Secretion Of GSH Research Articles

Abstract 5154: Developing Micro-fluidic Chip to understand Altruistic stem cell reprogramming

Abstract Background:Tumor microenvironment (TME) is a complex network of cancer and stromal cell interactions in specialized niches. Cancer stem cells (CSCs) and mesenchymal stem cells (MSCs) co-exist as significant components of TME that contribute to tumor progression. However, it is not clear whether MSCs compete or co-operate with CSCs for niche occupancy. In principle, stem cell competition eliminates the competing neighbors (e.g. p53 mutant cancer cells) while stem cell altruism enhances group-fitness in the niche. We found that the conditioned media of oral cancer CSCs, ABCG2+ cells, induced stem cell altruism in CD271+ MSCs (1). This indirect demonstration suggests the potential role of stem cell altruism in TME mediated tumor progression. Here we hypothesize that CSC mediated stem cell altruism of MSCs can be studied using an organ-on-chip microfluidic device. Methods:Naïve CD271+ MSCs were maintained in vitro by using an established culture method that we previously reported (2). Development of organ-on-chip microfluidic platform involved coating of 96 wells with polydimethylsiloxane (PDMS) and squalene to enhance cellular adhesion and delay stem cell differentiation. Two wells were linked by microfluidic channels to facilitate cell-to-cell interaction. We used 1000 ABCG2+ CSCs obtained from SCC-25 cell line (3) and equal numbers of naïve CD271+ MSCs. CSCs co-cultured with MSC in a Boyden-chamber assay was used as control population. After two weeks of culture, MSCs were evaluated for potential reprogramming to altruistic stem cell (ASC) phenotype as previously described. Results:We observed that MSCs collected from organ-on-chip exhibit stem cell altruism as indicated by loss of p53 and gain of HIF-2alpha expression as measured by In-Cell western assay (1). These MSCs also exhibited secretion of GSH, another characteristic of ASCs. Subsequently, these cells activated p53/MDM2 oscillation, and underwent apoptosis/differentiation, another important characteristic of ASCs. While, MSCs cultured in Boyden chamber assay, showed rapid proliferation and loss of CD271+ expression, and gain of markers associated with carcinoma-associated-fibroblasts including SMA and cytokeratin 8. Conclusion:Overall, our findings suggest that microfluidics-based organ-on-chip device can be used to understand stem cell altruism in TME. 1. PMID:22689594.2. PMID: 288841133. Talukdar J et al In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 920. Note: This abstract was not presented at the meeting. Citation Format: Bidisha Pal, Sorra Sandhya, Joyeeta Talukdar, Lekhika Pathak, Hong Li, Bikul Das. Developing Micro-fluidic Chip to understand Altruistic stem cell reprogramming [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5154.

OP-35 - Glutathione S-transferase P influences the Nrf2-dependent control of cellular glutathione metabolism

Glutathione-S-transferase P-1 (GSTP) was recently proposed to interact with Nrf2 thus regulating its transcriptional activity. The possible consequences of such functional interaction on the metabolism and redox of cellular GSH remain unexplored and were investigated for the first time in this study using both genetic and pharmacological manipulation models of GSTP in murine embryonic fibroblasts (MEFs). The GSTP-/- genotype was associated with a higher nuclear translocation of Nrf2 and apoptotic signaling of MEFs, and then with a marked increase of Cys uptake that resulted in a higher de novo biosynthesis and secretion of cellular GSH. Se-organic compounds (SOC) further stimulated these responses with a potency that correlated with the thiol peroxidase activity of the compounds. A defective expression of GSTP and SOC treatments also influenced the oxidation of GSH to GSSG and protein glutathionylation (PSSG) that was confirmed to be a GSTP-dependent process. The transient manipulation of Nrf2 gene interfered with the SOC-stimulated synthesis and secretion of GSH in the GSTP-/- aplotype, but not in GSTP+/+ cells. In conclusion, the GSTP-Nrf2 interaction strongly influences the metabolism and redox-signaling function of cellular GSH.

Abstract 42: MYC through HIF-2α regulates the altruistic stemness program in human leukemia stem cells

Abstract Leukemia is hierarchically organized into two distinct population of cancer cells, cancer stem cells (CSCs) and non-stem cancer cells (NSCC). Numerous studies suggest that CSCs may express embryonic stem (ES) cells related stemness genes including Nanog, Sox-2, and Oct-4. However, potential cell surface markers that could specifically enrich the leukemia stem cell (LSC) subfraction based on the expression of these stemness genes have not yet been identified. Previously, we reported that ABCG2, a drug efflux pump, could enrich a subpopulation of cells exhibiting very high level of Nanog, Sox-2, and Oct-4 in ES cells. Additionally, these ABCG2+ ES cells also exhibited high HIF-2α, a transcription factor, which in cooperation with MYC-regulated both Nanog and Sox-2. In addition to these features, ABCG2+ cells demonstrated highly cytoprotective altruistic behavior by secreting high levels of glutathione. In the present study, we evaluated whether ABCG2+ subfraction of human LSCs exhibits high level of Nanog, Sox2, and Oct4. We also investigated the altruistic behavior of the LSCs, a potential novel mechanism of drug resistance and disease relapse. Methods: ABCG2+ cells from cervical lymph node and peripheral blood of T- cell acute lymphoblastic lymphoma/leukemia (T-ALL) and chronic myeloid leukemia (CML) patients (n=12) were enriched using immunomagnetic sorting. These cells were then expanded and used for different experiments including flow cytometry, ChiP on ChiP assay, in vivo transplantation assay, and siRNA inhibition treatment, to demonstrate the role of HIF-2α and MYC in regulating the altruistic stemness program in human LSCs. Results: We were able to enrich ABCG2+ cell subfraction from human T-ALL (n=5) and CML (n=7) patients exhibiting high levels of stemness genes such as Nanog, Sox-2, and Oct-4 in addition to MYC and HIF-2α. Higher engraftment potential was observed in ABCG2+ cells in comparison to ABCG2- cells as indicated by in vivo transplantation assay in NOD/SCID mice. The inhibition of stemness genes Nanog and Sox2 by siRNA gene silencing led to the loss of expression of MYC and HIF-2α, indicating that these genes are regulated MYC and HIF-2α. We then investigated the altruistic stemness phenotype of the ABCG2+ cells by studying the distinct molecular signature of MYC binding to HIF-2α in these cells. siRNA HIF-2α treatment led to ABCG2+ cell loss of proliferative capacity and reduced GSH levels, suggesting reduction of cytoprotective altruistic behavior in vitro. Additionally, siRNA HIF-2α treated ABCG2+ cells, when injected in mice, exhibited increased survival. Importantly, treatment of ABCG2+ cell harboring mice with FM19G11, a HIF inhibitor, led to marked loss in the secondary engraftment in NOD/SCID mice, thus indicating loss of ABCG2+ cell self-renewal capacity. Then we found that siRNA MYC inhibition led to loss of HIF-2α expression, suggesting that MYC might be regulating HIF-2α mediated altruistic stemness program in the ABCG2+ cells. Conclusion: To summarize, we found that ABCG2 is an excellent marker to enrich the LSC subfraction having high expression of stemness genes. The ABCG2+ LSCs exhibit altruistic stemness phenotype that includes high secretion of GSH and the distinct binding of MYC to HIF-2a, Sox2, and Nanog. Thus, we now report that a subfraction of LSC could also be enriched such that it exhibits altruistic stemness phenotype which was previously reported by us in ES cells. Our findings may open up new understanding of the LSC-mediated cancer relapse and drug resistance. Citation Format: Bidisha Pal, Anupam Sarma, Joyeeta Talukdar, Seema Bhuyan, Sora Sandhya, Sukanya Gayan, Gayatri Gogoi, Debabrat Baishya, Amal Chandra Kataki, Dean W. Felsher, Bikul Das. MYC through HIF-2α regulates the altruistic stemness program in human leukemia stem cells [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr 42.

Abstract 251: Stem cell altruism may serve as a novel drug resistance mechanism in oral cancer

Abstract Background: The mechanism of oral squamous cell carcinoma resistance to platinum-based chemotherapy is not clearly known. Previous studies indicated that glutathione (GSH), a cellular antioxidant may detoxify cisplatinum (CDDP), a commonly used chemotherapy agent in oral cancer. Our previous research in human embryonic stem cells (hESCs) indicated the altruistic behavior of ABCG2+ hESCs that secrete high level of GSH to protect other hESCs exposed to oxidative stress (Das B et al. Stem Cells, 2012). Here we investigated if CDDP exposure lead to altruistic stem cell reprogramming of ABCG2+ oral squamous cell carcinoma cells and subsequent GSH-mediated resistance against CDDP. Methods: Two oral squamous cell cancer cell lines SCC-25 and SCC-15 were treated with 3-10 uM CDDP for three-days, and then subjected to flow cytometry and immunomagnetic sorting based evaluation of ABCG2+ cells. The conditioned media (CM) obtained from ABCG2+ cells were examined for GSH content. The CM treated cancer cell lines were examined for resistance against CDDP toxicity. Next, the post-CDDP treated ABCG2+ cells were examined for enhanced stemness phenotype that corresponds to altruistic stem cell phenotype (Das B et al, Stem Cells 2012). Results: We found that CDDP treatment increases the ABCG2+ self-renewal capacity of SCC-25 and SCC-15 cells as measured by serial transplantation assay. The CM of the post-CDDP treated cells exhibited high level of GSH. When the SCC-15 and SCC-25 cells were treated with CM plus CDDP, the cancer cells exhibited 10-15-fold increase in resistance against CDDP toxicity. Next, the post-CDDP treated SCC-25 and SCC-15 cells exhibited enhanced stemness reprogramming phenotype characterized by very high HIF-2alpha, Sox-2 and Nanog transcriptional activity. Furthemore, we found increased expression of EMT (epithelial mesenchymal transition) marker expression including Snail, Twist and vimentin as evaluated by flow cytometry. siRNA HIF-2alpha treatment led to marked loss in the in vivo self-renewal capacity of ABCG2+ SCC-25 and SCC-15 cells. We then noted that post-CDDP ABCG2+ cells exhibited reversible state, as after two weeks of culture, most of the cells either differentiated or underwent apoptosis. Conclusions: These results indicate that oral cancer cells exhibit altruistic defense mechanism to resist the toxicity of CDDP. The altruistic defense mechanism involved high secretion of GSH. Thus, we suggest that similar to bacterial altruism as a mechanism of drug resistance, stem cell altruism may also serve as a novel mechanism of drug resistance in cancer. Citation Format: Bidisha Pal, Reza Bayat-Mokhtari, Hong Li, Rashmi Bhuyan, Joyeeta Talukdar, Sora Sandhya, Anupam Sarma, Wael Tasabehji, Seema Bhuyan, Sukanya Gayan, Amal Ch Kataki, Debabrata Baishya, Herman Yeger, Dean W. Felsher, Bikul Das. Stem cell altruism may serve as a novel drug resistance mechanism in oral cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 251.

S-CMC-Lys-dependent stimulation of electrogenic glutathione secretion by human respiratory epithelium

Glutathione (GSH) is one of the most important defense mechanisms against oxidative stress in the respiratory epithelial lining fluid. Considering that GSH secretion in respiratory cells has been postulated to be at least partially electrogenic, and that the mucoregulator S-carbocysteine lysine salt monohydrate (S-CMC-Lys) can cause an activation of epithelial Cl(-) conductance, the purpose of this study was to verify whether S-CMC-Lys is able to stimulate GSH secretion. Experiments have been performed by patch-clamp technique, by high-performance liquid chromatography (HPLC) assay, and by Western blot analysis on cultured lines of human respiratory cells (WI-26VA4 and CFT1-C2). In whole-cell configuration, after cell exposure to 100 microM S-CMC-Lys, a current due to an outward GSH flux was observed, which was inhibitable by 5-nitro-2-(3-phenylpropylamino)-benzoate and glibenclamide. This current was not observed in CFT1-C2 cells, where a functional cystic fibrosis transmembrane conductance regulator (CFTR) is lacking. Inside-out patch-clamp experiments (GSH on the cytoplasm side, Cl(-) on the extracellular side) showed the activity of a channel, which was able to conduct current in both directions: the single channel conductance was 2-4 pS, and the open probability (P(o)) was low and voltage-independent. After preincubation with 100 microM S-CMC-Lys, there was an increase in P(o), in the number of active channels present in each patch, and in the relative permeability to GSH vs Cl(-). Outwardly directed efflux of GSH could also be increased by protein kinase A, adenosine 5'-triphosphate, and cyclic adenosine monophosphate (cAMP) added to the cytoplasmic side (whole-cell configuration). The increased secretion of GSH observed in the presence of S-CMC-Lys or 8-bromoadenosine-3',5'-cyclic monophosphate was also confirmed by HPLC assay of GSH on a confluent monolayer of respiratory cells. Western blot analysis confirmed the presence of CFTR in WI-26VA4 cells. This study suggests that S-CMC-Lys is able to stimulate a channel-mediated GSH secretion by human respiratory cells: electrophysiological and pharmacological characteristics of this channel are similar to those of the CFTR channel.

Impairment of conjunctival glutathione secretion and ion transport by oxidative stress in an adenovirus type 5 ocular infection model of pigmented rabbits

Conjunctival epithelial cells of pigmented rabbits secrete reduced glutathione (GSH) into the apical (mucosal) fluid. The aim of the current study was to determine the effect of oxidative stress resulting from viral infection and that of GSH supplementation on redox status, GSH, and ion transport in freshly excised conjunctival tissues and epithelial cell layers in primary culture (RCEC) of adenovirus type 5 (Ad5)-infected rabbits. Lipid peroxidation (LPO) products, nitric oxide (NO), and expression of nitric oxide synthase (NOS2) were quantitated as a function of time after viral inoculation. Unidirectional fluxes of [ 3H]GSH and changes in short-circuit current ( I sc) from mucosal supplementation of Ad5-inoculated conjunctival tissues with GSH and glutathione monoethyl ester (GSH-MEE) were also measured. Ad5 inoculation significantly decreased conjunctival GSH level by 19, 45, 48, and 50% at 8, 24, 48, and 72 h postinfection, respectively. LPO product and NO levels increased significantly (2- and 100-fold, respectively) above that of uninfected controls on Day 3 post-Ad5 inoculation, and co-treatment with GSH-MEE and tocopherol succinate abolished this effect. NO levels showed a progressive increase post-Ad5 inoculation, reaching 0.22 ± 0.06, 8.12 ± 0.91, and 2.05 ± 0.65 μM on Days 1, 3, and 5, respectively, and the highest level was observed on the day of maximal viral replication (Day 3). A very significant induction of the expression of NOS2 on Days 1, 3, and 5 post-Ad5 inoculation was observed. Uninfected control conjunctival tissues displayed a net serosal-to-mucosal GSH flux ( J sm), where the mucosal-to-serosal flux ( J ms) was ∼14 pmol h −1 cm −2 and the J sm was ∼22 pmol h −1 cm −2. In Ad5-inoculated rabbits similar GSH flux was observed in both the sm and ms directions, and the net GSH flux was negligible. I sc and potential difference (PD) across conjunctival tissues of Ad5-inoculated rabbits decreased by ≥50% compared with control, while the transepithelial electrical resistance (TEER) remained unchanged. Mucosal, but not serosal, superfusion of GSH or GSH-MEE in Ad5-inoculated conjunctival tissues increased the I sc by up to 40% in ∼100 min. Our results show that net secretion of GSH across rabbit conjunctiva is totally blocked after Ad5 inoculation and active ion transport rate decreased by ∼50%. Decreased net GSH secretion into mucosal fluid after Ad5 infection may have resulted from a decreased intracellular GSH pool due to oxyradical-induced changes in redox status and lower active ion transport. Mucosal treatment of Ad5-infected conjunctival tissues with pharmacological levels of GSH appears to transstimulate mucosal GSH secretion and restore active ion transport activity, suggesting a potentially useful therapeutic regimen for ocular infections.

Impairment of conjunctival glutathione secretion and ion transport by oxidative stress in an adenovirus type 5 ocular infection model of pigmented rabbits*1

Conjunctival epithelial cells of pigmented rabbits secrete reduced glutathione (GSH) into the apical (mucosal) fluid. The aim of the current study was to determine the effect of oxidative stress resulting from viral infection and that of GSH supplementation on redox status, GSH, and ion transport in freshly excised conjunctival tissues and epithelial cell layers in primary culture (RCEC) of adenovirus type 5 (Ad5)-infected rabbits. Lipid peroxidation (LPO) products, nitric oxide (NO), and expression of nitric oxide synthase (NOS2) were quantitated as a function of time after viral inoculation. Unidirectional fluxes of [3H]GSH and changes in short-circuit current (Isc) from mucosal supplementation of Ad5-inoculated conjunctival tissues with GSH and glutathione monoethyl ester (GSH-MEE) were also measured. Ad5 inoculation significantly decreased conjunctival GSH level by 19, 45, 48, and 50% at 8, 24, 48, and 72 h postinfection, respectively. LPO product and NO levels increased significantly (2- and 100-fold, respectively) above that of uninfected controls on Day 3 post-Ad5 inoculation, and co-treatment with GSH-MEE and tocopherol succinate abolished this effect. NO levels showed a progressive increase post-Ad5 inoculation, reaching 0.22 ± 0.06, 8.12 ± 0.91, and 2.05 ± 0.65 μM on Days 1, 3, and 5, respectively, and the highest level was observed on the day of maximal viral replication (Day 3). A very significant induction of the expression of NOS2 on Days 1, 3, and 5 post-Ad5 inoculation was observed. Uninfected control conjunctival tissues displayed a net serosal-to-mucosal GSH flux (Jsm), where the mucosal-to-serosal flux (Jms) was ∼14 pmol h−1 cm−2 and the Jsm was ∼22 pmol h−1 cm−2. In Ad5-inoculated rabbits similar GSH flux was observed in both the sm and ms directions, and the net GSH flux was negligible. Isc and potential difference (PD) across conjunctival tissues of Ad5-inoculated rabbits decreased by ≥50% compared with control, while the transepithelial electrical resistance (TEER) remained unchanged. Mucosal, but not serosal, superfusion of GSH or GSH-MEE in Ad5-inoculated conjunctival tissues increased the Isc by up to 40% in ∼100 min. Our results show that net secretion of GSH across rabbit conjunctiva is totally blocked after Ad5 inoculation and active ion transport rate decreased by ∼50%. Decreased net GSH secretion into mucosal fluid after Ad5 infection may have resulted from a decreased intracellular GSH pool due to oxyradical-induced changes in redox status and lower active ion transport. Mucosal treatment of Ad5-infected conjunctival tissues with pharmacological levels of GSH appears to transstimulate mucosal GSH secretion and restore active ion transport activity, suggesting a potentially useful therapeutic regimen for ocular infections.

Nifedipine modulation of biliary GSH and GSSG/ conjugate efflux in normal and regenerating rat liver.

Canalicular glutathione secretion provides the major driving force for bile acid-independent bile flow (BAIF), although the pathways involved are not established. The hypothesis that GSH efflux proceeds by a route functionally distinct from the high-affinity, low-capacity, mrp2-mediated pathway was tested by using perfused rat liver and three choleretic compounds that modify biliary secretion of GSH (the dihydropyridine nifedipine and organic anion probenecid) or GSSG [sodium nitroprusside (SNP)]. Whereas nifedipine (30 microM) stimulated GSH secretion and blocked SNP-stimulated GSSG efflux and choleresis, SNP (1 mM) was ineffective against nifedipine-stimulated GSH efflux or BAIF, suggesting that most GSSG exits through a GSH-inhibitable path independent of high-affinity GSSG/glutathione conjugate transport. Three observations support this proposal. SNP, but not nifedipine, significantly inhibited bromosulfophthalein (BSP, 1 microM) excretion. Probenecid (1 mM) blocked resting or nifedipine-stimulated GSH secretion but only weakly inhibited BSP excretion. Glutathione, but not BSP, efflux capacity was reduced following partial hepatectomy. We suggest GSH efflux is mediated by a high-capacity organic anion pathway capable of GSSG transport when its high-affinity route is saturated.

Hepatic and extra-hepatic stimulation of glutathione release into plasma by norepinephrine in vivo

Studies were conducted to determine the effect of norepinephrine (NE) on reduced glutathione (GSH) and oxidized glutathione (GSSG) export from hepatic and extra-hepatic tissues in vivo. Anesthetized Single Comb White Leghorn (SCWL) males were implanted with cannulae in the carotid artery, hepatic vein (HV) and hepatic portal veins (PV), and the left bile duct. In Experiment 1, GSH and GSSG in hepatic and portal venous plasma and bile were determined prior to, during, and following two 20-min infusions of NE (2 and 10 microg/min per kg BW) into the hepatic PV. The lower NE infusion rate increased hepatic venous GSH (indicative of increased GSH export into liver sinusoids) without affecting systemic or hepatic vascular pressures; however, it had no affect on portal venous GSH. The higher NE infusion rate increased GSH in the HV and hepatic PV (indicative of extra hepatic export of glutathione) as well as systemic pressure, hepatic and portal venous pressures, and the transhepatic pressure gradient. Biliary secretion of GSH and GSSG was unaffected by either rate of NE infusion in Experiment 1. In Experiment 2, pretreatment of birds with phentolamine, an alpha-adrenergic receptor blocker (alpha-block), abolished sinusoidal export GSH as well as the ability of NE to stimulate GSH release from hepatic and extra-hepatic tissue. Although HV and PV pressures were lower in alpha-block birds compared with controls, there were no differences in the transhepatic pressure gradient between groups. Plasma GSSG was below the limits of detection in Experiments 1 and 2. The combined results of Experiments 1 and 2 indicate that hepatic export of GSH was independent of changes in systemic or hepatic vascular pressures or changes in the transhepatic pressure gradient. The results of these studies are the first to demonstrate that export of GSH into plasma in vivo is mediated by an alpha-receptor-mediated mechanism in hepatic and extra-hepatic tissues. The findings may be particularly important with regard to antioxidant homeostasis of animals during periods of stress.

Induction of the multispecific organic anion transporter (cMoat/mrp2) gene and biliary glutathione secretion by the herbicide 2,4,5-trichlorophenoxyacetic acid in the mouse liver

The canalicular multispecific organic anion transporter, cMoat, is an ATP-binding-cassette protein expressed in the canalicular domain of hepatocytes. In addition to the transport of endo- and xenobiotics, cMoat has also been proposed to transport GSH into bile, the major driving force of bile-acid-independent bile flow. We have shown previously that the herbicide 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), a peroxisome-proliferator agent, significantly increases bile-acid-independent bile flow in mice. On this basis, the effect of the herbicide on cMoat gene expression was studied. A 3.6-fold increase in cMoat mRNA levels and a 2.5-fold increase in cMoat protein content were observed in the liver of mice fed on a diet supplemented with 0.125% 2,4,5-T. These effects were due to an increased rate of gene transcription (3.9-fold) and were not associated with peroxisome proliferation. Significant increases in bile flow (2.23±0.39 versus 1.13±0.15 μl/min per g of liver; P < 0.05) and biliary GSH output (7.40±3.30 versus 2.65±0.34 nmol/min per g of liver; P < 0.05) were observed in treated animals. The hepatocellular concentration of total glutathione also increased in hepatocytes of treated mice (10.95±0.84 versus 5.12±0.47 mM; P < 0.05), because of the induction (2.4-fold) of the heavy subunit of the γ-glutamylcysteine synthetase (GCS-HS) gene. This is the first model of co-induction of cMoat and GCS-HS genes in vivo in the mouse liver, associated with increased glutathione synthesis and biliary glutathione output. Our observations are consistent with the hypothesis that the cMoat transporter plays a crucial role in the secretion of biliary GSH.

Induction of the multispecific organic anion transporter (cMoat/mrp2) gene and biliary glutathione secretion by the herbicide 2,4,5-trichlorophenoxyacetic acid in the mouse liver

The canalicular multispecific organic anion transporter, cMoat, is an ATP-binding-cassette protein expressed in the canalicular domain of hepatocytes. In addition to the transport of endo- and xenobiotics, cMoat has also been proposed to transport GSH into bile, the major driving force of bile-acid-independent bile flow. We have shown previously that the herbicide 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), a peroxisome-proliferator agent, significantly increases bile-acid-independent bile flow in mice. On this basis, the effect of the herbicide on cMoat gene expression was studied. A 3.6-fold increase in cMoat mRNA levels and a 2.5-fold increase in cMoat protein content were observed in the liver of mice fed on a diet supplemented with 0.125% 2,4,5-T. These effects were due to an increased rate of gene transcription (3.9-fold) and were not associated with peroxisome proliferation. Significant increases in bile flow (2.23+/-0.39 versus 1.13+/-0.15 microl/min per g of liver; P<0.05) and biliary GSH output (7.40+/-3.30 versus 2.65+/-0.34 nmol/min per g of liver; P<0.05) were observed in treated animals. The hepatocellular concentration of total glutathione also increased in hepatocytes of treated mice (10.95+/-0.84 versus 5.12+/-0.47 mM; P<0.05), because of the induction (2.4-fold) of the heavy subunit of the gamma-glutamylcysteine synthetase (GCS-HS) gene. This is the first model of co-induction of cMoat and GCS-HS genes in vivo in the mouse liver, associated with increased glutathione synthesis and biliary glutathione output. Our observations are consistent with the hypothesis that the cMoat transporter plays a crucial role in the secretion of biliary GSH.

Glutathione efflux associated with a low γ-glutamyl transpeptidase activity in human melanoma cells

The cellular concentration of reduced glutathione (GSH) modulates the sensitivity of human melanoma cells to alkylating drugs in vitro. To investigate whether the membrane-associated enzyme gamma-glutamyl transpeptidase (gamma-GTP) involved in GSH breakdown was expressed in melanoma cells, the enzymatic activity of gamma-GTP as well as the secretion of GSH were measured in human melanoma cells from four different cell lines (Me8, JUSO, GLL19, Swift). All the cells showed low gamma-GTP activities (0-1 mU/mg protein) and released GSH in culture supernatants at significant rates. After incubation for 24 h in growth medium containing 0.1 mmol/L cystine, the levels of GSH in supernatants ranged from 56 to 111 nmol GSH/mg protein. The GSH metabolism of melanoma cells was also evaluated by measuring the levels of the melanogenesis intermediate 5-S-cysteinyldopa under different experimental conditions. The results of these experiments suggest that melanoma cells have a low ability to metabolize the tripeptide GSH, which appears to be responsible for GSH secretion and accumulation in culture supernatants.

Inhibitory Effect of Selenium on Biliary Secretion of Methyl Mercury in Rats

The inhibitory effect of sodium selenite on biliary secretion of methyl mercury was examined in rats. The biliary secretion of methyl mercury in rat treated with 1 μmol/kg of methyl mercury was significantly decreased by administration of selenite at doses of 0.05 μmol/kg or higher. In rats given 10 μmol/kg of methyl mercury, marked depression of biliary secretion of mercury was observed when selenite was injected at a dose of 0.2 μmol/kg. On the other hand, secretion of substantial amounts of selenium was observed when biliary secretion of mercury was depressed. When the concentration of selenium in the bile was higher than 5 nmol/ml, biliary secretion of mercury was markedly depressed independently of the dose of methyl mercury administered (1 μmol/kg or 10 μmol/kg). These results suggest that the degree of inhibitory effect of selenite may be determined by the selenium concentration in the liver or the bile after treatment with selenite rather than the molar ratio of the dose of methyl mercury and selenite. We concluded that the decrease in biliary secretion of methyl mercury induced by selenite may result from inhibition of pathway for secretion of methyl mercury from liver to bile rather than the direct formation of a compex between methyl mercury and selenium. Methyl mercury has been considered to be secreted from liver to bile as a complex with glutathione (GSH). However, administration of selenite did not affect biliary secretion of GSH or hepatic glutathione S-transferase activity. Moreover, gel filtration of liver cytosol demonstrated that the distribution pattern of hepatic methyl mercury between macromolecules and GSH was not significantly changed by administration of selenite. These results suggest that selenite does not affect complex formation of methyl mercury with GSH at least in the liver. Selenite might specifically inhibit the activity of the canalicular transporter(s) which transport complexes of methyl mercury and GSH from the liver to bile.

Secretion of cysteine and glutathione from mucosa to lumen in rat small intestine

Using a vascularly perfused rat intestinal preparation, we found that large quantities (i.e., 100-200 microM) of acid-soluble thiols accumulated in the jejunal lumen in 10-30 min and that the accumulation was largely unaffected by dietary food restriction for 24 or 48 h. Depending on the length of perfusion, cysteine comprised 20-40% of total luminal thiols, whereas glutathione (GSH) made up only 0-3%. To determine whether luminal cysteine accumulation resulted from mucosal secretion of GSH and subsequent degradation by brush-border gamma-glutamyltransferase (gamma-GT) and dipeptidases, acivicin or serine-borate was used to inhibit gamma-GT. Both agents inhibited gamma-GT activity by > 95%, reduced luminal cysteine by approximately 40-50%, and caused a modest elevation of luminal GSH to approximately 10-13 microM, indicating that GSH secretion does occur but cannot account for all of the luminal cysteine accumulation. Luminal thiol trapping experiments with Ellman's reagent supported this conclusion. Given that cysteine made up 15-20% of the mucosal thiol pool in jejunum, secretion of cysteine from mucosa to lumen likely accounted for the majority of luminal cysteine. Given the mucolytic nature of thiols and the role of cysteine in iron absorption, intestinal thiol secretion may be important in intestinal function.

Glutathione metabolism in the pancreas compared with that in the liver, kidney, and small intestine

The pancreas plays a major role, along with the kidney, liver, small intestine, and several other organs, in glutathione (GSH) metabolism, as evidenced by the large concentration of GSH in the pancreas, its rapid turnover rate, and the presence, at significant levels, of various enzymes involved in GSH metabolism. The pancreas appears to obtain much of the cysteine that is required for both GSH and protein synthesis by hydrolyzing plasma GSH to its constituent amino acids and then transporting cysteine into the cells. GSH hydrolysis is accomplished by the ectoenzymes gamma-glutamyl transferase (GGTase) and aminopeptidase N, both of which are present in the pancreas. Only the kidney has a greater GGTase activity. Although pancreatic GSH synthesis has not been directly demonstrated, pancreatic secretory protein synthesis is substantial, and these proteins contain significant amounts of cysteine as disulfides. The pancreas also contains significant levels of protein disulfide isomerase, glutathione peroxidase, and NADPH:GSH oxidoreductase. Protein disulfide isomerase, using oxidized glutathione generated by glutathione peroxidase, is important in the formation of disulfide bonds in secretory proteins in the pancreas. No other organ has a higher specific activity of protein disulfide isomerase. By analogy with kidney and liver, the pancreas presumably exhibits a rapid apical secretion of GSH. The purpose of this apical secretion is unknown in the kidney. In the liver, it is important in bile secretion. The large GGTase activity of apical plasma membranes in the pancreas is likely to be instrumental in the hydrolysis, and subsequent recovery of the constituent amino acids of apically secreted GSH, as occurs in the kidney and liver.

Renal tubular transport of glutathione in rat kidney.

Filtered glutathione (gamma-glutamyl-cysteinyl-glycine or GSH) is rapidly hydrolyzed by brush-border enzymes facing the tubular lumen and is reabsorbed in the form of the constituent amino acids. The first step of hydrolysis is catalyzed by gamma-glutamyltransferase (gamma-GT). We investigated localization and capacity of the rat renal glutathione degradation/reabsorption during elevation of the filtered load (intravenous infusion of 12 resp. 18 mumol GSH/min). Fractional excretion went up from about 0.003 to 0.31 +/- 0.02 SEM during infusion of the lower and to 0.49 +/- 0.03 SEM during infusion of the higher glutathione dose. GSH degradation/reabsorption took place along the entire proximal tubule and was partially saturated by a 150-200-fold elevation of the normal filtered load. Net reabsorption of GSH up to the last accessible superficial loop was significantly lower during infusion of 18 mumol GSH/min (0.3 mumol/min) than during infusion of 12 mumol GSH/min (1.6 mumol/min). In further experiments, infusion of 18 mumol GSH/min was preceded by the i.v. administration of acivicin (0.5 mmol/kg body wt.), an inhibitor of gamma-GT. In these experiments, fractional glutathione deliveries to late proximal and early distal tubules did not significantly differ from 1, fractional excretion of GSH at the same time was 1.46 +/- 0.11 SEM, revealing net secretion of GSH with the final urine. Tubular secretion of GSH in the acivicin-treated animals occurred either in distal tubules and/or collecting ducts or in the proximal tubules of deep nephrons which are not accessible to micropuncture.(ABSTRACT TRUNCATED AT 250 WORDS)

The mechanism of biliary secretion of reduced glutathione. Analysis of transport process in isolated rat-liver canalicular membrane vesicles.

Transport of reduced glutathione (GSH) was studied in isolated rat liver canalicular membrane vesicles by a rapid filtration technique. The membrane vesicles exhibit uptake of [2-3H]glycine--labeled GSH into an osmotically reactive intravesicular space. Although the canalicular membrane vesicles possess gamma-glutamyltransferase and aminopeptidase M, enzymes that hydrolyze glutathione into component amino acids, inactivation of the vesicle-associated transferase by affinity labeling with L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (AT-125) had no effect on the initial rate of GSH transport. Chemical analysis revealed that intact GSH accounted for most of vesicle-associated radioactivity. The initial rate of transport followed saturation kinetics with respect to GSH concentration; an apparent Km of 0.33 mM and V of 1.47 nmol/mg protein in 20 s were calculated. These results indicate that transport of GSH across the canalicular membranes is a carrier-mediated process. Replacement of NaCl in the transport medium by KCl, LiCl or choline chloride had no effect on the transport activity of the vesicles. The rate of GSH uptake by the vesicles was enhanced by valinomycin-induced K+-diffusion potential (vesicle inside-positive) and was inhibited by probenecid, indicating that GSH transport across the canalicular membranes is electrogenic and involves the transfer of negative charge. The transport of GSH was inhibited by oxidized glutathione or S-benzyl-glutathione. This transport system in canalicular plasma membranes may function in biliary secretion of GSH and its derivatives which are synthesized in hepatocytes by oxidative processes or glutathione S-transferase.

Biliary transport of glutathione and methylmercury

The hepatobiliary transport of glutathione (GSH) and methylmercury (MM) was investigated in male and female rats anesthetized with pentobarbital sodium. When bile flow was altered with either sodium dehydrocholate (DHC), hypertonic sucrose infusion, or by hypothermia, the absolute rates of GSH and MM secretion into bile were not affected, resulting in parallel concentration changes in the bile fluid for both GSH and MM. Indocyanine green and sulfobromophthalein (BSP), but not BSP-glutathione complex, inhibited the biliary secretion of free GSH. This inhibition was accompanied by a parallel inhibition of MM secretion into bile and occurred without any changes in liver GSH or MM levels. On the other hand, the intravenous administration of cysteine, GSH, and penicillamine was associated with an increase in the secretion rate of reduced sulfhydryl groups into bile and an increase in the biliary secretion rate of MM. The increased biliary secretion rate of MM after phenobarbital pretreatment was also associated with an increased rate of secretion of GSH into bile. In addition, sex differences and individual variability in the biliary secretion of MM were correlated with differing abilities to secrete GSH into bile. The results suggest the presence of a biliary transport system for GSH that determines the biliary secretion of MM.