Department of Medicinal Chemistry, College of Pharmacy, University of Florida, Gainesville, Florida 32610; and the Whitney Laboratory,University of Florida, St. Augustine, Florida 32086
Received March 7, 2000; accepted May 12, 2000
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ABSTRACT |
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Key Words: 9-hydroxybenzo(a)pyrene; lobster; bioavailability; sulfation of 9-hydroxybenzo(a)pyrene; glucose conjugation; accumulation of lipophilic xenobiotic.
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INTRODUCTION |
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American lobsters, Homarus americanus, that were environmentally exposed to PAH were shown to bioaccumulate PAH in the edible muscle and hepatopancreas tissues (Dunn and Fee, 1979; Uthe and Musial, 1986
). The hepatopancreas is a storage and digestive organ with functional similarity to the vertebrate liver (Cobb and Phillips, 1980
). A single oral or parenteral dose of BaP was very slowly eliminated from the American lobster, and was retained in the muscle and hepatopancreas for long periods, largely as parent BaP (James et al., 1995
). The estimated elimination half-life of BaP in the American lobster was 30 days to 2 months (Foureman et al., 1978
; James et al., 1995
). Low cytochrome P450-dependent monooxygenase activity with BaP was detected in the hepatopancreas of the American lobster (James, 1989
). The high lipophilicity of BaP, together with its slow rate of cytochrome P450-dependent biotransformation in the lobster hepatopancreas, appeared to be important factors contributing to the potential for bioaccumulation of BaP and other PAH in lobster tissues. In contrast to BaP, which must first be metabolized by cytochrome P450, compounds such as phenol (James et al., 1991) and 2-naphthol (Li and James, 1997
) were more readily excreted by the American lobster than BaP, as parent compound or conjugates. Phenolic groups may be conjugated with glucose and sulfate in the hepatopancreas and antennal gland of the lobster (Li and James, 1993
). These and other studies suggest that the potential for bioaccumulation of xenobiotics by the lobster may be dependent not only on lipophilicity, but also on the likelihood that biotransformation would be through phase-2 pathways. Previous in vivo studies in the lobster did not examine highly lipophilic hydroxylated compounds; the log P of phenol is 1.46 and that of 2-naphthol 2.84 (Hansch and Leo, 1979
). Like BaP, which has a log P of 6.0 (Mackay and Paterson, 1991
), the phenolic BaP metabolites, such as 3-OH-BaP and 9-OH-BaP, are highly lipophilic, with a calculated log P of about 5.3 (Leo et al., 1971
). The phenolic hydroxy group in 3- or 9-OH-BaP is weakly acidic and has a similar pKa, about 9.1, to that of 2-naphthol. Based on lipophilicity alone, it may be expected that BaP phenols would be retained in the lobster. In the environment, lobsters may be exposed to BaP phenols formed from BaP in prey such as small fish, which convert BaP to 3-hydroxyBaP, 7-hydroxyBaP, and 9-hydroxyBaP as well as other, predominantly benzo-ring, metabolites (James et al 1988
; James and Little, 1981
; Little et al., 1984
; Stegeman et al., 1984
; Ueng et al. 1994
). As discussed above, both 3- and 9-OH-BaP are of toxicological interest because they are precursors of mutagenic metabolites. It was decided to investigate 9-OH-BaP as a model BaP phenol in the lobster because it is more chemically stable than 3-OH-BaP, especially in the radiolabeled form. In the present study, the pharmacokinetics, oral bioavailability, and biotransformation of 9-OH-BaP were investigated in intermolt lobsters following oral or intrapericardial administration of [3H]-9-OH-BaP, 50, or 200 µg/kg. These doses were selected as being in the range to which lobsters may be exposed if they inhabit PAH-contaminated environments (Hattemer-Frey and Travis, 1991
; Uthe and Musial, 1986
).
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MATERIALS AND METHODS |
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Lobsters.
Experiments were conducted at the Whitney Laboratory, Florida. The treated animals were held singly in tanks with flowing (4 l/min) chilled seawater at 14 ± 2°C. All animals, weight 500 ± 60 g, were obtained from a restaurant supplier. Lobsters were held at the Whitney Laboratory in fresh, sand-filtered, flowing, full-strength natural seawater at 1214°C and maintained on a diet of squid for at least two weeks before use. Experimental lobsters were molt-staged before and after the experiment (Aiken, 1973 and Altman et al., 1994). At the time of dosing, all lobsters were in the intermolt stage.
Treatment and sampling.
For intravascular (iv) low-dose studies (approximately 50 µg/kg), undiluted [3H]-9-OH-BaP was injected into the pericardial sinus from DMSO solution (0.5 ml/kg). For iv high-dose studies (approximately 200 µg/kg), the [3H]-9-OH-BaP was diluted with unlabeled 9-OH-BaP to a specific activity of 210 mCi/mmol. Because it was necessary to withdraw hemolymph into the iv dose syringe to ensure correct placement, the exact dose given each lobster was corrected for the radioactivity remaining in the syringe after the dose. For the oral dose (approximately 200 µg/kg), undiluted [3H]-9-OH-BaP was used. Food was withheld for 3 days before dosing, the dose was administered intragastrically, combined with a shrimp paste, and food was given 2 h after the treatment. The shrimp paste dose was prepared by homogenizing shrimp with sufficient water to give a smooth consistency and adding a DMSO solution of the [3H]-9-OH-BaP such that 1 g paste contained 60 µCi (40 µg). After thorough mixing and assaying several samples of the paste to ensure even distribution of the [3H]-9-OH-BaP, lobsters were given, by syringe, 5 g paste/kg body weight. The exact dose was obtained by weighing the syringe before and after dosing.
Hemolymph samples, 12 ml, were taken from the appendages of the lobsters. The hemolymph volume of the lobsters used was estimated as 105 ± 11 ml (Cobb and Phillips, 1980), and there was no evidence that removal of hemolymph samples caused stress to the animals. Duplicate aliquots of each hemolymph sample (0.1 ml) were immediately pipetted into vials containing EcolumeTM scintillation cocktail (ICN Biochemical, Inc., Costa Mesa, CA), and the remainder of each sample was placed in a tube containing 0.1g N-ethylmaleimide to inhibit clotting (Durliat and Vranckx, 1981
; Martin et al., 1991
), flushed with nitrogen, then stored at 20°C before chemical analyses. At the time of sacrifice (16 days for iv low dose, 10 days for oral and iv high dose), animals were anesthetized by placing on ice before sacrifice, and the terminal hemolymph samples (36 ml) were removed from the pericardial sinus. Terminal urine was collected from the urinary papillae and other organs were dissected and stored at 20°C before analyses, as described previously (Barron et al., 1988
). Because previous studies had shown that the exoskeleton is an important site of initial distribution of xenobiotics in lobsters (James et al., 1995
; Little et al., 1985
), the shell and shell membrane were sampled. The shell membrane is a thin layer of tissue lining the shell, which eventually becomes the new exoskeleton.
Tissue distribution.
Hemolymph and urine samples were assayed for [3H] by direct scintillation spectrometry. Solid tissues and intestinal contents were digested in 0.5 ml 2 M NaOH and the digests neutralized prior to [3H]-spectrometry.
Chemical analyses.
The hemolymph samples were analyzed by solid-phase extraction with C18 SPICETM cartridges. The C18 SPICETM cartridge was washed sequentially with 6 ml 100% methanol, 6 ml deionized water, and 6 ml 10% methanol/deionized water solution before use. Hemolymph samples, 12 ml, were deproteinized by thoroughly mixing with 2 x 3 ml methanol/acetonitrile, 10:9. The pooled extracts were dried under nitrogen. An aliquot of 6 ml 10% methanol/deionized water solution was added to dissolve the residue and applied to a C18 SPICETM cartridge. After loading, the cartridge was washed sequentially with 6 ml of the same solution, then 2 x 6 ml of 45% methanol/deionized water solution, 6 + 3 ml of 65% methanol/deionized water solution, and 2 x 6 ml of 100% methanol. An aliquot of 1 or 2 ml of each eluent was directly assayed for [3H] by liquid scintillation spectrometry. The elution pattern of the unlabeled conjugates was determined by fluorescence scans of each fraction and of the radiolabeled conjugates by [3H] content.
The purity of each administered compound or substrate and the identity of each metabolite or reaction product were checked by HPLC (Beckman 125 analytical pump) with UV (Beckman 166 analytical UV detector set at 280 nm), fluorescence (Rainin Dynamax FL-2 fluorescence detector set excitation at 374 nm and emission at 434 nm), and radiochemical (IN/US, ß-RAM flow detector) detection. In some experiments where the radiochemical detector was not available, fractions were collected for scintillation counting. The stationary phase was an Altex 5 x 0.46 cm Ultrasphere octadecylsilane guard column coupled to a 25 x 0.46 cm analytical Ultrasphere octadecylsilane column. For analyses of 9-OH-BaP and its metabolites, a gradient system was used. The mobile phase was 45% methanol/deionized water solution by non-linear gradient to 100% methanol (45% methanol/deionized water for 3 min, linear gradient to 100% methanol for over 10 min, then held at 100% methanol for 12 min) and flow rate was 0.8 ml/min.
Metabolite identification and hydrolysis.
The metabolite compositions of intestinal contents, antennal gland, or hemolymph extracts were examined by HPLC analysis and by determining the stability of the metabolites to several hydrolytic enzymes.
Samples of C18 SPICETM eluents of intestinal contents, antennal gland, and hemolymph extracts were evaporated to dryness under vacuum and adjusted to the appropriate pH with one-tenth volume of 1 M sodium acetate buffer, pH 5, 1 M Tris/HCl, pH 7.4, or 1 M sodium carbonate and bicarbonate, pH 10.5. Samples were incubated with 0.78 units of ß-glucosidase type II from almond (pH 5) or 0.19 units of aryl sulfatase type VI from bacterium (Aerobacter aerogenes) (pH 7.4) or 1.5 units of alkaline phosphatase type III-N from Escherichia coli (pH 10.5) at 37°C overnight, then re-analyzed by HPLC.
The activity of each of the hydrolytic enzymes under the above incubation conditions was monitored by adding the appropriate 10 mM p-nitrophenyl conjugates to buffer solutions and monitoring enzyme-catalyzed liberation of p-nitrophenol. After incubation, an aliquot of 2.5 ml of 0.1 M NaOH was added, and the amount of p-nitrophenol formed was determined by comparison of the absorbance at 420 nm with a standard curve of similarly prepared samples of p-nitrophenol at known concentrations.
9-OH-BaP sulfotransferase (EC 2.8.2.1), 9-OH-BaP UDP-glucosyltransferase (EC 2.4.1.35), BaP 9-ß-D-glucoside ß-glucosidase, and BaP 9-sulfate sulfatase assays.
Microsomal and cytosolic subcellular fractions of the hepatopancreas, intestine, and antennal gland of control lobsters were prepared as described in Li and James (1993). 9-OH-BaP sulfotransferase and 9-OH-BaP UDP-glucosyltransferase activities were assayed in duplicate as described in James et al. (1997), except the incubation temperature was 12.5°C and incubation times were 40 min (UDP-glucosyltransferase), 35 min (intestinal sulfotransferase) and 30 min (antennal gland sulfotransferase). The method of Li and James, 1997, was used to assay BaP 9-ß-D-glucoside ß-glucosidase and BaP 9-sulfate sulfatase; enzyme activities were monitored at 12.5°C or 35°C and incubation time was 45 min. The formation of conjugates and the appearance of parent compound were monitored by fluorescence detection (9-OH-BaP: excitation 285 nm, emission 510 nm; BaP 9-ß-D-glucoside: excitation 295 nm, emission 415 nm; BaP 9-sulfate: excitation 285 nm, emission 410 nm).
Pharmacokinetics analysis.
RstripTM software was used to initially estimate parameters that were used to describe the disappearance of parent compound and the formation of metabolites. A 3-compartment model with bolus input, first-order output, and macro-constants as primary parameters from Win/NonlinTM (Statistical Consultants, Inc., Lexington, KY,) was used to fit the iv data for parent compound. For the oral data, a 2-compartment model with first-order input, first-order output, no lag time, and macro-constants as primary parameters was used (Gibaldi, 1991). The bioavailability of the 9-OH-BaP was calculated from the following equation:
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Statistical analysis.
ExcelTM software was used to analyze data for statistical significance by Student's t-test.
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RESULTS |
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Pharmacokinetics of [3H]-9-OH-BaP
A 3-compartment model best described the disposition of the parent [3H]-9-OH-BaP in the hemolymph after an iv dose. For orally dosed lobsters, the disappearance of the parent compound was best fit into a 2-compartment model with first-order input, first-order output, and no lag time. The parameters estimated from a 3-compartment model after iv low and high doses of [3H]-9-OH-BaP are summarized in Table 2 and the curves for disappearance of [3H]-9-OH-BaP from hemolymph after iv doses are shown in Figures 3A and 3B
. For lobsters treated orally with [3H]-9-OH-BaP the pharmacokinetic parameters are given in Table 3
and the concentration-time curves for [3H]-9-OH-BaP in hemolymph of males and females are shown in Figures 3C and 3D
. There was considerable individual variability in the terminal elimination half-lives of [3H]-9-OH-BaP. For some lobsters, the concentrations of [3H]-9-OH-BaP in hemolymph dropped only slightly between 96 h and 240 h. Because of the high variability, there were no significant differences in terminal elimination rates between males and females or between iv high dose and iv low dose values, however the AUC per dose was significantly higher in high dose lobsters (p < 0.01). The orally dosed lobsters had significantly shorter elimination half-lives than lobsters dosed iv. Pooling data from all iv-dosed lobsters, the terminal elimination half-life of [3H]-9-OH-BaP was 97.3 ± 36.3 h (mean ± SD, n = 14). For all orally dosed lobsters the elimination half-life was 56.5 ± 18.8 (n = 8).
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DISCUSSION |
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The amounts and concentrations of [3H]-9-OH-BaP and conjugates retained in lobster tissues at 10 to 16 days were similar to results with [14C]-BaP-7,8-dihydrodiol and differed from BaP. Total recoveries of BaP-7,8-dihydrodiol at 8 and 16 days after iv administration were 19.9% of the dose and 4% of the dose and after oral administration were 11% and 2% of the dose, respectively (James et al. 1989). In contrast, BaP was highly retained by the lobster: 62% of the dose was recovered in tissues 28 days after oral administration (James et al. 1995
). The pattern of distribution of [3H]-9-OH-BaP was also more similar to BaP-7,8-dihydrodiol than BaP, in that both hydroxylated BaP derivatives were present in muscle at similar concentrations to hemolymph, whereas BaP concentrations in muscle were about 10-fold higher than hemolymph concentrations, and showed no detectable decline between 3 and 28 days after the dose. In the present study, the time course of elimination from tissues was not determined. At the time points studied (10 and 16 days after exposure), the concentrations of [3H]-9-OH-BaP and metabolites in muscle were similar to those in hemolymph after intravenous administration, and were slightly higher in muscle than hemolymph after oral administration (Fig. 1
). Previous studies with other phenolic compounds, phenol and 2-naphthol, showed that concentrations in lobster muscle were lower than those of hemolymph, and residues decreased with time in concert with hemolymph (James et al., 1991, Li and James, 1997
). A relatively water-soluble drug studied in the lobster, sodium sulfadimethoxine, was present in muscle at similar or lower concentrations than hemolymph, and drug concentrations in muscle and hemolymph decreased with time in a parallel manner (Barron et al., 1988
; Barron and James, 1994
). By comparison with these other compounds, it seems likely that the residues of [3H]-9-OH-BaP will depurate from the edible muscle tissue at a similar rate as from hemolymph.
Although less than 1% of the administered dose of [3H]-9-OH-BaP was found in the gonads at sacrifice, it was of interest that in females, gonadal size determined the percentage of [3H]-9-OH-BaP in this tissue (Fig. 2). Similar findings were observed in lobsters treated with 2-naphthol (Li and James, 1997
) and BaP (James et al., 1995
). The toxicological implications of these findings in the lobster are not known, but they may indicate some potential for transfer of xenobiotics to offspring, or interference with reproduction. In another crustacean, the blue crab, triphenyltin, tributyltin, and hexachlorobiphenyl were found in the ovaries and oocytes of exposed crabs, and it was suggested that these lipophilic xenobiotics may interfere with the synthesis and assembly of lipovitellins (Lee, 1991
). Studies with fish and bird species have shown that xenobiotics in eggs transfer to the offspring and have biological effects in the young animal (Binder and Lech, 1984
, Gould et al. 1997
, Lorenzen et al. 1999
).
The pharmacokinetics of disappearance of the [3H]-9-OH-BaP from hemolymph after iv administration were best described by a traditional 3-compartment model and after oral administration by a 2-compartment model. Although it has an open circulatory system, the lobster still has an organized circulation and well-defined internal organs such as gills, stomach, antennal gland, and hepatopancreas. The cardiac output of the lobster was estimated as 2267 ml/kg/min and the turnover time for the whole volume of blood was 1 to 8 min (Burger and Smythe, 1953). After an iv dose, the parent compound will enter the central compartment, corresponding to hemolymph, then rapidly distribute to the intermediate compartment and the deep compartment. In these studies, the deep compartment probably corresponds to the hepatopancreas, which has a high fat content. Muscle and other tissues with relatively low fat content probably correspond to the intermediate compartment. When given orally, the parent compound will enter the central compartment, stomach and GI tract, then rapidly distribute to the deep compartments, such as hepatopancreas.
The terminal hemolymph elimination half-life of [3H]-9-OH-BaP showed considerable inter-animal variability (Fig. 3, Tables 2 and 3
), even though molt stage, an important determinant of excretion rate for 2-naphthol, was controlled (Li and James, 1997
). The overall terminal elimination half-life of parent [3H]-9-OH-BaP after iv administration (97.3 ± 36.3 h, mean ± SD, n = 14) was considerably longer than found previously for phenol: 14 min (James et al., 1991). Elimination of [3H]-9-OH-BaP from hemolymph was not significantly different from 2-naphthol in male lobsters (63.9 ± 15.4 h, mean ± SD, n = 4), although it was significantly slower than 2-naphthol in female lobsters (30.6 ± 6.9 h) (Li and James, 1997
). If lipophilicity alone determined elimination rates, [3H]-9-OH-BaP (log P of 5.34) should be eliminated much more slowly than 2-naphthol (log P of 2.84). These studies suggest that factors other than lipophilicity are important determinants of the rate of elimination of phenolic compounds: other important factors appear to be the rate of metabolism, the predominant conjugation pathway in vivo, and the rate of metabolite elimination or recycling back to parent compound. 2-Naphthylsulfate was a minor metabolite of 2-naphthol, and the major metabolite was the glucoside conjugate (Li and James, 1997
). The small amounts of 2-naphthylsulfate formed were readily excreted with an estimated half-life of 7 to 10 h, whereas 2-naphthyl-ß-D-glucoside was inefficiently excreted (hemolymph elimination half-life of 35 to 70 h, similar to parent 2-naphthol). Especially in males, 2-naphthyl-ß-D-glucoside was readily hydrolyzed back to 2-naphthol. The major metabolite of 9-OH-BaP was BaP-9-sulfate, with smaller amounts of BaP-9-ß-D-glucoside. The amount of BaP-9-ß-D-glucoside present in hemolymph was highest in the iv high-dose lobsters, where there were similar concentrations of both conjugates in some animals (Fig. 4
). As was found for 2-naphthol conjugates, the elimination half-life of [3H]-BaP-9-sulfate was shorter than parent compound, whereas elimination of [3H]-BaP-9-ß-D-glucoside was similar to that of [3H]-9-OH-BaP. Preferential formation of the sulfate conjugate appeared to be linked with more rapid overall elimination, perhaps because the anionic sulfate conjugate is more readily excreted than the unionized glucoside conjugate. More rapid formation of BaP-9-sulfate in the antennal glands of females may be linked with the smaller AUC per dose found in females after iv administration. The close retention times of BaP-9-ß-D-glucoside (17.3 min) and 9-OH-BaP (19.5 min) on reverse phase HPLC confirm that the glucoside conjugate was quite lipophilic (Garst and Wilson, 1984
). Thus, the glucoside would be expected to be retained in cells.
The oral bioavailability (F) for [3H]-9-OH-BaP calculated from the area under the hemolymph concentration curve was low, ranging from 5.5% to 23.1%, with considerable inter-animal variability (Table 4). Even including total metabolites, F ranged from 12.7% to 49.8%, i.e., apparently less than half of the dose was absorbed into hemolymph. The percent dose recovered in the body at sacrifice, however, was similar for orally and iv-dosed lobsters, suggesting more complete absorption than measurement of bioavailability from hemolymph concentrations indicated. Similarly, hemolymph concentrations of BaP, after oral administration to American lobsters, were lower than after iv administration, although the distribution data indicated complete absorption (James et al., 1995
). In lobsters, orally administered xenobiotics may be secreted directly from the stomach to the hepatopancreas (Cobb and Phillips, 1980
), whereas lipophilic xenobiotics and their metabolites may be partially sequestered and very slowly released to the intestine and to circulating hemolymph. Other considerations also suggest the likelihood of more complete absorption than indicated by the area under the hemolymph concentration curve. The pH of gastric fluid in the American lobster is 4.7 to 5 (Brockerhoff et al., 1970
) and upon feeding, it rises to 6.6 (Waterman, 1960
). At a pH of less than 6, greater than 99% of the 9-OH-BaP (pKa
9) will be unionized, as calculated from the Henderson-Hasselbalch equation. Based on physico-chemical properties alone, the lobsters should have readily absorbed orally dosed 9-OH-BaP.
Extensive first-pass metabolism is another factor that influences oral bioavailability. In vitro studies of the potential sites for conjugation of 9-OH-BaP showed that sulfation could occur in antennal gland and intestine, but no activity was found in hepatopancreas (Table 6). Glucose conjugation could occur in hepatopancreas and antennal gland and to a much lesser extent in intestine. Once formed, the conjugates could be hydrolyzed in antennal gland or hepatopancreas and excreted or re-conjugated. The in vivo role of the hepatopancreas in sulfation of 9-OH-BaP is unknown. Although no sulfotransferase activity was found in hepatopancreas cytosol, previous studies showed that hepatopancreas cytosol inhibited sulfotransferase activity (Schell and James, 1989
). The lack of measurable sulfotransferase activity may be due to inhibitors that are released during the destruction of the cell, and do not operate in vivo. The antennal gland, as an internal organ exposed to xenobiotics through the hemolymph, could not be a site of first-pass conjugation of orally administered 9-OH-BaP, although tissue distribution data and in vitro metabolism studies demonstrate its importance in the metabolism of absorbed 9-OH-BaP. Reconciling the enzymatic results with the in vivo findings suggests that the intestine may be a hitherto unrecognized site of conjugation of phenolic compounds in lobster. Although the intestine is a very small organ in the lobster, it may contribute to the first-pass metabolism of orally administered compounds. Since BaP-9-sulfate was a major metabolite in hemolymph of orally dosed lobsters, and can be formed in intestine, it is likely that at least some of the circulating BaP-9-sulfate was formed in intestine.
The intestine also appeared to play a role in excretion and metabolite cycling. Tissue distribution studies showed high concentrations of radioactivity in intestine and intestinal contents at the time of sacrifice. Greater than 90% of the radioactive material in intestinal contents was [3H]-BaP-9-sulfate. This suggests that the major routes of excretion of [3H]-9-OH-BaP and its metabolites were in feces (Figs. 1A and 1B). As mentioned above, antennal glands had high concentrations of radioactivity, mainly in the form of [3H]-BaP-9-sulfate, but urine contained little radioactivity, suggesting a role for metabolism, but not excretion. There were indications that a portion of the [3H]-BaP-9-sulfate may undergo metabolite cycling. Some of the iv-dosed lobsters showed no decrease in hemolymph concentrations of [3H]-9-OH-BaP between 192 and 240 or 384 h, raising the possibility of conjugate hydrolysis and redistribution. In vitro studies showed that BaP 9-sulfate was a substrate for the microsomal sulfatase, present in antennal gland and hepatopancreas. The product, 9-OH-BaP, could be released back to the circulation. Futile cycling has been observed for sulfate conjugates in isolated rat hepatocytes (Kauffman et al., 1991
) and in fish (skate) liver (Fricker et al., 1997
).
The minor conjugate, BaP 9-ß-D-glucoside, may be formed in the hepatopancreas, the antennal gland, or the intestine, as suggested from in vitro studies. BaP-9-ß-D-glucoside could be slowly hydrolyzed by ß-glucosidase in hepatopancreas cytosol, but there was no detectable hydrolytic activity with this substrate in the antennal gland. The importance of metabolite cycling of BaP-9-ß-D-glucoside is unknown. This contrasted with results for 2-naphthyl-ß-D-glucoside, which was readily hydrolyzed in the lobster hepatopancreas and antennal gland (Li and James, 1997). The shape of the hemolymph concentration curves for 2-naphthyl-ß-D-glucoside suggested that conjugation-deconjugation cycles took place for this metabolite. The present data for hemolymph concentrations of [3H]-BaP-9-ß-D-glucoside also show a tendency for flattening of the elimination curve at later times, suggesting conjugation-deconjugation occurs.
These studies have relevance to the desirability of lobsters, and perhaps other crustaceans, as human food, following environmental exposure of the lobsters to PAH and their metabolites. In PAH-polluted environments, hydroxylated PAH may be formed in small fish and consumed by lobsters. As discussed in the introduction, 9-OH-BaP and other hydroxylated BaP metabolites as well as BaP itself may be bioactivated in humans to reactive metabolites, raising the question of the food safety of lobsters from PAH-polluted environments. These studies showed that the lobster eliminated 9-OH-BaP more readily than BaP itself. Furthermore, the edible muscle did not concentrate 9-OH-BaP from hemolymph as it did BaP. Lobsters exposed to hydroxylated BaP may be expected to clear these metabolites from their systems by one month after exposure (approximately 8 half-lives). In the environment, however, lobsters will be co-exposed to BaP and metabolites, so safety considerations should be driven by the pharmacokinetics of BaP.
In summary, [3H]-9-OH-BaP was metabolized by the lobster to sulfate and glucoside conjugates after oral or intravascular administration. The rate of elimination of [3H]-9-OH-BaP was slower in iv-dosed lobsters, apparently due to saturation of the sulfation pathway. The sulfate conjugate was excreted more rapidly than parent [3H]-9-OH-BaP, but there were no significant differences in elimination rates of [3H]-9-OH-BaP and [3H]-BaP-9-ß-D-glucoside. The antennal gland and intestine appeared to be the major sites of sulfation, and in vitro studies suggested that [3H]-BaP-9-sulfate, and possibly the minor metabolite, [3H]-BaP-9-ß-D-glucoside, underwent some conjugation-deconjugation cycling. Only small amounts of [3H]-9-OH-BaP or conjugates were excreted in urine, and the feces were the major route of excretion. 9-OH-BaP was eliminated from the lobster more rapidly than BaP, but more slowly than the smaller, less lipophilic phenolic compound, phenol.
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ACKNOWLEDGMENTS |
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NOTES |
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