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

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.

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

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., n = 6) for young and adult rats, respectively, with inactivation significantly more rapid in adult rats, p < 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 (p < 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.

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

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 Kic 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.

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

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.

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

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.

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

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.

Slow O-demethylation of methyl triclosan to triclosan, which is rapidly glucuronidated and sulfonated in channel catfish liver and intestine.

The antibacterial personal care product triclosan is discharged in municipal waste, and converted in part by bacteria in sewage sludge and soil to its more lipid-soluble methyl ether, methyl triclosan. Triclosan and methyl triclosan have been detected in water, sediment, fish and invertebrates near sewage treatment facilities. Understanding the biotransformation of methyl triclosan and triclosan in a model food fish, the channel catfish, will be of value in assessing the likelihood that these compounds will bioaccumulate in exposed fish, and therefore potentially pass up the food chain. We hypothesize that cytochrome P450 will catalyze the O-demethylation of methyl triclosan to yield triclosan, which is likely to undergo glucuronidation or sulfonation of the phenolic hydroxyl group. Conversion of methyl triclosan to triclosan was measured by LC/MS/MS following aerobic incubation of varying concentrations of methyl triclosan with NADPH and hepatic and intestinal microsomes from untreated, 3-methylcholanthrene-treated (10 mg/kg, i.p.) or PCB-126-treated (0.1 mg/kg, i.p.) channel catfish (n=4 per treatment group). The K(m) values for methyl triclosan were similar for untreated, 3-methylcholanthrene-treated and PCB-126-treated catfish liver microsomes, ranging from 80 to 250 µM. V(max) values for O-demethylation ranged from 30 to 150 pmol/min/mg protein, with no significant differences between controls, PCB-126-treated or 3-methylcholanthrene-treated fish, suggesting that methyl triclosan O-demethylation was not a CYP1-catalyzed reaction. Methyl triclosan O-demethylation activities in intestinal microsomes were similar to or lower than those found with liver microsomes. The calculated rate of O-demethylation of methyl triclosan in catfish liver at 1 µM, a concentration reported in exposed fish, and 21°C, an early summer water temperature, is 0.10 pmol/min/mg protein. This slow rate of metabolism suggests that upon continued exposure, methyl triclosan may bioaccumulate in the channel catfish. Triclosan itself, however, was readily glucuronidated by hepatic and intestinal microsomes and sulfonated by hepatic and intestinal cytosol. Triclosan glucuronidation followed Michaelis-Menten kinetics when rates were measured across a concentration range of 5-1000 µM, whereas triclosan sulfonation exhibited substrate inhibition at concentrations above 10-20 µM in both intestinal and hepatic cytosol. Based on the enzyme kinetic constants measured in hepatic and intestinal fractions at 21°C, triclosan at 1 µM could be glucuronidated at rates of 23 and 3.2 pmol/min/mg protein respectively in liver and intestine, and sulfonated at rates of 277 (liver) and 938 (intestine) pmol/min/mg protein. These rates are much higher than the rates of demethylation of methyl triclosan, and suggest that triclosan would be rapidly cleared and unlikely to bioaccumulate in catfish tissues.

Triclosan is a potent inhibitor of estradiol and estrone sulfonation in sheep placenta.

The personal care product Triclosan, 5-chloro-2(2,4-dichlorophenoxy)-phenol, is widely used in consumer products as an antibacterial agent and is increasingly found in the environment as a contaminant of sewage sludge and wastewater. This compound has been identified in plasma and urine of people in the United States, Sweden and Australia. Triclosan is known to inhibit sulfonation of phenolic xenobiotics and is structurally related to inhibitors of estrogen sulfotransferase, such as polychlorobiphenylols. In pregnancy, the placenta is an important source of estrogen, which is needed for normal fetal development and successful parturition, and estrogen sulfotransferase is thought to play an important role in regulation of estrogen availability. In this study, we examined the effect of Triclosan on sheep placental cytosolic sulfotransferase activity with 17-beta-estradiol and estrone as substrates. For comparison, we studied the effects of 4-hydroxy-3,3',4',5-tetrachlorobiphenyl and 2'-hydroxytriclocarban on estradiol sulfonation. The apparent K(m) for placental cytosolic sulfotransferase activity with estradiol as substrate was 0.27 ± 0.06 nM (mean ± S.D., n = 3 individuals) and with estrone as substrate was 1.86 ± 0.22 nM. Partial substrate inhibition was observed with estradiol at concentrations higher than 10-20 nM, as is typical of estrogen sulfotransferases (SULT1E1) in other species. Studies of the effect of Triclosan on estrogen sulfotransferase activity were conducted with several concentrations (0.1-6 nM) of estradiol and with 2 nM estrone. Triclosan was a very potent inhibitor of both estradiol and estrone sulfonation. For estradiol the inhibition was shown to be mixed competitive/uncompetitive, with K(ic) of 0.09 ± 0.01 nM and K(iu) of 5.2 ± 2.9 nM. The IC(50) for inhibition of estrone sulfonation was 0.60 ± 0.06 nM. At an environmentally relevant concentration of 1 µM, Triclosan was not a substrate for glucuronidation in sheep placental microsomes. Triclosan could be sulfonated in placental cytosol with K(m) 1.14 ± 0.18 µM and V(max) 160 ± 26 pmol/min/mg protein, however the calculated rates of Triclosan sulfonation were negligible at the low nM concentrations that potently inhibit estrogen sulfonation. The high potency of Triclosan as an inhibitor of estrogen sulfotransferase activity raises concern about its possible effects on the ability of the placenta to supply estrogen to the fetus, and in turn on fetal growth and development.

Properties and regional expression of a CYP3A-like protein in channel catfish intestine.

Biotransformation in the intestine may influence the bioavailability and toxicity of ingested xenobiotics. The objective of this study was to examine the expression and catalytic properties of a constitutive cytochrome P450 (CYP) 3A-like protein along the intestine of channel catfish, Ictalurus punctatus. Fish were maintained on commercial chow or nutritionally complete semi-purified diets. Polyclonal antibodies generated against rainbow trout CYP3A proteins reacted strongly with catfish washed intestinal microsomes on Western blots showing a major protein band with MW of 59 kDa. In catfish maintained on a standard chow diet, the expression of this protein was higher in the proximal segment (0.101 +/- 0.031 units/mg protein, mean +/- S.D., n = 4) than in the distal part (0.032 +/- 0.023 units/mg protein). Microsomal testosterone 6beta-hydroxylation activity was monitored as the catalytic indicator of CYP3A, and was higher in proximal than distal catfish intestine (263 +/- 80.3 and 88.6 +/- 15.6 pmol/min/mg protein for proximal and distal, respectively, mean +/- S.D., n = 4). CYP3A protein levels and testosterone 6beta-hydroxylation activities were lower in microsomes from the proximal segment of intestine from catfish maintained on a semi-purified diet, compared with commercial chow, but again the proximal intestine had higher CYP3A and 6beta-hydroxylase activities than distal intestine. Testosterone 6beta-hydroxylase activities in all samples correlated with the CYP3A protein levels, r2 = 0.8. Testosterone 6beta-hydroxylation was inhibited by specific CYP3A inhibitors, ketoconazole (IC50 = 0.02 microM) and erythromycin (IC50 = 41 microM), as well as general CYP inhibitors, metyrapone (IC50 = 2.8 microM) and SKF-525A (IC50 = 25 microM). There was evidence for the involvement of CYP3A in the mono-oxygenation of benzo(a)pyrene and of (-)-benzo(a)pyrene-7,8-dihydrodiol in intestinal microsomes from catfish maintained on the semi-purified diet. Mono-oxygenation of both substrates was increased in a concentration-dependent manner by in vitro addition of alpha-naphthoflavone. Benzo(a)pyrene hydroxylase activities were higher in proximal than in distal intestine; 3.72 +/- 0.77 pmol/min/mg protein, mean +/- S.D., n = 5 and 1.45 +/- 0.42 in these respective segments. The results of this study strongly suggest that CYP3A is important in the first pass metabolism of dietary xenobiotics in untreated fish.