Oral Bioavailability and Pharmacokinetics of Elimination of 9-Hydroxybenzo[a]pyrene and Its Glucoside and Sulfate Conjugates after Administration to Male and Female American Lobsters, Homarus americanus

C.-L. J. Li and M. O. James1

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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The pharmacokinetics of [3H]-9-hydroxybenzo[a]pyrene (9-OH-BaP), a highly lipophilic primary metabolite of benzo(a)pyrene, were examined after intrapericardial (iv) or oral doses of 50 or 200 µg/kg to intermolt American lobsters, Homarus americanus. Combining data for all lobsters, the average terminal elimination half-life of parent 9-OH-BaP was 97.3 h after iv and 56.5 h after oral administration, considerably less than found previously for benzo(a)pyrene (720 h). The oral bioavailability of parent 9-OH-BaP, calculated from the area under the hemolymph concentration curve, was 15.9%. The low bioavailability and variable elimination rates were attributed to extensive first-pass conjugation and sequestration in the hepatopancreas. BaP-9-sulfate was the major metabolite. Hemolymph concentrations of BaP-9-sulfate increased up to one day after the dose, and then decreased, with a terminal elimination half-life of 45 h. BaP 9-ß-D-glucoside was a minor metabolite in most hemolymph and tissue samples; an exception was hemolymph from the iv high-dose group. Concentrations of 9-OH-BaP and metabolites in the edible muscle tissue were similar to those in hemolymph, and 9-OH-BaP residues at 10 to 16 days after the dose were 3 to 12 ng/g muscle. Sulfotransferase and UDP-glucosyltransferase (UGT) activities with 9-OH-BaP were found in the antennal gland, intestinal mucosa, and hepatopancreas (UGT only). Sulfatase activity with BaP-9-sulfate, found in both the hepatopancreas and the antennal gland, was thought to contribute to metabolite cycling. These studies showed that 9-OH-BaP was readily conjugated to sulfate and glucose in the lobster, and that despite their high lipophilicity, 9-OH-BaP and conjugates were excreted from the lobster hemolymph and tissues much more rapidly than benzo[a]pyrene.

Key Words: 9-hydroxybenzo(a)pyrene; lobster; bioavailability; sulfation of 9-hydroxybenzo(a)pyrene; glucose conjugation; accumulation of lipophilic xenobiotic.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Consumption of seafood from contaminated areas is a potential source of human exposure to environmental pollutants (James and Kleinow, 1994Go; James et al., 1989Go; Johansen et al., 1996Go; McElroy et al., 1991Go; McElroy and Sisson, 1990). This is especially true for lipophilic environmental chemicals such as polycyclic aromatic hydrocarbons (PAH) that are readily taken up by aquatic animals in polluted environments (Hattemer-Frey and Travis, 1991Go; Varanasi, 1989Go). Dietary exposure to PAH and PAH metabolites is of concern since these compounds are carcinogens in many animals. The model procarcinogen, benzo(a)pyrene (BaP), may be biotransformed in humans and animals to numerous phase-1 metabolites including 3-, 7- and 9-hydroxyBaP, BaP-diols, BaP-diol-epoxides, and BaP-quinones, and subsequently, to phase-2 metabolites such as glycoside, sulfate, and glutathione conjugates (Varanasi, 1989Go). Some phase-1 metabolites of BaP, such as BaP-7,8-dihydrodiol-9,10-oxide, 3-OH-BaP-7,8-dihydrodiol-9,10-oxide and 9-hydroxy-BaP-4,5-oxide are reactive electrophiles that can bind covalently to DNA and other macromolecules (Szeliga and Dipple, 1998Go; Glatt et al., 1987Go; Moorthy and Randerath, 1996Go). Thus, dietary exposure to BaP and its phase-1 metabolites is a potential risk factor in the development of cancer and other toxicities.

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, 1979Go; Uthe and Musial, 1986Go). The hepatopancreas is a storage and digestive organ with functional similarity to the vertebrate liver (Cobb and Phillips, 1980Go). 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., 1995Go). The estimated elimination half-life of BaP in the American lobster was 30 days to 2 months (Foureman et al., 1978Go; James et al., 1995Go). Low cytochrome P450-dependent monooxygenase activity with BaP was detected in the hepatopancreas of the American lobster (James, 1989Go). 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, 1997Go) 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, 1993Go). 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, 1979Go). Like BaP, which has a log P of 6.0 (Mackay and Paterson, 1991Go), 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., 1971Go). 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 1988Go; James and Little, 1981Go; Little et al., 1984Go; Stegeman et al., 1984Go; Ueng et al. 1994Go). 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, 1991Go; Uthe and Musial, 1986Go).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
G-[3H]-9-OH-BaP with initial specific activity of 400 mCi/mmol and greater than 96% radiochemical purity was purchased from SRI International (Menlo Park, CA) and purity was checked by reverse phase-HPLC before use. Unlabeled 9-OH-BaP, with greater than 93% purity, and BaP 9-sulfate potassium salt, with greater than 98% purity, were received from the NCI Chemical Carcinogen Reference Standard Repository, a function of the Division of Cancer Etiology, NCI, NIH, Bethesda, MD. Attempts to further purify the radiolabeled or unlabeled 9-OH-BaP were unsuccessful. BaP 9-ß-D-glucoside, [3H]-BaP 9-ß-D-glucoside, and [3H]-BaP 9-sulfate were synthesized enzymatically from 9-OH-BaP or [3H]-9-OH-BaP, according to Li and James (1993). The [3H]-labeled conjugate standards were purified as follows. The final incubation mixtures were adjusted as 10% methanol/deionized water solutions and subjected to C18 SPICETM cartridge chromatography (see Chemical Analyses). Fractions with the highest radioactivity were dried under N2, 6 ml of 10% methanol/deionized water solution were added, and samples were subjected to a second C18 SPICETM chromatography. The respective fractions with highest radioactivity after the second isolation were used as sulfate and glucoside conjugate standards. Unlabeled BaP-9-ß-D-glucoside was similarly prepared and purified. The synthesized metabolites were subjected to enzymatic hydrolysis with ß-glucosidase and aryl-sulfatase as appropriate, to confirm their identity. N-Ethylmaleimide, adenosine 3`-phosphate 5`-phosphosulfate (PAPS), uridine 5`-diphosphoglucose (UDP-glucose), glucosidase-free aryl sulfatase (EC 3.1.6.1) type VI from Aerobacter aerogenes, alkaline phosphatase (EC 3.1.3.1) type III-N from Escherichia coli, and aryl sulfatase-free ß-glucosidase (EC 3.2.1.21) type II from almond were obtained from Sigma Chemical Company. C18 SPICETM cartridges were purchased from Rainin Instruments. All solvents used were HPLC grade and obtained from Fisher Scientific Company.

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 12–14°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, 1–2 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, 1980Go), 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, 1981Go; Martin et al., 1991Go), 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 (3–6 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., 1988Go). Because previous studies had shown that the exoskeleton is an important site of initial distribution of xenobiotics in lobsters (James et al., 1995Go; Little et al., 1985Go), 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, 1–2 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, 1991Go). The bioavailability of the 9-OH-BaP was calculated from the following equation:

Statistical analysis.
ExcelTM software was used to analyze data for statistical significance by Student's t-test.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tissue Distribution of Radioactivity
At 16 days after the iv low dose (56 ± 4 µg/kg and 81.2 µ5.8 µCi/kg) or 10 days after the iv high dose (179 ± 10 µg/kg and 136 ± 7.6 µCi/kg) and the oral dose (215 ± 10 µg/kg and 311.6 ± 14.5 µCi/kg), groups of lobsters were dissected and all tissues analyzed for total radioactivity. The results for percent administered dose are shown in Table 1Go and for tissue concentrations in Figure 1Go(A and B). The total percent dose remaining in the body at sacrifice was quite variable within treatment groups, and there were no significant differences between similarly dosed males and females. Regardless of the actual percent dose recovered from all lobster tissues at the time of sacrifice (Table 1Go), 3 tissues, hepatopancreas, muscle, and hemolymph, accounted for most of the radioactivity that remained. The hepatopancreas (about 4% body weight) contained 28.3% to 61.7 % of the remaining radioactivity and the muscle (40% of the body weight) from 10.4% to 36.6%. In all treatment groups, there was a trend for the hepatopancreas in females to retain less of the dose than in males. The percent of remaining dose left in hemolymph (hemolymph volume accounted for 21% body weight) varied with the route of administration. Hemolymph from iv-dosed lobsters contained 7.2 to 21.3% of the radioactivity remaining in the animal, while hemolymph from lobsters dosed orally retained only 0.4 to 2.2% of the radioactivity left at sacrifice (significantly lower, p < 0.05). The percentage of radioactivity remaining in the muscle tissue was also significantly lower in orally dosed lobsters than in iv-dosed lobsters (p < 0.05).


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TABLE 1 Distribution of [3H] in the American Lobster (Homarus americanus), as Percentage of Administered Dose, after Treatment with [3H]-9-OH-BaP
 


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FIG. 1. Tissue concentrations of [3H] in the American lobsters dosed intrapericardially (iv) with 56 ± 4 µg/kg, 179 ± 10 µg/kg 9-OH-BaP or intragastrically (oral) with 215 ± 10 µg/kg 9-OH-BaP; (A) males, (B) females. Abbreviations are HP, hepatopancreas; INT, intestine; INT CT, intestinal contents; AG, antennal gland; HL, hemolymph; MS, muscle; and SM, shell membrane. Left Y-axis scale: HP, INT, INT CT, and AG; right Y-axis scale: HL, MS, and SM.

 
After iv or oral doses, the intestinal contents contained the highest concentrations of radioactivity, as shown in Figures 1A and 1BGo. In males, antennal glands had similar concentrations of radioactivity, as did intestinal contents. Concentrations of radioactivity in the hepatopancreas and intestine were about 20 times higher than that in hemolymph. Muscle and hemolymph contained similar, low concentrations of radioactivity. The terminal urine samples (0.5 to 6 ml total volume) from each lobster contained measurable radioactivity, but the concentrations were one-fifth to one-hundredth those of the corresponding hemolymph samples. The percent dose present in the female gonads correlated directly with the amount of gonadal tissue present in the lobster. Regression analysis gave a straight line with r2 = 0.76, p < 0.001 (Fig. 2Go).



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FIG. 2. Regression analysis of 9-OH-BaP-derived [3H] in female gonads of [3H]-9-OH-BaP–treated American lobsters. The data fit a straight line, r2= 0.76 (p < 0.002).

 
Analysis of Hemolymph, Intestinal Contents, and Antennal Gland
The [3H]-labeled 9-OH-BaP and its sulfate and glucoside conjugate standards were greater than 98%, retained by the C18 SPICETM cartridge. Elution of BaP-9-sulfate from the SPICETM cartridge, > 99%, was accomplished with 45% methanol/deionized water. BaP-9-ß-D -glucoside was over 90% eluted by 65% methanol/deionized water, and parent 9-OH-BaP was more than 99% eluted by 100% methanol. The HPLC retention time of 9-OH-BaP in the non-linear gradient system used was 19.5 min, BaP-9-ß-D-glucoside 17.3 min, and BaP-9-sulfate 11.6 min. The identities of the standard compounds in SPICETM cartridge eluent fractions were confirmed by HPLC, hydrolytic enzymes, and comparison of fluorescence scans. The composition of each hemolymph sample taken from [3H]-9-OH-BaP-dosed lobsters was quantitated by C18 SPICETM cartridge chromatography. HPLC analyses of the hemolymph C18 SPICETM cartridge eluent fractions confirmed that [3H]-9-OH-BaP, [3H]-BaP-9-ß-D-glucoside and [3H]-BaP-9-sulfate were present in hemolymph (data not shown). In both male and female lobsters, [3H]-BaP-9-sulfate was the major metabolite found in hemolymph and [3H]-BaP-9-sulfate concentrations peaked at one day after the dose. Analyses of antennal glands at 16 days (males, iv low-dose) or 10 days (both sexes, oral and iv high-dose) showed that [3H]-BaP-9-sulfate (73–89%) and smaller amounts of parent [3H]-9-OH-BaP and [3H]-BaP-9-ß-D-glucoside were present. The intestinal contents from both iv and oral samples contained primarily [3H]-BaP-9-sulfate and smaller amounts, less than 10% of the total radioactivity, were identified as [3H]-9-OH-BaP and [3H]-BaP-9-ß-D-glucoside. There was not enough radioactivity in urine to analyze for metabolites.

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 2Go and the curves for disappearance of [3H]-9-OH-BaP from hemolymph after iv doses are shown in Figures 3A and 3BGo. For lobsters treated orally with [3H]-9-OH-BaP the pharmacokinetic parameters are given in Table 3Go and the concentration-time curves for [3H]-9-OH-BaP in hemolymph of males and females are shown in Figures 3C and 3DGo. 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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TABLE 2 Pharmacokinetics Parameters Estimated from a 3-Compartment Model for Elimination of [3H]-9-OH-BaP from Hemolymph following an Intrapericardial Low Dose (56 ± 4 µg/kg) or High Dose (179 ± 10 µg/kg)
 


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FIG. 3. Concentration of 9-OH-BaP in hemolymph after iv administration [(A) and (B)] of a 56 ± 4 µg/kg dose (dark circle) or 179 ± 10 µg/kg dose (light circle) or an oral dose [(C) and (D)] of 215 ± 10 µg/kg to the intermolt American lobsters. The insets in (A) and (B) show details of the first 2 h after the dose.

 

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TABLE 3 Pharmacokinetic Parameters Estimated from a 2-Compartment Model with First-Order Input and First-Order Output for Elimination of 9-OH-BaP from Hemolymph following an Oral Dose
 
The oral bioavailability for parent [3H]-9-OH-BaP was calculated using data from only the low iv dose lobsters (Table 4Go). Data from low iv dose animals were used for these calculations because the AUC per dose showed dose-dependency, and examination of the hemolymph concentrations of [3H]-9-OH-BaP after oral administration clearly showed that there was incomplete bioavailability. As well as parent compound, the oral bioavailability of total radioactivity ([3H]-9-OH-BaP and metabolites) was calculated using AUC data from low-dose animals (Table 4Go).


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TABLE 4 Oral Bioavailability of Parent 9-OH-BaP and of 9-OH-BaP and Metabolites as Percentage
 
Elimination of BaP-Conjugates
The elimination t1/2 values for [3H]-BaP-9-sulfate were calculated from least-squares analyses of the hemolymph concentrations of [3H]-BaP-9-sulfate from 24 to 240 or 384 h. The elimination of metabolites from hemolymph is shown in Figure 4Go, and elimination phase half-lives for [3H]-BaP-9-sulfate, the major metabolite in hemolymph, are given in Table 5Go. There were no significant dose, route of administration, or sex-related differences in elimination of [3H]-BaP-9-sulfate and overall half-life for [3H]-BaP-9-sulfate was 45.5 ± 13.0 h (mean ± SD, n = 22). This was significantly shorter than that of intravenously administered [3H]-9-OH-BaP (p < 0.01). [3H]-Glucoside conjugate levels in hemolymph were very low after 24 h in samples from orally dosed lobsters and iv low-dose lobsters and approached the limit of quantitation in several samples. It was not possible to estimate elimination half-lives in these lobsters. For iv high-dose lobsters, which formed the most glucoside conjugate, there were no sex differences in terminal elimination half-life, and the overall elimination t1/2 of [3H]-BaP-9-glucoside was 67.9 ± 2.7 (mean ± SD, n = 6). This was not significantly lower that that of iv or oral 9-OH-BaP (p > 0.05).



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FIG. 4. Concentrations of [3H]-9-OH-BaP metabolites in hemolymph after iv administration of 56 ± 4 µg/kg (iv low dose) or 179 ± 10 µg/kg (iv high dose) or an oral dose of 215 ± 10 µg/kg to the intermolt American lobsters; diamonds, [3H]-BaP 9-ß-D-glucoside; circles, [3H]-BaP 9-sulfate.

 

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TABLE 5 The Elimination Half-Lives in Hours of [3H]-BaP-9-Sulfate and [3H]-BaP-9-ß-D-Glucoside
 
9-OH-BaP Sulfotransferase, 9-OH-BaP UDP-Glucosyltransferase, BaP-9-ß-D-Glucoside ß-Glucosidase and BaP-9-Sulfate Sulfatase Activities in Lobster Ttissues
Studies of UDP-glucosyltransferase (UGT) and sulfotransferase (ST) activities in lobster hepatopancreas, intestine, and antennal gland with 9-OH-BaP as substrate are summarized in Table 6Go. These studies were conducted at the seawater temperature (12.5°C) in which the lobsters were housed. Formation of BaP-9-ß-D-glucoside could be measured in microsomes from hepatopancreas, intestine, and antennal gland, although activity in the intestine was too low to quantitate, due in part to the very low microsomal yield and in part to the low activity. Formation of BaP-9-sulfate could be measured in cytosol from antennal gland or intestine, but no activity was found in hepatopancreas. The female antennal gland ST had significantly higher Vmax than the males (p < 0.05) and the female intestinal ST had significantly lower Vmax than the males (p < 0.05) (Table 6Go).


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TABLE 6 Apparent Km and Vmax of UDP-Glucosyltransferase or Sulfotransferase with 9-OH-BaP as Substrate, and Sulfatase Activities with BaP-9-Sulfate in Tissue Fractions from Male and Female American Lobsters
 
Aryl sulfatase activity with BaP-9-sulfate as substrate was found in the microsomal and cytosolic fractions from hepatopancreas and microsomal fraction from antennal gland. The apparent Km and Vmax could not be accurately determined, due in part to poor solubility of BaP-9-sulfate in the buffer solution at concentrations above 10 µM. Cytosolic sulfatase activity in hepatopancreas was about one-tenth that of microsomes. ß-Glucosidase activity with BaP-9-ß-D-glucoside as substrate was too low in hepatopancreas and antennal gland cytosol to measure accurately at 12.5°C. Accordingly, measurements were made at 35°C at the optimal pH, 4.5, established with other substrates (data not shown). Hepatopancreas cytosolic ß-glucosidase activity was 4.9 x pmol x min–1 x mg protein–1 at 0.18 µM BaP-9-glucoside substrate concentration. No activity was detected in antennal gland. Since BaP-9-ß-D-glucoside was a minor metabolite, no further studies of glucosidase activity were conducted.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In considering the fate of xenobiotics in food-producing animals, the distribution into edible tissues and the rates of elimination of the xenobiotic and its major metabolites are of interest. Although some people eat hepatopancreas, the muscle is more widely consumed. Ten to 16 days after a single dose, [3H]-9-OH-BaP and its conjugates were present at considerably higher concentrations in hepatopancreas, intestinal contents, and antennal gland than in hemolymph (Fig. 1Go and Table 1Go). Concentrations in muscle were similar to or slightly higher than those in hemolymph, and concentrations in urine were lower than in hemolymph. The high concentrations of [3H]-9-OH-BaP and conjugates in hepatopancreas probably relate to their lipophilicity and the high fat content of this organ. Other lipophilic xenobiotics including BaP and polychlorinated biphenyl also concentrate in the hepatopancreas (Dunn and Fee, 1979Go; James et al., 1995Go; King et al., 1996Go). The high concentrations in intestinal contents suggest that the major route of excretion of [3H]-9-OH-BaP and its conjugates is the feces. Finding high concentrations of radioactivity (primarily [3H]-BaP-9-sulfate) in the urine-forming organ, antennal gland, but not in urine suggests either there is no secretion into urine, or there is rapid resorption across the urinary bladder, as has been described for other xenobiotics in crustaceans (Pritchard and Miller, 1991Go). Antennal gland is also an important site of conjugation of 9-OH-BaP (Table 6Go).

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. 1989Go). 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. 1995Go). 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. 1Go). 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, 1997Go). 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., 1988Go; Barron and James, 1994Go). 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. 2Go). Similar findings were observed in lobsters treated with 2-naphthol (Li and James, 1997Go) and BaP (James et al., 1995Go). 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, 1991Go). 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, 1984Go, Gould et al. 1997Go, Lorenzen et al. 1999Go).

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 22–67 ml/kg/min and the turnover time for the whole volume of blood was 1 to 8 min (Burger and Smythe, 1953Go). 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. 3Go, Tables 2 and 3GoGo), even though molt stage, an important determinant of excretion rate for 2-naphthol, was controlled (Li and James, 1997Go). 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, 1997Go). 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, 1997Go). 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. 4Go). 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, 1984Go). 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 4Go). 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., 1995Go). In lobsters, orally administered xenobiotics may be secreted directly from the stomach to the hepatopancreas (Cobb and Phillips, 1980Go), 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., 1970Go) and upon feeding, it rises to 6.6 (Waterman, 1960Go). 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 6Go). 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, 1989Go). 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 1BGo). 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., 1991Go) and in fish (skate) liver (Fricker et al., 1997Go).

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, 1997Go). 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.


    ACKNOWLEDGMENTS
 
The authors are grateful to Dr. Sean Boyle for his assistance with animal handling and hemolymph sampling. This work was supported in part by the US Public Health Service, grant ES-05781.


    NOTES
 
1 To whom correspondence should be addressed at the Department of Medicinal Chemistry, College of Pharmacy, Building 475 JHMC, University of Florida, P.O. Box 100485, Gainesville, FL 32610-0485. Fax: (352) 846-1972. E-mail: mojames{at}ufl.edu. Back


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 DISCUSSION
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