Hydroxylated Polychlorinated Biphenyls Are Substrates and Inhibitors

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Chem. Res. Toxicol. 2006, 19, 1420-1425

Hydroxylated Polychlorinated Biphenyls Are Substrates and Inhibitors of Human Hydroxysteroid Sulfotransferase SULT2A1 Yungang Liu,† T. Idil Apak,† Hans-Joachim Lehmler,‡ Larry W. Robertson,‡ and Michael W. Duffel*,† DiVision of Medicinal and Natural Products Chemistry, College of Pharmacy, UniVersity of Iowa, Iowa City, Iowa 52242, and Department of Occupational and EnVironmental Health, UniVersity of Iowa, College of Public Health, Iowa City, Iowa 52242 ReceiVed July 13, 2006

Polychlorinated biphenyls (PCBs) are important persistent environmental contaminants. PCBs can be metabolically converted to their hydroxylated metabolites (OHPCBs), and in recent years, these OHPCBs have been observed to inhibit human sulfotransferases (SULTs) such as the phenol SULTs (SULT family1) involved in the metabolism of estrogen and various other endogenous and xenobiotic phenols. In the present study, we have investigated the hypothesis that OHPCBs interact with family 2 hydroxysteroid (alcohol) SULTs (e.g., human SULT2A1), enzymes that are physiologically important for the metabolic transformations of several key endogenous hydroxysteroids as well as xenobiotic alcohols. We have examined the interactions of three OHPCBs with purified recombinant human SULT2A1 (also known as either human DHEA-ST or ST2A3). These studies with SULT2A1 were carried out on 4′-hydroxy-2,5dichlorobiphenyl (4′-OH PCB 9), 4-hydroxy-2′,3,5-trichlorobiphenyl (4-OH PCB 34), and 4′-hydroxy2,3′,4,5′-tetrachlorobiphenyl (4′-OH PCB 68). Our results showed that 4-OH PCB 34 and 4′-OH PCB 68 were substrates for SULT2A1, and 4-OH PCB 34 exhibited substrate inhibition similar to that seen with the physiological substrate dehydroepiandrosterone (DHEA). Although the sulfation of 4-OH PCB 34 and 4′-OH PCB 68 represents a potential metabolic route for these compounds, these OHPCBs may also compete with other xenobiotic substrates as well as endogenous substrates for SULT2A1. The third OHPCB studied, 4′-OH PCB 9, was not a substrate for SULT2A1 but was an inhibitor of the enzyme. Thus, the interactions of OHPCBs with human SULT2A1 represent both a potential route of metabolism and a possible source of interference with sulfation reactions catalyzed by this enzyme. Introduction Polychlorinated biphenyls (PCBs)1 continue to be of worldwide concern because of the combination of their toxicity and their persistence in the environment. Although the lipophilic characteristics of PCBs enable them to accumulate in tissues, many undergo oxidative metabolism to hydroxylated PCBs (OHPCBs) in reactions catalyzed by cytochrome P450 (CYP) isoforms such as CYP2B and CYP1A (1-3). These OHPCBs persist in the blood, liver, adipose, and other tissues of humans (4-8). Indeed, there is evidence that OHPCBs may, in some cases, be present at substantially higher concentrations in blood than the corresponding parent PCBs (4). In recent years, OHPCBs have been observed to potently inhibit activities of cytosolic sulfotransferases (SULTs) that catalyze the sulfation of 3-hydroxy-benzo[a]pyrene in catfish (9) and humans (10), thyroid hormones in the rat (11, 12), and estrogen in humans (13, 14). These SULTs have been classified for many years as phenol sulfotransferases. On the basis of their * Corresponding author. Tel: 319-335-8840. Fax: 319-335-8766. Email: [email protected]. † Division of Medicinal and Natural Products Chemistry. ‡ Department of Occupational and Environmental Health. 1 Abbreviations: DHEA, dehydroepiandrosterone; (3β)-3-hydroxyandrost-en-17-one; DHEAS, dehydroepiandrosterone sulfate; HPLC, high performance liquid chromatography; IPTG, isopropyl-1-thio-D-galactopyranoside; LB, Luria broth; OHPCB, hydroxylated polychlorinated biphenyl; PAP, adenosine 3′,5′-diphosphate; PAPS, adenosine 3′-phosphate 5′phosphosulfate; PCB, polychlorinated biphenyl; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SULT, sulfotransferase.

sequence similarity, however, these particular SULTs are now all placed within the same gene family (i.e., family 1) by a recently proposed nomenclature system for the superfamily of cytosolic SULTs (15). There is, however, much less known about the potential effects of PCBs and OHPCBs on another major family of cytosolic SULTs, that is, family 2 or hydroxysteroid (alcohol) SULTs. This gap in our knowledge is significant because of the roles of hydroxysteroid SULTs in the homeostasis of steroid hormones, such as dehydroepiandrosterone (DHEA) and androsterone (16-18), the metabolism of bile acids (19), and the metabolic activation of mutagenic and carcinogenic hydroxyalkyl polycyclic aromatic hydrocarbons (20-22). SULT2A1 (also referred to as DHEA-ST and ST2A3) is a major family 2 sulfotransferase in humans, and we have, therefore, chosen this enzyme for the initial studies of the interaction of OHPCBs with a member of this enzyme family. The OHPCBs used in the present investigation include a di-, tri-, and tetra-chlorinated hydroxybiphenyl, and the structures are shown in Figure 1.

Materials and Methods Chemicals and Biochemicals. 4′-Hydroxy-2,5-dichlorobiphenyl (4′-OH PCB 9), 4-hydroxy-2′,3,5-trichlorobiphenyl (4-OH PCB 34), and 4′-hydroxy-2,3′,4,5′-tetrachlorobiphenyl (4′-OH PCB 68) (structures and abbreviated names shown in Figure 1) were synthesized as previously described (23). The analytical data for all of the compounds were in agreement with the indicated structures. The nomenclature for OHPCBs is based on the systematic nomenclature for the parent congeners described by Ballschmiter and co-workers

10.1021/tx060160+ CCC: $33.50 © 2006 American Chemical Society Published on Web 10/06/2006

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Figure 1. Structures and abbreviated names for the OHPCBs investigated in the study.

(24). Adenosine 3′-phosphate 5′-phosphosulfate (PAPS) was obtained from Sigma-Aldrich (St. Louis, MO) and further purified by a published procedure (25) to a purity greater than 99% as determined by HPLC. DHEA, 1-octylamine, and adenosine 3′,5′diphosphate (PAP)-agarose were from Sigma-Aldrich (St. Louis, MO). All other chemicals and reagents were of the highest purity commercially available. Expression and Purification of Human SULT2A1. The expression of human SULT2A1 in recombinant Escherichia coli BL21 (DE3) cells has been previously described (26), and cells were grown and extracted with the following minor modifications. Cells were inoculated into 3 mL of Luria broth (LB) medium that was supplemented with 50 µg/mL ampicillin and incubated at 29 °C on a reciprocating shaker (250 rpm) for 24 h. A 100 µL aliquot of the cell suspension was then transferred to each of four 20 mL medium-containing culture tubes, and incubated under the same conditions. After 24 h of incubation at 29 °C, each 20 mL culture was used to inoculate 400 mL of LB medium containing 50 µg/ mL ampicillin in a 1-L flask, and the cells were incubated at 29 °C on a reciprocating shaker (230 rpm). Following incubation of the cells for 1 h, 1 mM IPTG (isopropyl-1-thio-D-galactopyranoside) was added. After 24-28 h of incubation, cells were harvested by centrifugation at 10 000g for 20 min. The cell pellets (total wet weight of about 14 g) were suspended in 25 mL of ice-cold buffer A (10 mM tris-HCl buffer at pH 7.5, 250 mM sucrose, 10% (v/v) glycerol, 1 mM phenylmethylsulfonylfluoride, 1 µM pepstatin, 1 mM DTT, and 2 mg/L antipain). Cells in this suspension were then disrupted at 0-4 °C with a Digital Sonifier (model 450, Branson Ultrasonics Corp., Danbury, CT). The sonicator was programmed at 30% amplitude to provide 25 cycles of 0.2 s on and 0.2 s off. This sonication program was performed a total of 10 times for each homogenate with 40 s between each sonication period. The resulting cell homogenate was centrifuged at 24 000g for 30 min, and the resulting supernatant fraction (cell extract) was collected. Purification of human SULT2A1 was carried out using PAPagarose affinity column chromatography, with the following minor modifications of the previously described procedure (26). The cell extract (5.5 mL) was charged onto a column of PAP-agarose (10 mL) that had been equilibrated with buffer B (10 mM tris-HCl buffer at pH 7.5, 250 mM sucrose, 10% (v/v) glycerol, 1 µM pepstatin, 1 mM DTT, 2 mg/L antipain, and 0.05% (v/v) Tween 20). The column was then washed with 200 mL of buffer B. The recombinant protein was eluted with a linear gradient formed between 20 mL of buffer B and 20 mL of buffer B supplemented with 200 µM PAP. The fractions containing SULT2A1 were combined and concentrated by ultrafiltration (PM-10 membrane, NMWL:10,000; Millipore Corporation, Bedford, MA) to a volume of about 1.5 mL. PAP was then removed by chromatography using a PD-10 size-exclusion column (Amersham Biosciences, Piscataway, NJ). The protein was concentrated by ultrafiltration as described above, and ultrafiltration was repeated with 5 volumes

Chem. Res. Toxicol., Vol. 19, No. 11, 2006 1421 of buffer B. The resultant protein volume was 1.3 mL and stored at -70 °C. Verification of the removal of PAP from the protein was carried out by HPLC analysis using the chromatographic conditions described previously for the separation of PAP and PAPS (27, 28). Protein content was determined by the modified Lowry method (29) with bovine serum albumin as standard, and the enzymatic activity of SULT2A1 during purification steps was determined by the methylene blue paired ion extraction assay (28, 30). The purified SULT2A1 displayed a single band on SDSPAGE with Coomassie blue staining (data not shown). Assay of OHPCBs as Substrates of Human SULT2A1. Kinetic parameters describing the interactions of the three OHPCBs with SULT2A1 were obtained using the following procedure. Each 30 µL of reaction mixture contained 0.25 M potassium phosphate buffer at pH 7.0, 0.2 mM PAPS, 7.5 mM 2-mercaptoethanol (to maintain the reduced form of the enzyme), various indicated amounts of OHPCBs, 5% (v/v) acetone as solvent for OHPCBs, and 0.3 µg of protein of purified human SULT2A1. The reaction was carried out at 37 °C for 15 min and terminated by the addition of 30 µL of methanol. The concentration of PAP formed in each reaction was determined by HPLC as previously described (27, 28). Reaction velocities were calculated as the substrate-dependent formation of PAP from PAPS. The limit of detection for this assay method using purified recombinant human SULT2A1 is 0.1 nmol of PAP/min/mg of enzyme (26). The assay of SULT2A1 by the OHPCB-dependent formation of PAP (procedure above) was compared to the determination of the SULT2A1- and OHPCB-dependent formation of a product organic sulfate that forms a paired ion with methylene blue and is extracted into chloroform. The assay method utilizing methylene blue was based on the previously published procedure (28, 30). The methylene blue assay was carried out at 37 °C and at pH 7.0 with the amounts of enzyme and other components directly scaled to a reaction mixture 10 times larger than the one used for the determination of the OHPCB-dependent formation of PAP. A reaction time of 30 min was utilized for the methylene blue assay in order to further account for the decreased sensitivity of this procedure compared to the HPLC determination of the substratedependent formation of PAP. Solubility of OHPCBs in the Assay. In order to assess solubility under the specific conditions of these assays, the solubilities of the three OHPCBs as well as that of DHEA were determined at 37 °C in 0.25 M potassium phosphate buffer at pH 7.0, containing 5% (v/v) acetone and 7.5 mM 2-mercaptoethanol. Solubility was determined by light scattering at 400 nm using a Perkin-Elmer LS55 Luminescence Spectrometer according to a previously described procedure (31). Inhibition of Human SULT2A1 by 4′-OH PCB 9. An IC50 value for inhibition of SULT2A1 by 4′-OH PCB 9 was determined using a nonsaturating concentration of DHEA and the methylene blue paired ion extraction assay described previously (28, 30). Reactions were carried out in a total volume of 0.6 mL at pH 7.0 and 37 °C, with 0.2 mM PAPS and 5.0 µM DHEA, and varying concentrations of 4′-OH PCB 9. Acetone was used as a cosolvent for both DHEA and 4′-OH PCB 9 in a final assay concentration of 5% (v/v). Reactions of 15 min were followed by the addition of the methylene blue reagent and extraction with 1.5 mL of chloroform. Within the utilized concentration range of 4′-OH PCB 9, from 1.3 to 26% of the DHEA was sulfated under the reaction conditions. Reaction velocities were analyzed with SigmaPlot 8.0 to determine IC50 values.

Results Investigation of OHPCBs as Substrates for Human SULT2A1. In analyzing the potential for OHPCBs to serve as substrates for human SULT2A1, we first determined their solubility under the specific conditions of the assay (Materials and Methods). As seen in Figure 2, 4′-OH PCB 9 was soluble under the assay conditions at concentrations of 500 µM or less,

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Figure 2. Determination of the solubility of 4′-OH PCB 9, 4-OH PCB 34, and 4′-OH PCB 68 under the conditions of the assays for the activity of SULT2A1. Data points are the means ( standard deviation of three determinations.

4-OH-PCB 34 was soluble at concentrations up to and including 300 µM, and 4′-OH PCB 68 was soluble at concentrations of 50 µM or less. DHEA was soluble at concentrations at or below 200 µM (data not shown). SULT2A1 catalyzed the concentration-dependent sulfation of 4-OH PCB 34 and 4′-OH PCB 68 (Figures 3 and 4, respectively). The data for the SULT2A1catalyzed sulfation of 4-OH PCB 34 were fit to the following substrate inhibition equation: V ) Vmax/(1 + (Km/[S]) + ([S]/ Kis)), where V is the reaction velocity, Vmax is the maximal velocity, Km is the Michaelis constant for the reaction, [S] is the concentration of substrate, and Kis is the substrate inhibition constant. For 4-OH PCB 34, the kinetics of sulfation were described well by this equation with a Km of 46 ( 13 µM, a Vmax of 52 ( 8 nmol product/min/mg SULT2A1, and a substrate inhibition constant of 253 ( 85 µM (correlation coefficient of 0.929). As seen in Figure 3B, this compares favorably with the sulfation of DHEA catalyzed by SULT2A1 over a range of concentrations from 10 to 200 µM. Kinetic constants for the sulfation of DHEA catalyzed by the purified recombinant SULT2A1 under the same reaction conditions were the following: Km)25 ( 8 µM, Vmax)122 ( 24 nmol/min/mg SULT2A1, and a substrate inhibition constant of 112 ( 40 µM (correlation coefficient of 0.921) when fit to the substrate inhibition equation described above. For 4′-OH PCB 68, the limit of solubility made the determination of kinetic constants difficult. However, as seen in Figure 4, 4′-OH PCB 68 was clearly a substrate for SULT2A1, and the highest rate of sulfation was observed at the limit of solubility (i.e., 50 µM). Although sulfuric acid esters of these OHPCBs are not yet available as synthetic standards, confirmation of the assay methodology was obtained by comparing the rates of sulfation of 4-OH PCB 34 and 4′-OH PCB 68 using an assay for the formation of an organic sulfate that forms a paired ion with methylene blue for extraction into chloroform (28, 30). The comparison was carried out at concentrations where the optimum velocities of reaction had been observed by the assay for PAP formation (i.e., 150 µM 4-OH PCB 34 and 50 µM 4′-OH PCB 68), and the results were determined as the mean ( standard deviation of three assays. The rate of SULT2A1-catalyzed sulfation of 4-OH PCB 34 was 27.0 ( 1.2 nmol of product/ min/mg of enzyme by the methylene blue assay and 25.6 ( 1.0 nmol of product/min/mg of enzyme by the assay for PAP

Figure 3. Comparison of the sulfation of 4-OH PCB 34 (panel A) and DHEA (panel B) catalyzed by purified recombinant human SULT2A1. Data points are the means ( standard error of three determinations, and the curves are the calculated fit to the equation for substrate inhibition described in the Results section.

Figure 4. The sulfation of 4′-OH PCB 68 catalyzed by purified recombinant human SULT2A1. Data points are the mean ( standard error of three determinations.

formation. The rate of sulfation of 4′-OH PCB 68 was 17.5 ( 2.3 nmol of product/min/mg of SULT2A1 by the methylene blue assay and 15.9 ( 1.0 nmol of product/min/mg of enzyme by the assay for substrate-dependent formation of PAP. Thus, the assays for the formation of both the organic sulfate and PAP as reaction products were in agreement. Inhibition of Human SULT2A1 by 4′-OH PCB 9. In contrast to the two other OHPCBs, 4′-OH PCB 9 was not a substrate for purified recombinant human SULT2A1 when examined at a range of concentrations from 1 µM to 300 µM (data not shown). The 4′-OH PCB 9 was, however, a potent inhibitor of the SULT2A1-catalyzed sulfation of DHEA. As seen in Figure 5, 4′-OH PCB 9 displayed an IC50 value of 3.9 µM when examined at a substrate concentration of 5.0 µM DHEA.

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Figure 5. Inhibitory effect of 4′-OH PCB 9 on the sulfation of DHEA catalyzed by purified human SULT2A1. Assays for DHEA-dependent formation of PAP were conducted at pH 7.0 as described in Materials and Methods at a DHEA concentration of 5.0 µM and the indicated concentrations of 4′-OH PCB 9. Data points are the means ( standard error of duplicate determinations, and the calculated value of IC50 for 4′-OH PCB 9 was 3.9 µM under these conditions.

Discussion The hydroxysteroid (alcohol) SULTs, that is, family 2 SULTs, have significant functions in the metabolism of both endogenous and xenobiotic alcohols. Much attention has been focused on the role of family 2 enzymes in the toxicology of xenobiotic alcohols such as the hydroxyalkyl polycyclic aromatic hydrocarbons (20-22). Another function of hydroxysteroid (alcohol) SULTs (e.g., SULT2A1) is the catalysis of sulfation of DHEA to form DHEA-sulfate (DHEAS), and this is one component of the regulation of the complex dynamic equilibrium that exists between these two steroid hormones (18). In addition to their role as precursors to androgens and estrogens, extensive studies in both humans and other animals indicate numerous less understood potential effects of DHEA and DHEAS on immunological, cardiovascular, metabolic, and neurological function (32). Recent evidence also points to a potentially important role of SULT2A1 in the metabolism and function of steroid hormones through the sulfation of androsterone; androsterone sulfate is an abundant circulating 5R-reduced androgen in human serum and a major androgen metabolite in urine (16). Other physiological functions of hydroxysteroid SULTs relate to their role in the detoxication of bile acids such as lithocholic acid (33). Thus, there is the potential for xenobiotics that alter the sulfation of endogenous molecules catalyzed by hydroxysteroid SULTs to have effects on physiological processes. Although such alterations in the sulfation of endogenous molecules might come about by the action of enzyme inhibitors, alternate substrates (e.g., some OHPCBs) may also compete with endogenous substrates for the active site of a SULT. Our interest in hydroxylated metabolites of semi-volatile PCBs, which contain lower numbers of chlorine atoms, led us to investigate the possibility that OHPCBs might serve as substrates or inhibitors of the human hydroxysteroid (alcohol) sulfotransferase SULT2A1. The specific OHPCBs chosen for these preliminary studies were selected to provide molecules with 2, 3, and 4 chlorine atoms but without regard to specific chlorine substitution patterns. Thus, while our results provide a firm basis for the interaction of OHPCBs with a human sulfotransferase in gene family 2, they clearly point to the need for future examinations of structure-activity relationships for the sulfation of OHPCBs catalyzed by hydroxysteroid (alcohol) SULTs. Nonetheless, several characteristics of the interactions

of these three OHPCBs with SULT2A1 have emerged from this preliminary investigation. Two of the model OHPCBs, 4-OH PCB 34 and 4′-OH PCB 68, are substrates for SULT2A1. Of these two, 4-OH PCB 34 exhibits substrate inhibition at high concentrations, similar to the kinetic behavior seen with DHEA at high concentrations. Our examination of the sulfation of both 4-OH PCB 34 and DHEA under identical assay conditions provides a direct and straightforward basis for comparison because reported calculations of Km and Vmax values for the SULT2A1-catalyzed sulfation of DHEA are often difficult to compare because of differing assay conditions, source and purity of the enzyme preparations, and kinetic assumptions made about the impact of substrate inhibition on the reaction. Although similar comparisons with 4′OH PCB 68 are limited by the solubility of this congener, it is clear that it is also a substrate for SULT2A1. Thus, our findings suggest a potential role for this enzyme in the metabolic sulfation of OHPCBs. Moreover, good substrates for SULT2A1, such as 4-OH PCB 34 and 4′OH PCB 68, might be expected to compete with either xenobiotic or endogenous substrates for the active site of the enzyme, resulting in a decrease in the rate of sulfation of the non-OHPCB substrate. This mode of interference with the metabolism of either xenobiotic or endogenous molecules would be in addition to the ability of other OHPCBs to serve purely as inhibitors of the enzyme. In the case of 4′-OH PCB 9, an inhibitor that does not serve as a substrate, the IC50 value of 3.9 µM observed for the inhibition of the sulfation of 5.0 µM DHEA catalyzed by SULT2A1 indicates that its interactions with the enzyme are at concentrations comparable to those seen for the interactions of DHEA with the enzyme. Thus, inhibition of the sulfation of DHEA by 4′-OH PCB 9 would depend upon the relative intracellular concentrations of the OHPCB and DHEA in the vicinity of the enzyme. In evaluating the potential toxicological significance of our results obtained with SULT2A1, it is important to consider several factors; among these are the in vivo concentrations of OHPCBs, concentrations of endogenous substrates such as DHEA and androsterone, and the expression of SULT2A1 in specific tissues. As noted above, OHPCBs have been detected in humans (4-8), and they may be present in substantially higher concentrations in blood than in the parent PCBs (4). Direct comparisons of blood and/or serum levels of PCBs and OHPCBs with tissue concentrations are relatively rare and are complicated by differences in the way in which these are measured and reported. That is, blood and serum levels are often determined on the basis of serum or plasma fresh weights, whereas tissue levels are often reported on the basis of lipid weight. Nonetheless some approximations can be made to assist in comparing potential serum concentrations of OHPCBs with those of DHEA and DHEA sulfate. Assuming an average molecular weight of 300 for OHPCBs, human values of 0.082 - 0.328 ng/g (6) and 0.117-11.6 ng/g (8) based on whole blood wet weight of total OHPCBs would represent an approximate concentration range from 0.3 - 40 nM. In comparison, the serum concentration of DHEA in humans varies with age and differs between men and women but is within the general range of 5-24 nM (34). Human serum concentrations of androsterone have been reported to be in the range of 2.5 - 5.0 nM (34). In contrast, DHEA sulfate (1.5 - 11.5 µM) and androsterone sulfate (0.3 - 1.4 µM) are present in human serum at much higher concentrations than those of their parent alcohols (34). So, in general, OHPCBs in human serum have been reported in concentrations very similar to those of circulating steroid

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substrates for SULT2A1. However, it should be emphasized that because the SULT2A1 is an intracellular enzyme, the blood or serum concentrations of physiological or xenobiotic substrates and inhibitors present an incomplete picture of the potential concentration in the vicinity of the enzyme within the tissue. Thus, in addition to serum concentrations of OHPCBs, it is essential to consider the potential intratissue concentrations of these compounds. Experiments in rats have shown that there is a selective concentration of OHPCBs in blood, liver, and other tissues, and this is consistent with blood samples analyzed from humans and from environmentally exposed gray seals (4). Moreover, other studies have shown that although the levels of PCBs in the livers of humans are similar to those in adipose tissue, OHPCBs actually exhibit higher concentrations in liver than in adipose tissue (35). The selective concentration of OHPCBs in liver could be particularly relevant to the results obtained in the present investigation because of the expression of SULT2A1 in this tissue. Additional studies will be needed to clarify the tissue concentrations of specific OHPCBs that may be reached under both acute and chronic exposures; however, it is clear that liver and other tissues may selectively concentrate OHPCBs, with the resulting concentration being significantly higher than that observed in blood or serum samples. Moreover, our results indicate that the kinetic constants for either the sulfation of a OHPCB catalyzed by SULT2A1 or the inhibition of the enzyme by a OHPCB are of similar magnitude to those obtained with DHEA, one of the most effective substrates known for SULT2A1. This result suggests that if intracellular concentrations of OHPCBs approach or exceed those of endogenous substrates (e.g., DHEA), their interaction with SULT2A1 may affect physiological sulfation reactions catalyzed by the enzyme. Further studies will be required to explore both the intratissue concentrations of the OHPCBs and SULT2A1 as well as the potential toxicological significance. As noted in the Introduction, studies on the interactions of OHPCBs with sulfotransferases have been previously focused on the family 1 sulfotransferases (9-14). In general, this family of sulfotransferases has a broad tissue distribution and often act on phenolic substrates. Although most attention has been placed on inhibition of family 1 sulfotransferases by OHPCBs, OHPCBs are also substrates for the SULT1A1 (14, 36), SULT1B1 (36), and SULT1E1 (14) isoforms of this family of enzymes. Because the family 1 enzymes have often been characterized as phenol sulfotransferases, it might be easy to assume that sulfation of all phenols is primarily catalyzed by SULT1A1, SULT1E1, and other SULT1 forms. However, SULT2A1 has also been found to catalyze the sulfation of a few phenols. For example, phenols such as estradiol and estrone are relatively weak substrates for human SULT2A1 (17), and 1-hydroxypyrene is a substrate for this isoform (37). As a result of our findings that some OHPCBs are good substrates for SULT2A1, future studies on the sulfation of OHPCBs in tissues such as liver, where both SULT1 and SULT2 isoforms are expressed, will be needed to determine the relative contribution of these sulfotransferases. In summary, we have determined that OHPCBs can interact with a human hydroxysteroid (family 2) sulfotransferase, SULT2A1, as substrates and inhibitors. When the kinetic characteristics for these interactions are considered in conjunction with the potential for intratissue concentration of OHPCBs to levels significantly higher than those observed in blood or serum, it is clear that the potential exists for some OHPCBs to be metabolically sulfated in reactions catalyzed by SULT2A1. However, the extent to which this is a detoxication reaction for

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OHPCBs remains to be determined. Those OHPCBs that serve either as competing substrates or inhibitors of SULT2A1 may also have the potential for inhibition of other sulfation reactions catalyzed by the enzyme. Although the particular OHPCBs examined in this study were chosen for preliminary examination of a di-, tri-, and tetra-chlorinated hydroxybiphenyl, they were not chosen to be representative of all OHPCBs. Indeed, some other OHPCBs may potentially be even more effective as substrates or inhibitors of SULT2A1. A complete determination of structure-activity relationships for the interactions of OHPCBs with the human SULT2A1 is clearly warranted as a result of these findings. Such studies will elucidate more fully the possible roles of this enzyme in the metabolism of OHPCBs as well as the potential that interactions of OHPCBs with SULT2A1 as either substrates or inhibitors might contribute to the physiological consequences of exposure to PCBs. Acknowledgment. This study was supported by the National Institutes of Health through research grant R01 CA38683 (to M.W.D.) from the National Cancer Institute and research grants P42 ES013661 (to L.W.R., M.W.D., and H.J.L.) and K25 ES012475 (to H.J.L.) from the National Institute of Environmental Health Sciences. We also acknowledge programmatic support through the University of Iowa Environmental Health Sciences Research Center (NIEHS/NIH P30 ES05605). The contents of this paper are solely the responsibility of the authors and do not necessarily represent the official views of the NIH.

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