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Polychlorinated biphenyls (PCBs) are persistent toxic chemicals with both legacy sources (e.g., Aroclors) and new sources (e.g., unintentional contaminants in some pigments and varnishes). PCB sulfates are derived from further metabolism of hydroxylated PCBs (OH-PCBs), which are oxidative metabolites of PCBs. While OH-PCBs and PCB sulfates are implicated in multiple toxicological effects, studies of PCB sulfates in human serum have been limited by available analytical procedures. We have now developed a method for extraction of PCB sulfates from serum followed by differential analysis with, and without, sulfatase-catalyzed hydrolysis to OH-PCBs. A sulfatase from Helix pomatia was purified by affinity chromatography, and it displayed broad specificity for PCB sulfates without contaminant glucuronidase activity. Following sulfatase-catalyzed hydrolysis of the PCB sulfates extracted from serum, the corresponding OH-PCBs were derivatized to methoxy-PCBs and quantitated by GC-MS/MS. In a pooled sample of human serum, we identified 10 PCB sulfates, with three PCB sulfate congeners exhibiting the highest concentrations from 1200 to 3970 pg/g of serum. In conclusion, we have developed a sensitive and specific method for the determination of PCB sulfates in human serum.

Serum samples from 24 subjects (6 mother-daughter and 6 mother-son dyads) in a rural community (Columbus Junction, Iowa) and 24 subjects (6 mother-daughter and 6 mother-son dyads) in an urban community (East Chicago, Indiana) were analyzed for 74 sulfated metabolites of polychlorinated biphenyls (PCBs). We detected significantly higher mean concentrations of total assessed PCB sulfates in the urban group (110-8900 ng/g fresh weight of serum, mean = 3400 ng/g, standard error = 300) than in the rural cohort (530-6700 ng/g fresh weight of serum, mean = 1800 ng/g, standard error = 500). Eight PCB sulfate congeners (4-PCB 2 sulfate, 4'-PCB 2 sulfate, 2'-PCB 3 sulfate, 4'-PCB 3 sulfate, 4-PCB 11 sulfate, 4'-PCB 18 sulfate, 4'-PCB 25 sulfate, and 4-PCB 52 sulfate) contributed over 90% of the total assessed PCB sulfates in most individuals. The serum samples were enriched in PCB sulfates with fewer than 5 chlorine atoms, and this congener distribution differed from those of PCBs and hydroxylated PCBs in previous studies in the same communities. Regression analysis indicated several significant congener-specific correlations in mother-child dyads, and these relationships differed by location and by mother-daughter or mother-son dyads. This is the first study reporting a broad range of PCB sulfates in populations from urban and rural areas.

Polychlorinated biphenyls (PCBs) are persistent environmental contaminants that continue to be of concern due to their varied toxicities. Upon human exposure, many PCBs with lower numbers of chlorine atoms are metabolized to hydroxylated derivatives (OH-PCBs), and cytosolic sulfotransferases can subsequently catalyze the formation of PCB sulfates. Recent studies have indicated that PCB sulfates bind reversibly with a high affinity to human serum proteins, and that they are also taken up by cells and tissues. Since PCB sulfates might be hydrolyzed to the more toxic OH-PCBs, we have investigated the ability of human hepatic microsomal sulfatase to catalyze this reaction. Twelve congeners of PCB sulfates were substrates for the microsomal sulfatase with catalytic rates exceeding that of dehydroepiandrosterone sulfate as a comparison substrate for steroid sulfatase (STS). These results are consistent with an intracellular mechanism for sulfation and de-sulfation that may contribute to retention and increased time of exposure to OH-PCBs.

Despite increasing evidence for a major role for sulfation in the metabolism of lower-chlorinated polychlorinated biphenyls in vitro and in vivo, and initial evidence for potential bioactivities of the resulting sulfate ester metabolites, the formation of PCB sulfates in PCB exposed human populations had not been explored. The primary goal of this study was to determine if PCB sulfates, and potentially other conjugated PCB derivatives, are relevant classes of PCB metabolites in the serum of humans with known exposures to PCBs. In order to detect and quantify dichlorinated PCB sulfates in serum samples of 46 PCB-exposed individuals from either rural or urban communities, we developed a high-resolution mass spectrometry-based protocol using 4-PCB 11 sulfate as a model compound. The method also allowed the preliminary analysis of these 46 human serum extracts for the presence of other metabolites, such as glucuronic acid conjugates and hydroxylated PCBs. Sulfate ester metabolites derived from dichlorinated PCBs were detectable and quantifiable in more than 20% of analyzed serum samples. Moreover, we were able to utilize this method to detect PCB glucuronides and hydroxylated PCBs, albeit at lower frequencies than PCB sulfates. Altogether, our results provide initial evidence for the presence of PCB sulfates in human serum. Considering the inability of previously employed analytical protocols for PCBs to extract these sulfate ester metabolites and the concentrations of these metabolites observed in our current study, our data support the hypothesis that total serum levels of PCB metabolites in exposed individuals may have been underestimated in the past. Keywords: Polychlorinated biphenyls, PCB, Metabolism, Exposure assessment, Sulfation

Polychlorinated biphenyls (PCBs) pose significant risk to the developing human brain; however, mechanisms of PCB developmental neurotoxicity (DNT) remain controversial. Two widely posited mechanisms are tested here using PCBs identified in pregnant women in the MARBLES cohort who are at increased risk for having a child with a neurodevelopmental disorder (NDD). As determined by gas chromatography-triple quadruple mass spectrometry, the mean PCB levels in maternal serum was 2.22 ng/mL. The 12 most abundant PCBs were tested singly and as a mixture mimicking the congener profile in maternal serum for activity at the thyroid hormone receptor (THR) and ryanodine receptor (RyR). Neither the mixture nor the individual congeners (2 fM to 2 µM) exhibited agonistic or antagonistic activity in a THR reporter cell line. However, as determined by equilibrium binding of [(3)H]ryanodine to RyR1-enriched microsomes, the mixture and the individual congeners (50 nM to 50 μM) increased RyR activity by 2.4-19.2-fold. 4-Hydroxyl (OH) and 4-sulfate metabolites of PCBs 11 and 52 had no TH activity; but 4-OH PCB 52 had higher potency than the parent congener towards RyR. These data support evidence implicating RyRs as targets in environmentally-triggered NDDs and suggest that PCB effects on the THR are not a predominant mechanism driving PCB DNT. These findings provide scientific rationale regarding a point of departure for quantitative risk assessment of PCB DNT, and identify in vitro assays for screening other environmental pollutants for DNT potential.

Although neurotoxicity and hepatotoxicity have long been associated with exposure to polychlorinated biphenyls (PCBs), less is known about the selective toxicity of those hydroxylated PCBs (OH-PCBs) and PCB sulfates that are metabolites derived from exposure to PCBs found in indoor air. We have examined the toxicity of OH-PCBs and PCB sulfates derived from PCBs 3, 8, 11, and 52 in two neural cell lines (N27 and SH-SY5Y) and an hepatic cell line (HepG2). With the exception of a similar toxicity seen for N27 cells exposed to either OH-PCB 52 or PCB 52 sulfate, these OH-PCBs were more toxic to all three cell-types than their corresponding PCB or PCB sulfate congeners. Differences in the distribution of individual OH-PCB and PCB sulfate congeners between the cells and media, and the ability of cells to interconvert PCB sulfates and OH-PCBs, were important components of cellular sensitivity to these toxicants.

Polychlorinated biphenyls (PCBs) are ubiquitous environmental contaminants that are associated with varied adverse health effects. Lower chlorinated PCBs are prevalent in indoor and outdoor air and can be metabolized to their hydroxylated derivatives (OH-PCBs) followed by sulfation to form PCB sulfates. Sulfation is also a means of signal termination for steroid hormones. The human estrogen sulfotransferase (SULT1E1) and alcohol/hydroxysteroid sulfotransferase (SULT2A1) catalyze the formation of steroid sulfates that are inactive at steroid hormone receptors. We investigated the inhibition of SULT1E1 (IC50s ranging from 7.2 nM to greater than 10 μM) and SULT2A1 (IC50s from 1.3 μM to over 100 μM) by five lower-chlorinated OH-PCBs and their corresponding PCB sulfates relevant to airborne PCB-exposure. Several congeners of lower chlorinated OH-PCBs relevant to airborne PCB exposures were potent inhibitors of SULT1E1 and SULT2A1 and thus have the potential to disrupt regulation of intracellular concentrations of the receptor-active steroid substrates for these enzymes.

Small molecules that bind with high affinity to thyroxine (T4) binding sites on transthyretin (TTR) kinetically stabilize the protein's tetrameric structure, thereby efficiently decreasing the rate of tetramer dissociation in TTR related amyloidoses. Current research efforts aim to optimize the amyloid inhibiting properties of known inhibitors, such as derivatives of biphenyls, dibenzofurans and benzooxazoles, by chemical modification. In order to test the hypothesis that sulfate group substituents can improve the efficiencies of such inhibitors, we evaluated the potential of six polychlorinated biphenyl sulfates to inhibit TTR amyloid fibril formation in vitro. In addition, we determined their binding orientations and molecular interactions within the T4 binding site by molecular docking simulations. Utilizing this combined experimental and computational approach, we demonstrated that sulfation significantly improves the amyloid inhibiting properties as compared to both parent and hydroxylated PCBs. Importantly, several PCB sulfates were of equal or higher potency than some of the most effective previously described inhibitors.

Polychlorinated biphenyls (PCBs), a group of 209 congeners that differ in the number and position of chlorines on the biphenyl ring, are anthropogenic chemicals that belong to the persistent organic pollutants (POPs). For many years, PCBs have been a topic of interest because of their biomagnification in the food chain and their environmental persistence. PCBs with fewer chlorine atoms, however, are less persistent and more susceptible to metabolic attack, giving rise to chemicals characterized by the addition of one or more hydroxyl groups to the chlorinated biphenyl skeleton, collectively known as hydroxylated PCBs (OH-PCBs). In animals and plants, this biotransformation of PCBs to OH-PCBs is primarily carried out by cytochrome P-450-dependent monooxygenases. One of the reasons for infrequent detection of lower chlorinated PCBs in serum and other biological matrices is their shorter half-lives, and their metabolic transformation, resulting in OH-PCBs or their conjugates, such as sulfates and glucuronides, or macromolecule adducts. Recent biomonitoring studies have reported the presence of OH-PCBs in human serum. The occurrence of OH-PCBs, the size of this group (there are 837 mono-hydroxyl PCBs alone), and their wide spectra of physical characteristics (pKa’s and log P’s ranging over 5 to 6 orders of magnitude) give rise to a multiplicity of biological effects. Among those are bioactivation to electrophilic metabolites that can form covalent adducts with DNA and other macromolecules, interference with hormonal signaling, inhibition of enzymes that regulate cellular concentrations of active hormones, and interference with the transport of hormones. This new information creates an urgent need for a new perspective on these often overlooked metabolites.

Toxicological studies use “specialty chemicals” and, thus, should assess and report both identity and degree of purity (homogeneity) of the chemicals (or toxicants) under investigation to ensure that other scientists can replicate experimental results. Although detailed reporting criteria for the synthesis and characterization of organic compounds have been established by organic chemistry journals, such criteria are inconsistently applied to the chemicals used in toxicological studies. Biologically active trace impurities may lead to incorrect conclusions about the chemical entity responsible for a biological response, which in turn may confound risk assessment. Based on our experience with the synthesis of PCBs and their metabolites, we herein propose guidelines for the “authentication” of synthetic PCBs and, by extension, other organic toxicants, and provide a checklist for documenting the authentication of toxicants reported in the peer-reviewed literature. The objective is to expand guidelines proposed for different types of biomedical and pre-clinical studies to include a thorough authentication of specialty chemicals, such as PCBs and their derivatives, with the goal of ensuring transparent and open reporting of scientific results in toxicology and the environmental health sciences.

Chlorophenols are an important class of persistent environmental contaminants and have been implicated in a range of adverse health effects, including cancer. They are readily conjugated and excreted as the corresponding glucuronides and sulfates in the urine of humans and other species. Here we report the synthesis and characterization of a series of ten chlorophenol sulfates by sulfation of the corresponding chlorophenols with 2,2,2-trichloroethyl (TCE) chlorosulfate using N,N-dimethylaminopyridine (DMAP) as base. Deprotection of the chlorophenol diesters with zinc powder/ammonium formate yielded the respective chlorophenol sulfate ammonium salts in good yield. The molecular structure of three TCE-protected chlorophenol sulfate diesters and one chlorophenol sulfate monoester were confirmed by X-ray crystal structure analysis. The chlorophenol sulfates were stable for several months if stored at −20 °C and, thus, are useful for future toxicological, environmental and human biomonitoring studies.

Mouse models are powerful tools to study the developmental neurotoxicity of polychlorinated biphenyls (PCBs); however, studies of the oxidation of chiral PCB congeners to potentially neurotoxic hydroxylated metabolites (OH-PCBs) in mice have not been reported. Here we investigate the atropselective oxidation of chiral PCB 91 (2,2',3,4',6-pentachlorobiphenyl), PCB 95 (2,2',3,5',6-pentachlorobiphenyl), PCB 132 (2,2',3,3',4,6'-hexachlorobiphenyl), PCB 136 (2,2',3,3',6,6'-hexachlorobiphenyl) and PCB 149 (2,2',3,4',5',6-hexachlorobiphenyl) to OH-PCBs in liver tissue slices prepared from female mice. The metabolite profile of PCB 136 typically followed the rank order 5-OH-PCB > 4-OH-PCB > 4,5-OH-PCB, and metabolite levels increased with PCB concentration and incubation time. A similar OH-PCB profile was observed with the other PCB congeners, with 5-OH-PCB:4-OH-PCB ratios ranging from 2 to 12. More 5-OH-PCB 136 was formed in liver tissue slices obtained from animals pretreated with phenobarbital (P450 2B inducer) or, to a lesser extent, dexamethasone (P450 2B and 3A enzyme inducer) compared to tissue slices prepared from vehicle-pretreated animals. The apparent rate of 5-OH-PCBs formation followed the approximate rank order PCB 149 > PCB 91 > PCB 132 ~ PCB 136 > PCB 95. Atropselective gas chromatography revealed a congener-specific atropisomeric enrichment of major OH-PCB metabolites. Comparison of our results with published OH-PCB patterns and chiral signatures (i.e., the direction and extent of the atropisomeric enrichment) from rat liver microsomal revealed drastic differences between both species, especially following induction of P450 2B enzymes. These species differences in the metabolism of chiral PCBs should be considered in developmental neurotoxicity studies of PCBs.

Human hydroxysteroid sulfotransferase (hSULT2A1) catalyzes the sulfation of a broad range of environmental chemicals, drugs, and other xenobiotics in addition to endogenous compounds that include hydroxysteroids and bile acids. Polychlorinated biphenyls (PCBs) are persistent environmental contaminants, and oxidized metabolites of PCBs may play significant roles in the etiology of their adverse health effects. Quinones derived from the oxidative metabolism of PCBs (PCB-quinones) react with nucleophilic sites in proteins and also undergo redox cycling to generate reactive oxygen species. This, along with the sensitivity of hSULT2A1 to oxidative modification at cysteine residues, led us to hypothesize that electrophilic PCB-quinones react with hSULT2A1 to alter its catalytic function. Thus, we examined the effects of four phenylbenzoquinones on the ability of hSULT2A1 to catalyze the sulfation of the endogenous substrate, dehydroepiandrosterone (DHEA). The quinones studied were 2'-chlorophenyl-2,5-benzoquinone (2'-Cl-BQ), 4'-chlorophenyl-2,5-benzoquinone (4'-Cl-BQ), 4'-chlorophenyl-3,6-dichloro-2,5-benzoquinone (3,6,4'-triCl-BQ), and phenyl-2,5-benzoquinone (PBQ). At all concentrations examined, pretreatment of hSULT2A1 with the PCB-quinones decreased the catalytic activity of hSULT2A1. Pretreatment with low concentrations of PBQ, however, increased the catalytic activity of the enzyme, while higher concentrations inhibited catalysis. A decrease in substrate inhibition with DHEA was seen following preincubation of hSULT2A1 with all of the quinones. Proteolytic digestion of the enzyme followed by LC/MS analysis indicated PCB-quinone- and PBQ-adducts at Cys55 and Cys199, as well as oxidation products at methionines in the protein. Equilibrium binding experiments and molecular modeling suggested that changes due to these modifications may affect the nucleotide binding site and the entrance to the sulfuryl acceptor binding site of hSULT2A1.

Background: The displacement of l-thyroxine (T4) from binding sites on transthyretin (TTR) is considered a significant contributing mechanism in polychlorinated biphenyl (PCB)-induced thyroid disruption. Previous research has discovered hydroxylated PCB metabolites (OH-PCBs) as high-affinity ligands for TTR, but the binding potential of conjugated PCB metabolites such as PCB sulfates has not been explored. Objectives: We evaluated the binding of five lower-chlorinated PCB sulfates to human TTR and compared their binding characteristics to those determined for their OH-PCB precursors and for T4. Methods: We used fluorescence probe displacement studies and molecular docking simulations to characterize the binding of PCB sulfates to TTR. The stability of PCB sulfates and the reversibility of these interactions were characterized by HPLC analysis of PCB sulfates after their binding to TTR. The ability of OH-PCBs to serve as substrates for human cytosolic sulfotransferase 1A1 (hSULT1A1) was assessed by OH-PCB–dependent formation of adenosine-3´,5´-diphosphate, an end product of the sulfation reaction. Results: All five PCB sulfates were able to bind to the high-affinity binding site of TTR with equilibrium dissociation constants (Kd values) in the low nanomolar range (4.8–16.8 nM), similar to that observed for T4 (4.7 nM). Docking simulations provided corroborating evidence for these binding interactions and indicated multiple high-affinity modes of binding. All OH-PCB precursors for these sulfates were found to be substrates for hSULT1A1. Conclusions: Our findings show that PCB sulfates are high-affinity ligands for human TTR and therefore indicate, for the first time, a potential relevance for these metabolites in PCB-induced thyroid disruption.

The human cytosolic sulfotransferase hSULT2A1 catalyzes the sulfation of a broad range of xenobiotics, as well as endogenous hydroxysteroids and bile acids. Reversible modulation of the catalytic activity of this enzyme could play important roles in its physiologic functions. Whereas other mammalian sulfotransferases are known to be reversibly altered by changes in their redox environment, this has not been previously shown for hSULT2A1. We have examined the hypothesis that the formation of disulfide bonds in hSULT2A1 can reversibly regulate the catalytic function of the enzyme. Three thiol oxidants were used as model compounds to investigate their effects on homogeneous preparations of hSULT2A1: glutathione disulfide, 5,5′-dithiobis(2-nitrobenzoic acid), and 1,1’-azobis(N,N-dimethylformamide) (diamide). Examination of the effects of disulfide bond formation with these agents indicated that the activity of the enzyme is reversibly altered. Studies on the kinetics of the hSULT2A1-catalyzed sulfation of dehydroepiandrosterone (DHEA) showed the effects of disulfide bond formation on the substrate inhibition characteristics of the enzyme. The effects of these agents on the binding of substrates and products, liquid chromatography-mass spectrometry identification of the disulfides formed, and structural modeling of the modified enzyme were examined. Our results indicate that conformational changes at cysteines near the nucleotide binding site affect the binding of both the nucleotide and DHEA to the enzyme, with the specific effects dependent on the structure of the resulting disulfide. Thus, the formation of disulfide bonds in hSULT2A1 is a potentially important reversible mechanism for alterations in the rates of sulfation of both endogenous and xenobiotic substrates.

The cytosolic sulfotransferase hSULT2A1 is the major hydroxysteroid (alcohol) sulfotransferase in human liver, and it catalyzes the 3′-phosphoadenosine-5′-phosphosulfate (PAPS)-dependent sulfation of various endogenous hydroxysteroids as well as many xenobiotics that contain alcohol and phenol functional groups. The hSULT2A1 often displays substrate inhibition, and we have hypothesized that a key element in this response to increasing substrate concentration is the formation of non-productive ternary dead-end enzyme complexes involving the nucleotide product, adenosine 3′,5′-diphosphate (PAP). One of these substrates for hSULT2A1 is dehydroepiandrosterone (DHEA), a major circulating steroid hormone in humans that serves as precursor to both androgens and estrogens. We have utilized DHEA in both initial velocity studies and equilibrium binding experiments in order to evaluate the potential role of ternary complexes in substrate inhibition of the enzyme. Our results indicate that hSULT2A1 forms non-productive ternary complexes that involve either DHEA or dehydroepiandrosterone sulfate, and the formation of these ternary complexes displays negative cooperativity in the binding of DHEA.

Hydroxylated metabolites of polychlorinated biphenyls (OHPCBs) interact with rat sulfotransferase 1A1 (rSULT1A1) as substrates and inhibitors. Previous studies have shown that there are complex and incompletely understood structure–activity relationships governing the interaction of rSULT1A1 with these molecules. Furthermore, modification of the enzyme with glutathione disulfide (GSSG) results in the conversion of some OHPCBs from inhibitors to substrates. We have now examined estimated values for the acid-dissociation constant (Ka) and the octanol–water distribution coefficient (D), as well as experimentally determined dissociation constants for enzyme complexes, to assist in the prediction of interactions of OHPCBs with rSULT1A1. Under reducing conditions, initial velocities for rSULT1A1-catalyzed sulfation exhibited a positive correlation with pKa and a negative correlation with log D of the OHPCBs. IC50 values of inhibitory OHPCBs decreased with decreasing pKa values for both the glutathione (GSH)-pretreated and GSSG-pretreated forms of rSULT1A1. Comparison of GSH- and GSSG-pretreated forms of rSULT1A1 with respect to binding of OHPCB in the presence and absence of adenosine 3′,5′-diphosphate (PAP) revealed that the dissociation constants with the two redox states of the enzyme were similar for each OHPCB. Thus, pKa and log D values are useful in predicting the binding of OHPCBs to the two redox forms of rSULT1A1 as well as the rates of sulfation of those OHPCBs that are substrates. However, the differences in substrate specificity for OHPCBs that are seen with changes in redox status of the enzyme are not directly related to specific structural effects of individual OHPCBs within inhibitory enzyme–PAP–OHPCB complexes.

Polychlorinated biphenyls (PCBs), a major class of persistent organic pollutants, are metabolized to hydroxylated PCBs. Several hydroxylated PCBs are substrates of cytosolic phase II enzymes, such as phenol and hydroxysteroid (alcohol) sulfotransferases; however, the corresponding sulfation products have not been isolated and characterized. Here we describe a straightforward synthesis of a series of ten PCB sulfate monoesters from the corresponding hydroxylated PCBs. The hydroxylated PCBs were synthesized by coupling chlorinated benzene boronic acids with appropriate brominated (chloro-)anisoles, followed by demethylation with boron tribromide. The hydroxylated PCBs were sulfated with 2,2,2-trichloroethyl chlorosulfate using DMAP as base. Deprotection with zinc powder/ammonium formate yielded the ammonium salts of the desired PCB sulfate monoesters in good yields when the sulfated phenyl ring contained no or one chlorine substituent. However, no PCB sulfate monoesters were isolated when two chlorines were present ortho to the sulfated hydroxyl group. To aid with future quantitative structure activity relationship studies, the structures of two 2,2,2-trichloroethyl-protected PCB sulfates were verified by X-ray diffraction. Keywords: Biaryls, Polychlorinated biphenyls (PCBs), Sulfates, 2,2,2-Trichloroethyl (TCE) group, Metabolites

Hydroxylated metabolites of polychlorinated biphenyls (OH-PCBs) are inhibitors and substrates for various human sulfotransferases (SULTs). Although the rat is often used in toxicological studies on PCBs, the interactions of OH-PCBs with rat SULTs are less well understood. In the present study, 15 OH-PCBs were investigated as potential substrates or inhibitors of purified recombinant rSULT1A1 and rSULT2A3, the major family 1 and family 2 SULTs present in rat liver, respectively. None of these OH-PCBs were substrates for rSULT2A3, 11 weakly inhibited rSULT2A3-catalyzed sulfation of dehydroepiandrosterone, and 4 had no effect on the reaction. With rSULT1A1, 4-OH-PCB 8, 4′-OH-PCB 3, 9, 12, 35, and 6′-OH-PCB 35 were substrates, whereas 4′-OH-PCB 6, 4-OH-PCB 14, 4′-OH-PCB 25, 4′-OH-PCB 33, 4-OH-PCB 34, 4-OH-PCB36, 4′-OH-PCB 36, 4′-OH-PCB 68, and 4-OH-PCB 78 inhibited the sulfation of 2-naphthol catalyzed by this enzyme. OH-PCBs with a 3,5-dichloro-4-hydroxy substitution were the most potent inhibitors of rSULT1A1, and the placement of chlorine atoms in the ortho- and meta-positions on either ring of para-OH-PCBs resulted in significant differences in activity as substrates and inhibitors. The specificity of rSULT1A1 for several inhibitory OH-PCBs was altered by pretreatment of the enzyme with oxidized glutathione (GSSG). Four OH-PCBs that were inhibitors of rSULT1A1 under reducing conditions became substrates after pretreatment of the enzyme with GSSG. This alteration in specificity of rSULT1A1 for certain OH-PCBs suggests that conditions of oxidative stress may significantly alter the sulfation of some OH-PCBs in the rat.

Pentachlorophenol (PCP) is a persistent chemical contaminant which has been extensively investigated in terms of its toxicology and metabolism. Similar to PCP, other chlorinated phenol derivatives are also widely present in the environment from various sources. Even though some of the chlorine-substituted phenols, and particularly PCP, are well known inhibitors of phenol sulfotransferases (SULTs), these compounds have been shown to undergo sulfation in humans. In order to investigate the enzymatic basis for sulfation of PCP in humans, we have studied the potential for PCP as well as the mono-, di-, tri-, and tetra-chlorinated phenols to serve as substrates for human hydroxysteroid sulfotransferase, hSULT2A1. Our results show that all of these compounds are substrates for this isoform of sulfotransferase and the highest rates of sulfation are obtained with PCP, trichlorophenols, and tetrachlorophenols. Much lower rates of sulfation were obtained with isomers of monochlorophenol and dichlorophenol as substrates for hSULT2A1. Thus, the sulfation of polychlorinated phenols catalyzed by hSULT2A1 may be a significant component of their metabolism in humans.

Determining the relationships between the structures of substrates and inhibitors and their interactions with drug-metabolizing enzymes is of prime importance in predicting the toxic potential of new and legacy xenobiotics. Traditionally, quantitative structure activity relationship (QSAR) studies are performed with many distinct compounds. Based on the chemical properties of the tested compounds, complex relationships can be established so that models can be developed to predict toxicity of novel compounds. In this study, the use of fluorinated analogues as supplemental QSAR compounds was investigated. Substituting fluorine induces changes in electronic and steric properties of the substrate without substantially changing the chemical backbone of the substrate. In vitro assays were performed using purified human cytosolic sulfotransferase hSULT2A1 as a model enzyme. A mono-hydroxylated polychlorinated biphenyl (4-OH PCB 14) and its four possible mono-fluoro analogues were used as test compounds. Remarkable similarities were found between this approach and previously published QSAR studies for hSULT2A1. Both studies implicate the importance of dipole moment and dihedral angle as being important to PCB structure in respect to being substrates for hSULT2A1. We conclude that mono-fluorinated analogues of a target substrate can be a useful tool to study the structure activity relationships for enzyme specificity.

Polychlorinated biphenyls (PCBs) with lower numbers of chlorine atoms exhibit a greater susceptibility to metabolism than their higher-chlorinated counterparts. Following initial hydroxylation of these lower chlorinated PCBs, metabolic sulfation to form PCB sulfates is increasingly recognized as an important component of their toxicology. Since procedures for the quantitative analysis of PCB sulfates in tissue samples have not been previously available, we have now developed an efficient, LC-ESI-MS/MS based, protocol for the quantitative analysis of 4-PCB 11 sulfate in biological samples. This procedure was used to determine the distribution of 4-PCB 11 sulfate in liver, kidney, lung, and brain, as well as its excretion profile, following its intravenous administration to male Sprague-Dawley rats. Following initial uptake of 4-PCB 11 sulfate, its concentration in these tissues and serum declined within the first hour following injection. Although biliary secretion was detected, analysis of 24 hour collections of urine and feces revealed recovery of less than 4% of the administered 4-PCB 11 sulfate. High-resolution LC-MS analysis of bile, urine, and feces showed metabolic products derived from 4-PCB 11 sulfate. Thus, 4-PCB 11 sulfate at this dose was not directly excreted in the urine, but was, instead, re-distributed to tissues and/or subjected to further metabolism.

Tamoxifen is successfully used for both treatment and prevention of estrogen-dependent breast cancer, yet side effects and development of resistance remain problematic. Endoxifen is a major active metabolite of tamoxifen that is being investigated for clinical use. We hypothesized that endoxifen and perhaps other major metabolites of tamoxifen may affect the ability of human estrogen sulfotransferase 1E1 (hSULT1E1) and human phenol sulfotransferase 1A1 isoform 1 (hSULT1A1*1) to catalyze the sulfation of estradiol, an important mechanism in termination of estrogen signaling through loss of activity at estrogen receptors. Our results indicated that endoxifen, N-desmethyltamoxifen (N-desTAM), 4-hydroxytamoxifen (4-OHTAM), and tamoxifen-N-oxide were weak inhibitors of hSULT1E1 with Ki values ranging from 10 μM to 38 μM (i.e., over 1000 times higher than the 8.1 nM Km value for estradiol as substrate for the enzyme). In contrast to the results with hSULT1E1, endoxifen and 4-OHTAM were significant inhibitors of the sulfation of 2.0 µM estradiol catalyzed by hSULT1A1*1, with IC50 values (9.9 μM and 1.6 μM, respectively) that were similar to the Km value (1.5 μM) for estradiol as substrate for this enzyme. Additional investigation of the interaction of these metabolites with the two sulfotransferases revealed that endoxifen, 4-OHTAM, and N-desTAM were substrates for hSULT1E1 and hSULT1A1*1, although the relative catalytic efficiencies varied with both the substrate and the enzyme. These results may assist in future elucidation of cell- and tissue-specific effects of tamoxifen and its metabolites.

The metabolism of polychlorinated biphenyls (PCBs) is complex and has an impact on toxicity, and thereby on the assessment of PCB risks. A large number of reactive and stable metabolites are formed in the processes of biotransformation in biota in general, and in humans in particular. The aim of this document is to provide an overview of PCB metabolism, and to identify the metabolites of concern and their occurrence. Emphasis is given to mammalian metabolism of PCBs and their hydroxyl, methylsulfonyl, and sulfated metabolites, especially those that persist in human blood. Potential intracellular targets and health risks are also discussed.

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 family-1) 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-2,5-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). 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.

Tamoxifen (TAM) is a nonsteroidal antiestrogenic drug that is widely used for the treatment of estrogen receptor-dependent breast cancer. An increased risk of endometrial cancer in some patients treated with TAM has been linked to the metabolic formation of alpha-hydroxytamoxifen (alpha-OHTAM) and its subsequent sulfation. Alpha-OHTAM has been found to be a substrate for rat and human hydroxysteroid sulfotransferases (STa and SULT2A1, respectively). Since stereochemistry plays an important role in the interactions of hydroxysteroid sulfotransferases with their substrates, we have now investigated the interactions of each of the stereoisomers of alpha-OHTAM with highly purified recombinant STa and SULT2A1. Methods for the preparation of the enantiomers of E- and Z-alpha-OHTAM were developed. When each of the four enantiomers was examined with rat STa, E-(+)-alpha-OHTAM was the only substrate for the enzyme, whereas E-(-)-alpha-OHTAM, Z-(+)-alpha-OHTAM, and Z-(-)-alpha-OHTAM were inhibitors of the sulfation of E-(+)-alpha-OHTAM catalyzed by STa. The dissociation constants for the alpha-OHTAM enantiomers indicated that they bound to STa with similar affinity, but only the E-(+)-enantiomer was a substrate. In contrast to the results obtained with rat hydroxysteroid sulfotransferase STa, all enantiomers of alpha-OHTAM were substrates for the human SULT2A1. Moreover, kcat/Km values with SULT2A1 were higher with the Z enantiomers than with the E enantiomers. As a result of the potential for interconversion of the E and Z geometric isomers upon metabolism, the sulfation of the Z isomers may be of greater concern in human tissues than has been previously assumed.

Hydroxysteroid (alcohol) sulfotransferases catalyze the sulfation of several endogenous steroids and many hydrophobic xenobiotic alcohols. The substrate stereoselectivities of sulfotransferases may be critically important in determining their overall roles in metabolism of drugs, carcinogens, and other xenobiotics. In the present work, stereoselectivity of the human hydroxysteroid sulfotransferase ST2A3 (also variously named as SULT2A1 or human DHEA-ST) was examined through analysis of its catalytic activities with the enantiomers of 1-naphthyl-1-ethanol and 2-naphthyl-1-ethanol. The kcat/Km value for sulfation of the R-(+)-enantiomer of 1-naphthyl-1-ethanol catalyzed by ST2A3 was 3.3 min-1mM-1, whereas the S-(-)-enantiomer was not a substrate for the enzyme. S-(-)-1-naphthyl-1-ethanol did however interact with ST2A3 as an inhibitor of the sulfation of dehydroepiandrosterone. This substrate stereospecificity was not present with the enantiomers of 2-naphthyl-1-ethanol, since both were substrates for the enzyme. Such differences between the sulfation of 1- and 2-naphthyl-1-ethanol are consistent with the importance of steric interactions between the ethanol group and a hydrogen atom at the peri-position (C8) on the naphthyl ring in 1-naphthyl-1-ethanol that combine with the topology of the enzyme9s active site to determine stereospecificity.

Although tamoxifen is a successful agent for treatment and prevention of estrogen-dependent breast cancer, its use has been limited by the low incidence of endometrial cancer. Human hydroxysteroid sulfotransferase 2A1 (hSULT2A1) catalyzes the formation of an α-sulfooxy metabolite of tamoxifen that is reactive toward DNA, and this has been implicated in its carcinogenicity. Also, hSULT2A1 functions in the metabolism of steroid hormones such as dehydroepiandrosterone (DHEA) and pregnenolone (PREG). These roles of hSULT2A1 in steroid hormone metabolism and in generating a reactive metabolite of tamoxifen led us to examine its interactions with tamoxifen and several of its major metabolites. We hypothesized that metabolites of tamoxifen may regulate the catalytic activity of hSULT2A1, either through direct inhibition or through serving as alternate substrates for the enzyme. We found that 4-hydroxy-N-desmethyltamoxifen (endoxifen) is a potent inhibitor of hSULT2A1-catalyzed sulfation of PREG and DHEA, with Ki values of 3.5 and 2.8 μM, respectively. In the hSULT2A1-catalyzed sulfation of PREG, 4-hydroxytamoxifen (4-OHTAM) and N-desmethyltamoxifen (N-desTAM) exhibited Ki values of 12.7 and 9.8 μM, respectively, whereas corresponding Ki values of 19.4 and 17.2 μM were observed with DHEA as substrate. A Ki value of 9.1 μM was observed for tamoxifen-N-oxide with DHEA as substrate, and this increased to 16.9 μM for the hSULT2A1-catalyzed sulfation of PREG. Three metabolites were substrates for hSULT2A1, with relative sulfation rates of 4-OHTAM > N-desTAM > > endoxifen. These results may be useful in interpreting ongoing clinical trials of endoxifen and in improving the design of related molecules.

Polychlorinated biphenyls (PCBs) are persistent environmental contaminants, and exposure to PCBs and their hydroxylated metabolites (OHPCBs) has been associated with various adverse health effects. The mammalian cytosolic sulfotransferases (SULTs) catalyze the sulfation of OHPCBs, and the interaction of OHPCBs with both the SULT1 and SULT2 families of these enzymes has received attention both with respect to metabolic disposition of these molecules and the potential mechanisms for their roles in endocrine disruption. We have previously shown that OHPCBs interact with human hydroxysteroid sulfotransferase hSULT2A1, an enzyme that catalyzes the sulfation of dehydroepiandrosterone (DHEA), other alcohol-containing steroids, bile acids, and many xenobiotics. The objective of our current studies is to investigate the mechanism of inhibition of hSULT2A1 by OHPCBs by combining inhibition kinetics with determination of equilibrium binding constants and molecular modeling of potential interactions. Examination of the effects of fifteen OHPCBs on the sulfation of DHEA catalyzed by hSULT2A1 showed predominantly noncompetitive inhibition patterns. This was observed for OHPCBs that were substrates for sulfation reactions catalyzed by the enzyme as well as those that solely inhibited the sulfation of DHEA. Equilibrium binding experiments and molecular modeling studies indicated that the OHPCBs bind at the binding site for DHEA on the enzyme, and that the observed noncompetitive patterns of inhibition are consistent with binding in more than one orientation to more than one enzyme complex. These results have implications for the roles of SULTs in the toxicology of OHPCBs, while also providing molecular probes of the complexity of substrate/inhibitor interactions with hSULT2A1.

Hydroxysteroid (alcohol) sulfotransferase catalyzes numerous reactions that are important to our understanding of the metabolism of both endogenous steroids and exogenous alcohols. Here we report a method for prokaryotic expression and rapid purification of the recombinant hydroxysteroid sulfotransferase STa, a major isoform of hydroxysteroid sulfotransferase in the rat. The cDNA encoding STa was cloned into a pET-3c vector and expressed in Escherichia coli BL21 cells. After disruption of the cells by sonication, the enzyme was purified in one step by affinity chromatography on adenosine 3',5'-diphosphate-agarose. The purified recombinant STa had a relative molecular mass on SDS-PAGE that was identical with the native hepatic STa in rat liver. The expressed enzyme displayed similar substrate inhibition characteristics with dehydroepiandrosterone as have been noted previously with the native enzyme purified from rat liver. Furthermore, the catalytic efficiency in sulfation of 7-hydroxymethyl-12-methylbenz[a]anthracene, as well as the stereoselectivity of sulfation of the enantiomers of 1-phenyl-1-heptanol and 1-naphthyl-1-ethanol, catalyzed by the recombinant STa were consistent with characteristics of the STa isolated from rat liver.

The sulfotransferases that are active in the metabolism of xenobiotics represent a large family of enzymes that catalyze the transfer of the sulfuryl group from 3'-phosphoadenosine 5'-phosphosulfate to phenols, to primary and secondary alcohols, to several additional oxygen-containing functional groups, and to amines. Restriction of this review to the catalytic processes of phenol or aryl sulfotransferases does not really narrow the field, because these enzymes have overlapping specificity, not only for specific compounds, but also for multiple functional groups. The presentation aims to provide an overview of the wealth of phenol sulfotransferases that are available for study but concentrates on the enzymology of rat and human enzymes, particularly on the predominant phenol sulfotransferase from rat liver. The kinetics and catalytic mechanism of the rat enzyme is extensively reviewed and is compared with observations from other sulfotransferases.

Esters of ethacrynic acid and partial structural analogs were synthesized and evaluated for topical antiglaucoma activity in rabbits. Maximum activity was shown by analogs 2 and 6 (34% and 30% reduction in intraocular pressure recovery rate, respectively). Among the esters, only the ethyl ester (2) was found to be active; the methyl and n-propyl esters (1 and 3) were inactive. Analogs 1-3 were subjected to an estimation of physicochemical properties and chemical stability. However, no correlation was found to exist between the biological activity/inactivity and the physicochemical properties of the analogs. The analogs were evaluated for ex vivo hydrolysis using rabbit aqueous humor (AH), corneal (C) homogenate and iris-ciliary body (ICB) homogenate. For all tissues, the rate of enzymatic hydrolysis increased significantly with an increasing ester chain length. The ICB-mediated hydrolysis was the fastest among the three tissues for all of the analogs. The relationship between the rate constants for the tissue-mediated hydrolyses were: analog 1, ICB>C>AH; analog 2, ICB>C=AH and analog 3, ICB>AH>C. Apparent Michaelis-Menten kinetic parameters were determined for the three analogs using corneal homogenate. Analog 2 showed the highest v0 for all substrate concentrations studied. The conventional Michaelis-Menten equation did not fit the data as well as a sigmoidal model. Both fits of the data showed the fastest enzyme-mediated hydrolysis for analog 2. The parameters of the sigmoidal fit of the data correlated with the activity/inactivity of the analogs. The data indicate that the major factors responsible for the observed activity/inactivity are the differences in the corneal enzymatic hydrolysis of the esters in conjunction with the rapid dynamics of ocular prodrug absorption.

Hydroxysteroid (alcohol) sulfotransferases catalyze the sulfation of polycyclic aromatic hydrocarbons (PAHs) that contain benzylic hydroxyl functional groups. This metabolic reaction is often a critical step in the activation of a hydroxyalkyl-substituted PAH to form an electrophilic metabolite that is capable of forming covalent bonds at nucleophilic sites on DNA, RNA, and proteins. Since hydroxyalkyl-substituted PAHs are often metabolically formed by the stereoselective enzymatic hydroxylation of a benzylic position on an alkyl-substituted PAH, we have investigated the possibility that the sulfation of hydroxyalkyl aromatic hydrocarbons is also stereoselective. Homogeneous preparations of rat hepatic hydroxysteroid (alcohol) sulfotransferase STa were utilized to investigate the stereoselectivity of its catalytic function with the enantiomers of model 1-arylethanols. While only minimal stereoselectivity was observed for the catalytic efficiency of STa with the enantiomers of 1-(2-naphthyl)ethanol and 1-acenaphthenol, the enzyme was stereospecific for (R)-(+)-1-(1-naphthyl)ethanol, (R)-(+)-1-(1-pyrenyl)ethanol, and (R)-(+)-1-(9-phenanthryl)ethanol as substrates. Moreover, (S)-(-)-1-(1-naphthyl)ethanol, (S)-(-)-1-(1-pyrenyl)ethanol, and (S)-(-)- 1-(9-phenanthryl)ethanol were competitive inhibitors of STa. Structural and conformational analyses of these 1-arylethanols indicated that steric interactions between the substituents on the benzylic carbon and the hydrogen in the peri-position on the aromatic ring system were important determinants of the stereospecificity of the enzyme with these molecules. The findings presented here have implications for the more accurate prediction of the ability of hydroxyalkyl-substituted PAHs to be activated via metabolic formation of electrophilic sulfuric acid esters.

Active site-directed affinity labeling was utilized to elucidate peptide sequences at the binding site for sulfuryl acceptors in rat hepatic aryl sulfotransferase (AST) IV (also known as tyrosine-ester sulfotransferase, EC 2.8.2.9). The affinity labeling reagent, N-bromoacetyl-4-hydroxyphenylamine, was designed on the basis of substrate specificity studies with para-substituted phenols, utilization of a bromoacetamido group for reactivity with active site amino acid residues and its similarity to acetaminophen, a known substrate for aryl (phenol) sulfotransferases. AST IV utilized N-bromoacetyl-4-hydroxyphenylamine as a substrate with kinetic constants that compared favorably to those obtained with acetaminophen. Incubation of AST IV with N-bromoacetyl-4-hydroxyphenylamine at pH 7.0 in the absence of PAPS and other substrates resulted in an irreversible inactivation of the enzyme that was both time- and concentration-dependent. [14C]-N-bromoacetyl-4-hydroxyphenylamine was synthesized and used to analyze the regions of protein sequence that were involved in the binding of the affinity label. AST IV was incubated with [14C]-N-bromoacetyl-4-hydroxyphenylamine, hydrolyzed with endoproteinase Lys-C and the labeled peptides were purified by HPLC. Control incubations of AST IV with the affinity label in the presence of 4-propylphenol and PAP were utilized to ascertain the specificity of the interaction. Sequence analysis of the labeled peptides, carried out by automated Edman degradation, revealed labeling sites on cysteine (Cys-232, Cys-283 and Cys-289) and lysine (Lys-286) residues near the C-terminus of the protein. The locations of these labeling sites were further evaluated both by sequence-alignment with other sulfotransferases and by theoretical calculations on predicted secondary structure.

1. Chiral polychlorinated biphenyls (PCBs) such as PCB 136 enantioselectively sensitize the ryanodine receptor (RyR). In light of recent evidence that PCBs cause developmental neurotoxicity via RyR-dependent mechanisms, this suggests that enantioselective PCB metabolism may influence the developmental neurotoxicity of chiral PCBs. However, enantioselective disposition of PCBs has not been fully characterized. 2. The effect of sex and cytochrome P450 (P450) enzyme induction on the enantioselective metabolism of PCB 136 was studied using liver tissue slices prepared from naïve control (CTL), phenobarbital (PB; CYP2B inducer) or dexamethasone (DEX; CYP3A inducer) pretreated adult Sprague-Dawley rats. PCB 136 metabolism was also examined in hippocampal slices derived from untreated rat pups. 3. In liver tissue slices, hydroxylated PCB (OH-PCB) profiles depended on sex and inducer pretreatment, and OH-PCB levels followed the rank orders male > female and PB > DEX > CTL. In contrast, the enantiomeric enrichment of PCB 136 and its metabolites was independent of sex and inducer pretreatment. Only small amounts of PCB 136 partitioned into hippocampal tissue slices and no OH-PCB metabolites were detected. 4. Our results suggest that enantioselective metabolism, sex and induction status of P450 enzymes in the liver may modulate the neurotoxic outcomes of developmental exposure to chiral PCBs.

The title compound, C14H10Cl4O4S, is an inter­mediate in the synthesis of the PCB sulfate monoester of 4′-chloro-biphenyl-4-ol. Both the sulfate monoester and 4′-chloro-biphenyl-4-ol are metabolites of PCB 3 (4-chloro­biphen­yl). There are two mol­ecules with different conformations in the asymmetric unit. The solid state dihedral angles between the benzene rings are 18.52 (10) and 41.84 (16)° in the two mol­ecules, whereas the dihedral angles between the least-squares plane of the sulfated benzene ring and O—S (Ar—C—O—S) are 66.2 (3) and 89.3 (3)°. The crystal was an inversion twin with a refined component fraction of 0.44 (7).

Sulfotransferase a (STa) is an isoform of hydroxysteroid (alcohol) sulfotransferase that catalyzes the formation of sulfuric acid esters from both endogenous and xenobiotic alcohols. Among its various functions in toxicology, STa is the major form of hepatic sulfotransferase in the rat that catalyzes the formation of genotoxic and carcinogenic sulfuric acid esters from hydroxymethyl polycyclic aromatic hydrocarbons. The goal of the present study was to elucidate fundamental quantitative relationships between substrate structure and catalytic activity of STa that would be applicable to these and other xenobiotics. We have modified previous procedures for purification of STa in order to obtain sufficient amounts of homogeneous enzyme for determination of k(cat)/K-m values, a quantitative measure of catalytic efficiency. We determined the catalytic efficiency of STa with benzyl alcohol and eight benzylic alcohols that were substituted with n-alkyl groups (CnH2n+1, where n = 1-8) in the para position, and the optimum value for k(cat)/K-m in these reactions was obtained with n-pentylbenzyl alcohol. Correlations between logarithms of k(cat)/K-m values and logarithms of partition coefficients revealed that hydrophobicity of the substrate was a major factor contributing to the catalytic efficiency of STa. Primary n-alkanols (CnH2n+1OH, where n = 3-16) exhibited an optimum k(cat)/K-m for C-9-C-11 and a linear decrease in upsilon(max) of the reaction for C-3-C-14; 15- and 16-carbon n-alkanols were not substrates for STa. These results indicated limits to the length of the extended carbon chain in substrates. Such limits may also apply to hydroxysteroids, since cholesterol was inactive as either substrate or inhibitor of STa. Furthermore, the importance of steric effects on the catalytic efficiency of STa was also evident with a series of linear, branched, and cyclic seven-carbon aliphatic alcohols. In conclusion, our results provide fundamental quantitative relationships between substrate structure and catalytic efficiency that yield insight into the specificity of STa for both endogenous and xenobiotic alcohols.

Polychlorinated biphenyls (PCBs) are legacy pollutants that exert toxicities through various mechanisms. In the recent years exposure to PCBs via inhalation has been recognized as a hazard. Those PCBs with lower numbers of chlorine atoms (LC-PCBs) are semi-volatile, and have been reported in the urban air, as well as in the indoor air of older buildings. LC-PCBs are bioactivated to phenols and further to quinone electrophiles with genotoxic/carcinogenic potential. We hypothesized that phenolic LC-PCBs are subject to conjugation and excretion in the urine. PCB3, often present in high concentrations in air, is a prototypical congener for the study of the metabolism and toxicity of LC-PCBs. Our objective was to identify metabolites of PCB3 in urine that could be potentially employed in the estimation of exposure to LC-PCBs. Male Sprague Dawley rats (150–175 g) were housed in metabolism cages and received a single intraperitoneal injection of 600 µmol/kg body weight of PCB3. Urine was collected every four hours; rats were euthanized at 36 h and serum was collected. LC-MS analysis of urine before and after incubation with β-glucuronidase and sulfatase showed that sulfate conjugates were in higher concentrations than glucuronide conjugates and free phenolic forms. At least two major metabolites, and two minor metabolites were identified in urine that could be attributed to mercapturic acid metabolites of PCB3. Quantitation by authentic standards confirmed that approximately 3% of the dose was excreted in the urine as sulfates over 36 hours; with peak excretion occurring at 10–20 h after exposure. The major metabolites were 4’PCB3 sulfate, 3’PCB3 sulfate, 2’PCB3 sulfate, and presumably a catechol sulfate. The serum concentration of 4’PCB3 sulfate was 6.18±2.16 µg/mL. This is the first report that sulfated metabolites of PCBs are formed in vivo. These findings suggest a prospective approach for exposure assessment of LC- PCBs by analysis of phase II metabolites in urine.

Developmental exposure to multiple-ortho substituted polychlorinated biphenyls (PCBs) causes adverse neurodevelopmental outcomes in laboratory animals and humans by mechanisms involving the sensitization of Ryanodine receptors (RyRs). In the case of PCB 136, the sensitization of RyR is enantiospecific, with only (-)-PCB 136 being active. However, the role of enantioselective metabolism in the developmental neurotoxicity of PCB 136 is poorly understood. The present study employed hepatic microsomes from phenobarbital (PB-), dexamethasone (DEX-) and corn oil (VEH-)treated male Sprague-Dawley rats to investigate the hypothesis that PCB 136 atropisomers are enantioselectively metabolized by P450 enzymes to potentially neurotoxic, hydroxylated PCB 136 metabolites. The results demonstrated the time- and isoform-dependent formation of three metabolites, with 5-OH-PCB 136 (2,2',3,3',6,6'-hexachlorobiphenyl-5-ol) being the major metabolite. The formation of 5-OH-PCB 136 increased with the activity of P450 2B enzymes in the microsomal preparation, which is consistent with PCB 136 metabolism by rat P450 2B1. The minor metabolite 4-OH-PCB 136 (2,2',3,3',6,6'-hexachlorobiphenyl-4-ol) was produced by a currently unidentified P450 enzymes. An enantiomeric enrichment of (-)-PCB 136 was observed in microsomal incubations due to the preferential metabolism of (+)-PCB 136 to the corresponding 5-OH-PCB 136 (2,2',3,3',6,6'-hexachlorobiphenyl-5-ol) atropisomer. 4-OH-PCB 136 displayed an enrichment of the atropisomer formed from (-)-PCB 136; however, the enrichment of this metabolite atropisomer didn't affect the enantiomeric enrichment of the parent PCB because 4-OH-PCB 136 is only a minor metabolite. Although the formation of 5- and 4-OH-PCB 136 atropisomers increased with time, the enantioselective formation of the OH-PCB metabolites resulted in constant enantiomeric enrichment, especially at later incubation times. These observations not only demonstrate that the chiral signatures of PCBs and their metabolites in wildlife and humans are due to metabolism by P450 enzymes, but also suggest that the enantioselective formation of neurotoxic PCB 136 metabolites, such as 4-OH-PCB 136, may play a role in the developmental neurotoxicity of PCBs.

Chiral PCB congeners are major components of PCB mixtures and undergo enantioselective biotransformation to hydroxylated (OH-)PCBs by cytochrome P450 enzymes. While it is known that biotransformation results in an enantiomeric enrichment of the parent PCB, it is currently unknown if OH-PCBs are formed enantioselectively. The present study screened seven commercial capillary gas chromatography columns containing modified β- or γ-cyclodextrins for their potential to separate the atropisomers of methylated derivatives of OH-PCB. The atropisomers of 3-, 4- and 5-methoxy derivatives were at least partially separated on one or more columns. A subsequent biotransformation study was performed with rat liver microsomes to assess if hydroxylated metabolites are formed enantioselectively from PCBs 91, 95, 132, and 149. The OH-PCBs were extracted from the microsomal incubations, derivatized with diazomethane and analyzed as the respective methoxylated (MeO-)PCB derivatives using selected columns. The 5-hydroxylated metabolites of PCBs 91, 95, 132, and 149 were the major metabolites, which is consistent with PCB's biotransformation by cytochrome P450 2B enzymes. All 5-hydroxylated metabolites displayed a clear, congener-specific enantiomeric enrichment. Overall, this study demonstrates for the first time that chiral PCBs, such as PCB 91, 95, 132, and 149, are enantioselectively metabolized to OH-PCBs by cytochrome P450 enzymes.

The iodination of chlorinated aromatic compounds using Ag(2)SO(4)/I(2), AgSbF(6)/I(2), AgBF(4)/I(2) and AgPF(6)/I(2) offers access to iodoarenes that are valuable intermediates in organic synthesis. Specifically, iodination of phenols, anisoles and anilines with a 3,5-dichloro substitution pattern preferentially yielded the ortho, para and para iodinated product, respectively. In the case of chlorobenzene and 3-chlorotoluene, AgSbF(6)/I(2), AgBF(4)/I(2) and AgPF(6)/I(2), but not Ag(2)SO(4)/I(2), selectively introduced the iodine in para position to the chlorine substituent.

A series of sterically hindered (methoxylated) polychlorinated biphenyl derivatives was synthesized using the Suzuki and the Ullmann coupling reaction. The Suzuki coupling with Pd(dba)(2)/2-dicyclohexylphosphino-2',6'-dimethoxybiphenyl (DPDB) gave better yields (65-98%) compared to the classic Ullmann coupling reaction (20-38%). Despite the reactive catalyst system, no significant coupling with aromatic chlorine substituents was observed. Crystal structure analysis of four PCB derivatives revealed solid state dihedral angles ranging from 69.7° to 81.0°, which indicates that these highly ortho substituted PCB derivatives have some conformational flexibility.

Two methods are presented for determining the catalytic activity of aryl and alcohol sulfotransferases. The first assay is a simple and rapid procedure that is based on the extraction of a paired ion formed between the product organic sulfate and methylene blue. The second method employs HPLC analysis of the substrate-dependent formation of PAP, an assay that is particularly useful when the sulfuric acid ester product is chemically unstable.

More than three decades after their discovery and implementation in medicine, essentially nothing is known about the metabolism or the implications of metabolism in mechanism of action or toxicity of the Catharanthus alkaloids. The frustrating paucity of information about pathways of metabolism has limited a major source of structure-activity relationship information and has blocked a critical avenue necessary for the logical development of new and more useful Catharanthus alkaloids. Microbial transformations, peroxidases, copper oxidases, mouse and rat cytochrome P-450 systems, and mouse brain and bovine liver monoamine oxidase (MAO) preparations have been explored in the study of Catharanthus alkaloid metabolism. In this report, we present results which have clarified the involvement of enzymatic and chemically catalyzed one-electron oxidations that yield nitrogen-centered cation radicals, iminium, and carbinolamine intermediates, all of which explain how new carbon-carbon and carbon-oxygen bonds form, or break and rearrange. The dimeric Catharanthus alkaloids are recalcitrant to oxidations catalyzed by monoamine oxidases and to both normal and induced P-450 rat microsomal preparations. However, the Catharanthus alkaloids appear to be selective reversible inhibitors of MAO-B. Chemical and biochemical aspects of the metabolic transformations of dimeric Catharanthus alkaloids are reviewed together with the implications of our findings.

1. Although neither the (+)- nor (-)-enantiomer of trans-benzo[a]pyrene-7,8-dihydrodiol was a substrate for aryl sulphotransferase IV from rat liver, both enantiomers inhibited the enzyme-catalysed sulphation of 1-naphthalene-methanol with Ki values of 3.7 +/- 0.4 microM for the (+)-enantiomer, and 4.4 +/- 0.3 microM for the (-)-enantiomer. 2. Based on the magnitude of the Ki values, the binding affinity of these dihydrodiols for the aryl sulphotransferase was significantly greater than that for the corresponding phenolic derivatives of benzo[a]pyrene. That is 7-hydroxybenzo[a]pyrene and 8-hydroxybenzo[a]pyrene were both substrates for aryl sulphotransferase IV, with apparent Km values of 280 +/- 41 microM and 370 +/- 72 microM, respectively. 3. Both (+)- and (-)-trans-naphthalene-1,2-dihydrodiols were also inhibitors of aryl sulphotransferase IV, but with higher Ki values than would be expected from previously determined apparent Km and Ki values for (R)-(-)- and (S)-(+)-1,2,3,4-tetrahydro-1-naphthols, respectively.