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Department of Medicinal Chemistry, College of Pharmacy, University of Florida, Gainesville Florida
(Received March 5, 2005; Accepted June 8, 2005)
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Abstract |
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).
The lipophilicity and inherent chemical stability of persistent organic pollutants renders them excellent candidates for absorption through biological membranes as well as accumulation in both organisms and their environment. Many persistent organic pollutants have been shown to biomagnify in food webs to potentially toxic levels in top predators such as the polar bear (Ursus maritimus), whose diet mainly consists of ringed seal (Phoca hispida) blubber (Kucklick et al., 2002).
Since the sulfonation of xenobiotics has never been studied in the polar bear, the objective of this study was to investigate the efficiency of this route of detoxification on a select group of known environmental pollutants: 4'-hydroxy-3,3',4,5'-tetrachlorobiphenyl (4'OH-PCB79), 4'-hydroxy-2,3,3',4,5,5'-hexachlorobiphenyl (4'-OH-PCB159), 4'-hydroxy-2,3,3',5,5',6-hexachlorobiphenyl (4'-OH-PCB165), pentachlorophenol (PCP), tris(4-chlorophenyl)-methanol (TCPM), 2-(4-methoxyphenyl)-2-(4-hydroxyphenyl)-1,1,1-trichloroethane (OHMXC), 3-hydroxybenzo[a]pyrene (3-OH-B[a]P), and triclosan (2,4,4'-trichloro-2'-hydroxydiphenyl ether) (Fig. 1).
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), have been shown to be present in relatively high concentrations in polar bears (Sandau and Norstrom, 1998; Sandau et al., 2000). The abundance of these hydroxylated metabolites may be due to cytochrome P450 induction (Letcher et al., 1996), inefficient phase II detoxication, and inhibition of their own biotransformation. The 4'-OH-PCB79 (an oxidation product of PCB congener 77) is a potent inhibitor of the sulfonation of several substrates, including 3-OH-B[a]P in channel catfish intestine and human liver (van den Hurk et al., 2002; Wang et al., 2005), 3,5-diiodothyronine (T2) in rat liver (Schuur et al., 1998), and estradiol by recombinant human SULT1E1 (Kester et al., 2000). Both 4'-OH-PCB159 and 4'-OH-PCB165 have been shown to inhibit the sulfonation of 3-OH-B[a]P by human SULT (Wang et al., 2005). Another compound detected in polar bears is PCP (Sandau and Norstrom, 1998), a commonly used wood preservative that has been implicated in thyroid hormone disruption in Arctic Inuit populations (Sandau et al., 2002). TCPM is a globally distributed organochlorine compound of uncertain origin, which was reported in human adipose tissue (Minh et al., 2000). Polar bear liver contains 4000 to 6800 ppb lipid weight TCPM, the highest levels recorded for this compound in all species studied (Jarman et al., 1992). TCPM is a potent androgen receptor antagonist in vitro (Schrader and Cooke, 2002). OHMXC, formed by demethylation of the organochlorine pesticide methoxychlor, is an estrogen receptor 
agonist, an estrogen receptor ß antagonist, and an androgen receptor antagonist (Gaido et al., 2000). The ubiquitous environmental pollutant benzo[a]pyrene is mainly metabolized to 3-OH-B[a]P, a procarcinogen that can be eliminated via sulfonation (Tong and James, 2000). Together with its 7,8-dihydrodiol-9,10-oxide and 7,8-oxide metabolites, 3-OH-B[a]P can form adducts with macromolecules and initiate carcinogenesis (Ribeiro et al., 1986). Triclosan is an antimicrobial agent that has been detected in human plasma and breast milk (Adolfsson-Erici et al., 2002). In vitro studies have shown that triclosan inhibits various biotransformation enzymes, including SULT and UDP-glucuronosyltransferases (UGTs) (Wang et al., 2004).
The fact that 3-OH-B[a]P, triclosan, OHMXC, 4'-OH-PCB79, 4'-OH-PCB159, and 4'-OH-PCB165 have not been reported as environmental contaminants in polar bears to date may be due to nonsignificant levels in the Arctic environment or efficient metabolism via, for example, sulfonation. On the other hand, the presence of PCP and, particularly, high amounts of TCPM in these Arctic carnivores may indicate poor sulfonation of these substrates. The polychlorobiphenylols 4'-OH-PCB159 and 4'-OH-PCB165 are of interest since, though they have not been detected in polar bears, they are structurally similar to 4'-OH-PCB172, one of the major OH-PCBs found in polar bear plasma (Sandau et al., 2000). It is thus possible that these compounds are sulfonated with similar efficiencies. The other major phase II biotransformation pathway for the above-mentioned compounds is glucuronidation. Polar bear liver efficiently glucuronidated 3-OH-B[a]P and several OH-PCBs (Sacco and James, 2004).
This study determined that polar bear hepatic SULT activity was highest for 3-OH-B[a]P and triclosan, followed by 4'-OH-PCB79. The other compounds studied, 4'-OH-PCB159, 4'-OH-PCB165, PCP, TCPM, and OHMXC, were poor substrates. This is the first time, to our knowledge, that the sulfation of TCPM was investigated for any species.
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Materials and Methods |
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). Triclosan and sulfatase (type VI from Aerobacter, S1629) were purchased from Sigma, while methoxychlor and TCPM were purchased from MP Biomedicals (Irvine, CA) and Lancaster Synthesis, Inc. (Pelham, NH), respectively. The OHMXC was prepared by the demethylation of methoxychlor and purified by recrystallization (Hu and Kupfer, 2002). Tetrabutyl ammonium hydrogen sulfate PIC-A low UV reagent was obtained from Waters (Milford, MA). Other reagents were the highest grade available from Fisher Scientific Co. (Pittsburgh, PA) and Sigma. Animals. The samples used in this study were a kind donation from Dr. S. Bandiera (University of British Columbia, Vancouver) and Dr. R. Letcher (Environment Canada, Quebec). They were derived from the distal portion of the right lobe of livers of three adult male bears G, K, and X. Bears G and K were collected as part of a legally controlled hunt by Inuit in the Canadian Arctic in April 1993 near Resolute Bay, Northwest Territories, whereas bear X was collected in November 1993 near Churchill, Manitoba, just after the fasting period. Liver samples were removed within 10 to 15 min after death, cut into small pieces, and frozen at –196°C in liquid N2. The samples were subsequently stored at –80°C.
Cytosol and Microsome Preparation. Prior to homogenization, the frozen polar bear liver samples (
2 g) were gradually thawed in a few milliliters of homogenizing buffer. Homogenizing buffer consisted of 1.15% KCl, 0.05 M K3PO4, pH 7.4, and 0.2 mM phenylmethylsulfonyl fluoride, added from concentrated ethanol solution just before use. Resuspension buffer consisted of 0.25 M sucrose, 0.01 M Hepes, pH 7.4, 5% glycerol, 0.1 mM dithiothreitol, 0.1 mM ethylenediaminetetraacetic acid, and 0.1 mM phenylmethylsulfonyl fluoride. The liver was placed in a volume of fresh ice-cold buffer equal to 4 times the weight of the liver sample. The cytosol and microsomal fractions were obtained using a procedure described previously (Wang et al., 2004). Microsomal and cytosolic protein contents were measured by the Lowry assay (Lowry et al., 1951), using bovine serum albumin (BSA) as standard.
Sulfotransferase Assays. Fluorometric Method. The activity was measured on the basis that at alkaline pH, the benzo[a]pyrene-3-O-sulfate has different wavelength optima for fluorescence excitation and emission (294/415 nm) from the benzo[a]pyrene-3-O-phenolate anion (390/545 nm) (James et al., 1997). Saturating concentrations of PAPS were determined by performing the assay at 1 µM 3-OH-B[a]P. The reaction mixture for detecting the sulfation of 3-OH-B[a]P by polar bear liver cytosol consisted of 0.1 M Tris-HCl buffer (pH 7.6), 0.4% BSA, PAPS (0.02 mM), 25 µg of polar bear hepatic cytosolic protein, and 3-OH-B[a]P (0.05–25 µM) in a total reaction volume of 1.0 ml. SULT activity (pmol/min/mg) was calculated from a standard curve prepared with B[a]P-3-O-sulfate standards. Substrate consumption did not exceed 10%.
Radiochemical Extraction Method. This method, based on the method of Wang et al. (2004), was used in the study of the sulfonation of 4'-OH-PCB79, 4'-OH-PCB159, 4'-OH-PCB165, triclosan, PCP, TCPM, and OHMXC. Cytosolic protein concentrations and incubation time were optimized for every test substrate to ensure that the reaction was linear during the incubation period. Substrate consumption did not exceed 5%. The incubation mixture consisted of 0.1 M Tris-HCl buffer (pH 7.0), 0.4% BSA in water, 20 µM PAPS (10% labeled with 35S), 0.1 mg of polar bear hepatic cytosolic protein, or 0.005 mg in the case of 4'-OH-PCB79 and triclosan, and substrate in a total reaction volume of 0.1 ml, or 0.5 ml in the case of TCPM. The OH-PCBs, triclosan, and OHMXC were added to tubes from methanol solutions, and the methanol was removed under N2 prior to addition of other components. The TCPM was dissolved in DMSO, the solvent being present at a concentration not exceeding 1% in the final assay volume. Control determinations utilizing 1% DMSO had no inhibitory effect on sulfonation. Aqueous solutions of sodium pentachlorophenolate were utilized in the case of PCP. Tubes containing all components except the cosubstrate were placed in a water bath at 37°C, and PAPS was added to initiate the reaction. Incubation times were 5 min (TCPM), 20 min (4'-OH-PCB79, triclosan), 30 min (PCP), and 40 min (OHMXC, 4'-OH-PCB159, 4'-OH-PCB165). The incubation was terminated by the addition of an equal volume of a 1:1 mixture of 2.5% acetic acid and PIC-A and water. The sulfated product was extracted with 3.0 ml of ethyl acetate as described previously (Wang et al., 2004) and the phases were separated by centrifugation. Duplicate portions of the ethyl acetate phase were counted for quantitation of sulfate conjugates.
Radiochemical TLC Method. Since the ethyl acetate phase contains sulfate conjugates formed from both the substrate of interest and substrates already present in polar bear liver, TLC was used to quantify substrate sulfation in cases where SULT activity was similar in samples and substrate blanks. After evaporating 2 ml of ethyl acetate extract from the SULT assay under N2, the solutes were reconstituted in 40-µl methanol. For 4'-OH-PCB159, 4'-OH-PCB165, PCP, and OHMXC, the substrate conjugates were separated on RP-18F254s reverse-phase TLC plates with a fluorescent indicator (Merck, Darmstadt, Germany) using methanol/water (80:20). For TCPM, Whatman KC18F reverse-phase 200-µm TLC plates with a fluorescent indicator in conjunction with a developing solvent system consisting of methanol/water/0.28 M PIC-A (40:60:1.9 by volume) were used. Electronic autoradiography (PerkinElmer Instant Imager) was used to identify and quantify the radioactive bands separated on the TLC plate. The counts representing the substrate sulfate conjugate products were expressed as a fraction of the total radioactivity determined by scintillation counting, thus enabling the radioactivity due to the substrate conjugate to be accurately determined.
The identity of the conjugate of TCPM as a sulfate ester was verified by studying its sensitivity to sulfatase. Polar bear cytosol (0.5 mg) was incubated for 75 min with or without 200 µM TCPM. The incubation was terminated and the product extracted into ethyl acetate as above. The ethyl acetate was evaporated to dryness and dissolved in 0.25 ml of Tris buffer, pH 7.5, containing 0 or 0.08 unit of sulfatase. After an overnight incubation at 35°C, the reaction was stopped by the addition of methanol and the tubes were centrifuged. The supernatants were evaporated to dryness, reconstituted in methanol, and analyzed by TLC as described above.
UDP-Glucuronosyltransferase Assay. The fluorometric assay for 3-OH-B[a]P was performed according to a method described previously (James et al., 1997). Preliminary studies established the conditions for linearity of reaction with respect to time, protein, and detergent concentrations, at the same time ensuring that substrate consumption did not exceed 10%. The apparent Km for UDPGA was determined by performing experiments at a fixed concentration of 3-OH-B[a]P (10 µM). Saturating UDPGA concentrations were used to determine 3-OH-B[a]P glucuronidation kinetics.
Kinetic Analysis. Duplicate values for the rate of conjugate formation at each substrate concentration were used to calculate kinetic parameters using Prism v 4.0 (GraphPad Software Inc., San Diego, CA). Equations used to fit the data were the Michaelis-Menten hyperbola for one-site binding (eq. 1), the Hill plot (eq. 2), substrate inhibition for one-site binding (eq. 3) (Houston and Kenworthy, 2000), and partial substrate inhibition due to binding at an allosteric site (eq. 4) (Zhang et al., 1998).
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Optimum conditions for the glucuronidation of 3-OH-B[a]P by polar bear microsomes were found to be 5 µg of microsomal protein and a 20-min incubation. A concentration of 4 mM UDPGA was determined to be suitable for providing saturating concentrations of the cosubstrate. The binding of UDPGA to UGT at 10 µM 3-OH-B[a]P was shown to be biphasic, with a 5-fold reduction in affinity at higher UDPGA concentrations (Table 1, Glucuronidation). The kinetic parameters for the cosubstrate were calculated by deconvoluting the curvilinear data in the Eadie-Hofstee plot (Fig. 3). In the presence of 4 mM UDPGA, the formation of B[a]P-3-O-glucuronide followed Michaelis-Menten kinetics (Table 1, Glucuronidation).
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The total TCPM sulfate conjugate production formed after 5 min under initial rate conditions did not exceed 30 pmol. TLC, followed by autoradiography, was thus used to distinguish the TCPM-sulfate band (Rf 0.54) from other sulfate conjugates (Rf 0.05 and 0.72) originating from compounds in the polar bear liver cytosol (Fig. 6). The data obtained followed hyperbolic kinetics (Table 2). Even though the TLC from the kinetic experiments showed a TCPM concentration-dependent increase of the band corresponding to the purported TCPM-sulfate, and this band was absent in the substrate blank, the fact remained that we were apparently looking at the only instance ever reported of a successful sulfonation of a tertiary alcohol. Thus, additional experiments were performed to verify the identity of this conjugate. The purity of the TCPM was tested in the event that the additional band was caused by an impurity in the substrate. However, the substrate used was found to be free of contaminants by high-performance liquid chromatography (C18 reverse-phase column, with detection at 268 and 220 nm, using 90% methanol in water and a flow rate of 1 ml/min). A single peak was recorded at 7.3 min. Another experiment involved a 60-min incubation performed with 100 µM TCPM and 0.1 mg of cytosolic protein from polar bear, channel catfish, and human liver. For each of the three species, we detected a conjugate at Rf = 0.54. The substrate blanks showed no band at the same position (Fig. 6). The TCPM sulfate conjugate from polar bear could be hydrolyzed by sulfatase (Fig. 7), providing further evidence of the sulfonation of this alcohol.
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Inhibition of sulfonation of substrates already present in the polar bear liver was noted upon adding 1 µM PCP (Fig. 8). The data for PCP sulfonation fitted the nonlinear Hill plot (eq. 2) (Table 2).
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) and humans (Wang et al., 2004). Benzo[a]pyrene-3-glucuronide has been shown to be produced by fish (James et al., 1997), rats (Lilienblum et al., 1987), and humans (Wang et al., 2004). There are, however, few studies investigating the kinetics of these conjugation reactions. Glucuronidation of 3-OH-B[a]P was more efficient in polar bear liver than in human liver or catfish intestine. On the other hand, the efficiency of sulfonation was similar to that shown in human liver but around 3 times less than that in catfish intestine (James et al., 2001; Wang et al., 2004). From the limited comparative data available, it can be surmised that, in general, polar bear liver is an important site of 3-OH-B[a]P detoxication, particularly with respect to glucuronidation.
Substrate inhibition for the sulfonation of 3-OH-B[a]P has been observed at relatively low concentrations of the xenobiotic in other species such as catfish and human (Tong and James, 2000; Wang et al., 2005). Data from the polar bear sulfonation assay fitted a two-substrate model developed for the sulfonation of 17ß-estradiol by SULT1E1 (Zhang et al., 1998). This model was also used to explain the sulfonation profile observed for the biotransformation of 1-hydroxypyrene, a compound structurally similar to 3-OH-B[a]P, by SULTs 1A1 and 1A3 (Ma et al., 2003). In the original model, SULT1E1 was saturated with PAPS, and each of the estradiol substrate molecules bound independently to the enzyme. The estradiol binding sites were proposed to consist of a catalytic site, and an allosteric site that regulates turnover of the substrate (Zhang et al., 1998). The substrate inhibition observed with polar bear liver cytosol at higher 3-OH-B[a]P concentrations (>0.75 µM) can thus be explained by the binding of a second substrate molecule to an allosteric site, which leads to a 2-fold decrease in affinity and an 8-fold decrease in Vmax.
SULTs are generally high-affinity, low-capacity biotransformation enzymes that operate effectively at low substrate concentrations. Thus, typical Km values for the sulfonation of xenobiotic substrates are usually significantly lower than Km values for the same substrates undergoing biotransformation by low-affinity, high-capacity glucuronosyltransferases (UGTs). In polar bear liver, both pathways showed similar apparent affinities for 3-OH-B[a]P, with Km values of 0.4 and 1.4 µM for sulfonation and glucuronidation, respectively, suggesting that these two pathways of phase II metabolism compete at similar 3-OH-B[a]P concentrations. However, the apparent maximal rate of sulfonation was about 7.5 times lower than the rate of glucuronidation.
It was previously reported that the maximum rate of glucuronidation of 3-OH-B[a]P by polar bear liver was 1.26 nmol/min/mg, or around half the Vmax value obtained in this study (Sacco and James, 2004). However, the preceding study utilized 0.2 mM UDPGA, which, as seen from Table 1, Glucuronidation, is equivalent to the Km (for UDPGA) of the low-affinity enzyme and, thus, does not represent saturating concentrations of the cosubstrate. The affinity of the enzyme for 3-OH-B[a]P did not change significantly with a 20-fold increase in UDPGA concentrations, suggesting that substrate binding is independent of the binding of cosubstrate. The binding of UDPGA was biphasic, indicating that multiple hepatic UGTs may be responsible for the biotransformation. Biphasic UDPGA kinetics have also been demonstrated in human liver and kidney for 1-naphthol, morphine, and 4-methylumbelliferone (Miners et al., 1988a,b; Tsoutsikos et al., 2004). Although Vmax was similar for both components, there was a 5-fold decrease in enzyme affinity for UDPGA as the cosubstrate concentration was increased. The involvement of at least two enzymes can be physiologically advantageous since it enables the maintenance of a high turnover rate even as UDPGA is consumed. Although physiological UDPGA concentrations in polar bear liver are unknown, mammalian hepatic UDPGA has been determined to be around 200 to 400 µM (Zhivkov et al., 1975; Cappiello et al., 1991), implying that the observed nonlinear kinetics in the polar bear may operate in vivo.
The rate of triclosan sulfonation was the highest of all the substrates studied; apparent Vmax was twice as high as for 3-OH-B[a]P. However, the overall efficiency of sulfonation of the hydroxylated poly-aromatic hydrocarbon was still 13 times higher than for triclosan sulfonation. The presence of three chlorine substituents (though none flanking the phenol group) does not hinder the sulfonation of triclosan when compared with the "chlorine-free" 3-OH-B[a]P. Triclosan sulfonation in polar bear liver was similar to that in human liver with respect to enzyme affinity; however the maximum rate was 10-fold higher in polar bears than in humans (Wang et al., 2004). This may be one reason why triclosan has not been detected in polar bear plasma or liver to date.
Our data fitted a model that indicates that the substrate inhibition observed for 4'-OH-PCB79 may be due to a second substrate molecule interacting with the enzyme-substrate complex at the active site rather than an allosteric site, resulting in a dead-end complex. Unlike 3-OH-B[a]P, sulfonation can only proceed via the single substrate-SULT complex. Models of SULT1A1 and 1A3, with two molecules of p-nitrophenol or dopamine at the active site, respectively, have been proposed as a mechanism of substrate inhibition (Gamage et al., 2003; Barnett et al., 2004), whereas the crystal structure of human estrogen sulfotransferase containing bound 4,4'-OH-3,3',5,5'-tetrachlorobiphenyl at the active site has not provided any evidence of an allosteric site (Shevtsov et al., 2003). The slower sulfonation of 4'-OH-PCB79 compared with 3-OH-B[a]P may result from the inductive effect of the chlorines flanking the phenolic group rather than steric hindrance (Duffel and Jakoby, 1981). However, polar bear liver sulfonated 4'-OH-PCB79 more rapidly than the other OH-PCB substrates studied.
The inclusion of two additional chlorine substituents on the nonphenol ring (with respect to 4'-OH-PCB79) resulted in both 4'-OH-PCB159 and 4'-OH-PCB165 being very poor substrates. Inefficient sulfonation may be one reason why the related compound 4'-OH-PCB172 accumulates in polar bears. Some degree of substrate inhibition may also be expected to contribute to this accumulation, as was observed with 4'-OH-PCB165.
Sulfonation was not an efficient pathway of OHMXC detoxification. The rate of OHMXC-sulfonate formation was around 7 times lower than for 4'-OH-PCB79. Since resonance delocalization of negative charge on the phenolic oxygen by the flanking chlorines in chlorophenols may decrease Vmax by increasing the energy of the transition state of the reaction (Duffel and Jakoby, 1981), it is possible that in the case of OHMXC (with no chlorines flanking the phenolic group), product release, rather than sulfonate transfer, may have been the rate-limiting step.
TCPM was a poor substrate for sulfonation, and this may be one reason why it has been measured in such high amounts in polar bear liver. To our knowledge, sulfonation of acyclic tertiary alcohols has not been reported in the literature. Despite the considerable steric hindrance of three phenyl groups, the alcohol group could be sulfonated. Although the alcohol in TCPM is not of the benzylic type, the presence of three proximal phenyl groups may give this group some benzylic character, rendering sulfonation of the alcohol possible. Both SULT1E1 and SULT2A1 have been shown to sulfonate benzylic alcohol groups attached to large molecules (Glatt, 2000). Sulfation of the benzylic hydroxyl group leads to an unstable sulfate conjugate that readily degrades to the reactive carbocation or spontaneously hydrolyzes back to the alcohol. Attempts to recover TCPM-O-sulfonate from TLC plates resulted in recovery of TCPM from the conjugate band, perhaps because of the conjugate's instability (data not shown).
A study of the sulfonation of PCP was complicated by the fact that it is a known SULT inhibitor, often with Ki values in the submicromolar range. In our experiments, this was seen as a 74% decrease in formation of the unidentified sulfonate conjugates (band shown at the solvent front in Fig. 8) upon addition of 1 µM PCP. Although PCP was a strong inhibitor of SULT1E1 (Kester et al., 2000) and has been postulated to be a dead-end inhibitor for phenol sulfotransferases (Duffel and Jakoby, 1981), it was possible that polar bear SULT 1A isoforms were not completely inhibited by PCP, or that other SULT isoform(s) were responsible for the limited sulfonation activity observed. Thus, we have shown that, in vitro, at least, one mammalian species is capable of limited PCP sulfonation. Even though the tertiary alcohol of TCPM was a poor candidate for sulfonation, it was metabolized at twice the efficiency of PCP, which has a phenolic group that is usually more susceptible to sulfonation. This demonstrates the extent of the decreased nucleophilicity on the phenolic oxygen due to the resonance delocalization afforded by the five chlorine substituents.
In summary, this study has demonstrated that, in polar bear liver, 3-OH-B[a]P was a good substrate for sulfonation and glucuronidation. Other, chlorinated substrates were biotransformed with less efficiency, implying that reduced rates of sulfonation may contribute to the persistence of compounds such as hexachlorinated OH-PCBs, TCPM, and PCP in polar bear tissues.
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
ABBREVIATIONS: SULT, sulfotransferase; BSA, bovine serum albumin; PAPS, 3'-phosphoadenosine-5'-phosphosulfate; OH-PCB, polychlorinated biphenylol; 4'-OH-PCB79, 4'-hydroxy-3,3',4,5'-tetrachlorobiphenyl; 4'-OH-PCB159, 4'-hydroxy-2,3,3',4,5,5'-hexachlorobiphenyl; 4'-OH-PCB165, 4'-hydroxy-2,3,3',5,5',6-hexachlorobiphenyl; PCP, pentachlorophenol; TCPM, tris(4-chlorophenyl)-methanol; OHMXC, 2-(4-methoxyphenyl)-2-(4-hydroxyphenyl)-1,1,1-trichloroethane; 3-OH-B[a]P, 3-hydroxybenzo[a]pyrene; UDPGA, uridine 5'-diphosphoglucuronic acid; UGT, UDP-glucuronosyltransferase; TLC, thin-layer chromatography.
Address correspondence to: Margaret O. James, Department of Medicinal Chemistry, College of Pharmacy, University of Florida, P.O. Box 100485, Gainesville, FL 32610. E-mail: mojames{at}cop.ufl.edu
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and ß and androgen receptor: structure-activity studies. Mol Pharmacol 58: 852–858.This article has been cited by other articles:
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  D Pugazhendhi, K A Watson, S Mills, N Botting, G S Pope, and P D Darbre Effect of sulphation on the oestrogen agonist activity of the phytoestrogens genistein and daidzein in MCF-7 human breast cancer cells J. Endocrinol., June 1, 2008; 197(3): 503 - 515. [Abstract] [Full Text] [PDF]
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 J. C. Sacco, H.-J. Lehmler, L. W. Robertson, W. Li, and M. O. James Glucuronidation of Polychlorinated Biphenylols and UDP-Glucuronic Acid Concentrations in Channel Catfish Liver and Intestine Drug Metab. Dispos., April 1, 2008; 36(4): 623 - 630. [Abstract] [Full Text] [PDF]
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