Publications by authors named "James M O"

131 Publications

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Drug Metab Dispos 2021 Apr 2. Epub 2021 Apr 2.

Department of Pharmacodynamics, University of Florida, United States of America.

Sodium dichloroacetate (DCA) is an investigational drug that shows promise in the treatment of acquired and congenital mitochondrial diseases, including myocardial ischemia and failure. DCA activates the pyruvate dehydrogenase complex (PDC) to increase energy production and decrease circulating lactate concentrations. In a sheep model of chronic hypercortisolemia in pregnancy, PDK4 mRNA was increased and fetal bradycardia was evident at birth in the affected fetuses. The current study shows DCA crosses the placenta and can be measured in fetal blood, following its intravenous administration to pregnant ewes during late gestation and in labor. Sustained administration of DCA to the mother over 72h decreased the hepatic expression of the DCA-metabolizing enzyme GSTZ1 in the fetuses exposed to the drug, leading to higher fetal plasma DCA concentrations during continued dosing, and reduced plasma lactate levels. Multi-compartmental pharmacokinetics modeling indicated drug metabolism in the fetal and maternal compartments is best described by the autoinhibition of metabolism in both compartments. We provide the first evidence that DCA can cross the placental compartment to enter the fetal circulation, inhibit its own hepatic metabolism and decrease fetal plasma lactate concentrations. This study was the first to administer Sodium Dichloroacetate (DCA) to pregnant animals (sheep). It showed that DCA administered to the mother can cross the placental barrier and achieve concentrations in fetus to decrease the fetal lactate concentrations. Like other reported species, DCA mediated inhibition of GSTZ1 was also observed in ewes which resulted in the reduced metabolism of DCA after a prolonged administration.
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http://dx.doi.org/10.1124/dmd.120.000330DOI Listing
April 2021

Effects of Multiple Doses of Dichloroacetate on GSTZ1 Expression and Activity in Liver and Extrahepatic Tissues of Young and Adult Rats.

Drug Metab Dispos 2020 11 1;48(11):1217-1223. Epub 2020 Sep 1.

Departments of Medicinal Chemistry (E.J.S., M.G.S., L.R.-F., M.O.J.), Medicine (L.P.H., P.W.S.), and Biochemistry and Molecular Biology (P.W.S.), University of Florida, Gainesville, Florida

Glutathione transferase zeta 1 (GSTZ1), expressed in liver and several extrahepatic tissues, catalyzes dechlorination of dichloroacetate (DCA) to glyoxylate. DCA inactivates GSTZ1, leading to autoinhibition of its metabolism. DCA is an investigational drug for treating several congenital and acquired disorders of mitochondrial energy metabolism, including cancer. The main adverse effect of DCA, reversible peripheral neuropathy, is more common in adults treated long-term than in children, who metabolize DCA more quickly after multiple doses. One dose of DCA to Sprague Dawley rats reduced GSTZ1 expression and activity more in liver than in extrahepatic tissues; however, the effects of multiple doses of DCA that mimic its therapeutic use have not been studied. Here, we examined the expression and activity of GSTZ1 in cytosol and mitochondria of liver, kidney, heart, and brain 24 hours after completion of 8-day oral dosing of 100 mg/kg per day sodium DCA to juvenile and adult Sprague Dawley rats. Activity was measured with DCA and with 1,2-epoxy-3-(4-nitrophenoxy)propane (EPNPP), reported to be a GSTZ1-selective substrate. In DCA-treated rats, liver retained higher expression and activity of GSTZ1 with DCA than other tissues, irrespective of rodent age. DCA-treated juvenile rats retained more GSTZ1 activity with DCA than adults. Consistent with this finding, there was less measurable DCA in tissues of juvenile than adult rats. DCA-treated rats retained activity with EPNPP, despite losing over 98% of GSTZ1 protein. These data provide insight into the differences between children and adults in DCA elimination under a therapeutic regimen and confirm that the liver contributes more to DCA metabolism than other tissues. SIGNIFICANCE STATEMENT: Dichloroacetate (DCA) is one of few drugs exhibiting higher clearance from children than adults, after repeated doses, for reasons that are unclear. We hypothesized that juveniles retain more glutathione transferase zeta 1 (GSTZ1) than adults in tissues after multiple DCA doses and found this was the case for liver and kidney, with rat as a model to assess GSTZ1 protein expression and activity with DCA. Although 1,2-epoxy-3-(4-nitrophenoxy)propane was reported to be a selective GSTZ1 substrate, its activity was not reduced in concert with GSTZ1 protein.
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http://dx.doi.org/10.1124/dmd.120.000142DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7589944PMC
November 2020

Exposure of Rats to Multiple Oral Doses of Dichloroacetate Results in Upregulation of Hepatic Glutathione Transferases and NAD(P)H Dehydrogenase [Quinone] 1.

Drug Metab Dispos 2020 11 1;48(11):1224-1230. Epub 2020 Sep 1.

Departments of Medicinal Chemistry (E.J.S., R.M., M.O.J.), Medicine (L.P.H., P.W.S.), and Biochemistry and Molecular Biology (P.W.S.), University of Florida, Gainesville, Florida

Dichloroacetate (DCA) is an investigational drug that is used in the treatment of various congenital and acquired disorders of energy metabolism. Although DCA is generally well tolerated, some patients experience peripheral neuropathy, a side effect more common in adults than children. Repetitive DCA dosing causes downregulation of its metabolizing enzyme, glutathione transferase zeta 1 (GSTZ1), which is also critical in the detoxification of maleylacetoacetate and maleylacetone. (-/-) knockout mice show upregulation of glutathione transferases (GSTs) and antioxidant enzymes as well as an increase in the ratio of oxidized glutathione (GSSG) to reduced glutathione (GSH), suggesting GSTZ1 deficiency causes oxidative stress. We hypothesized that DCA-mediated depletion of GSTZ1 causes oxidative stress and used the rat to examine induction of GSTs and antioxidant enzymes after repeated DCA exposure. We determined the expression of alpha, mu, pi, and omega class GSTs, NAD(P)H dehydrogenase [quinone] 1 (NQO1), gamma-glutamylcysteine ligase complex (GCLC), and glutathione synthetase (GSS). GSH and GSSG levels were measured by liquid chromatography-tandem mass spectrometry. Enzyme activity was measured in hepatic cytosol using 1-chloro-2,4-dinitrobenzene, 1,2-dichloro-4-nitrobenzene, and 2,6-dichloroindophenol as substrates. In comparison with acetate-treated controls, DCA dosing increased the relative expression of GSTA1/A2 irrespective of rodent age, whereas only adults displayed higher levels of GSTM1 and GSTO1. NQO1 expression and activity were higher in juveniles after DCA dosing. GSH concentrations were increased by DCA in adults, but the GSH:GSSG ratio was not changed. Levels of GCLC and GSS were higher and lower, respectively, in adults treated with DCA. We conclude that DCA-mediated depletion of GSTZ1 causes oxidative stress and promotes the induction of antioxidant enzymes that may vary between age groups. SIGNIFICANCE STATEMENT: Treatment with the investigational drug, dichloroacetate (DCA), results in loss of glutathione transferase zeta 1 (GSTZ1) and subsequent increases in body burden of the electrophilic tyrosine metabolites, maleylacetoacetate and maleylacetone. Loss of GSTZ1 in genetically modified mice is associated with induction of glutathione transferases and alteration of the ratio of oxidized to reduced glutathione. Therefore, we determined whether pharmacological depletion of GSTZ1 through repeat administration of DCA produced similar changes in the liver, which could affect responses to other drugs and toxicants.
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http://dx.doi.org/10.1124/dmd.120.000143DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7589945PMC
November 2020

Age-Related Changes in miRNA Expression Influence GSTZ1 and Other Drug Metabolizing Enzymes.

Drug Metab Dispos 2020 07 1;48(7):563-569. Epub 2020 May 1.

Departments of Medicinal Chemistry (S.C.J., C.J.W., M.O.J.), Pharmacotherapy and Translational Research (T.Y.L.), Medicine (P.W.S.), Biochemistry and Molecular Biology (P.W.S.), and Molecular Genetics and Microbiology (L.A.G., R.R.), University of Florida, Gainesville, Florida

Previous work has shown that hepatic levels of human glutathione transferase zeta 1 (GSTZ1) protein, involved in tyrosine catabolism and responsible for metabolism of the investigational drug dichloroacetate, increase in cytosol after birth before reaching a plateau around age 7. However, the mechanism regulating this change of expression is still unknown, and previous studies showed that mRNA levels did not correlate with GSTZ1 protein expression. In this study, we addressed the hypothesis that microRNAs (miRNAs) could regulate expression of GSTZ1. We obtained liver samples from donors aged less than 1 year or older than 13 years and isolated total RNA for use in a microarray to identify miRNAs that were downregulated in the livers of adults compared with children. From a total of 2578 human miRNAs tested, 63 miRNAs were more than 2-fold down-regulated in adults, of which miR-376c-3p was predicted to bind to the 3' untranslated region of mRNA. There was an inverse correlation of miR-376c-3p and GSTZ1 protein expression in the liver samples. Using cell culture, we confirmed that miR-376c-3p could downregulate GSTZ1 protein expression. Our findings suggest that miR-376c-3p prevents production of GSTZ1 through inhibition of translation. These experiments further our understanding of GSTZ1 regulation. Furthermore, our array results provide a database resource for future studies on mechanisms regulating human hepatic developmental expression. SIGNIFICANCE STATEMENT: Hepatic glutathione transferase zeta 1 (GSTZ1) is responsible for metabolism of the tyrosine catabolite maleylacetoacetate as well as the investigational drug dichloroacetate. Through examination of microRNA (miRNA) expression in liver from infants and adults and studies in cells, we showed that expression of GSTZ1 is controlled by miRNA. This finding has application to the dosing regimen of the drug dichloroacetate. The miRNA expression profiles are provided and will prove useful for future studies of drug-metabolizing enzymes in infants and adults.
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http://dx.doi.org/10.1124/dmd.120.090639DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7289049PMC
July 2020

Efficacy data of halogenated phenazine and quinoline agents and an NH125 analogue to veterinary mycoplasmas.

BMC Vet Res 2020 Apr 6;16(1):107. Epub 2020 Apr 6.

Department of Infectious Diseases and Immunology, College of Veterinary Medicine, University of Florida, Gainesville, Florida, USA.

Background: Mycoplasmas primarily cause respiratory or urogenital tract infections impacting avian, bovine, canine, caprine, murine, and reptilian hosts. In animal husbandry, mycoplasmas cause reduced feed-conversion, decreased egg production, arthritis, hypogalactia or agalactia, increased condemnations, culling, and mortality in some cases. Antibiotics reduce transmission and mitigate clinical signs; however, concerning levels of antibiotic resistance in Mycoplasma gallisepticum and M. capricolum isolates exist. To address these issues, we evaluated the minimum inhibitory concentrations (MICs) of halogenated phenazine and quinoline compounds, an N-arylated NH125 analogue, and triclosan against six representative veterinary mycoplasmas via microbroth or agar dilution methods. Thereafter, we evaluated the minimum bactericidal concentration (MBC) of efficacious drugs.

Results: We identified several compounds with MICs ≤25 μM against M. pulmonis (n = 5), M. capricolum (n = 4), M. gallisepticum (n = 3), M. alligatoris (n = 3), M. agassizii (n = 2), and M. canis (n = 1). An N-arylated NH125 analogue, compound 21, served as the most efficacious, having a MIC ≤25 μM against all mycoplasmas tested, followed by two quinolines, nitroxoline (compound 12) and compound 20, which were effective against four and three mycoplasma type strains, respectively. Nitroxoline exhibited bactericidal activity among all susceptible mycoplasmas, and compound 21 exhibited bactericidal activity when the MBC was able to be determined.

Conclusions: These findings highlight a number of promising agents from novel drug classes with potential applications to treat veterinary mycoplasma infections and present the opportunity to evaluate preliminary pharmacokinetic indices using M. pulmonis in rodents as an animal model of human infection.
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http://dx.doi.org/10.1186/s12917-020-02324-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7137434PMC
April 2020

Mitochondrial Glutathione Transferase Zeta 1 Is Inactivated More Rapidly by Dichloroacetate than the Cytosolic Enzyme in Adult and Juvenile Rat Liver.

Chem Res Toxicol 2019 10 1;32(10):2042-2052. Epub 2019 Oct 1.

Dichloroacetate (DCA) has potential for treating mitochondrial disorders and cancer by activating the mitochondrial pyruvate dehydrogenase complex. Repeated dosing of DCA results in reduced drug clearance due to inactivation of glutathione transferase ζ1 (GSTZ1), its metabolizing enzyme. We investigated the time-course of inactivation of GSTZ1 in hepatic cytosol and mitochondria after one oral dose of 100 mg/kg DCA to female Sprague-Dawley rats aged 4 weeks (young) and 52 weeks (adult) as models for children and adults, respectively. GSTZ1 activity with both DCA and an endogenous substrate, maleylacetone (MA), as well as GSTZ1 protein expression were rapidly reduced in cytosol from both ages following DCA treatment. In mitochondria, loss of GSTZ1 protein and activity with DCA were even more rapid. The cytosolic in vivo half-lives of the loss of GSTZ1 activity with DCA were 1.05 ± 0.03 and 0.82 ± 0.02 h (mean ± S.D., = 6) for young and adult rats, respectively, with inactivation significantly more rapid in adult rats, < 0.001. The mitochondrial inactivation half-lives were similar in young (0.57 ± 0.02 h) and adult rats (0.54 ± 0.02 h) and were significantly ( < 0.0001) shorter than cytosolic inactivation half-lives. By 24 h after DCA administration, activity and expression remained at 10% or less than control values. The in vitro GSTZ1 inactivation half-lives following incubation with 2 mM DCA in the presence of physiological chloride (Cl) concentrations (cytosol = 44 mM, mitochondria = 1-2 mM) exhibited marked differences between subcellular fractions, being 3 times longer in the cytosol than in the mitochondria, regardless of age, suggesting that the lower Cl concentration in mitochondria explained the faster degradation of GSTZ1. These results demonstrate for the first time that rat mitochondrial GSTZ1 is more readily inactivated by DCA than cytosolic GSTZ1, and cytosolic GSTZ1 is inactivated more rapidly in adult than young rats.
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http://dx.doi.org/10.1021/acs.chemrestox.9b00207DOI Listing
October 2019

Dichloroacetate-induced peripheral neuropathy.

Int Rev Neurobiol 2019 11;145:211-238. Epub 2019 Jun 11.

Department of Pathology, University of California San Diego, La Jolla, CA, United States.

Dichloroacetate (DCA) has been the focus of research by both environmental toxicologists and biomedical scientists for over 50 years. As a product of water chlorination and a metabolite of certain industrial chemicals, DCA is ubiquitous in our biosphere at low μg/kg body weight daily exposure levels without obvious adverse effects in humans. As an investigational drug for numerous congenital and acquired diseases, DCA is administered orally or parenterally, usually at doses of 10-50mg/kg per day. As a therapeutic, its principal mechanism of action is to inhibit pyruvate dehydrogenase kinase (PDK). In turn, PDK inhibits the key mitochondrial energy homeostat, pyruvate dehydrogenase complex (PDC), by reversible phosphorylation. By blocking PDK, DCA activates PDC and, consequently, the mitochondrial respiratory chain and ATP synthesis. A reversible sensory/motor peripheral neuropathy is the clinically limiting adverse effect of chronic DCA exposure and experimental data implicate the Schwann cell as a toxicological target. It has been postulated that stimulation of PDC and respiratory chain activity by DCA in normally glycolytic Schwann cells causes uncompensated oxidative stress from increased reactive oxygen species production. Additionally, the metabolism of DCA interferes with the catabolism of the amino acids phenylalanine and tyrosine and with heme synthesis, resulting in accumulation of reactive molecules capable of forming adducts with DNA and proteins and also resulting in oxidative stress. Preliminary evidence in rodent models of peripheral neuropathy suggest that DCA-induced neurotoxicity may be mitigated by naturally occurring antioxidants and by a specific class of muscarinic receptor antagonists. These findings generate a number of testable hypotheses regarding the etiology and treatment of DCA peripheral neuropathy.
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http://dx.doi.org/10.1016/bs.irn.2019.05.003DOI Listing
January 2020

Sulfonation and glucuronidation of hydroxylated bromodiphenyl ethers in human liver.

Chemosphere 2019 Jul 20;226:132-139. Epub 2019 Mar 20.

Department of Medicinal Chemistry, University of Florida, Gainesville, FL, 32610-0485, USA. Electronic address:

Hydroxylated bromodiphenyl ethers (OH-BDEs) can arise from monooxygenation of anthropogenic BDEs or through natural biosynthetic processes in marine organisms, and several OH-BDEs have been shown to be toxic. OH-BDEs are expected to form sulfate and glucuronide conjugates that are readily excreted, however there is little information on these pathways. We examined the human hepatic glucuronidation and sulfonation of 6-OH-BDE47, 2-OH-BDE68, 4-OH-BDE68 and 2-OH-6'methoxy-BDE68. Human liver microsomes and cytosol were from de-identified female and male donors aged 31 to 75 under an exempt protocol. Recombinant human SULT1A1, 1B1, 1E1 and 2A1 enzymes were prepared from bacterial expression systems. Sulfonation and glucuronidation of each OH-BDE were studied using radiolabeled co-substrates, 3'phosphoadenosine-5'phospho-S-sulfate or uridine diphospho-β-D-C-glucuronic acid in order to quantify the sulfated or glucuronidated products. The OH-BDEs studied were more efficiently glucuronidated than sulfonated. Of the compounds studied, 2-OH-BDE68 was the most readily conjugated, and exhibited an efficiency (V/K) of glucuronidation of 0.274 ± 0.125 mL/min/mg protein, mean ± S.D., n = 3, while that for sulfonation was 0.179 ± 0.030 mL/min/mg protein. For both pathways, all K values were in the low μM range. Studies with human SULT enzymes showed that sulfonation of these four substrates was readily catalyzed by SULT1B1 and SULT1E1. Much lower activity was found with SULT1A1 and SULT2A1. Assuming that the glucuronide and sulfate conjugates are non-toxic and readily excreted, as is the case for most such conjugates, these studies suggest that OH-BDEs should not accumulate in people to the same extent as the parent BDEs.
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http://dx.doi.org/10.1016/j.chemosphere.2019.03.103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6516465PMC
July 2019

Phase II metabolism of betulin by rat and human UDP-glucuronosyltransferases and sulfotransferases.

Chem Biol Interact 2019 Apr 15;302:190-195. Epub 2019 Feb 15.

Alkali Soil Natural Environmental Science Center, Northeast Forestry University, Key Laboratory of Saline-alkali Vegetation Ecology Restoration in Oil Field, Ministry of Education, Harbin, 150040, China. Electronic address:

The natural product betulin is under investigation for several therapeutic indications, however little is known about its metabolism. In the present study, the glucuronidation and sulfation of betulin in human and rat liver microsomes and cytosol were tested. We further identified the main UDP-glucuronosyltransferases (UGTs) and sulfotransferases (SULTs) involved in these two metabolism pathways. Results showed that one betulin glucuronide metabolite was observed after incubation with human and rat liver microsomes. The glucuronidation of betulin in human liver microsomes had a K value of 21.1 ± 5.93 μM and a V value of 6.39 ± 0.66 pmol/min/mg protein. The glucuronidation activity in rats was too low to get enzyme kinetic parameters. Among the 11 recombinant UGT enzymes investigated, UGT1A3 and UGT1A4 were identified as the major enzymes catalyzing the glucuronidation of betulin [K values of 10.12 ± 8.09 and 8.04 ± 3.96 μM, V values of 6.71 ± 1.51 and 5.98 ± 0.76 nmol/min/(mg protein)]. Two betulin sulfate metabolites were found in human and rat liver cytosols. Human and rat liver had similar affinity for the formation of these two metabolites, the apparent V for betulin sulfate I was higher than that for betulin sulfate II in both species. Among the SULT isoforms studied, SULT2A1 was the major isoenzyme involved in the betulin sulfation metabolism in human liver cytosol. The results suggest that glucuronidation and sulfation are important metabolism pathways for betulin, and UGT1A3, UGT1A4 and SULT2A1 play the major roles in betulin glucuronidation and sulfation.
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http://dx.doi.org/10.1016/j.cbi.2019.02.009DOI Listing
April 2019

Turning the Tide against Antibiotic Resistance by Evaluating Novel, Halogenated Phenazine, Quinoline, and NH125 Compounds against Species Clinical Isolates and Type Strains.

Antimicrob Agents Chemother 2019 03 26;63(3). Epub 2019 Feb 26.

Department of Infectious Diseases and Immunology, College of Veterinary Medicine, University of Florida, Gainesville, Florida, USA

Escalating levels of antibiotic resistance in mycoplasmas, particularly macrolide resistance in and , have narrowed our antibiotic arsenal. Further, mycoplasmas lack a cell wall and do not synthesize folic acid, rendering common antibiotics, such as beta-lactams, vancomycin, sulfonamides, and trimethoprim, of no value. To address this shortage, we screened nitroxoline, triclosan, and a library of 20 novel, halogenated phenazine, quinoline, and NH125 analogues against species and clinical isolates from urine. We tested a subset of these compounds ( = 9) against four mycoplasma type strains (, , , and ) using a validated broth microdilution or agar dilution method. Among 72 species clinical isolates, nitroxoline proved most effective (MIC, 6.25 µM), followed by an -arylated NH125 analogue (MIC, 12.5 µM). NH125 and its analogue had significantly higher MICs against isolates than against isolates, whereas nitroxoline did not. Nitroxoline exhibited bactericidal activity against isolates but bacteriostatic activity against the majority of isolates. Among the type strains, the compounds had the greatest activity against and , with 8 (80%) and 5 (71.4%) isolates demonstrating MICs of ≤12.5 µM, respectively. Triclosan also exhibited lower MICs against and Overall, we identified a promising range of quinoline, halogenated phenazine, and NH125 compounds that showed effectiveness against and and found that nitroxoline, approved for use outside the United States for the treatment of urinary tract infections, and an -arylated NH125 analogue demonstrated low MICs against species isolates.
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http://dx.doi.org/10.1128/AAC.02265-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6395908PMC
March 2019

Age-Related Changes in Expression and Activity of Human Hepatic Mitochondrial Glutathione Transferase Zeta1.

Drug Metab Dispos 2018 08 31;46(8):1118-1128. Epub 2018 May 31.

Department of Medicinal Chemistry (G.Z., M.O.J., M.G.S., S.C.J.), Department of Pharmacotherapy and Translational Research (T.L.), Center for Pharmacogenomics (T.L.), and Departments of Medicine and Biochemistry and Molecular Biology (P.W.S.), University of Florida, Gainesville, Florida; and Department of Pediatrics, Medical College of Wisconsin, Milwaukee, Wisconsin (P.S.).

Glutathione transferase zeta1 (GSTZ1) catalyzes glutathione (GSH)-dependent dechlorination of dichloroacetate (DCA), an investigational drug with therapeutic potential in metabolic disorders and cancer. GSTZ1 is expressed in both hepatic cytosol and mitochondria. Here, we examined the ontogeny and characterized the properties of human mitochondrial GSTZ1. GSTZ1 expression and activity with DCA were determined in 103 human hepatic mitochondrial samples prepared from livers of donors aged 1 day to 84 years. DNA from each sample was genotyped for three common GSTZ1 functional single nucleotide polymorphisms. Expression of mitochondrial GSTZ1 protein increased in an age-dependent manner to a plateau after age 21 years. Activity with DCA correlated with expression, after taking into account the somewhat higher activity of samples that were homo- or heterozygous for GSTZ1A. In samples from livers with the GSTZ1C variant, apparent enzyme kinetic constants for DCA and GSH were similar for mitochondria and cytosol after correcting for the loss of GSH observed in mitochondrial incubations. In the presence of 38 mM chloride, mitochondrial GSTZ1 exhibited shorter half-lives of inactivation compared with the cytosolic enzyme ( = 0.017). GSTZ1 protein isolated from mitochondria was shown by mass spectrometry to be identical to cytosolic GSTZ1 protein in the covered primary protein sequence. In summary, we report age-related development in the expression and activity of human hepatic mitochondrial GSTZ1 does not have the same pattern as that reported for cytosolic GSTZ1. Some properties of cytosolic and mitochondrial GSTZ1 differed, but these were not related to differences in amino acid sequence or post-translationally modified residues.
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http://dx.doi.org/10.1124/dmd.118.081810DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6038028PMC
August 2018

Administration of low dose triclosan to pregnant ewes results in placental uptake and reduced estradiol sulfotransferase activity in fetal liver and placenta.

Toxicol Lett 2018 Sep 26;294:116-121. Epub 2018 May 26.

Department of Physiology and Functional Genomics, University of Florida, Gainesville, FL, 32610, USA.

Sulfonation is a major pathway of estrogen biotransformation with a role in regulating estrogen homeostasis in humans and sheep. Previous in vitro studies found that triclosan is an especially potent competitive inhibitor of ovine placental estrogen sulfotransferase, with K of <0.1 nM. As the placenta is the main organ responsible for estrogen synthesis in pregnancy in both women and sheep, and the liver is another site of estrogen biotransformation, this study examined the effects of triclosan exposure of pregnant ewes on placental and hepatic sulfotransferase activity. Triclosan, 0.1 mg/kg/day, or saline vehicle was administered to late gestation fetal sheep for two days either by direct infusion into the fetal circulation or infusion into the maternal blood. On the third day, fetal liver and placenta were harvested and analyzed for triclosan and for cytosolic estradiol sulfotransferase activity. Placenta contained higher concentrations of triclosan than liver in each individual sheep in both treatment groups. There was a negative correlation between triclosan tissue concentration (pmol/g tissue) and cytosolic sulfotransferase activity (pmol/min/mg protein) towards estradiol. These findings demonstrated that in the sheep exposed to very low concentrations of triclosan, this substance is taken up into placenta and reduces estrogen sulfonation.
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http://dx.doi.org/10.1016/j.toxlet.2018.05.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6026481PMC
September 2018

Regulation of dichloroacetate biotransformation in rat liver and extrahepatic tissues by GSTZ1 expression and chloride concentration.

Biochem Pharmacol 2018 06 5;152:236-243. Epub 2018 Apr 5.

Department of Medicinal Chemistry, University of Florida, Gainesville, FL 32610, United States. Electronic address:

Biotransformation of dichloroacetate (DCA) to glyoxylate by hepatic glutathione transferase zeta 1 (GSTZ1) is considered the principal determinant of the rate of plasma clearance of the drug. However, several other organismal and subcellular factors are also known to influence DCA metabolism. We utilized a female rat model to study these poorly understood processes. Rats aged 4 weeks (young) and 42-52 weeks (adult) were used to model children and adults, respectively. Hepatic chloride concentrations, which influence the rate of GSTZ1 inactivation by DCA, were lower in rat than in human tissues and rats did not show the age dependence previously seen in humans. We found GSTZ1 expression and activity in rat brain, heart, and kidney cell-free homogenates that were age-dependent. GSTZ1 expression in brain was higher in young rats than adult rats, whereas cardiac and renal GSTZ1 expression levels were higher in adult than young rats. GSTZ1 activity with DCA could not be measured accurately in kidney cell-free homogenates due to rapid depletion of glutathione by γ-glutamyl transpeptidase. Following oral administration of DCA, 100 mg/kg, to rats, GSTZ1 expression and activity were reduced in all rat tissues, but chloride concentrations were not affected. Together, these data extend our understanding of factors that determine the in vivo kinetics of DCA.
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http://dx.doi.org/10.1016/j.bcp.2018.04.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5960630PMC
June 2018

Model Informed Dose Optimization of Dichloroacetate for the Treatment of Congenital Lactic Acidosis in Children.

J Clin Pharmacol 2018 02 15;58(2):212-220. Epub 2017 Sep 15.

Center for Pharmacometrics & Systems Pharmacology, College of Pharmacy, University of Florida, Orlando, FL, USA.

Dichloroacetate (DCA) is an investigational drug used to treat congenital lactic acidosis and other mitochondrial disorders. Response to DCA therapy in young children may be suboptimal following body weight-based dosing. This is because of autoinhibition of its metabolism, age-dependent changes in pharmacokinetics, and polymorphisms in glutathione transferase zeta 1 (GSTZ1), its primary metabolizing enzyme. Our objective was to predict optimal DCA doses for the treatment of congenital lactic acidosis in children. Accordingly, a semimechanistic pharmacokinetic-enzyme turnover model was developed in a step-wise approach: (1) a population pharmacokinetic model for adults was developed; (2) the adult model was scaled to children using allometry and physiology-based scaling; and (3) the scaled model was externally qualified, updated with clinical data, and optimal doses were projected. A 2-compartment model accounting for saturable clearance and GSTZ1 enzyme turnover successfully characterized the DCA PK in adults and children. DCA-induced inactivation of GSTZ1 resulted in phenoconversion of all subjects into slow metabolizers after repeated dosing. However, rate and extent of inactivation was 2-fold higher in subjects without the wild-type EGT allelic variant of GSTZ1, resulting in further phenoconversion into ultraslow metabolizers after repeated DCA administration. Furthermore, DCA-induced GSTZ1 inactivation rate and extent was found to be 25- to 30-fold lower in children than in adults, potentially accounting for the observed age-dependent changes in PK. Finally, a 12.5 and 10.6 mg/kg twice-daily DCA dose was optimal in achieving the target steady-state trough concentrations (5-25 mg/L) for EGT carrier and EGT noncarrier children, respectively.
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http://dx.doi.org/10.1002/jcph.1009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5967850PMC
February 2018

Celecoxib affects estrogen sulfonation catalyzed by several human hepatic sulfotransferases, but does not stimulate 17-sulfonation in rat liver.

J Steroid Biochem Mol Biol 2017 09 25;172:46-54. Epub 2017 May 25.

Department of Medicinal Chemistry, College of Pharmacy, University of Florida, Gainesville, FL 32610-0485, USA. Electronic address:

Celecoxib is known to alter the preferred position of SULT2A1-catalyzed sulfonation of 17β-estradiol (17β-E2) and other estrogens from the 3- to the 17-position. Understanding the effects of celecoxib on estrogen sulfonation is of interest in the context of the investigational use of celecoxib to treat breast cancer. This study examined the effects on celecoxib on cytosolic sulfotransferases in human and rat liver and on SULT enzymes known to be expressed in liver. Celecoxib's effects on the sulfonation of several steroids catalyzed by human liver cytosol were similar but not identical to those observed previously for SULT2A1. Celecoxib was shown to inhibit recombinant SULT1A1-catalyzed sulfonation of 10nM estrone and 4μM p-nitrophenol with IC values of 2.6 and 2.1μM, respectively, but did not inhibit SULT1E1-catalyzed estrone sulfonation. In human liver cytosol, the combined effect of celecoxib and known SULT1A1 and 1E1 inhibitors, quercetin and triclosan, resulted in inhibition of 17β-E2-3-sulfonation such that the 17-sulfate became the major metabolite: this is of interest because the 17-sulfate is not readily hydrolyzed by steroid sulfatase to 17β-E2. Investigation of hepatic cytosolic steroid sulfonation in rat revealed that celecoxib did not stimulate 17β-E2 17-sulfonation in male or female rat liver as it does with human SULT2A1 and human liver cytosol, demonstrating that rat is not a useful model of this effect. In silico studies suggested that the presence of the bulky tryptophan residue in the substrate-binding site of the rat SULT2A homolog instead of glycine as in human SULT2A1 may explain this species difference.
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http://dx.doi.org/10.1016/j.jsbmb.2017.05.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5554727PMC
September 2017

A multi-year study of hepatic biomarkers in coastal fishes from the Gulf of Mexico after the Deepwater Horizon Oil Spill.

Mar Environ Res 2017 Aug 27;129:57-67. Epub 2017 Apr 27.

Department of Medicinal Chemistry, University of Florida, Gainesville, FL, USA. Electronic address:

Following the 2010 Gulf of Mexico oil spill, concerns were raised regarding exposure of fish to crude oil components, particularly polycyclic aromatic hydrocarbons (PAHs). This three year study examined hepatic enzymes in post-mitochondrial supernatant fractions from red snapper (Lutjanus campechanus) and gray triggerfish (Balistes capriscus) collected in the north central Gulf of Mexico between 2011 and 2014. Biomarker activities evaluated included benzo(a)pyrene hydroxylase (AHH), ethoxyresorufin O-deethylase (EROD), glutathione transferase (GST), and glutathione peroxidase (GPx). Mean EROD activity was higher in gray triggerfish (12.97 ± 7.15 pmol/min/mg protein [mean ± SD], n = 115) than red snapper (2.75 ± 1.92 pmol/min/mg protein, n = 194), p < 0.0001. In both species, EROD declined over time between 2011 and 2014. Declines in GST and GPx activities were also noted over this time period for both species. Gray triggerfish liver was fatty, and heptane extracts of the liver fat contained fluorescent substances with properties similar to known PAHs, however the origin of these PAHs is unknown.
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http://dx.doi.org/10.1016/j.marenvres.2017.04.015DOI Listing
August 2017

A Mechanism-Based Pharmacokinetic Enzyme Turnover Model for Dichloroacetic Acid Autoinhibition in Rats.

J Pharm Sci 2017 05 3;106(5):1396-1404. Epub 2017 Feb 3.

Division of Pharmaceutics and Translational Therapeutics, College of Pharmacy, University of Iowa, Iowa 52242. Electronic address:

Dichloroacetic acid (DCA), a halogenated organic acid, is a pyruvate dehydrogenase kinase inhibitor that has been used to treat congenital or acquired lactic acidosis and is currently in early-phase clinical trials for cancer treatment. DCA was found to inhibit its own metabolism by irreversibly inactivating glutathione transferase zeta 1 (GSTZ1-1), resulting in nonlinear kinetics and abnormally high accumulation ratio after repeated dosing. In this analysis, a semi-mechanistic pharmacokinetic enzyme turnover model was developed for the first time to capture DCA autoinhibition, gastrointestinal region-dependent absorption, and time-dependent change in bioavailability in rats. The maximum rate constant for DCA-induced GSTZ1-1 inactivation is estimated to be 0.96/h, which is 110 times that of the rate constant for GSTZ1-1 natural degradation (0.00875/h). The model-predicted DCA concentration that corresponds to 50% of maximum enzyme inhibition (EC) is 4.32 mg/L. The constructed pharmacokinetic enzyme turnover model, when applied to human data, could be used to predict the accumulation of DCA after repeated oral dosing, guide selection of dosing regimens in clinical studies, and facilitate clinical development of DCA.
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http://dx.doi.org/10.1016/j.xphs.2017.01.032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5553688PMC
May 2017

Therapeutic applications of dichloroacetate and the role of glutathione transferase zeta-1.

Pharmacol Ther 2017 02 19;170:166-180. Epub 2016 Oct 19.

Department of Medicine, University of Florida, Gainesville, FL 32610-0226, United States; Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL 32610, United States.

Dichloroacetate (DCA) has several therapeutic applications based on its pharmacological property of inhibiting pyruvate dehydrogenase kinase. DCA has been used to treat inherited mitochondrial disorders that result in lactic acidosis, as well as pulmonary hypertension and several different solid tumors, the latter through its ability to reverse the Warburg effect in cancer cells and restore aerobic glycolysis. The main clinically limiting toxicity is reversible peripheral neuropathy. Although administration of high doses to rodents can result in liver cancer, there is no evidence that DCA is a human carcinogen. In all studied species, including humans, DCA has the interesting property of inhibiting its own metabolism upon repeat dosing, resulting in alteration of its pharmacokinetics. The first step in DCA metabolism is conversion to glyoxylate catalyzed by glutathione transferase zeta 1 (GSTZ1), for which DCA is a mechanism-based inactivator. The rate of GSTZ1 inactivation by DCA is influenced by age, GSTZ1 haplotype and cellular concentrations of chloride. The effect of DCA on its own metabolism complicates the selection of an effective dose with minimal side effects.
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http://dx.doi.org/10.1016/j.pharmthera.2016.10.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5274567PMC
February 2017

Genomic Effect of Triclosan on the Fetal Hypothalamus: Evidence for Altered Neuropeptide Regulation.

Endocrinology 2016 07 4;157(7):2686-97. Epub 2016 May 4.

Centro de Excelencia en Procesos y Productos de Córdoba (M.B.R.), National Scientific and Technical Research Council, Córdoba, Argentina X5164; Department of Physiology and Functional Genomics (E.I.C., C.E.W.), College of Medicine, University of Florida, Gainesville, Florida 32610; and Departments of Medicinal Chemistry (M.O.J.) and Pharmacodynamics (E.M.R., M.K.-W.), College of Pharmacy, University of Florida, Gainesville, Florida 32610.

Triclosan (TCS), an antibacterial compound commonly added to personal care products, could be an endocrine disruptor at low doses. Although TCS has been shown to alter fetal physiology, its effects in the developing fetal brain are unknown. We hypothesize that exposure to TCS during fetal life could affect fetal hypothalamic gene expression. The objective of this study was to use transcriptomics and systems analysis to identify significantly altered biological processes in the late gestation ovine fetal hypothalamus after direct or indirect exposure to low doses of TCS. For direct TCS exposure, chronically catheterized late gestation fetal sheep were infused with vehicle (n = 4) or TCS (250 μg/d; n = 4) iv. For indirect TCS exposure, TCS (100 μg/kg · d; n = 3) or vehicle (n = 3) was infused into the maternal circulation. Fetal hypothalami were collected after 2 days of infusion, and gene expression was measured through microarray. Hierarchical clustering of all samples according to gene expression profiles showed that samples from the TCS-treated animals clustered apart from the controls. Gene set enrichment analysis revealed that fetal hypothalamic genes stimulated by maternal and fetal TCS infusion were significantly enriching for cell cycle, reproductive process, and feeding behavior, whereas the inhibited genes were significantly enriching for chromatin modification and metabolism of steroids, lipoproteins, fatty acids, and glucose (P < .05). In conclusion, short-term infusion of TCS induces vigorous changes in the fetal hypothalamic transcriptomics, which are mainly related to food intake pathways and metabolism. If these changes persist to postnatal life, they could result in adverse consequences in adulthood.
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http://dx.doi.org/10.1210/en.2016-1080DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4929550PMC
July 2016

Pharmacogenetic considerations with dichloroacetate dosing.

Pharmacogenomics 2016 05 4;17(7):743-53. Epub 2016 May 4.

Department of Medicine, College of Medicine, University of Florida, Gainesville, FL 32610-0485, USA.

The investigational drug dichloroacetate (DCA) is a metabolic regulator that has been successfully used to treat acquired and congenital metabolic diseases and, recently, solid tumors. Its clinical use has revealed challenges in selecting appropriate doses. Chronic administration of DCA leads to inhibition of DCA metabolism and potential accumulation to levels that result in side effects. This is because conversion of DCA to glyoxylate is catalyzed by one enzyme, glutathione transferase zeta 1 (GSTZ1-1), which is inactivated by DCA. SNPs in the GSTZ1 gene result in expression of polymorphic variants of the enzyme that differ in activity and rates of inactivation by DCA under physiological conditions: these properties lead to considerable variation between people in the pharmacokinetics of DCA.
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http://dx.doi.org/10.2217/pgs-2015-0012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5558523PMC
May 2016

GSTZ1 expression and chloride concentrations modulate sensitivity of cancer cells to dichloroacetate.

Biochim Biophys Acta 2016 Jun 2;1860(6):1202-10. Epub 2016 Feb 2.

Department of Medicinal Chemistry, University of Florida, Gainesville, FL 32610-0485, United States. Electronic address:

Dichloroacetate (DCA), commonly used to treat metabolic disorders, is under investigation as an anti-cancer therapy due to its ability to reverse the Warburg effect and induce apoptosis in tumor cells. While DCA's mechanism of action is well-studied, other factors that influence its potential as a cancer treatment have not been thoroughly investigated. Here we show that expression of glutathione transferase zeta 1 (GSTZ1), the enzyme responsible for conversion of DCA to its inactive metabolite, glyoxylate, is downregulated in liver cancer and upregulated in some breast cancers, leading to abnormal expression of the protein. The cellular concentration of chloride, an ion that influences the stability of GSTZ1 in the presence of DCA, was also found to be abnormal in tumors, with consistently higher concentrations in hepatocellular carcinoma than in surrounding non-tumor tissue. Finally, results from experiments employing two- and three-dimensional cultures of HepG2 cells, parental and transduced to express GSTZ1, demonstrate that high levels of GSTZ1 expression confers resistance to the effect of high concentrations of DCA on cell viability. These results may have important clinical implications in determining intratumoral metabolism of DCA and, consequently, appropriate oral dosing.
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http://dx.doi.org/10.1016/j.bbagen.2016.01.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4837035PMC
June 2016

Celecoxib influences steroid sulfonation catalyzed by human recombinant sulfotransferase 2A1.

J Steroid Biochem Mol Biol 2015 Aug 7;152:101-13. Epub 2015 May 7.

Department of Medicinal Chemistry, College of Pharmacy, University of Florida, Gainesville, FL 32610-0485, USA. Electronic address:

Celecoxib has been reported to switch the human SULT2A1-catalyzed sulfonation of 17β-estradiol (17β-E2) from the 3- to the 17-position. The effects of celecoxib on the sulfonation of selected steroids catalyzed by human SULT2A1 were assessed through in vitro and in silico studies. Celecoxib inhibited SULT2A1-catalyzed sulfonation of dehydroepiandrosterone (DHEA), androst-5-ene-3β, 17β-diol (AD), testosterone (T) and epitestosterone (Epi-T) in a concentration-dependent manner. Low μM concentrations of celecoxib strikingly enhanced the formation of the 17-sulfates of 6-dehydroestradiol (6D-E2), 17β-dihydroequilenin (17β-Eqn), 17β-dihydroequilin (17β-Eq), and 9-dehydroestradiol (9D-E2) as well as the overall rate of sulfonation. For 6D-E2, 9D-E2 and 17β-Eqn, celecoxib inhibited 3-sulfonation, however 3-sulfonation of 17β-Eq was stimulated at celecoxib concentrations below 40 μM. Ligand docking studies in silico suggest that celecoxib binds in the substrate-binding site of SULT2A1 in a manner that prohibits the usual binding of substrates but facilitates, for appropriately shaped substrates, a binding mode that favors 17-sulfonation.
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http://dx.doi.org/10.1016/j.jsbmb.2015.05.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4501872PMC
August 2015

Chloride concentrations in human hepatic cytosol and mitochondria are a function of age.

Biochem Biophys Res Commun 2015 Apr 3;459(3):463-8. Epub 2015 Mar 3.

Department of Medicinal Chemistry, University of Florida, Gainesville, FL 32610-0485, United States. Electronic address:

We recently reported that, in a concentration-dependent manner, chloride protects hepatic glutathione transferase zeta 1 from inactivation by dichloroacetate, an investigational drug used in treating various acquired and congenital metabolic diseases. Despite the importance of chloride ions in normal physiology, and decades of study of chloride transport across membranes, the literature lacks information on chloride concentrations in animal tissues other than blood. In this study we measured chloride concentrations in human liver samples from male and female donors aged 1 day to 84 years (n = 97). Because glutathione transferase zeta 1 is present in cytosol and, to a lesser extent, in mitochondria, we measured chloride in these fractions by high-performance liquid chromatography analysis following conversion of the free chloride to pentafluorobenzylchloride. We found that chloride concentration decreased with age in hepatic cytosol but increased in liver mitochondria. In addition, chloride concentrations in cytosol, (105.2 ± 62.4 mM; range: 24.7-365.7 mM) were strikingly higher than those in mitochondria (4.2 ± 3.8 mM; range 0.9-22.2 mM). These results suggest a possible explanation for clinical observations seen in patients treated with dichloroacetate, whereby children metabolize the drug more rapidly than adults following repeated doses, and also provide information that may influence our understanding of normal liver physiology.
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http://dx.doi.org/10.1016/j.bbrc.2015.02.128DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4380591PMC
April 2015

The influence of human GSTZ1 gene haplotype variations on GSTZ1 expression.

Pharmacogenet Genomics 2015 May;25(5):239-45

aDepartment of Pharmacotherapy and Translational Research, Center for Pharmacogenomics, College of Pharmacy bDepartment of Medicinal Chemistry, College of Pharmacy cDepartment of Medicine, College of Medicine dDepartment of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida, USA eDepartment of Clinical Pharmacy, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt.

Background/objectives: The zeta-1 family isoform of GST biotransforms the investigational drug dichloroacetate (DCA) and certain other halogenated carboxylic acids. Haplotype variability in GSTZ1 influences the kinetics and, possibly, the toxicity of DCA. DCA metabolism correlates with expression of the GSTZ1 protein, so it is important to document variables that affect expression. Following up on a limited previous study, we tested the hypothesis that a coding single nucleotide polymorphism (SNP), the lysine (K) amino acid (E32>K) in GSTZ1 haplotypes linked to a promoter region SNP results in lower hepatic expression of GSTZ1.

Materials And Methods: The influence of K carrier and non-K carrier haplotypes on GSTZ1 expression was determined by analyzing 78 liver samples from individuals aged 7-84 years of various racial and ethnic backgrounds. GSTZ1 expression data were analyzed on the basis of the presence or absence of lysine 32.

Results: GSTZ1 protein expression differed significantly between K carrier and non-K carrier haplotypes (P=0.001) in Whites, but not in African-Americans (P=0.277). We attribute this difference in GSTZ1 expression among K carrier haplotypes in Whites to the linkage disequilibrium between the K or A allele from the G>A SNP (rs7975), within the promoter G>A-1002 SNP (rs7160195) A allele. There is no linkage disequilibrium between these two polymorphisms in African-Americans.

Conclusion: We conclude that the lower expression of GSTZ1 in Whites who possess the K carrier haplotype results in lower enzymatic activity and slower metabolism of DCA, compared with those who possess the non-K carrier haplotype. These results further define safe, genetics-based dosing regimens for chronic DCA administration.
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http://dx.doi.org/10.1097/FPC.0000000000000129DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4382440PMC
May 2015

Preliminary X-ray crystallographic analysis of glutathione transferase zeta 1 (GSTZ1a-1a).

Acta Crystallogr F Struct Biol Commun 2014 Feb 21;70(Pt 2):187-9. Epub 2014 Jan 21.

Department of Biochemistry and Molecular Biology, University of Florida, PO Box 100245, Gainesville, FL 32610, USA.

Glutathione transferase zeta 1 (GSTZ1-1) is a homodimeric enzyme found in the cytosol and mitochondrial matrix of the liver and other tissues. It catalyzes the glutathione-dependent isomerization of maleylacetoacetate to fumarylacetoacetate in the tyrosine catabolic pathway and can metabolize small halogenated carboxylic acids. GSTZ1a-1a crystals diffracted to a resolution of 3.1 Å and belonged to space group P1, with unit-cell parameters a = 42.0, b = 49.6, c = 54.6 Å, α = 82.9, β = 69.9, γ = 73.4°, with a calculated Matthews coefficient of 2.1 Å(3) Da(-1) assuming a dimer in the crystallographic asymmetric unit. Refinement of the structure is currently in progress.
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http://dx.doi.org/10.1107/S2053230X13033591DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3936459PMC
February 2014

Chloride and other anions inhibit dichloroacetate-induced inactivation of human liver GSTZ1 in a haplotype-dependent manner.

Chem Biol Interact 2014 May 13;215:33-9. Epub 2014 Mar 13.

Department of Medicinal Chemistry, University of Florida, Gainesville, FL 32610-0485, United States. Electronic address:

The in vivo elimination rate of dichloroacetate (DCA), an investigational drug; is determined by the rate of its biotransformation to glyoxylate, catalyzed by glutathione transferase ζ1 (GSTZ1). DCA is a mechanism-based inactivator of GSTZ1, thus elimination of DCA is slowed with repeated dosing. We observed that chloride, a physiologically important anion, attenuated DCA-induced GSTZ1 inactivation in human liver cytosol in a concentration and GSTZ1 haplotype-dependent way. In the absence of chloride, incubation with 0.5mM DCA resulted in inactivation of GSTZ1 with a half-life of 0.4h (samples with the KRT haplotype) to 0.5h (EGT haplotype). At the hepatic physiological chloride concentration, 38mM, samples with the EGT haplotype retained more activity (80%) following a 2-h incubation with 0.5mM DCA than those possessing the KRT haplotype (55%). The chloride concentration that protected 50% of the GSTZ1 activity following 2-h incubation with 0.5mM DCA (EC50) was 15.0±3.1mM (mean±S.D., n=3) for EGT samples and 36.2±2.2mM for KRT samples. Bromide, iodide and sulfite also protected GSTZ1 from inactivation by DCA, however fluoride, sulfate, carbonate, acetate, cyanide did not. Protection by bromide varied by GSTZ1 haplotype: EC50 was 1.3±0.3mM for the EGT haplotype and 5.0±0.60mM for the KRT haplotype. The EC50 values for iodide and sulfite in liver cytosol samples with EGT haplotype were respectively 0.14±0.06mM and 9.6±1.1mM (mean±S.D., n=3). Because the in vivo half-life of DCA is determined by the fraction of active GSTZ1 in the liver, identifying factors that regulate GSTZ1 activity is important in determining appropriate DCA dosing in humans.
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http://dx.doi.org/10.1016/j.cbi.2014.02.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4019600PMC
May 2014

Enzyme kinetics of conjugating enzymes: PAPS sulfotransferase.

Authors:
Margaret O James

Methods Mol Biol 2014 ;1113:187-201

Department of Medicinal Chemistry, University of Florida, Gainesville, FL, USA.

The sulfotransferase (SULT) enzymes catalyze the formation of sulfate esters or sulfamates from substrates that contain hydroxy or amine groups, utilizing 3'-phosphoadenosyl-5'-phosphosulfate (PAPS) as the donor of the sulfonic group. The rate of product formation depends on the concentrations of PAPS and substrate as well as the sulfotransferase enzyme; thus, if PAPS is held constant while varying substrate concentration (or vice versa), the kinetic constants derived are apparent constants. When studied over a narrow range of substrate concentrations, classic Michaelis-Menten kinetics can be observed with many SULT enzymes and most substrates. Some SULT enzymes exhibit positive or negative cooperativity during conversion of substrate to product, and the kinetics fit the Hill plot. A characteristic feature of most sulfotransferase-catalyzed reactions is that, when studied over a wide range of substrate concentrations, the rate of product formation initially increases as substrate concentration increases, then decreases at high substrate concentrations, i.e., they exhibit substrate inhibition or partial substrate inhibition. This chapter gives an introduction to sulfotransferases, including a historical note, the nomenclature, a description of the function of SULTs with different types of substrates, presentation of examples of enzyme kinetics with SULTs, and a discussion of what is known about mechanisms of substrate inhibition in the sulfotransferases.
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http://dx.doi.org/10.1007/978-1-62703-758-7_10DOI Listing
September 2014

Interactions of cytosolic sulfotransferases with xenobiotics.

Drug Metab Rev 2013 Nov;45(4):401-14

Department of Medicinal Chemistry, University of Florida, Gainesville , FL , USA.

Cytosolic sulfotransferases are a superfamily of enzymes that catalyze the transfer of the sulfonic group from 3'-phosphoadenosine-5'-phosphosulfate to hydroxy or amine groups in substrate molecules. The human cytosolic sulfotransferases that have been most studied, namely SULT1A1, SULT1A3, SULT1B1, SULT1E1 and SULT2A1, are expressed in different tissues of the body, including liver, intestine, adrenal, brain and skin. These sulfotransferases play important roles in the sulfonation of endogenous molecules such as steroid hormones and neurotransmitters, and in the elimination of xenobiotic molecules such as drugs, environmental chemicals and natural products. There is often overlapping substrate selectivity among the sulfotransferases, although one isoform may exhibit greater enzyme efficiency than other isoforms. Similarly, inhibitors or enhancers of one isoform often affect other isoforms, but typically with different potency. This means that if the activity of one form of sulfotransferase is altered (either inhibited or enhanced) by the presence of a xenobiotic, the sulfonation of endogenous and xenobiotic substrates for other isoforms may well be affected. There are more examples of inhibitors than enhancers of sulfonation. Modulators of sulfotransferase enzymes include natural products ingested as part of the human diet as well as environmental chemicals and drugs. This review will discuss recent work on such interactions.
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http://dx.doi.org/10.3109/03602532.2013.835613DOI Listing
November 2013

Pharmacokinetics of oral dichloroacetate in dogs.

J Biochem Mol Toxicol 2013 Dec 13;27(12):522-5. Epub 2013 Sep 13.

Small Animal Clinical Sciences, University of Florida, Gainesville, FL, 32610, USA.

We characterized the pharmacokinetics and dynamics of dichloroacetate (DCA), an investigational drug for mitochondrial diseases, pulmonary arterial hypertension, and cancer. Adult Beagle dogs were orally administered 6.25 mg/kg q12h DCA for 4 weeks. Plasma kinetics was determined after 1, 14, and 28 days. The activity and expression of glutathione transferase zeta 1 (GSTZ1), which biotransforms DCA to glyoxylate, were determined from liver biopsies at baseline and after 27 days. Dogs demonstrate much slower clearance and greater inhibition of DCA metabolism and GSTZ1 activity and expression than rodents and most humans. Indeed, the plasma kinetics of DCA in dogs is similar to humans with GSTZ1 polymorphisms that confer exceptionally slow plasma clearance. Dogs may be a useful model to further investigate the toxicokinetics and therapeutic potential of DCA.
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http://dx.doi.org/10.1002/jbt.21518DOI Listing
December 2013