Department of Environmental and Molecular Toxicology, Oregon State University, Corvallis, Oregon 97331
Received April 17, 2003; accepted June 30, 2003
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ABSTRACT |
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Key Words: benzo[a]pyrene; biliary excretion; dieldrin; in vivo metabolism; rainbow trout.
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INTRODUCTION |
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Dieldrin pretreatment of rainbow trout altered tissue distribution of a 14C-dieldrin challenge dose and stimulated biliary excretion of polar metabolites. Bioaccumulation of dieldrin in rainbow trout was measured following waterborne and dietary exposures for 16 weeks (Shubat and Curtis, 1986). When normalized for lipid content, whole-body dieldrin concentrations increased through 8 weeks. However, at week 16 whole-body concentrations decreased unexpectedly to concentrations observed in trout exposed for 2 weeks. Subsequent work confirmed this phenomenon and demonstrated that altered disposition, following dieldrin pretreatment, was not specific to a subsequent dose of dieldrin, but also occurred with the PAH 7,12-dimethylbenz[a]anthracene (DMBA) (Donohoe et al., 1998
; Gilroy et al., 1993
). Feeding rainbow trout 0.3 or 0.4 mg dieldrin/kg/day for 912 weeks stimulated the biliary excretion of a subsequent dose of 14C-dieldrin by 500% and 3H-DMBA by 240%. The same exposure also significantly increased the disposition of 14C-dieldrin to liver and mesenteric fat and elevated the levels of 3H-DMBA in liver. In another study, pretreatment (0.4 mg dieldrin/kg/day in the diet for 1012 weeks) stimulated a two-fold increase in the uptake of 14C-dieldrin by precision-cut liver slices, as well as an increased uptake and efflux of 3H-DMBA (Gilroy et al., 1996
).
Dieldrin and other nonplanar OCs interact with the mammalian consititutive androstane receptor (CAR) and the pregnane X receptor (PXR) to induce cytochrome P450 (CYP) 2B or CYP3A family enzymes, respectively (Goodwin et al., 2002; Sueyoshi and Negishi, 2001
). However, dieldrin-stimulated biliary excretion of the 14C-dieldrin or 3H-DMBA challenge dose was unexpected because fish are refractory to CYP by nonplanar OCs (Vodicnik et al., 1981
). In vitro work with hepatic microsomes, from control and dieldrin-pretreated fish, detected no induction of the CYP system or conjugative enzyme activities. Total CYPs, exthoxyresorufin-O-deethylase (EROD), and pentoxyresorufin-O-deethylase (PROD) activities were not significantly different (Gilroy et al., 1993
). There were also no differences in glutathione S-transferase (GST) or UDP glucuronosyltransferase (UDPGT) activities (Gilroy et al., 1993
). Substrates well characterized in mammals were used. Isoform-specific substrate selectivities for rainbow trout conjugative enzymes were unknown. Western blot analysis revealed no changes in six hepatic CYP isozymes (Gilroy et al., 1996
). In addition, hepatic microsomes from control and dieldrin-pretreated fish contained equivalent aryl hydrocarbon hydroxylase (AHH) activities toward 14C-BP and 3H-DMBA (Gilroy et al., 1996
).
Increased performance of cytosolic binding proteins, putatively involved in intracellular trafficking to sites of metabolism, and induction of proteins involved in the excretion of xenobiotics into bile were also examined (Curtis et al., 2000). Hepatic cytosolic binding of 3H-DMBA doubled in dieldrin-fed trout after 10 weeks of exposure. However, immunohistochemistry of liver revealed no changes in multidrug resistance (i.e., adenosine binding cassette) proteins.
This research examined in vivo metabolism and disposition of BP in rainbow trout pretreated with dieldrin. BP was selected for three reasons. First, previous research demonstrated increased biliary excretion of DMBA in dieldrin-fed fish (Donohoe et al., 1998). This study assessed whether a similar increase occurred with a subsequent dose of BP. Second, BP is the ligand being used in ongoing protein studies to identify the mechanism by which dieldrin pretreatment enhances biliary excretion. Third, researchers have extensively characterized the complex metabolism of BP. Therefore, this compound was used to observe the in vivo state of the CYP system, UDP-glucuronyltransferases, and sulfotransferases. The biliary polar metabolite profile of 14C-BP in control and dieldrin-fed fish was compared to assess preferential stimulation of a pathway not identified using in vitro methods. Conversion of dieldrin to the diol was a potential biotransformation pathway. Therefore, epoxide hydrolase activities in hepatic cytosol and microsomes were evaluated. The hepatic concentration of dieldrin was determined, so altered BP disposition was interpreted in context of target organ concentration.
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MATERIALS AND METHODS |
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Animals.
Shasta strain rainbow trout (Oncorhynchus mykiss) were provided by the Marine/Freshwater Biomedical Center at Oregon State University (Corvallis, OR). Fish were held in continuous-flow (approximately 6 l/min; 13°C) circular tanks (88.9 l; 80 fish/tank) and kept on a 15-h light/9-h dark photoperiod.
Dieldrin exposure.
Fish (~2 g) were fed a growth ration (4% dry weight diet/dry weight fish) of Oregon Test Diet (Sinnhuber et al., 1977) with or without dieldrin (15 ppm; 0.324 mg dieldrin/kg/day) for 9 weeks. Due to signs of toxicity, fish were fed a maintenance ration (2% dry weight diet/dry weight fish) without dieldrin for the remaining 3 weeks.
Liver preparation.
Following 3, 6, 9, and 12 weeks of dieldrin pretreatment, fish were euthanized (200 mg/l tricane methane sulfonate), weighed, and livers excised. Liver is approximately 1% of the body weight in rainbow trout. To obtain sufficient mass for analysis it was necessary to composite tissue. Pooled intact liver samples were frozen at -20°C for dieldrin residue analysis (samples were pooled into 3 groups for each time-treatment combination to yield 0.51.0 g of tissue per group; 24 groups total). Remaining livers were analyzed for microsomal and cytosolic epoxide hydrolase (mEH and cEH) activities. Samples were pooled as described above, homogenized in 3 volumes of buffer (10 mM potassium phosphate, 0.15 M potassium chloride [KCL], 1 mM EDTA, 0.1 mM butylhydroxytoluene [BHT], 0.1 mM phenylmethanesulfonyl fluoride [PMSF], pH 7.5) and centrifuged (10,000 x g for 23 min, followed by a second centrifugation at 100,000 x g for 90 min). Supernatant was collected as the cytosolic fraction, and the microsomal pellet was resuspended in buffer (0.1 M potassium phosphate, 1 mM EDTA, 0.1 mM PMSF, 20% glycerol, pH 7.4). Microsomes and cytosol were frozen at -80°C until use.
Dieldrin residue analysis.
Livers were ground in a glass mortar with 5 g sodium sulfate. The mixture was poured into a chromatographic column, filled with sodium sulfate and dichloromethane : hexane (1:1). Sodium sulfate (an additional 2 g) was added to the mortar and then the column to remove residual sample. Eluates were evaporated to 1 ml, and solvent exchange (dichloromethane/hexane) eliminated dichloromethane. The volume was adjusted to 10 ml with hexane and diluted by a factor of 10. Two-microliter samples were analyzed by gas chromatography with electron capture detection (Varian Star 3400 Cx, Autosampler 8200), using a ramped temperature program (initial temperature 250°C, column temperature 150°C for 2 min, 255°C for 14 min, and 270°C for 15 min).
Depletion of glutathione by dialysis and enzyme assays.
Reduced and oxidized glutathione (GSH and GSSG) were depleted by dialysis to prevent conjugation of stilbene oxides in the microsomal and cytosolic epoxide hydrolase assays. Hepatic cytosol and microsomes (0.5 ml) were thawed and dialyzed for 2 h at 4°C in 1 l of a 10 mM potassium phosphate buffer (150 mM KCL, 1 mM EDTA, 0.1 mM BHT, pH 7.5). Efficiency was determined by paired analyses of dialyzed and nondialyzed samples. Glutathione concentrations were measured by HPLC with ultraviolet (UV) detection and 2,4 dinitrofluorobenzene (FDNB) (Reed and Will, 1999). Samples were derivatized, with initial formation of S-carboxymethyl derivatives of free thiols by a reaction with iodoacetic acid. The primary amines were converted to 2,4 dinitrophenyl (DNP) derivatives, after a second derivatization with Sangers reagent and FDNB.
Following dialysis, protein concentrations were determined using the BCA assay (bovine serum albumin served as the standard). Preliminary enzymatic assays at different pHs (6.5, 7.0, 7.5, 8.0, 8.5, 9.0, and 9.5) and temperatures (4, 15, 23, and 30°C) optimized conditions for subsequent work. 3H-trans-stilbene oxide and 3H-cis-stilbene oxide were used as substrates for cEH and mEH, respectively. Activities of mEH and cEH were measured radiometrically (in preparations from weeks 3, 6, 9, and 12 at optimum pH and temperature) utilizing differential partitioning of the epoxide into dodecane and the diol metabolites into the aqueous phase (Gill et al., 1983). Thin layer chromatography (TLC) assessed partitioning of stilbene oxide, stilbene diol, and the absence of GSH conjugates of stilbene oxide (Gill et al., 1983
). Diol metabolites in the remaining aqueous phase were removed following extraction with hexanol. The organic phase was concentrated under nitrogen, and 25 µl was counted for diols by liquid scintillation counting (LSC). Fifty microliters were applied to silicate prelayered plates developed in toluene : n-propanol (19:1) and dried overnight. Bands of silica-gel plates (1 cm) were scraped, added to LSC cocktail (1 ml), and counted by LSC. The presence of mercapturate conjugates was assessed by LSC of the remaining aqueous phase.
14C-BP exposure.
Tissue distribution of 14C-BP was examined in fish fed control (n = 9) or 15 ppm dieldrin (n = 9) for 9 weeks, fish fed 15 ppm dieldrin for 9 weeks followed by control diet for 3 weeks (n = 9), and fish fed control diet for 12 weeks (n = 9). Fish were transferred to polypropylene buckets containing clean well-water (static conditions) and submersed charcoal filters. Fish were not fed for 24 h prior to intraperitoneal (ip) injection with 10 µmol 14C-BP/kg in menhaden oil (10 ml/kg). Fish were killed 24 h after the ip injection and gallbladder/bile, liver, and dissectible visceral fat were removed.
Tissue preparation.
Gallbladder/bile was stored in amber microcentrifuge tubes without solvent to minimize spontaneous oxidation to quinone metabolites. Bile subsamples were removed and the remainder transferred to -200C for subsequent metabolite analysis. Polar and nonpolar metabolites in the subsamples were separated using a methanol/water-chloroform extraction system (Bligh and Dyer, 1959). The aqueous and chloroform fractions were evaporated, Cytoscint ESTM added, and radioactivity analyzed by LSC. Liver and fat tissues were digested with NCS II Tissue Solubilizer at 40°C for 48 h. Cytoscint ESTM was added and radioactivity analyzed by LSC.
HPLC analysis of biliary metabolites.
Gallbladder/bile samples frozen for metabolite analysis were extracted using the method described by Willet et al.(2000), with the following modifications. Samples were combined with 1 ml buffer (potassium phosphate with 1.0% (w/v) bovine serum albumin, pH 6.8 at 37°C) and extracted with 2 ml ethyl acetate to isolate parent 14C-BP and unconjugated oxidized metabolites. The aqueous phase was incubated with ß-glucuronidase for 6 h at 37°C (1000 units; 8.4 µl) and extracted with 2 ml ethyl acetate to isolate cleaved glucuronide conjugates. The aqueous phase was then adjusted from pH 6.8 to 5 (using HCl) and incubated with 19 units arylsulfatase (dissolved in sodium chloride immediately before use; negligible ß-glucuronidase activity) for 6 h at 37°C. Cleaved sulfate conjugates were extracted with 2 ml ethyl acetate. Organic fractions (50 µl duplicates) were analyzed for radioactivity by LSC, and the remainder was stored at -20°C for HPLC analysis. The remaining aqueous phase (50 µl duplicates) was also analyzed by LSC for residual polar material (e.g., glutathione, sulfate, or glucuronide conjugates refractory to cleavage). Blanks and standards were processed with samples for HPLC analysis.
The organic fractions, containing BP and unconjugated metabolites, were transferred to amber microcentrifuge tubes. Ethyl acetate was evaporated to dryness under a nitrogen stream, and residue was resuspended in 50 µl methanol (Varanasi et al., 1982). Twenty microliters were injected onto a C-18 reverse-phase HPLC column (Vydac 218TP54, Vydac, Hesperia, CA) and the remainder stored at -20°C. A two-step gradient was used to separate BP and its metabolites, as described by Willet et al.(2000) with the following modifications: methanol : water : acetic acid (50:49.5:0.5) to methanol : water (83.5:16.5) in 30 min to 100% methanol in 60 min. Final conditions were maintained for an additional 5 min before the gradient was returned to initial settings. The flow rate was lowered (0.4 ml/min) during sample injection and then held at 1 ml/min.
Eluent from the column was analyzed by fluorescence spectrophotometry; however, radioactivity provided a more sensitive method of detection and was used for data analysis (Nishimoto et al., 1992; Varanasi et al., 1986
). HPLC fractions were collected at 3-min intervals using a fraction collector (FC 203, Gilson Inc., Middleton, WI). Duplicates (300 µl) from each fraction were pipetted into Deep-Well LumaPlateTM microplates that contained solid scintillator (Bornsen, 2000
). The plates were evaporated, sealed, and placed in the TopCount® microplate scintillation and luminescence counter (Packard Instrument Company, Meriden, CT) (Bornsen, 2000
).
Statistical analysis.
Statgraphics Plus 5.0 was used for all statistical analyses. Two-way analysis of variance compared multiple means and determined significant time or treatment effects. A two-sample comparison, or t-test, compared two means for significant treatment effects. Significance was determined using a 95.0% confidence level (p < 0.05). Nonparametric alternatives were used when assumptions of normality or equality of variance were not met.
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RESULTS |
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Preliminary assays that verified optimal pHs and temperatures (data not shown) for mEH (pH 8 to 8.5 and 23°C) and cEH (pH 6.5 to 7.5 and 23°C) yielded results similar to previous work for rainbow trout (Lauren et al., 1989). Differential partitioning of the epoxide into dodecane yielded 90% of the radioactivity; therefore, the enzyme was not substrate limited. The activity of mEH, the form of the enzyme active toward arene oxides (Wixtrom and Hammock, 1984
), was unaffected by dieldrin (Fig. 3
). The activity of cEH was also unaffected in dieldrin-fed fish (overall grand mean 3.91 ± 0.28 nmol/min/mg protein).
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The nmol of 14C-BP polar metabolites in each fraction were divided by the total nmol recovered in bile for each fish. This yielded the percent radioactivity in each fraction containing phase 1 metabolites, cleaved glucuronide conjugates, cleaved sulfate conjugates, and residual polar material. Statistical analysis revealed no significant time or treatment effects. Therefore, the averages of control and treated fish at weeks 9 and 12 were pooled into one average. Of the total radioactivity in bile, the majority was detected as cleaved glucuronide conjugates (24%). Only a small proportion of free metabolites (6%) and cleaved sulfate conjugates (2%) were recovered. Parent compound, assumed to be contamination on the outside of the gallbladder as a result of the ip injection, represented 25% of the total radioactivity.
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DISCUSSION |
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Approximately 90% of total 14C-dieldrin equivalents in bile of control and dieldrin-fed fish were polar metabolites (Gilroy et al., 1993). Induction of hepatic xenobiotic metabolizing enzymes was investigated as the basis for stimulated biliary excretion of 14C-dieldrin. In vitro work demonstrated no induction of the CYP system or increased activity of several conjugating enzymes (Gilroy et al., 1996
). Hepatic microsomal metabolism of a variety of substrates selective for different CYP isoforms and Western blotting for 6 CYP isoforms revealed no differences between control and dieldrin-fed fish. Hepatic glucuronyl transferase and GSH transferase activities were also the same in control and dieldrin-fed fish. As an epoxide, dieldrin was a potential substrate of mEH (Wixtrom and Hammock, 1984
). However, neither hepatic mEH, nor cEH activities increased in dieldrin-fed fish (Fig. 3
).
Uptake and efflux of 3H-DMBA was significantly higher in liver slices from dieldrin-fed rainbow trout than those from controls (Gilroy et al., 1996). The DMBA binding capacity of hepatic cytosol from dieldrin-fed fish was twice that of controls (Curtis, 2000
). In addition, biliary excretion of DMBA approximately doubled in dieldrin-fed rainbow trout (Donohoe et al., 1998
). Therefore, altered hepatic elimination of a lipophilic xenobiotic, induced by prolonged dieldrin treatment, was not specific to that cyclodiene. A significant interaction occurred with the PAH, DMBA.
The present research examined the effects of dieldrin pretreatment on the metabolism and disposition of another PAH, BP. This substrate, which undergoes complex metabolism, characterized the in vivo state of the CYP system, UDP-glucuronyltransferases, and sulfotransferases. This assessed alteration of a particular CYP isoform(s) for which selective substrates or antibodies were not developed.
In the current study fish were fed 0.3 mg dieldrin/kg/day for 3, 6, or 9 weeks. The dieldrin-fed rainbow trout exhibited some, although not statistically significant, signs of toxicity after 9 weeks of treatment (Fig. 1). Some enlargement and vacuolization of hepatocytes was observed in rainbow trout fed 0.3 mg dieldrin/kg/day for 10 weeks (Curtis et al., 2000
). These results indicated 0.3 mg dieldrin/kg/day approximated a threshold for subchronic toxicity in rainbow trout. There were no signs of toxicity in fish fed control diet for 3 weeks after 9 weeks of dieldrin treatment (Fig. 1
). The peak hepatic dieldrin concentration of 1.2 ug/g (3.2 nmol/g) at 9 weeks decreased to 0.4 ug/g (1.0 nmol/g) after 3 weeks on control diet (Fig. 2
). Rapid hepatic elimination of dieldrin during 3 weeks on control diet was consistent with stimulated biliary excretion of this cyclodiene, reported after prolonged treatment (Gilroy et al., 1993
).
There were no statistically significant differences in 14C-BP concentrations in liver or fat associated with dieldrin pretreatment (Fig. 4). This was true for fish fed dieldrin for 9 weeks and for fish fed dieldrin 9 weeks, followed by control diet for 3 weeks (12 weeks). The liver concentration of 14C-BP was significantly higher after 12 weeks in control and dieldrin-fed fish, than after 9 weeks (Fig. 4A
). However, the 14C-BP concentration in visceral fat was significantly higher in control and dieldrin-fed fish at 9 weeks, compared to 12 weeks (Fig. 4B
). Triacylglycerols are incorporated into lipoproteins by the liver for secretion into the circulation and transportation to various tissues. The liver is the main regulator of lipid homeostasis and incorporates BP into lipoproteins (Aarstad et al., 1987
). A change in ration between weeks 9 and 12 (4% growth ration vs. 2% maintenance ration, respectively) probably influenced 14C-BP tissue distribution. Energy storage as fat was favored during the first 9 weeks at high ration. When fish were placed on a lower ration, synthesis of fat probably decreased by week 12. Therefore, lipoprotein export by the liver to transport fat to adipose tissue probably decreased. As a result, accumulation of 14C-BP by liver increased, while distribution to fat decreased.
Consistent with earlier work using DMBA (Donohoe et al., 1998), dieldrin pretreatment for 9 weeks stimulated biliary excretion of 14C-BP (Fig. 5
). Stimulated biliary excretion of 14C-BP persisted in fish fed control diet for 3 weeks after 9 weeks of dieldrin treatment (12 weeks). This occurred despite markedly reduced hepatic dieldrin concentrations after 3 weeks on control diet (Fig. 2
). Dieldrin pretreatment significantly elevated the concentration of 14C-BP in bile (142% and 200% at 9 and 12 weeks, respectively) (Fig. 5A
). Extraction of bile subsamples with methanol/water-chloroform confirmed dieldrin pretreatment significantly stimulated total biliary excretion of 14C-BP polar metabolites (244% and 221% at week 9 and 12, respectively; Fig. 5B
).
Evaluation of biliary polar metabolite profiles of 14C-BP revealed no significant differences between control and dieldrin-fed fish (Figs. 6, 7
, and 8
). There was no selective enhancement of any particular metabolite or formation of a novel metabolite with dieldrin pretreatment. Many of the biliary metabolites increased in dieldrin-fed fish, which suggested no particular CYP was altered. Of the total radioactivity in bile, the majority was detected as cleaved glucuronide conjugates (24%), while only a small proportion of free metabolites (6%) and cleaved sulfate conjugates (2%) were recovered. Recovery of parent compound (25%) was assumed to be contamination on the outside of the gallbladder, as a result of the ip injection.
In agreement with other studies (Nishimoto et al., 1992; Varanasi et al., 1982
, 1986
; Willett et al., 2000
), glucuronide conjugates predominated in gallbladder bile, while only a small proportion of sulfate conjugates were recovered (Figs. 7
and 8
). There may be several reasons why detection of sulfate conjugates is low. Lower water solubility and recognition by organic anion transport systems for sulfate compared to glucuronide conjugates decreased their elimination (Parkinson, 1996
). Another potential explanation was directly related to sulfation reactions, catalyzed by sulfotransferases. Transfer of the sulfate group, from the cofactor 3'-phosphoadenosine 5'-phosphosulfate, to hydroxyl groups on PAHs introduced a good leaving group, which formed a reactive, electrophilic carbocation species (Parkinson, 1996
). Instability of sulfate conjugates probably contributed to the small percentage recovered in bile.
Approximately 43% of the total radioactivity remained in the final aqueous phase. This residual polar material contained water-soluble conjugates including glutathione and glucuronide, or sulfate conjugates not hydrolyzed during the ß-glucuronidase or arylsulfatase reactions. Standards were chosen based on earlier studies that demonstrated 3-hydroxy is one of the primary BP metabolites formed, which was consistent with our results (Nishimoto et al., 1992; Varanasi et al., 1982
; Willett et al., 2000
).
Polar metabolites of 14C-BP (unconjugated oxidized metabolites, cleaved glucuronide conjugates, cleaved sulfate conjugates, or residual polar material) excreted into bile were not significantly increased in dieldrin-fed fish (Fig. 9). At week 12, all polar metabolites were elevated in dieldrin-fed fish. However, this response was not apparent at week 9. Oxidation products created during storage and sample processing explained why this data detected insignificantly enhanced biliary excretion of 14C-BP polar metabolites. Analysis of bile subsamples, immediately following each experiment, provided a better estimate of total biliary excretion than reconstruction of data following the metabolite analysis.
In conclusion, chronic dieldrin exposure stimulated biliary excretion in rainbow trout given subsequent doses of 14C-dieldrin, 3H-DMBA, and 14C-BP. This study confirmed that the interaction was not explained by induction of xenobiotic metabolizing enzymes. Therefore, the mechanism by which dieldrin pretreatment enhances biliary excretion of lipophilic compounds requires further examination. Biliary excretion of xenobiotics is complex and involves hepatic processes other than metabolism (Donohoe et al., 1998): uptake across the plasma membrane, intracellular trafficking to sites of metabolism and elimination, and excretion into bile (Curtis et al., 2000
; Donohoe et al., 1998
; Gilroy et al., 1993
, 1996
). Immunohistochemistry with several antibodies to adenosine binding cassette proteins revealed no increased hepatic content of these proteins in sinusoidal or canalicular plasma membrane domains of dieldrin-fed fish (Curtis et al., 2000
). Alternative approaches to assessing involvement of this protein family are warranted. Specific binding for 3H-DMBA doubled in hepatic cytosol from dieldrin-fed fish (Curtis et al., 2000
). The current hypothesis is that prolonged dieldrin treatment increases capacity of hepatic intracellular trafficking proteins (Curtis et al., 2000
; Donohoe et al., 1998
; Gilroy et al., 1993
, 1996
). Ongoing research focuses on BP binding proteins.
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ACKNOWLEDGMENTS |
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NOTES |
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