GSTZ1 Expression Research Articles

Immunohistochemical approach to obesity disease in terms of expression levels of glutathione s-transferase (sigma, zeta, theta) isozymes

Objectives: Obesity is a complex multifactorial disease with recently increasing prevalence and incidence. Several studies have been conducted to explain the ethiology, pathophysiology, epidemiology, molecular and genetic mechanisms, and effective treatments of obesity. Glutathione S-transferase (GST) S1, GSTZ1, and GSTT1 are essential enzymes for oxidative stress and metabolism-related disorders. For this purpose, we aimed to reveal the role of GSTS1, GSTZ1, and GSTT1 in obesity. Methods: The gastric tissue samples were taken from the patients diagnosed with obesity who underwent bariatric surgery in Ankara Keçiören Training and Research Hospital General Surgery Clinic between 2017 and 2019. Immunostaining was performed on paraffin-embedded tissues to evaluate GSTS1, GSTZ1, and GSTT1 expressions. Laboratory data of the patients were recorded. All the results were analyzed statistically. Results: Weak GSTS1 expression was observed in 38.1% of tissues and moderate in 6.3%. 37.3% of the tissues presented weak GSTZ1 expression, and 11 (8.7%) displayed moderate. There were weak GSTT1 expressions in 7.1% of the tissues and moderate 0.8% of them. A positive and statistically significant correlation was observed between GSTS1 and GSTT1 expression levels ((r)=0.028, p = 0.010; p < 0.05). There were no significant differences between expression levels and gender, age, comorbidities, and medication usage (p > 0.05). Conclusions: GSTs, in particular GSTS1, GSTT1, and GSTZ1, might contribute to molecular mechanisms and the progression of obesity. In our study, GSTS1, GSTT1, and GSTZ1 were found to be moderately expressed in gastric tissues taken from obese patients. However, new studies using more samples and advanced techniques are needed to elucidate the relationship.

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.

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

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 GSTZ1 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 GSTZ1 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.

GSTZ1 deficiency promotes hepatocellular carcinoma proliferation via activation of the KEAP1/NRF2 pathway

BackgroundGlutathione S-transferase zeta 1 (GSTZ1) is the penultimate enzyme in phenylalanine/tyrosine catabolism. GSTZ1 is dysregulated in cancers; however, its role in tumorigenesis and progression of hepatocellular carcinoma (HCC) is largely unknown. We aimed to assess the role of GSTZ1 in HCC and to reveal the underlying mechanisms, which may contribute to finding a potential therapeutic strategy against HCC.MethodsWe first analyzed GSTZ1 expression levels in paired human HCC and adjacent normal tissue specimens and the prognostic effect of GSTZ1 on HCC patients. Thereafter, we evaluated the role of GSTZ1 in aerobic glycolysis in HCC cells on the basis of the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). Furthermore, we assessed the effect of GSTZ1 on HCC proliferation, glutathione (GSH) concentration, levels of reactive oxygen species (ROS), and nuclear factor erythroid 2-related factor 2 (NRF2) signaling via gain- and loss- of GSTZ1 function in vitro. Moreover, we investigated the effect of GSTZ1 on diethylnitrosamine (DEN) and carbon tetrachloride (CCl4) induced hepatocarcinogenesis in a mouse model of HCC.ResultsGSTZ1 was downregulated in HCC, thus indicating a poor prognosis. GSTZ1 deficiency significantly promoted hepatoma cell proliferation and aerobic glycolysis in HCC cells. Moreover, loss of GSTZ1 function depleted GSH, increased ROS levels, and enhanced lipid peroxidation, thus activating the NRF2-mediated antioxidant pathway. Furthermore, Gstz1 knockout in mice promoted DEN/CCl4-induced hepatocarcinogenesis via activation of the NRF2 signaling pathway. Furthermore, the antioxidant agent N-acetylcysteine and NRF2 inhibitor brusatol effectively suppressed the growth of Gstz1-knockout HepG2 cells and HCC progression in Gstz1−/− mice.ConclusionsGSTZ1 serves as a tumor suppressor in HCC. GSH depletion caused by GSTZ1 deficiency elevates oxidative stress, thus constitutively activating the NRF2 antioxidant response pathway and accelerating HCC progression. Targeting the NRF2 signaling pathway may be a promising therapeutic approach for this subset of HCC.

Influence of Dichloroacetate Treatment on the Contributions of Rodent Brain, Heart, Liver and Kidney in the Expression of GSTZ1

Glutathione transferase zeta 1 (GSTZ1)/maleylacetoacetate isomerase (MAAI) is a critical enzyme in tyrosine catabolism via its trans‐isomerization of maleylacetoacetate (MAA) and maleylacetone (MA). GSTZ1 also catalyzes the metabolism of dichloroacetate (DCA), an investigational drug utilized in the treatment of cancer and mitochondrial diseases. The pharmacology and toxicity of DCA are influenced, in part, by subject age. DCA is a mechanism‐based inactivator of GSTZ1, and repetitive DCA dosing results in the auto‐inhibition of DCA metabolism. Consequently, the chronic use of DCA in adults can lead to development of reversible peripheral neuropathy. However this effect is seldom observed in children, who metabolize DCA more quickly than adults. The tissue accumulation of DCA, MAA, and MA, due to GSTZ1 degradation, may be a mechanism responsible for DCA toxicity. Although liver is the main site of GSTZ1 expression and activity, extrahepatic tissues also express GSTZ1. Following a single dose of DCA to female rats, GSTZ1 expression and activity were reduced in liver to a greater extent than in kidney, brain and heart. However, the role of extrahepatic tissues in metabolism of DCA, MAA, and MA following repeated doses of DCA that mimic therapy has not been studied. Therefore, we examined the relative tissue expression of GSTZ1 following multiple doses of DCA. Adult female Sprague Dawley rats, 8 per group, and juvenile female Sprague Dawley rats, 4 per group, were treated with sodium DCA (100 mg/kg) or sodium acetate (100 mg/kg) by oral gavage daily for 8 consecutive days. On the 9th day the tissues were removed and used to prepare cytosolic and mitochondrial subcellular fractions. GSTZ1 expression was then assessed via Western blot using a polyclonal antibody to rat GSTZ1. In comparison to the same tissue in acetate‐treated adults, the expression of cytosolic GSTZ1 remaining in the liver, kidney, heart and brain of DCA‐treated adults was 2.8%, 9.8%, 8.6% and 5.7%, respectively, with liver retaining the highest expression at 0.030 ± 0.009 ng GSTZ1/μg protein (mean ± S.E.). Similarly, the expression of cytosolic GSTZ1 in DCA‐treated juveniles was greatest in liver (0.044 ± 0.014), whereas extrahepatic tissues were 20‐fold lower. Expression of mitochondrial GSTZ1 in DCA‐treated animals was highest in juvenile liver (0.016 ± 0.005) and adult kidney (0.048 ± 0.007). Considering the higher protein yield and larger size of the liver, compared with extrahepatic tissues, most DCA metabolism is expected to occur in the liver, even after repeated dosing. Given the greater proportion of liver to body weight in young rats, the hepatic metabolism of DCA is predicted to be augmented within this age group. This is consistent with the higher clearance of DCA in young populations, as children may retain a higher capacity for the GSTZ1‐catalyzed metabolism of DCA due to relative liver size.Support or Funding InformationSupported in part by the US Public Health Service GM 099871This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Hepatic GSTZ1 Expression in Pregnant Ewes and Their Offspring: Influence of Treatment with Dichloroacetate

Dichloroacetate (DCA) is an investigational drug that can stimulate mitochondrial energy metabolism by inhibiting pyruvate dehydrogenase kinase. We hypothesize that DCA could be used in high‐risk pregnancy to reduce perinatal mitochondrial deficiency and resulting cardiac dysfunction in at‐risk fetuses and are testing this postulate in a pregnant sheep model. DCA is metabolized by the enzyme glutathione transferase zeta1 (GSTZ1) to glyoxylate. GSTZ1 is not expressed in human fetal liver, but expression rises after birth. Furthermore, DCA inactivates GSTZ1, resulting in altered pharmacokinetics with repeated dosing. The objectives of this research were (1) to investigate the relative expression of GSTZ1 in maternal and fetal sheep liver from untreated controls close to full term (140 days gestation) and (2) to determine if treatment of sheep with DCA resulted in reduced expression of GSTZ1. We administered DCA intravenously to either the ewe or the fetus at daily divided doses of 25 mg/kg for three to five days prior to sacrifice 24 h after the last dose. We used samples of maternal and fetal liver to prepare cytosol and mitochondria and employed a custom‐made polyclonal antibody to rat GSTZ1 to measure expression relative to a single rat hepatic cytosol standard by Western blot. The sheep liver samples exhibited good cross‐reactivity to the rat antibody. Surprisingly, expression of GSTZ1 in the full‐term fetal liver cytosol fractions from controls was similar to that in the control maternal liver. Treatment with DCA resulted in reduced expression in both maternal and fetal liver cytosol fractions to less than 10% of the control values. In control fetuses, mitochondrial expression of GSTZ1 was 17.6 ± 7.6% of the cytosolic expression. In the DCA‐treated group, mitochondrial expression decreased and levels were undetectable in one‐half of the fetal sheep. These studies demonstrate that (1) GSTZ1 expression in liver cytosol and mitochondria in the late fetal period varies with animal species and (2) repeated treatment with DCA results in reduced GSTZ1 expression, which is likely to alter DCA pharmacokinetics.Support or Funding InformationSupported in part by the US Public Health Service, grant R21HD91599This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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

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 (P = 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.

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.

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

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.

Abstract A62: Chemoresistance through the intratumoral metabolism of dichloroacetate

Abstract Dichloroacetate (DCA), a small molecule 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. While DCA's mechanism of action is well-studied, other factors that influence its success as a cancer treatment have not been thoroughly studied. Here we present data showing that expression of glutathione transferase zeta 1 (GSTZ1), the enzyme responsible for the metabolism of DCA, is often misregulated in cancer, resulting in expression in tissues where there generally is none and a lack of expression in sites that have a high level of basal expression. Expression of GSTZ1 in a tumor would be expected to reduce intratumoral DCA concentrations. As we see a disconnect between GSTZ1 mRNA and protein expression in these tumors, control of expression is likely through miRNA. We also report that chloride concentrations, which strongly impact the stability of GSTZ1 in the presence of DCA, may be abnormal in tumors, further influencing DCA metabolism, concentration, and treatment success. Both two- and three-dimensional cell culture of cells expressing or not expressing GSTZ1 provide a proof of principle that GSTZ1 expression confers a level of resistance to DCA. These results have important clinical implications as GSTZ1 expression and chloride concentration could be used as markers to predict outcomes of DCA anticancer therapy. Citation Format: Stephan C. Jahn, Mohamed H. Solaymen, Ryan J. Lorenzo, Taimour Langaee, Peter W. Stacpoole, Margaret O. James. Chemoresistance through the intratumoral metabolism of dichloroacetate. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A62.

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

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. 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. 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. 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.

Prenatal and Postnatal Expression of Glutathione Transferase ζ 1 in Human Liver and the Roles of Haplotype and Subject Age in Determining Activity with Dichloroacetate

Glutathione transferase ζ 1 (GSTZ1), also known as maleylacetoacetate isomerase, catalyzes the penultimate step of tyrosine catabolism and metabolizes several α-halocarboxylic acids, including dichloroacetic acid (DCA), an investigational drug used for lactic acidosis and, recently, solid tumors. Age-related differences have been suggested in DCA pharmacotoxicology, but no information is available on GSTZ1 ontogeny in humans. Here, we investigated the cytosolic GSTZ1 developmental expression pattern and the influence of haplotype on GSTZ1 activity with DCA by using human livers from donors between 10 weeks gestation and 74 years. GSTZ1 expression was very low in fetal livers (<2 pmol of GSTZ1/mg cytosol). The expression began to increase after birth in an age-dependent manner until age 7 years. GSTZ1 was then sustained at stable, yet variable, levels (median, 20.0 pmol/mg cytosol; range, 4.8-47.3 pmol/mg cytosol) until age 74 years. GSTZ1 activity with DCA was strongly associated with haplotype and expression level. Samples homozygous or heterozygous for GSTZ1A exhibited ∼3-fold higher DCA dechlorinating activity than samples carrying other alleles at a given level of expression. The correlations (r²) between activity and expression were 0.90 and 0.68, respectively, for GSTZ1A carriers (n = 11) and noncarriers (n = 61). GSTZ1 is expressed in mitochondria in addition to cytosol. The GSTZ1A allele exhibited similar effects in the mitochondrial fraction by conferring a higher activity with DCA. In summary, we report a neonatal onset and an age-related increase in GSTZ1 protein expression during human liver development. Haplotype influenced GSTZ1 activity with DCA but not protein expression.

Polymorphisms in the human glutathione transferase zeta promoter

The zeta-class glutathione transferase GSTZ1-1 catalyses the glutathione-dependent isomerization of maleylacetoacetate to fumarylacetoacetate in the tyrosine catabolic pathway and the biotransformation of alpha-halo acids, including dichloroacetic acid (DCA). Genetic polymorphisms in the coding sequence of GSTZ1 result in significant changes in enzyme function, and deficiency of GSTZ1-1 in mice causes induction of a range of Phase-II enzymes. In this study, the potential for polymorphisms in regulatory sequences to alter gene transcription was investigated. A total of 10 single-nucleotide polymorphisms (SNP) were identified in African and Australian European subjects in a region extending 1.5-kb upstream of the GSTZ1 start of transcription. These SNPs formed at least 10 haplotypes and only two were shared between the two population samples. The effect of these SNPs on gene expression was evaluated by the transient expression of specific alleles fused to a luciferase reporter gene. Of the 10 SNPs identified, only -1002 G>A and -289 C>T caused significant changes in promoter activity. The -1002 G>A SNP converts a v-Myb site to a S8 homeodomain (Prx2) site, and the -289 C>T SNP abolishes an Egr1 binding site. These SNPs may alter GSTZ1 expression, which may alter the pharmacokinetics of DCA, which is used therapeutically for the treatment of lactic acidosis.