivity) were collected, evaporated to dryness under nitrogen, dissolved in methanol, and coinjected with authentic 3-OH-TCDF. Fractions were collected and radioassayed as described previously. Environmental health and safety. TCDF is a dioxin-related compound with a toxicity (in rodents) estimated to be about 0.1 times that of TCDD (Safe, 1990). Fish were treated and maintained in a laboratory specifically approved for the use of hazardous and radioactive materials. The laboratory was provided with cotton and charcoal filters for the effluent water from the fish tanks. The tanks were in a large enclosed space designed to contain any possible overflow from the tanks and equipped with leak detectors. All wastes, including carcasses, were labeled with regard to the concentration of TCDF and stored pending approved disposal. So far as possible, the concentration of TCDF in all wastes was kept to less than 1 ppb. Some wastes containing more than 1 ppb of TCDF [e.g., from HPLC purification of stock [3H]TCDF] were concentrated for optimum storage. Materials containing low concentrations of [3H]TCDF were handled in the hood under laboratory hygiene protocols traditional for the use of radiolabeled materials. Transfers of microgram quantities of solid TCDF to be used in the preparation of stock solutions were done with great care in an enclosed box. A stock solution, containing 300–500 mg TCDF in acetone, was aliquoted into vials at 50 mg per vial. The acetone was evaporated and the vials were stored at 0807C.

FIG. 1. Whole body elimination of TCDF equivalents from trout treated with 1 mg TCDF/kg. The data represent geometric means { 95% confidence levels for n Å 4–6 fish. The half-life (t12) between 3 and 19 days was determined to be 14.8 days (95% CI, 10.5–25.3 days). [t 12 Å (0ln 2/k), where k/day is the slope of the regression line.]

AID

TOX 7876

/

6h0f$$$422

07-10-96 08:10:56

TABLE 1 Half-Lives for the Elimination of TCDF Equivalentsa from Trout

Whole body Liver Muscle Blood

Half-life

95% Confidence interval

14.8 8.64 15.2 12.5

10.5–25.3 5.43–21.1 8.24–98.0 5.29– `

a

In days, calculated from data obtained at 3–19 days (for whole body half-life) or at 3, 7, and 14 days posttreatment (for the tissues). See Fig. 1 legend.

Statistics. As described in the companion paper (Steward et al., 1996), the concentrations of TCDF equivalents in tissues are expressed as geometric means with 68 or 95% confidence intervals equivalent to 1.0 or 2.0 SE, respectively.

RESULTS

Whole body half-life. The elimination of TCDF-derived radioactivity from trout was biphasic: The terminal phase of elimination, starting at the 3-day time point, had a half-life of 14.8 days [95% confidence interval (CI, 10.5–25.3 days) (Fig. 1; Table 1). Based on the assumption that postassimilation elimination was first order, the assimilation efficiency was estimated, as the zero-time intercept of the Phase II regression line, to be 48.5% of the administered dose. During the first 3 days, the 51.5% of the dose that was not assimilated was excreted (presumably unchanged) with an estimated half-life of 0.84 days [determined from the data of Fig. 1 by the method of residuals (Gibaldi and Perrier, 1976)]. Biliary elimination. The first substantial amount of TCDF-derived radioactivity (0.87% of the administered dose) found in gallbladder bile accumulated between 8 and 24 hr posttreatment. (During the first 8 hr, only 0.013% of the dose accumulated in gallbladder bile.) The average concentration of TCDF equivalents secreted in the bile during the 8- to 24-hr time interval, prior to dilution with the bile already in the gallbladder, was estimated to have been 10.5 ng/ml (Fig. 2). The final observed concentration in bile collected at the 1-day time point was 3.7 ng/ml. During the next 2 days, the concentration of TCDF equivalents secreted into the bile decreased rapidly, and then more slowly after the 3-day time point (Fig. 2). The total amount of TCDF equivalents secreted into the bile during the first 14 days, estimated from Fig. 2 as the area under the curve, amounted to 60 ng/kg body wt (i.e., 6.0% of the administered dose) (Fig. 3). For comparison, the total amount of TCDF equivalents eliminated from the fish postassimilation through the 14-day time point was estimated from Fig. 1 to have been 233 ng/kg body wt [the administered dose (1000 ng/kg)

toxas

AP: Tox

DISPOSITION AND METABOLISM OF TCDF BY TROUT

421

FIG. 4. Changes with time in the concentrations of TCDF equivalents in trout liver, kidney, and blood. The data represent geometric means { 68% confidence intervals for n Å 3 or 4 fish. FIG. 2. TCDF equivalents in trout bile. The cumulative bile secretion was estimated as 1.25 ml/kg/day (Schmidt and Weber, 1973). The bars represent means { SE for the measured concentrations of TCDF equivalents (for Days 3–14). Inasmuch as the bile collected at Day 1 had been diluted with bile not containing [3H]TCDF, the concentration of TCDF equivalents excreted between 8 and 24 hr was estimated as the amount of TCDF equivalents collected (8.7 { 3.2 ng/kg fish) divided by the volume of bile secreted during this time period (0.67 day 1 1.25 ml/kg/day), or 10.5 { 3.9 ng/ml bile. The widths of the bars (for Days 3–14) represent the volume of bile collected at each time point [2.16 { 0.09 ml/kg (mean { SE, n Å 15 fish)]. For Day 1, the volume of bile secreted containing TCDF equivalents was estimated as 0.83 ml/kg (see above).

multiplied by the difference between the assimilation efficiency (48.5%) and the amount remaining at 14 days (25.2%)]. Thus, biliary excretion accounted for only about

FIG. 3. TCDF equivalents (i) secreted into bile and (ii) eliminated from the fish following uptake from the gastrointestinal tract. The cumulative amount secreted into the bile was estimated as the area under the curve of Fig. 2. The total amount eliminated (postabsorption) was estimated from Fig. 1 as the difference between the y intercept (48.5%) and the amount remaining in the fish at the given time point.

AID

TOX 7876

/

6h0f$$$423

07-10-96 08:10:56

26% of the total amount eliminated (postassimilation) (Fig. 3). Furthermore, the proportion of the incremental excretion (Dy, determined between two time points) that was found in the bile decreased from 39% at Day 1 to 17% calculated between Days 7 and 14 (Fig. 3). Thus, there appeared to have been a second route of elimination that increased in prominence with time. Tissue distribution. Initially, the tissue with the highest concentration of TCDF equivalents was the liver, in which the highest concentration (recorded at 8 hr) was 0.91 ng/g, decreasing rapidly to 0.37 ng/g at 3 days, and more slowly thereafter (Fig. 4). TCDF equivalents in the blood reached a peak concentration (of 0.14 ng/ml) between 8 and 24 hr, decreased to 0.03 ng/ml at 7 days, and changed very little thereafter. Concentrations of TCDF equivalents in kidney were intermediate between those in liver and blood. The calculated half-lives of TCDF equivalents in liver and blood were 8.6 and 12.5 days, respectively (Table 1). In contrast to liver, kidney, and blood, which had already reached peak or near-peak concentrations of TCDF equivalents by 8 hr posttreatment (Fig. 4), muscle reached a peak concentration of 0.23 ng/g by 24 hr. This concentration remained constant until 7 days post-treatment and decreased slowly thereafter (Fig. 5). TCDF equivalents in abdominal fat reached a peak concentration of 1.7 ng/g at 3 days. This concentration remained constant until 7 days, decreased somewhat between 7 and 14 days, and then actually increased (2.6-fold) between 14 and 28 days (Fig. 5). This apparent equilibration with time of TCDF equivalents between muscle and fat (Table 2; Fig. 5) was consistently observed (p õ 0.05). As a result of these changes, the overall tissue distribution of TCDF and its derivatives changed with time. At 3 days, 85% of the amount of TCDF-derived radioactivity recovered

toxas

AP: Tox

422

STEWARD ET AL.

FIG. 5. Changes with time in the concentrations of TCDF equivalents in trout muscle and abdominal fat. The data represent geometric means { 68% confidence intervals for n Å 3 or 4 fish.

in eight tissues (Table 2) was found in the identified tissue depots (i.e., muscle plus abdominal fat), with 8.9% in intestines plus bile and 2.2% in liver. At 28 days posttreatment, the proportional amount in the tissue depots had increased to 94%, whereas the amounts in intestines plus bile had

decreased to 1.9% and in liver to 1.2% of the recovered radioactivity (Table 2). At all time points, a substantial fraction of the TCDF equivalents (estimated from the whole body elimination data), amounting to about 56% of the body burden at the 3and 14-day time points, was not recovered in the tissues analyzed (Table 2). To clarify this possible discrepancy, carcasses from two fish terminated at the 8-hr time point were homogenized and radioassayed for total TCDF equivalents. The results were corrected by subtracting a portion of the total TCDF equivalents determined for muscle (estimated as 55% of the body weight) remaining in the carcass. The remaining TCDF equivalents, which can be ascribed to carcass devoid of muscle (representing 36% of the body weight), amounted to 44% of the absorbed body burden of TCDF-derived radioactivity [estimated from the extrapolated regression line for Phase II of whole body elimination (Fig. 1) as 47.8% of the administered dose]. Thus, the substantial fraction of the total body burden of TCDF equivalents that was not recovered in the tissues analyzed (Table 2) apparently represented well-perfused lipid deposits in the carcass, which included such highly perfused tissues as gill filaments, brain, eye, and anterior kidney. Fish carcass is known to contain high concentrations of lipid and to take up and retain

TABLE 2 Relative Distribution of [3H]TCDF-Derived Radioactivity in Trout Tissues Days

Liver Biled Intestines / bile Kidney Bloode Abdominal fat Musclee Total in eight tissues Estimated body burden f

3

14

28

0.37 (0.32–0.43)a 0.88b 0.95 (0.75–1.20) 2.25 1.43 (1.13–1.92) 3.39 0.05 (0.04–0.06) 0.12 0.32 (0.20–0.51) 0.76 2.36 (2.18–2.55) 5.60 13.4 (10.9–16.4) 31.8 18.7 (16.4–21.3) 42.2

0.15 (0.13–0.18) 0.60 0.18 (0.08–0.38) 0.71 0.31 (0.20–0.53) 1.23 0.04 (0.03–0.04) 0.16 0.16 (0.14–0.18) 0.64 1.59 (1.18–2.14) 6.31 8.31 (7.35–9.38) 33.0 11.0 (10.0–12.0) 25.2

0.12 (0.09–0.16) NAc 0.11 (0.00–0.34) NA 0.19 (0.14–0.28) NA 0.03 (0.02–0.03) NA 0.16 (0.13–0.19) NA 4.18 (3.55–4.94) NA 5.99 (5.34–6.73) NA 10.9 (10.1–11.8) NA

a Percentage of administered dose. The values are geometric means and 68% confidence intervals for n Å 4 fish (at 3 and 14 days) or 3 fish (at 28 days). b Percentage of estimated body burden. c NA, not available. d Not included in the totals. e Estimated at 5 and 55% of body weight for blood and muscle, respectively (Gingerich et al., 1990). f Estimated from Fig. 1.

AID

TOX 7876

/

6h0f$$$423

07-10-96 08:10:56

toxas

AP: Tox

DISPOSITION AND METABOLISM OF TCDF BY TROUT

FIG. 6. HPLC analysis of TCDF equivalents extracted from trout liver (3 days post-treatment). The extract was methylated, mixed with authentic standards, and then resolved on a Zorbax ODS column eluted for 25 min with 80% methanol, followed by a 20-min gradient to 88% methanol (see Materials and Methods). Fractions were collected and radioassayed.

high concentrations of lipophilic compounds (Kuehl et al., 1986; Kleeman et al., 1986). TCDF-derived radioactivity in liver. When homogenates (pH 7.4) prepared from livers of trout terminated 1 day posttreatment were extracted with ethyl acetate, 2.7 { 1.6% (mean { SD, n Å 3) of the radioactivity remained in the aqueous phase. Aliquots of the ethyl acetate extracts were evaporated. The residues were dissolved in 0.10 M KOH and extracted with hexane. Under these conditions, 35.8 { 2.9% of the extracted radioactivity (34.8% of total liver radioactivity) remained in the aqueous phase, indicating the presence of one or more acidic (presumably phenolic) derivatives of TCDF. When the original ethyl acetate extract was methylated and then analyzed by HPLC (Fig. 6), two radioactive peaks were resolved, each containing 38% of the injected radioactivity (37% of the total TCDF equivalents present in 1-day liver). One peak coeluted with TCDF, whereas the other coeluted with 4-methoxy-TCDF and 3MeO-TCDF (which were not resolved in this system). This peak was confirmed as 4-MeO-TCDF, derived by methylation from 4-OH-TCDF, by HPLC analysis of a cleaned-up aliquot of unmethylated liver extract mixed with authentic 3-OH-TCDF (see Materials and Methods). The metabolite peak eluted with a retention time of 4.5 min, whereas the 3-OH-TCDF eluted at 10.0 min, confirming the identification of the metabolite as 4-OH-TCDF (rather than 3-OH-TCDF). Similar results revealing equal amounts of TCDF and 4OH-TCDF (each amounting to approximately 40% of total extracted radioactivity) were obtained with extracts of liver representing the 0.3-, 1-, 3-, and 7-day time points.

AID

TOX 7876

/

6h0f$$$423

07-10-96 08:10:56

423

TCDF derivatives in bile. When bile pooled from four trout terminated at 3 days was extracted with ethyl acetate (at pH 6.5), 64% of the TCDF-derived radioactivity remained in the aqueous phase. Preliminary experiments revealed that up to 75% of this ‘‘aqueous’’ radioactivity could be subsequently extracted with ethyl acetate following a 5-hr incubation under control conditions (i.e., pH 5.0, 377C, and no added enzymes). The initial interpretation of these data was that relatively unstable conjugated derivatives of TCDF had become nonspecifically hydrolyzed under these acidic conditions. HPLC analysis later suggested an alternative interpretation (see below). However, the hydrolysis protocol that was developed emphasized the use of mild and consistent conditions (i.e., pH 5.5, incubation temperature 287C, 4-hr incubation times, and a constant pH of 6.5 for all extractions). The importance of extracting at constant pH was underscored by data showing a dramatic increase in the extractability of TCDF equivalents from bile as the pH of the medium being extracted was decreased from 7.8 to 5.5 (Steward et al., 1996). To maintain consistent conditions, it was later found necessary to add SAL to some control incubations to control for the presence of this acidic inhibitor in the aryl sulfatase incubations. The protocol was optimized for maximum extraction of TCDF-derived radioactivity following hydrolysis with b-glucuronidase and arylsulfatase, combined with minimum extraction of TCDF equivalents following a control (i.e., nonenzymatic) incubation under the same conditions. That further optimization may prove possible is suggested by later studies on the analysis of TCDF derivatives in catfish bile (Steward et al., 1996). Thus, a study was done to compare the results obtained under the following incubation conditions (Table 3): incubation of bile samples following a preliminary extraction with ethyl acetate (samples CX , CX,SAL , SX,SAL , and GX) vs incubation with no preextraction (samples CNX , CNX,SAL , SNX,SAL , and GNX); incubation with glucurase (GX and GNX), arylsulfatase (SX,SAL and SNX,SAL), or under control conditions without enzyme (samples CX , CX,SAL , CNX , and CNX,SAL); and incubation in the presence of the inhibitor SAL (CX,SAL , SX,SAL , CNX,SAL , and SNX,SAL) and in its absence (CX , GX , CNX , and GNX). In this experiment, 23.7% of the total biliary TCDFderived radioactivity was initially extracted with ethyl acetate from the bile (SX0) (Table 3). Incubation with b-gluronidase substantially increased the amount of TCDF-derived radioactivity that was subsequently extracted with ethyl acetate. The increase in the amount of extracted radioactivity under these conditions amounted to 59–62% of total biliary radioactivity and could be accounted for by increases in the amounts of radioactivity in peaks B1, B2, and the peak coeluting with 3- and 4-MeO-TCDF (Fig. 7). The total amount of radioactivity obtained in the preextracts, combined with the amount of radioactivity extracted following

toxas

AP: Tox

424

STEWARD ET AL.

TABLE 3 HPLC Analysis of 7-Day Bile Following Enzymatic Hydrolysis % of TCDF-derived radioactivity in bilea Samples extracted with EtOAc prior to hydrolysis Peak A1 A2 B1 B2 3-/4-MeO-TCDF TCDF Se

Samples not extracted prior to hydrolysis

Retention time (min)

Preextract X0b

CXc,d

CX,SAL

SX,SAL

GX

CNX

CNX,SAL

SNX,SAL

GNX

1.8 4.8 7.3 9.5 30–31 33–35

4.8 2.3 — — 9.6 1.9 23.7

0.4 3.5 — — 0.4 1.3 8.0

0.4 17.0 — — — 2.0 19.4

0.4 12.0 — — 2.2 3.1 22.2

0.7 0.9 13.6 8.0 37.9 1.9 66.9

13.2 6.1 — — 3.3 3.4 28.7

13.6 13.4 — — 6.2 3.3 39.8

0.9 12.1 — — 20.2 4.3 43.3

15.1 1.0 12.8 7.6 44.2 3.0 91.1

a

All extracts were methylated with diazomethane and analyzed by HPLC as shown in Fig. 7. X0 , ethyl acetate extract obtained from bile prior to hydrolysis. c C, control samples (incubated at pH 5.5); S, incubated with arylsulfatase; G, incubated with b-glucuronidase. d Subscripts: X, extracted prior to enzymatic hydrolysis; NX, not extracted prior to hydrolysis; SAL, saccharic acid-1,4-lactone added as an inhibitor of b-glucuronidase. e S, Percentage of total biliary TCDF-derived radioactivity in extract prior to preparation for HPLC. b

incubation (under each of four conditions) of these preextracted samples, agreed well with the amount of radioactivity recovered under the same incubation conditions without preextraction (Table 3). However, some results were surprising. Rather than increasing in magnitude (or remaining the same) following enzymatic hydrolysis, peak A1, which was substantial in control sample CNX,SAL , almost disappeared in the corresponding sample incubated with arylsulfatase (sample SNX,SAL) (Table 3; Fig. 7). Likewise, peak A2 largely disappeared in the sample incubated with b-glucuronidase (sample GNX compared with sample CNX). A proposed interpretation of these results is that these polar peaks may represent conjugates that were partially extracted by ethyl acetate: Peak A1 might represent a sulfate that was hydrolyzed by arylsulfatase. Peak A2, on the other hand, might represent a glucuronide that was hydrolyzed by b-glucuronidase. The presence of the 3-/4-MeO-TCDF peak in sample SNX,SAL (compared to sample CNX,SAL) provides evidence of the presence of sulfates, amounting to 14% of biliary radioactivity. In contrast, the presence of a substantial amount of sulfates is not apparent from a comparison of the total amounts of TCDF-derived radioactivity present in these two extracts. Nor is the presence of sulfates apparent in the samples that had been preextracted with ethyl acetate (comparing total amounts and peaks for samples SX,SAL vs CX,SAL). Apparently, sulfates had been removed by the preliminary extraction and were possibly not quantitatively recovered in the X0 extract [because sulfates in ethyl acetate extracts tend to partition into the aqueous phase when the extract is dried over Na2SO4

AID

TOX 7876

/

6h0f$$$423

07-10-96 08:10:56

(A. R. Steward, unpublished observations)]. The major metabolite peak is much more prominent in SNX,SAL than in SX,SAL , presumably for the same reason. If this interpretation is correct, substantial amounts of TCDF-derived radioactivity that are extracted from bile following a control incubation are unhydrolyzed conjugates rather than hydrolysis products of unstable conjugates, as initially thought. Another unexpected result was the substantially larger amount of radioactivity extracted from control samples incubated in the presence of SAL compared to the corresponding samples incubated in its absence (CNX,SAL compared to CNX and CX,SAL compared to CX) (Table 3). These results are particularly clear in the data for peak A2 (proposed to represent glucuronides). The interpretation of these data is not clear at this time. Conceivably, SAL either increases the extractability of glucuronides by ethyl acetate or inhibits their hydrolysis even under control conditions. These results indicate that for optimum quantitation SAL must be added to appropriate control samples. As a result of this experiment, it was determined that although glucuronide conjugates of metabolites other than peaks A and B could be determined either with or without a preextraction with ethyl acetate, i.e., as either GX 0 CX or GNX 0 CNX , sulfates could be determined only in samples that had not been preextracted, i.e., only as SNX,SAL 0 CNX,SAL . The reliable determination of either glucuronides or sulfates requires chromatographic analysis of the extracts. The major primary metabolite in bile (retention time following methylation, 30 to 31 min) coeluted with 4-MeOTCDF and 3-MeO-TCDF, which were not resolved in this

toxas

AP: Tox

DISPOSITION AND METABOLISM OF TCDF BY TROUT

chromatography system (Fig. 7A). The identification of this metabolite as 4-OH-TCDF was confirmed by chromatography in solvent system B (containing acetonitrile instead of methanol). The resolution of 4-MeO-TCDF and 3-MeOTCDF in this solvent system is shown in Fig. 7E. The (methylated) biliary metabolite coeluted with 4-MeO-TCDF. Thus, the major metabolite in trout bile was TCDF-4-Oglucuronide, which amounted to 27–39% of TCDF equivalents in bile obtained at the 1-, 3-, and 7-day time points (Table 4). TCDF equivalents in muscle. At least 99% of the TCDFderived radioactivity in muscle was extracted with ethyl acetate. These extracts were evaporated and the residues were dissolved in 0.1 M KOH and extracted with hexane. Less than 1% of the radioactivity remained in the aqueous phase.

425

The identity of the hexane-extractable compound, representing over 98% of the TCDF equivalents in muscle, was confirmed by HPLC to be unmetabolized TCDF. A single peak, coeluting with authentic TCDF, represented 87% of the injected radioactivity (data not shown). DISCUSSION

The kinetics of the tissue distribution of TCDF equivalents is fully consistent with published data on the relative rates of perfusion of the respective tissues (Nichols et al., 1990, 1991). Kidney and liver, which are well perfused, reach peak concentrations within the first 8 hr. At this early time point, the liver was the tissue with the highest concentration of [3H]TCDF-derived radioactivity, as expected for a first-pass

FIG. 7. HPLC analysis of [3H]TCDF-derived radioactivity in a pool of trout bile (from four fish) collected 7 days after treatment. Resolution of authentic standards (A) and extracts of bile obtained following incubation (see Table 3) (B) without enzyme but with saccharic acid-1,4-lactone (SAL) (sample CNX,SAL), (C) with arylsulfatase plus SAL (sample SNX,SAL), and (D) with b-glucuronidase (sample GNX). Bile was not extracted prior to these incubations. The extracts of B–D were methylated, mixed with authentic standards, and then chromatographed under the same conditions as described in the legend to Fig. 6, except that the final concentration of methanol was 90%. (E and F) Confirmation of the major metabolite peak seen in D as the methylated derivative of 4-OH-TCDF by chromatography on a Zorbax ODS column eluted for 25 min with 75% acetonitrile, followed by a 20-min gradient to 90% acetonitrile.

AID

TOX 7876

/

6h0f$$$423

07-10-96 08:10:56

toxas

AP: Tox

426

STEWARD ET AL.

TABLE 4 TCDF Derivatives in Trout Liver and Bile % of TCDF-derived radioactivity ina

Peaks Bd Unconjugated Glucuronides Sulfates 4-OH-TCDF f Unconjugated Glucuronide Sulfated Water-soluble (liver) TCDFg

Liverb

Bilec

0 NDe ND

0–1.2 1.8–21 0–1.6

37 ND ND 3 37

0.2–10 27–39 0–14 2.5–3.8

a All extracts were methylated with diazomethane and analyzed by HPLC as shown in Fig. 7. b From fish terminated 1 day posttreatment. c Range of five hydrolysis experiments on three pools of bile, each pool representing four fish. The three groups of fish were terminated 1, 3, and 7 days posttreatment. Two hydrolysis experiments were done on each of the 1- and 7-day pooled bile. Each datum was corrected for the corresponding HPLC fraction from the appropriate control incubated without hydrolytic enzymes. d Data for Peaks B (retention times, 7.3–9.5 min) and for TCDF-4-Osulfate are ranges for three hydrolysis experiments, one experiment at each time point. e ND, Not determined. f Retention time of the methylated derivative, 30 or 31 min. g Retention time, 33–35 min.

distribution to this organ. Muscle and fat, in contrast, which are less well perfused, reached peak concentrations between 1 and 3 days. It has been observed (Van den Berg et al., 1994) that in fish, in contrast to mammals, PCDDs and PCDFs (together designated PCDXs) partition among the tissues predomi-

nantly in proportion to the lipid content of the respective tissues. The partitioning of TCDF equivalents from the blood into the liver was greater than would have been predicted from the lipid contents of blood and liver (as much as twofold greater at 8 hr) (Table 5; Nichols et al., 1991). In contrast, at later time points (e.g., 14–28 days) TCDF equivalents appeared to be in equilibrium among the lipids of blood, liver, and kidney (Table 5). The accumulation of substantial amounts of polar TCDF metabolites in the liver (about 40% of total TCDF equivalents at the 1-day time point) could account in part for the apparently disproportionate partitioning of TCDF-derived radioactivity into liver at early time points. The accumulation in the liver of large amounts of unconjugated 4-OH-TCDF, which was present at only low concentrations in bile (Table 4), suggests that the conversion of 4-OH-TCDF to water-soluble compounds is a rate-limiting step compared to the excretion of these water-soluble derivatives into bile, which occurs rapidly. A considerable fraction of the TCDF metabolites, which increased with time, apparently was excreted from the fish via extrabiliary pathways (Fig. 3). This could perhaps be attributed to transluminal excretion of unmetabolized TCDF directly into the intestines, as has been proposed for other polyhalogenated aromatic hydrocarbons (Kedderis et al., 1993). Excretion of 4OH-TCDF (formed in the liver) via the gills or (following conjugation) via kidney (Pritchard and Bend, 1984) are alternative possibilities. In contrast to the data for liver, there is little evidence that polar TCDF derivatives accumulated in the kidney (Fig. 4; Table 5). These results contrast sharply with corresponding data obtained for channel catfish (Steward et al., 1996) in which kidney did appear to accumulate TCDF-derived radioactivity at the early time points, despite the fact that a much larger proportion of the whole body elimination of TCDF appeared to occur via biliary excretion. The tissue depots (i.e., muscle and abdominal fat) had lower (lipid normalized) concentrations of TCDF than did

TABLE 5 Concentrations of TCDF Equivalents in Trout Tissues, Normalized to Lipid Content Days posttreatment Lipid (%)a Liver Kidney Blood Fat Muscle

4.7 5.0 1.5 94.2 3.0

{ { { { {

0.3 19.4b 5.34 9.36 0.14 2.08

0.3 0.3 0.1 1.0 0.2

1

(12.6–29.7)c (3.63–7.87) (7.49–11.7) (0.10–0.20) (1.06–4.08)

15.9 5.20 9.50 1.17 7.63

(12.3–20.5) (4.14–6.52) (7.37–12.3) (0.76–1.79) (5.92–9.83)

3 7.95 3.26 4.11 1.85 7.78

(6.52–9.69) (2.64–4.01) (2.56–6.59) (1.71–2.00) (6.34–9.54)

a

From Nichols et al. (1991). ng TCDF equivalents/g lipid. c 68% confidence interval for n Å 3 or 4 fish. b

AID

TOX 7876

/

6h0f$$$423

07-10-96 08:10:56

toxas

AP: Tox

14 3.00 1.70 2.05 1.25 4.83

(2.41–3.73) (1.55–1.86) (1.78–2.36) (0.93–1.68) (4.27–5.46)

28 2.30 1.07 2.01 3.28 3.49

(1.68–3.15) (0.89–1.29) (1.66–2.44) (2.78–3.87) (3.11–3.92)

427

DISPOSITION AND METABOLISM OF TCDF BY TROUT

TABLE 6 TCDF in Liver Compared to Body Burden: Species Comparisons TCDF in liver Species Rainbow trout Rainbow trout Rainbow trout Rainbow trout Rainbow trout Rainbow trout Channel catfish Channel catfish Mouse (C57/BL6) Mouse (DBA/2) Rat Monkey a b

Dose, route (mg/kg)

Time point (day)

Cba (mg/kg)

Half-life (days)

1.0, po 1.0, po 0.91 ppb diet 9.2 ppb diet 9.2 ppb diet 9.2 ppb diet 1.0, po 1.0, po 31, iv 31, iv 31, iv

3 14 140 10 30 140 3 14 3 3 3

0.42 0.25 0.45 0.76 1.25 2.83 0.36 0.036 9.2 14.6 5.7 0.055

15 15 47 54 54 54 3.6 2 4 1.3

% of Cb 0.87 0.60 0.75 2.35 2.09 0.90 18.3 1.88 75.4 35.1 31.6 21.5

mg/kg liver

0.37 0.14 0.25 0.84 1.34 1.29 5.7 0.04 125b1 96b2 50b3

Reference This study This study Muir et al. (1992) Muir et al. (1992) Muir et al. (1992) Muir et al. (1992) Steward et al. (1996) Steward et al. (1996) Decad et al. (1981) Decad et al. (1981) Birnbaum et al. (1980) Neubert et al. (1990)

Body burden of TCDF. Maximum concentrations of TCDF in liver were (in mg/kg liver): 328 (at 15 min)b1, 226 (3 hr)b2; 348 (3 hr)b3.

blood at the earliest time point (Table 5). Muscle lipids required 1–3 days to reach equilibrium with blood lipids, whereas abdominal fat required 14–28 days (Table 5). At the latest time point, the lipid-corrected concentrations of TCDF equivalents in muscle and fat were similar and significantly greater than those in blood, suggesting that TCDF may actually be more soluble in the nonpolar lipids of fat and muscle tissue than in the lipoproteins of blood and liver. Trout liver, at least under the conditions of this experiment, appears to play a much less significant role in the distribution, metabolism, and elimination of TCDF than has been reported for mammalian liver (Carrier et al., 1995a; Decad et al., 1981; Birnbaum et al., 1980) or which was observed in similar experiments with channel catfish (Steward et al., 1996). For example, the fraction of the total body burden of TCDF found in trout liver in these studies was almost two orders of magnitude less than the fraction of the body burden previously reported in mammalian liver (Table 6). The accumulation of PCDXs in mammalian liver appears to result from the presence of high concentrations of hepatic PCDX-binding proteins, which are present at low levels in untreated animals and are induced to high levels by PCDXs (Carrier et al., 1995a,b). These binding proteins include cytochrome P450 IA2, the enzymatic activity of which appears to be conspicuously absent in trout liver (Steward et al., 1995; Elmarakby et al., 1995). This lack of cytochrome P450 IA2 and/or other binding proteins may help to explain the relatively low concentration of TCDF in trout liver compared to rat and mouse liver (Table 6). A consequence of the induction, and subsequent deinduction, of these binding proteins in mammalian liver is a pattern of whole body elimination, under quasi-steady-state condi-

AID

TOX 7876

/

6h0f$$$423

07-10-96 08:10:56

tions, that is not first order and in which the rate of elimination consequently decreases (and the whole body half-life increases) as the body burden decreases (Carrier et al., 1995a,b). If data from studies such as this one are to be useful in assessing the risk (both to humans and to environmental predators) of eating contaminated fish, it is important to know whether the depuration rate of TCDF and related compounds in fish also depends on the body burden. This question was addressed by Muir and co-workers (1992), who examined the effects of different concentrations of TCDF in fish diet on the depuration rate and whole body half-life of TCDF. Some of these data are summarized in Table 6. These workers found that at a very high concentration of TCDF in the feed (43 ppb) (not shown), the whole body half-life was reduced somewhat (from 54 to 40 days), consistent with the concept of the metabolism of TCDF being induced at this high dose. However, no consistent dose–response relationship was apparent (Table 6). The difference between the TCDF half-lives determined in a 140-day feeding study (Muir et al., 1992) (i.e., 40–54 days) and the half-life found in this 19-day study (i.e., 14.8 days) cannot be attributed to differences in body burden of TCDF in the respective investigations (Table 6). However, the difference might be explainable by the appearance of a third phase of elimination as the system approaches quasisteady state. Criteria for such a steady state include constant ratios of TCDF concentration in tissues vs the central compartment (i.e., blood) that are determined by the lipid content of the respective tissues or some other partition coefficient. Based on the data of Table 5, such a steady state was being approached at 28 days posttreatment. The whole body elimination study, upon which the half-life determination of 14.8

toxas

AP: Tox

428

STEWARD ET AL.

days is based, extended for only 19 days (Fig. 1), which may have been too short to reveal such a steady-state elimination phase. However, the half-life of TCDF in trout can also be estimated from the tissue distribution study. Based on the total recovery of TCDF-derived radioactivity in the tissues examined (Table 2), the whole body half-lives determined between 3 and 14 days and between 7 and 28 days were 13.7 and 35.1 days, respectively. The latter value approaches the half-lives reported by Muir et al. (1992). The large amount of TCDF retained by trout muscle [32 or 33% of the body burden of TCDF 3–14 days posttreatment (Table 2)] contrasts sharply with the comparatively small amounts of the body burden of TCDF found in Fisher rat and C57/BL6 mouse muscle (5 and 1.5%, respectively) 3 days posttreatment (Decad et al., 1981; Birnbaum et al., 1980). By simulation of data obtained in several mammalian species (including humans), nearly all (§95%) of the body burden of PCDXs was found to be distributed between liver proteins and the lipid content of all tissues (Carrier et al., 1995a), indicating that these compounds occur almost entirely either bound to liver proteins or dissolved in lipid. In trout at steady state, binding of TCDF to liver proteins appears to be insignificant compared to solvation in lipid, including lipids that are an integral part of trout muscle. Thus, the retention of TCDF by muscle appears to be a major factor limiting the metabolism and elimination of TCDF by trout. Despite these species differences in the kinetics and dynamics of tissue distribution and elimination of TCDF, the major primary metabolite of TCDF identified in rats (Burka et al., 1990), rainbow trout (Figs. 6 and 7; Table 4), and channel catfish (Steward et al., 1996) is the same: 4-OHTCDF. In rainbow trout, as in the other species, this primary metabolite was conjugated and excreted in the bile, primarily as TCDF-4-O-glucuronide. The method of bile analysis and data interpretation, here reported in detail, is critically important because without close attention to these details no interpretable quantitative data can be obtained. To the best of our knowledge, these details have not been previously reported. The results of this investigation reveal two features of the physiology of rainbow trout that contribute to the hazard of TCDF contamination to birds and mammals that eat this fish: (i) Unmetabolized TCDF is highly retained by trout muscle, thereby making TCDF relatively unavailable for uptake, metabolism, and elimination by liver; and (ii) the TCDF that is taken up by trout liver is metabolized only very slowly. The inherently slow rate of metabolism of TCDF by trout liver cytochrome P450 IA1, the enzyme believed responsible for the initial oxidative metabolism of TCDF (Tai et al., 1993; Olson et al., 1994), is underscored by observations made with hepatocytes isolated from trout pre-

AID

TOX 7876

/

6h0f$$$423

07-10-96 08:10:56

treated with 3-methylcholanthrene to induce this enzyme. These hepatocytes, which metabolized benzo[a]pyrene rapidly, nevertheless metabolized [3H]TCDF only extremely slowly to form derivatives that included 4-OH-TCDF and water-soluble products (A. R. Steward and H. Sikka, manuscript in preparation). It is important to note that the rate of metabolism determined in the present in vivo study designed to determine the capability of trout to eliminate a single dose of TCDF over a relatively short time period resulted in an estimated half-life of TCDF that appears to be much shorter than half-lives that might be applicable under environmentally relevant conditions (Muir et al., 1992). Similar considerations may also apply to the elimination of TCDF by mammals (Olson et al., 1994). ACKNOWLEDGMENTS We thank Dr. Stephen Safe of Texas A & M University, College Station, Texas, for a gift of [3H]TCDF and Dr. Leo T. Burka, of the National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, for a gift of authentic TCDF metabolite standards.

REFERENCES Birnbaum, L. S., Decad, G. M., and Matthews, H. B. (1980). Disposition and excretion of 2,3,7,8-tetrachlorodibenzofuran in the rat. Toxicol. Appl. Pharmacol. 55, 342–352. Burka, L. T., and Overstreet, D. (1989). Synthesis of possible metabolites of 2,3,7,8-tetrachlorodibenzofuran. J. Agric. Food Chem. 37, 1528–1532. Burka, L. T., McGown, S. R., and Tomer, K. B. (1990). Identification of the biliary metabolites of 2,3,7,8-tetrachlorodibenzofuran in the rat. Chemosphere 21, 1231–1242. Carrier, G., Brunet, R. C., and Brodeur, J. (1995a). Modeling of the toxicokinetics of polychlorinated dibenzo-p-dioxins and dibenzofurans in mammalians, including humans. I. Nonlinear distribution of PCDD/PCDF body burden between liver and adipose tissues. Toxicol. Appl. Pharmacol. 131, 253–266. Carrier, G., Brunet, R. C., and Brodeur, J. (1995b). Modeling of the toxicokinetics of polychlorinated dibenzo-p-dioxins and dibenzofurans in mammalians, including humans. II. Kinetics of absorption and disposition of PCDDs/PCDFs. Toxicol. Appl. Pharmacol. 131, 266–276. Cook, P. M., Kuehl, D. W., Walker, M. K., and Peterson, R. E. (1991). Bioaccumulation and toxicity of TCDD and related compounds in aquatic ecosystems. In Biological Basis for Risk Assessment of Dioxins and Related Compounds (M. A. Gallo, R. J. Scheuplein, and K. A. van der Heijden, Eds.), Banbury Report, Vol. 35. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Decad, G. M., Birnbaum, L. S., and Matthews, H. B. (1981). Distribution and excretion of 2,3,7,8-tetrachlorodibenzofuran in C57BL/6J and DBA/ 2J mice. Toxicol. Appl. Pharmacol. 59, 564–573. Elmarakby, S. A., Shappell, N. W., Kumar, S., and Sikka, H. C. (1995). Comparative metabolism of 2-acetylaminofluorene by rainbow trout and rat liver subcellular fractions. Aquat. Toxicol. 33, 1–15. Fletcher, C. L., and McKay, W. A. (1993). Polychlorinated dibenzo-pdioxins (PCDDs) and dibenzofurans (PCDFs) in the aquatic environment—A literature review. Chemosphere 26, 1041–1069. Gibaldi, M., and Perrier, D. (1975). Pharmacokinetics. Dekker, New York.

toxas

AP: Tox

DISPOSITION AND METABOLISM OF TCDF BY TROUT Gingerich, W. H., Pityer, R. A., and Rach, J. J. (1990). Whole body and tissue blood volumes of two strains of rainbow trout (Oncorhynchus mykiss). Comp. Biochem. Physiol. A 97, 615–620. Henderson, R. J., and Tocher, D. R. (1987). The lipid composition and biochemistry of freshwater fish. Prog. Lipid Res. 26, 281–347. Kedderis, L. B., Mills, J. J., Andersen, M. E., and Birnbaum, L. S. (1993). A physiologically based pharmacokinetic model for 2,3,7,8-tetrabromodibenzo-p-dioxin (TBDD) in the rat: Tissue distribution and CYP 1A induction. Toxicol. Appl. Pharmacol. 121, 87–98. Kleeman, J. M., Olson, J. R., Chen, S. M., and Peterson, R. E. (1986). Metabolism and disposition of 2,3,7,8-tetrachlorodibenzo-p-dioxin in rainbow trout. Toxicol. Appl. Pharmacol. 83, 391–401. Kuehl, D. W., Cook, P. M., and Batterman, A. R. (1986). Uptake and depuration studies of PCDDS and PCDFS in freshwater fish. Chemosphere 15, 2023–2026. Kuehl, D. W., Cook, P. M., Batterman, A. R., Lothenbach, D., and Butterworth, B. C. (1987). Bioavailability of polychlorinated dibenzo-p-dioxins and dibenzofurans from contaminated Wisconsin River sediment to carp. Chemosphere 16, 667–679. Maslanka, R., Steward, A. R., Pangrekar, J., and Sikka, H. C. (1992). Disposition and metabolism of 2,3,7,8-tetrachlorodibenzofuran in rainbow trout. Mar. Environ. Res. 34, 255–259. Muir, D. C. G., Yarechewski, A. L., Metner, D. A., and Lockhart, W. L. (1992). Dietary 2,3,7,8-tetrachlorodibenzofuran in rainbow trout: Accumulation, disposition, and hepatic mixed-function oxidase enzyme induction. Toxicol. Appl. Pharmacol. 117, 65–74. Neubert, D., Wiesmu¨ller, T., Abraham, K., Krowke, R., and Hagenmaier, H. (1990). Persistence of various polychlorinated dibenzo-p-dioxin and dibenzofurans (PCDDs and PCDFs) in hepatic and adipose tissue of Marmoset monkeys, Arch. Toxicol. 64, 431–442. Nichols, J. W., McKim, J. M., Andersen, M. E., Gargas, M. L., Clewell, H. J., III, and Erickson, R. J. (1990). A physiologically based toxicokinetic model for the uptake and disposition of waterborne organic chemicals in fish. Toxicol. Appl. Pharmacol. 106, 433–447. Nichols, J. W., McKim, J. M., Lien, G. J., Hoffman, A. D., and Bertelsen, S. L. (1991). Physiologically based toxicokinetic modeling of three waterborne chloroethanes in rainbow trout (Oncorhynchus mykiss). Toxicol. Appl. Pharmacol. 110, 374–389. Olson, J. R., McGarrigle, B. P., Gigliotti, P. J., Kumar, S., and McReynolds, J. H. (1994). Hepatic uptake and metabolism of 2,3,7,8-tetrachlorodibenzo-p-dioxin and 2,3,7,8-tetrachlorodibenzofuran. Fundam. Appl. Toxicol. 22, 631–640. Opperhuizen, A., and Sijm, D. T. H. M. (1990). Bioaccumulation and

AID

TOX 7876

/

6h0f$$$424

07-10-96 08:10:56

429

biotransformation of polychlorinated dibenzo-p-dioxins and dibenzofurans in fish. Environ. Toxicol. Chem. 9, 175–186. Pritchard, J. B., and Bend, J. B. (1984). Mechanisms controling the renal excretion of xenobiotics in fish: Effects of chemical structure. Drug Metab. Rev. 15, 655–671. Safe, S. (1990). Polychlorinated biphenyls (PCBs), dibenzo-p-dioxins (PCDDS), dibenzofurans (PCDFs), and related compounds: Environmental and mechanistic considerations which support the development of toxic equivalency factors (TEFs). Crit. Rev. Toxicol. 21, 51–88. Schmidt, D. C., and Weber, L. J. (1973). Metabolism and biliary excretion of sulfobromophthalein by rainbow trout (Salmo gairdneri). J. Fish. Res. Bd. Can. 30, 1301–1308. Stalling, D. L., Norstrom, R. J., Smith, L. M., and Simon, M. (1985). Patterns of PCDD, PCDF, and PCB contamination in Great Lakes fish and birds and their characterization by principal components analysis. Chemosphere 14, 627–643. Steward, A. R., Maslanka, R., Kumar, S., and Sikka, H. C. (1992). Metabolism of 2,3,7,8-tetrachlorodibenzofuran by rainbow trout (Oncorhynchus mykiss). Chemosphere 25, 1215–1220. Steward, A. R., Elmarakby, S. A., Stuart, K. G., Kumar, S., and Sikka, H. C. (1995). Metabolism of 2-acetylaminofluorene by hepatocytes isolated from rainbow trout. Toxicol. Appl. Pharmacol. 130, 188–196. Steward, A. R., Maslanka, R., Stuart, K. G., Kumar, S., and Sikka, H. C. (1996). Disposition and metabolism of 2,3,7,8-tetrachlorodibenzofuran by channel catfish (Ictalurus punctatus). Toxicol. Appl. Pharmacol. 139, 430–436. Tai, H. L., McReynolds, J. H., Goldstein, J. A., Eugster, H. P., Senstag, C., Alworth, W. L., and Olson, J. R. (1993). Cytochrome P-450 1A1 mediates the metabolism of 2,3,7,8-tetrachlorodibenzofuran (TCDF) in the rat and human. Toxicol. Appl. Pharmacol. 123, 34–42. Van Cantfort, J., DeGraeve, J., and Gielen, J. E. (1977). Radioactive assay for aryl hydrocarbon hydroxylase. Improved method and biological importance. Biochem. Biophys. Res. Commun. 79, 505–512. Van den Berg, M., De Jongh, J., Poiger, H., and Olson, J. R. (1994). The toxicokinetics and metabolism of polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) and their relevance for toxicity. Crit. Rev. Toxicol. 24, 1–74. Van der Weiden, M. E. J., Tibosch, H. J. H., Bleumink, R., Sinnige, T. L., van de Guchte, C., Seinen, W., and van den Berg, M. (1993). Cytochrome P450 IA induction in the common carp (Cyprinus carpio) following exposure to contaminated sediments with halogenated polyaromatics. Chemosphere 27, 1297–1309. Whittle, D. M., Sergeant, D. B., Huestis, S. Y., and Hyatt, W. H. (1992). Foodchain accumulation of PCDD and PCDF isomers in the Great Lakes aquatic community. Chemosphere 25, 1559–1563.

toxas

AP: Tox