Michael W. Duffel

The mammalian cytosolic sulfotransferases (SULTs) catalyze the sulfation of endocrine hormones as well as a broad array of drugs, environmental chemicals, and other xenobiotics. Many endocrine-disrupting chemicals (EDCs) interact with these SULTs as substrates and inhibitors, and thereby alter sulfation reactions responsible for metabolism and regulation of endocrine hormones such as estrogens and thyroid hormones. EDCs or their metabolites may also regulate expression of SULTs through direct interaction with nuclear receptors and other transcription factors. Moreover, some sulfate esters derived from EDCs (EDC-sulfates) may serve as ligands for endocrine hormone receptors. While the sulfation of an EDC can lead to its excretion in the urine or bile, it may also result in retention of the EDC-sulfate through its reversible binding to serum proteins and thereby enable transport to other tissues for intracellular hydrolysis and subsequent endocrine disruption. This mini-review outlines the potential roles of SULTs and sulfation in the effects of EDCs and our evolving understanding of these processes.

Polychlorinated biphenyls (PCBs) are persistent organic toxicants derived from legacy pollution sources and their formation as inadvertent byproducts of some current manufacturing processes. Metabolism of PCBs is often a critical component in their toxicity, and relevant metabolic pathways usually include their initial oxidation to form hydroxylated polychlorinated biphenyls (OH-PCBs). Subsequent sulfation of OH-PCBs was originally thought to be primarily a means of detoxication; however, there is strong evidence that it may also contribute to toxicities associated with PCBs and OH-PCBs. These contributions include either the direct interaction of PCB sulfates with receptors or their serving as a localized precursor for OH-PCBs. The formation of PCB sulfates is catalyzed by cytosolic sulfotransferases, and, when transported into the serum, these metabolites may be retained, taken up by other tissues, and subjected to hydrolysis catalyzed by intracellular sulfatase(s) to regenerate OH-PCBs. Dynamic cycling between PCB sulfates and OH-PCBs may lead to further metabolic activation of the resulting OH-PCBs. Ultimate toxic endpoints of such processes may include endocrine disruption, neurotoxicities, and many others that are associated with exposures to PCBs and OH-PCBs. This review highlights the current understanding of the complex roles that PCB sulfates can have in the toxicities of PCBs and OH-PCBs and research on the varied mechanisms that control these roles.

Polychlorinated biphenyls (PCBs) are a class of legacy, and also current inadvertently produced, toxic persistent chemicals found in building materials and consumer products. PCB sulfates are metabolic products derived from hydroxylated metabolites of PCBs (OH-PCBs). Both OH-PCBs and PCB sulfates exert multiple toxicological effects on human health such as disrupting thyroid hormone transport and inhibiting steroid sulfotransferases (SULT1E1 and SULT2A1). Although PCB 11 sulfate has been previously detected in human serum samples, the lack of a generally applicable method for a broad range of PCB sulfates in human serum has limited our understanding of their prevalence and importance. This thesis describes studies on how we successfully developed a method for extraction and quantification of PCB sulfates from human serum. Our method employs acetonitrile extraction of the PCB sulfates from serum followed by differential analysis with, and without, hydrolysis to OH-PCBs catalyzed by a purified sulfatase from crude Helix pomatia juice using a tyrosine ethyl ester (TEE)-Sepharose affinity column. Our results on the specificity of the purified enzyme for PCB sulfates indicate the feasibility of its use for their quantitative hydrolysis. OH-PCBs are quantified by GC/MS/MS after derivatization to their corresponding methoxy PCBs. In a pooled human serum sample, we identified ten PCB sulfate congeners above the limit of quantitation with statistical evidence to validate their significance, with three PCB sulfate congeners exhibiting the highest concentrations from 1200-3970 pg/g of serum. Application of our method in serum samples collected from 24 individuals (6 mother-daughter and 6 mother-son dyads) from a rural community (Columbus Junction, Iowa) and 24 subjects (6 mother-daughter and 6 mother-son dyads) from an urban community (East Chicago, Indiana) also brought us valuable information. We found 8 PCB sulfate congeners that contributed the most to the total PCB sulfates in these samples, and these were all lower-chlorinated 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. The urban community was known to have a history of PCB-utilizing industries, whereas the rural community had no known current or past industrial uses of PCBs. We detected significantly higher levels of total PCB sulfates in the urban group (110-8900 ng/g fresh weight of serum, mean = 3351 ng/g, S.E.= 300, N=24) than in the rural group (530-6700 ng/g fresh weight of serum, mean = 1785 ng/g, S.E.=500, N=24), however, based on previous studies, the total serum PCB levels of individuals from these two areas have no significant difference. This suggests that the serum levels of many PCBs do not fully reflect the actual composition of PCB exposures in humans. Our work contributes to understanding of PCB metabolism in humans as well as guiding future research on the relationship of PCB exposure to their metabolite concentrations from biological samples. Also, the results from our study provide clues for future research directions such as PCB metabolite determination in human urine samples and how this may affect steady-state concentrations and toxicities of OH-PCBs and PCB sulfates in serum and tissues.

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.

G protein-coupled receptors (GPCRs) are the largest family of protein targets for approved drugs and are the most commonly targeted by FDA approved drugs. GPCR signaling is facilitated by a number of downstream proteins and effectors that create an intracellular response to extracellular stimuli. Two protein families that mediate GPCR signaling are the regulator of G protein signaling (RGS) proteins and the adenylyl cyclase (AC) proteins. This work focuses on furthering our understanding of novel regulating partners for RGS17 and identifying small molecules capable of inhibiting the association of AC type 1 (AC1) with its regulatory partner Ca2+/Calmodulin (CaM). RGS proteins regulate GPCR signaling by increasing the rate of GTP hydrolysis for Gα proteins, in effect inhibiting further signaling and returning the Gα protein to an inactive state. As certain Gα proteins are known to stimulate (Gαs) or inhibit (Gαi/o) AC mediated production of cAMP, RGS protein activity can directly or indirectly regulate AC activity. RGS proteins as downstream regulators of GPCR signaling pathways have attracted significant interest as potential drug targets. RGS17 in particular has been identified as overactive/overexpressed in a number of cancer types. As a member of the RZ subfamily of RGS proteins, RGS17 inhibits Gαo, Gαz, and Gαi1-3 protein signaling, which includes Ca2+-dependent signaling pathways. A number of high throughput screening efforts have identified small molecules capable of inhibiting the RGS17/Gα protein-protein interaction (PPI), but poor RGS protein selectivity or potency for these compounds have prompted alternative screening approaches. One such approach utilized nuclear magnetic resonance (NMR) spectroscopy to screen a fragment library, leading to the identification of several fragments capable of binding RGS17. In pursuit of binding site information for these fragments, a high-resolution crystal structure of RGS17 with multiple Ca2+ ions bound was obtained. Binding between RGS17 and Ca2+ was confirmed with NMR. Further, RGS17 was found to preferentially bind Ca2+ over Mg2+, and RGS17 exhibited a higher affinity for Ca2+ when compared with RGS4 and RGS2. Finally, the presence of Ca2+ promoted the interaction between RGS17 and active Gα proteins, decreased the Km for GTP hydrolysis, and altered the RGS17-Gα binding mechanism. These results indicate Ca2+ can increase RGS17 activity, potentially adding to an existing feedback mechanism for RZ-mediated inhibition of Ca2+ signaling. Finally, preclinical studies have identified neuronal AC1 as a novel target for the treatment of chronic inflammatory pain. In order to develop any protein into a viable therapeutic target it is important to understand its activity and how it is regulated within the body. For AC1, its activity is significantly stimulated by the signaling protein CaM. Therein lies the avenue for developing AC1 as a therapeutic target for treating chronic pain, as inhibiting the AC1/CaM PPI would greatly diminish the activity of AC1. In search of small molecule inhibitors of the AC1/CaM PPI we developed biochemical and cell-based assays, and a pilot screen of ~22,000 compounds yielded 54 hit compounds. Ultimately, 21 compounds were identified as being capable of inhibiting the AC1/CaM PPI in cells.

Polychlorinated biphenyls (PCBs) are widespread persistent environmental contaminants. PCB126 is an aryl hydrocarbon receptor (AhR) agonist and the most potent dioxin-like (DL) toxicant among the 209 different congeners of PCBs. AhR is a ligand-activated transcription factor involved in the regulation of biological responses to planar aromatic hydrocarbons, and many xenobiotic-metabolizing enzymes, such as CYP1A1. People are exposed to PCB126 through food, specifically consumption of PCB126 contaminated fish. As DL PCBs have adverse effects on female reproduction system, so pregnant mothers, children, and women of childbearing age may be highly vulnerable for PCB126 exposure. Considering the ongoing exposure to dioxin and DL compounds, the overall goal of this study is the identification of targets, understanding the mechanisms of toxicity, and examination the involvement of AhR in this toxicity that will form a basis for our understanding of PCB126 risks to human health and their amelioration. We hypothesized that PCB126 causes reproductive and developmental toxicity in rats, and these toxicities are mediated through the AhR. We therefore investigated the effects of PCB126 on pregnant and nonpregnant rats for in utero and adult exposure, and whether these effects are mediated through the AhR by performing separate animal studies. First, we investigated the developmental toxicity of PCB126 exposure in utero. For that, a timed pregnant rat study and three different doses (0.2, 0.5, 5µmol/kg) of PCB126 were used to understand the dose dependent effects of PCB126 for prenatal exposure to pregnant rats (dams), placentae, and fetuses. After only 6 days exposure, PCB126 significantly reduced weight gain, increased liver weights and decreased thymus weights in dams. It also imbalanced some important hormones like estradiol and leptin that may affect fetal development. PCB126 also caused placental toxicity by reducing placental weights, altering the thickness of placental layers, and causing necrosis that may disrupt placental normal function to provide sufficient nutrition and energy to the fetuses. In addition, PCB126 caused adverse pregnancy outcomes (e.g. low birth weights, resorption of fetuses, and fetal deaths) in high dose (5µmol/kg) group suggesting developmental toxicity even in a short-term exposure. Most of these effects were found to be dose dependent. Further, we examined the role of AhR in the toxicity of PCB126 exposure in AhR knock out (AhR KO) rat model who received a single IP dose of PCB126 (5µmol/kg) and necropsy was performed 28 days later. In this study, effects of PCB126 on liver energy homeostasis and female reproductive system were examined. Only control PCB126-exposed rats had significant hypoglycemia which was a result of downregulation of transcript levels of genes related to gluconeogenesis, glycogenolysis and fatty acid oxidation, necessary for liver energy homeostasis. In addition, reproductive toxicity including low ovarian reserve (reduced ovarian weight, low number of follicles in ovary, and low anti-Mullerian hormone), reduced uterine gland number and altered metabolic and inflammatory gene transcription were observed only in WT rats after PCB126 exposure, but not in AhR-KO animals. These data suggest that most, if not all, toxic effects of PCB126 are mediated through the AhR. Further, we elucidated that not only reproductive toxicity is mediated through the AhR, but also developmental toxicity is mediated by AhR. In this study, there was no significant toxicity in AhR-KO pregnant mothers, fetuses and placentae observed after PCB126 exposure. In addition, we investigated if pregnant animals are more sensitive to in utero exposure to PCB126 than non-pregnant animals. For that, the comparison was made to the toxicity of PCB126 in pregnant and non-pregnant animals with same age, dose, and exposure time. This study reported the different adverse effects of PCB126 in energy homeostasis for pregnant and non-pregnant animals, and pregnant dams are more vulnerable to PCB126 exposure than non-pregnant animals. These results helped to better understand the role of AhR activation in the toxicity of PCB126 exposure, gain insight into the mechanisms of toxicity including effects mediated through placental impairment, and evaluate whether pregnancy is a risk factor for female rats.

Topoisomerases (topos) are enzymes responsible for managing DNA topology and linking, making them critical cellular machinery for the management of DNA. Due to their key roles in cellular maintenance and replication, topo inhibitors are used clinically as antimicrobials and anticancer agents. Fluoroquinolones are clinically successful bacterial type II topo inhibitors or poisons used to treat bacterial infections and several clinically successful cancer chemotherapy agents are inhibitors or poisons of human topos. Quinazoline-2,4-diones (diones) are structural mimics of fluoroquinolones that may mimic the topo inhibiting activity of either antibacterial or anticancer fluroquinolones. These diones show promise both as antimicrobials for antibiotic-resistant bacteria or as anticancer chemotherapy agents, but for both applications there is an apparent problem with cellular accumulation of diones. This work describes the exploration of two strategies for increasing the cellular accumulation of antibiotic or anticancer diones. In the clinic, fluoroquinolones face both target-mediated and plasmid-mediated resistance (efflux resistance). Quinazoline 2,4-dione dimers were proposed as a solution to both mechanisms of resistance. Past work in the Kerns lab has shown that C7-linked fluoroquinolone dimers overcome efflux-based fluoroquinolone resistance in Gram-positive bacteria with upregulated efflux pumps while maintaining or having improved antibacterial activity with different Gram-positive organisms. It was hypothesized that dione dimers would also maintain antibacterial activity and evade efflux. In pursuit of these goals, three C7-piperazenyl-linked antimicrobial dione dimers and one C7-linked dione/fluoroquinolone asymmetric dimer were synthesized, and all but the asymmetric dimer were poor bacterial topo inhibitors. N1-biphenyl fluoroquinolones are human topo inhibitors with unique properties that set them apart from conventional fluoroquinolones and anticancer fluoroquinolones. Unlike conventional fluoroquinolones, N1-biphenyl fluoroquinolones target type I topos over type II topos and are catalytic inhibitors rather than topo poisons. N1-biphenyl diones are the same and have equal human topo I inhibition to N1-biphenyl fluoroquinolones. Despite the similar topo I inhibition of a representative fluoroquinolone and a representative dione, the results of NCI 60 cancer cell lines screening were significantly different. In multiple cell lines N1-biphenyl fluoroquinolones had greater growth inhibition than N1-biphenyl diones. This suggests that there is a difference in cellular uptake between the fluoroquinolone and dione. It was hypothesized that this difference was related to the charge and physical properties of these diones and quinolones. A panel of N1-biphenyl diones with altered charged groups at C7 and N3 were synthesized to evaluate whether changes in the charged groups at these locations could alter the ability of diones to enter cells, evade efflux, and inhibit topo I. The diones were predicted to have lower partition coefficients than their cognate quinolones. Topo I DNA relaxation, cell viability, and DNA binding were evaluated for these compounds. It was determined that DNA binding, enzyme inhibition, and cell viability inhibition track well together, which suggests that, at least in the cell types studied, cell penetration does not appear to be a major issue. The DNA binding assays also confirm that DNA binding is an important factor in topo I inhibition for these compounds.

Benoxacor is a chiral dichloroacetamide safener that is often co-formulated with the chloroacetanilide herbicide metolachlor. The main function of benoxacor is to induce metabolic enzymes such as cytochrome P450s (CYPs), carboxylesterases (CESs) and glutathione S-transferases (GSTs) within crops, such as maize, to provide protection from metolachlor toxicity. While previous studies have determined that benoxacor along with other dichloroacetamide safeners are found in the surface water in the Midwest United States, the enantioselective metabolism and toxicity of benoxacor has not been previously studied in mammalian systems. Benoxacor was incubated in microsomes and cytosol from rats, monkeys, humans, and mice. The co-factors nicotinamide adenine dinucleotide phosphate (NADPH) and glutathione (GSH) were added to the incubations to determine the metabolism of benoxacor by CYPs and GSTs respectively. CES metabolism was monitored in incubations without the addition of co-factor. These incubations studies showed that benoxacor was enantioselectively metabolized by CYPs, CESs, and GSTs in all of the species tested; however, many interspecies and sex differences were noted. Benoxacor was also tested for toxicity in C57Bl/6 mice following an acute oral exposure. Following benoxacor exposure there were significant changes to the bodyweight-adjusted weights of the testes and livers of the exposed animals and 163 genes were differentially expressed. The profile of differentially expressed genes closely matched the profile of expression as a result of dichloroacetic acid which is a likely metabolite of benoxacor hydrolysis. Minimal overall toxicity was noted in this study, but the results from this study warrant further investigation. While these studies provided a foundation for understanding the metabolism and potential toxicity of benoxacor, many further studies need to be conducted to determine the metabolites of benoxacor and further understand any toxic effects.

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.