Biometals DOI 10.1007/s10534-014-9722-y
Arsenic trioxide induced indirect and direct inhibition of glutathione reductase leads to apoptosis in rat hepatocytes Atish Ray • Sarmishtha Chatterjee • Sandip Mukherjee • Shelley Bhattacharya
Received: 4 January 2014 / Accepted: 17 February 2014 Ó Springer Science+Business Media New York 2014
Abstract Glutathione reductase (GR) is an essential enzyme which maintains the reduced state of a cell. Therefore GR malfunction is closely associated with several disorders related to oxidative damage. The present study reports toxic manifestation of arsenic trioxide in respect of GR leading to apoptosis. Isolated rat hepatocytes exposed to arsenic trioxide were analyzed for GR expression and activity. Arsenic resulted in a time dependent inhibition of GR mediated by the superoxide anion. The cellular demand of functional enzyme is achieved by concomitant rise in gene expression. However, direct inhibition of GR by arsenic trioxide was also evident. Furthermore, arsenic induced free radical mediated inhibition of GR was found to be partially uncompetitive and associated with time dependent decrease in the substrate binding rate. Externalization of phosphatidylserine, nuclear degradation, apoptosis inducing factor leakage, apoptosome formation, caspase activation, DNA damage and break down of PARP suggest consequential
A. Ray S. Chatterjee S. Mukherjee S. Bhattacharya (&) Environmental Toxicology Laboratory, Department of Zoology, Centre for Advanced Studies, Visva-Bharati (A Central University), Santiniketan 731235, India e-mail: [email protected] Present Address: A. Ray Immunobiology Group, Department of Zoology, University of Delhi, Delhi, India
induction of apoptosis due to inhibition of GR. The implication of GR was further established from the reduced rate of caspase activation in the arsenic trioxide treated cell, supplemented with complete and incomplete enzyme systems. Keywords Apoptosis Glutathione reductase NAC Free radical Nrf2
Introduction Glutathione reductase (GR) is an enzyme of the flavoprotein disulfide oxidoreductase family that catalyzes the reaction leading to reduction of GSH from GS–SG. The reaction is essentially connected with maintenance of reduced environment in the cells during a toxic exposure. Earlier reports from our research group have already demonstrated that GSH abnormality is associated with the target organ dysfunction and GR is important in the maintenance of redox ratio (Roy and Bhattacharya 2006; Maity et al. 2008; Bhattacharya et al. 2007). The clinical significance of glutathione system is well known and it has been shown that elevation of glutathione dependent enzymes is associated with compensatory process of phase 2 detoxication systems (Loginov et al. 1997). Metal induced free radical generation is of immense importance in cellular damage, DNA breakage and
123
Biometals
cancer (Valko et al. 2006). To demonstrate the significance of GR, it is necessary to consider the series reaction of detoxication process. During formation of excessive amount of reactive oxygen species (ROS) super oxide dismutase (SOD) and catalases produce lipid and hydro peroxides (Mei et al. 2012). Glutathione peroxidase (GPX) detoxifies peroxides with GSH acting as electron donor producing GS–SG as the end product. The restoration reaction of GSSG is catalyzed by GR. Therefore, altered GR expression is associated with a number of pathological conditions, including hepatitis. Different types of cancers and aging are also directly related with altered GR status (Townsend and Tew 2003). Recently, cellular glutathione and GR system is also found to be directly coupled with non hepatic disorder such as Parkinson’s disease and rheumatoid arthritis (Martin and Teismann 2009). Further in joint disease, GR activity protects joint tissue collagen against degradative action of ROS (Sredzinska et al. 2009). Taken together, it is established that GR is of immense importance in disruption of the redox status and consequent disease progression. The present study is therefore focused on arsenic induced alteration in GR expression and activity. Arsenic is of prime importance from the present day perspective because with increasing load of environmental contaminants arsenic poisoning is a serious global issue. Even sub-chronic arsenic exposure is found to affect the levels of trace elements in mice brain, where Fe, Se and Cr levels decreased and that of Cu increased (Wang et al. 2013). Systemic deposition due to slow unavoidable chronic exposure results in severe lesions including certain forms of cancers without exhibiting any immediate effects. On the other hand, arsenic trioxide at a comparatively high dose is often used as a potent chemotherapeutic agent in treating certain forms of cancer including acute promyelocytic leukemia (APL) (Estey et al. 2006; Gore et al. 2010) as well as hepatocellular carcinoma where the weekly accumulative load per adult individual is as high as around 100 mg (Lin et al. 2007). The problems become severe in case of chemo-resistance that may increase the probability of normal cell toxicity without the desired outcome (Montero et al. 2008). It has already been demonstrated with zinc that a bivalent transition metal induces disruption of glutathione metabolism leading to endothelial apoptosis (Wiseman et al. 1999). We have demonstrated manifestation of arsenic toxicity leading
123
to arsenic induced apoptosis in hepatocytes (Ray et al. 2008). The present investigation establishes the association between mechanism of differential GR inhibition and promotion of apoptosis in arsenic treated normal rat hepatocytes. Results depicted here provide a novel comprehensive insight into the significance of altered GR status in arsenic induced oxidative stress/apoptosis of normal differentiated hepatocytes, where arsenic challenge causes loss of GR functionality via bidirectional inhibition.
Materials and methods Chemicals Cell culture medium was procured from Invitrogen Corporation, (Carlsbad, California, USA). All primary antibodies except anti GR antibody were purchased from Santa Cruz Biotechnology Inc., Madison, Wisconsin, USA. Anti GR antibody, mouse and rabbit secondary antibodies, purified GR enzyme, Annexin V-Cy3/CFDA apoptosis detection kit (APO-AC), Hoechst Stain (Bisbenzimide H 33258), other fine chemicals and kits were procured from Sigma Chemical Co. (St Louis, MO, USA). PCR primers were procured from Sigma-Aldrich Corporation. First strand cDNA Synthesis kit and accessory chemicals including Taq DNA polymerase and reverse transcriptase and transfection reagents were procured from Fermentas Life Sciences. All other fine chemicals of analytical grade were purchased from Sisco Research Laboratories (Mumbai, India) and E. Merck (Mumbai, India). Animals Male Swiss albino rats of Sprague–Dawley strain were maintained according to Inglis (1980). All experiments were carried out in accordance with the regulations of the Institutional Animal Ethics Committee. Isolation of hepatocytes Hepatocytes were isolated by collagenase digestion method. Briefly, livers were perfused with 200 mL of Ca??-free Hanks balanced salt solution (HBSS), minced and incubated in 50 mL of Ca??-HBSS containing 0.1 % collagenase type IV for 60 min at
Biometals
37 °C, filtered through 60-lm nylon mesh, and centrifuged at 509g for 1 min. Pellets formed were used as a population of parenchymal cells containing differentiated hepatocytes. Arsenic treatment regimens of isolated hepatocytes Arsenic concentration was selected on the basis of earlier studies which demonstrated that apoptosis in rat hepatocyte is initiated at 10 lM arsenic exposure (Ray et al. 2008). 2 9 106 number of cells per 2 mL of modified basal medium DMEM supplemented with 10 % fetal calf serum (FCS) was plated in 24-well culture plates. Hepatocytes were treated with 10 lM of As2O3 for 0 min 1, 2, 4 and 6 h at 37 °C in a CO2 incubator set at 5 % along with concurrent controls with or without inhibitors. N acetyl cysteine (NAC), exogenous GSH and buthionine sulfoximine (BSO) were used as inhibitors of the de novo GSH synthetic pathway. Exogenous supplementation of glutathione reductase enzyme Glutathione reductase was introduced in the cell population using cationic protein transfection reagent according to the manufacturer’s (Fermentas) instruction. Replicates were used for conventional supplementation by mild permeabilization of the cells with 0.1 % Triton-100 transient shock treatment. Assessment of cytotoxicity and generation of ROS Cell viability was checked by Trypan blue dye exclusion and MTT assays. ROS production was assessed by NBT reduction assay at 0 min, 1, 2, 4 and 6 h in As treated and NAC pre-treated cells (Datta et al. 2009).
Estimation of glutathione reductase activity Cytosol was prepared by ultracentrifugation in a Beckman L 90 K ultracentrifuge. GR activity was measured by monitoring NADPH oxidation rate using NADPH and GSSG as substrate. Reaction mixture was constituted with 0.1 mM NADPH and 1 mM GSSG and the enzyme source (the cytosolic preparation) in 50 mM phosphate buffer (pH 7.6). Decrease in absorbance was recorded at 340 nm for 5 min. Activity was calculated from extinction coefficient of NADPH (6.22 9 103 M-1 cm-1) and expressed in terms of molar fraction of NADPH oxidized min-1 mg protein-1. GR activity was also recorded measuring the rate of GSH production from GSSG by Ellman’s reagent and expressed as lg GSH produced min-1 mg protein-1. For the cell free assay, cytosol was placed in the reaction mixture containing adequate NADPH (100 lM) with varying GSSG concentration. Enzyme kinetics was represented as Lineweaver–Burk Plots. Substrate binding assay and co-immuno precipitation For concentration and time dependent substrate binding assay, cytosol was isolated from arsenic untreated cells. Aliquots of isolated cytosol were incubated with different concentrations of arsenic trioxide (10, 20, 40 lM) for 10 min at 37 °C and formation of enzyme– substrate complex was assessed by Western Blot analysis from co-immuno precipitated (Co-IP) samples. Co-IP was performed using the protocol provided by Abcam plc. Anti GR monoclonal antibody (Sigma) was used to precipitate the enzyme and the bound GSSG fraction was detected using the antibody raised against glutathione (Millipore) by Western blot. Protein was estimated following the method of Lowry et al. (1951) Western blot
Estimation of reduced glutathione GSH was estimated from protein-free clear supernatant, after trichloro-acetic acid (TCA) extraction, using 5,5-dithiobis-2-nitrobenzoic acid (DTNB) at k 405 nm in a Beckman DU 730 spectrophotometer and ophthalaldehyde (OPA) fluorometric method (k excitation = 350, k emission = 430) (Senft et al. 2000).
An aliquot of cytosol containing 100 lg protein was run through 10 % sodium dodecyl sulphate—polyacrylamide gel electrophoresis (SDS PAGE) at a constant voltage (60 V) for 2 h and transferred on a polyvinylidene fluoride (PVDF) membrane (Roy and Bhattacharya 2006). The blotted membranes were incubated in a SNAP i.d. system (Millipore) with
123
Biometals
primary antibodies and with alkaline phosphatase conjugated rabbit IgG as secondary antibody (diluted to 1:2,000). The blots were developed using 5-bromo4-chloro-3-indolylphosphate/nitro blue tetrazolium (BCIP/NBT) as substrate. b actin was considered as loading control.
Immunofluorescence Respective cell populations fixed in 4 % paraformaldehyde were smeared on poly-L-lysine coated cover slips. After permeabilization with ice cold PBS containing 0.25 % Triton X-100 and blocking with PBST with 2.5 % bovine serum albumin (BSA) samples were incubated with primary and secondary antibodies (dilution 1:1,000) for 8 h at 4 °C and 2 h at room temperature respectively and analyzed under a fluorescence microscope (Olympus) with appropriate filters.
Visualization of Annexin-CY3/CFDA stained cells
DNA ladder formation Genomic DNA was isolated from control and treated cells following phenol–chloroform-isoamyl alcohol solvent extraction method. The DNA was precipitated with 3 M sodium acetate and ice cold ethanol (Sambrook et al. 1989). Formation of DNA ladder was investigated in 1.2 % Agarose gel after staining with ethidium bromide. RT-PCR analysis Total RNA from rat hepatocytes was isolated using Tri Reagent (Sigma-Aldrich) as per the manufacturer’s guideline. First strand complementary DNA was synthesized from total RNA as per the protocol provided. PCR was performed according to manufacturer’s instruction for 35 cycles. All test samples were amplified simultaneously from equal quantity of initial template with the particular primer pair using a PCR master mix. PCR reactions were run in a programmable Thermal cycler (Applied Biosystem) with simultaneous NTC (No template control) and GAPDH (internal control). Statistical and image analysis
Freshly harvested cells were incubated in binding buffer and stained with Annexin V-CY3/CFDA according to the manufacturer’s guideline (APO-AC, Sigma). The same fields were studied in green (CFDA) and red fluorescence (Annexin-CY3) using appropriate filter set. Normal cells appeared positive only in green fluorescence and apoptotic cells appeared positive in both green and red fluorescence.
Statistical analysis was done following paired t test (Snedecor and Cochran, 1967). Sigma Plot (SPSS) was used for graphical representation and Image J (available at http://rsbweb.nih.gov/ij/) and Quantity One (Bio Rad) were utilized for image analysis.
Results Cellular deformation and nuclear breakdown Stress imposed on the cell Cellular deformation was studied in routine eosin haematoxylin stained slides. Briefly, smears of 4 % paraformaldehyde fixed cells on poly-L-lysine coated slides were passed through appropriate alcohol gradients and stained with haematoxylin for 15 min and eosin for 1 min. For nuclear degradation studies 1 mg mL-1 Hoechst solution was overlaid on smears prepared from freshly harvested cells. Intact nuclei had regular rounded shape under UV filter in a fluorescence microscope whereas degraded nuclei appeared multi lobed and irregular in shape. The magnitude of nuclear degradation was studied and indexed through random screening.
123
Stress imposed on the cells was investigated by NBT reduction assay. Time dependent superoxide anion (SOA) generation is sufficiently prominent attaining the peak at 1 h (Fig. 1a). Expression profile of glutathione reductase associated with Nrf-2 status GR was found to be induced in arsenic treated cells at 1 h and remained remarkably high as compared to control till the end of the experiment. NAC pre administration exhibits reduction in GR level although
Biometals
Fig. 1 a NBT reduction assay exhibits generation of superoxide anion (SOA) in 10 lM arsenic treated hepatocytes. Magnitude of superoxide anion induction is remarkable at 2–6 h of incubation. (*p \ 0.05). b Elevated level of cellular glutathione reductase (GR) in 10 lM arsenic treated cells. NAC pre-treatment demonstrates reduction in GR level as compared to the cells treated with arsenic alone. c Nuclear translocation of
Nrf2 is associated with decreased cytosolic Keap level in arsenic treated cells. d Generation of superoxide anion, gsr expression, and nuclear translocation of Nrf-2 profile in arsenic treated as well as NAC and BSO pre-treated hepatocytes is concomitant with degree of apoptosis index. Apoptotic index was calculated from magnitude cell death and level of superoxide anion and express as percent of control
it remained higher than control (Fig. 1b). Nuclear translocation of Nrf-2 increased against control in arsenic treated cells concomitantly with an increased GR level and a substantial decrease in Keap1 (Fig. 1c). Toxicity of As to hepatocyte is adequately clear from induced GSH level paralleled to induced SOA generation and apoptotic index which was further investigated in presence of exogenous NAC and BSO. NAC enriched reduced environment prevents induction of intracellular glutathione by scavenging the free radicals along with substantial decrease in apoptotic index. On the other hand, blocking of GSH synthesis with BSO results in GSH depleted cells with profound SOA and augmented apoptotic index. Gsr expression profile demonstrates opposite pattern between NAC and BSO
pre-treated cells. NAC pre-treatment retains gsr expression level lower than arsenic treated cells whereas robust expression is noteworthy in case of BSO pre-treatment. Induction of SOA driven GR level and gsr gene expression was also substantiated from the present study. NAC supplement maintains basal GR level via prevention of gsr over expression. The profile is directly correlated with reduced nuclear Nrf-2 level in GSH pre- administered cells and enhanced NRf-2 translocation in BSO pre-treated cells (Fig. 1d). Inhibition of GR activity Time kinetics study reveals substantial inhibition of GR activity in response to arsenic exposure against
123
Biometals
Fig. 2 Time kinetics of GR inhibition. a Inhibition of GR activity in arsenic treated hepatocytes as detected by NADPH oxidation rate. (*p \ 0.05). b NAC pre-treatment exhibits increased GR activity as compared to NAC untreated cells. (*Significant difference from control, #Significant difference from arsenic treatment; p \ 0.05). c Time dependent substrate
ES (GR–GSSG) complex formation rate in arsenic treated cells. d Lineweaver–Burk (double reciprocal) plot of GR activity against varying substrate (GS–SG) concentration in control, arsenic treated and NAC pre treated cells. e Lineweaver–Burk (double reciprocal) plot of GR activity against varied NADPH concentration in control and arsenic treated cells
untreated cells (Fig. 2a). NAC pre administration maintains the enzyme activity nearer to control (Fig. 2b). Although formation of enzyme–substrate complex (ES) is elevated in treated cells as compared to control time dependent decrease in rate is significant and concomitant with inhibition of GR activity (Fig. 2c). Furthermore, varying concentrations of both the GSSG (Fig. 2d) and NADPH (Fig. 2e) exhibit a
remarkable decrease in the reaction velocity (Vmax) and reduced Km of GR. Direct role of arsenic in GR inhibition is investigated with the cytosol incubated with arsenic trioxide. Result demonstrates reduction both in Vmax and Km in arsenic treated cells as compared to control. Magnitude of decrease in Vmax is significantly higher as compared to decrease in Km (Fig. 3a, b). Concentration (AsIII) dependent decrease
123
Biometals Fig. 3 Modulation of GR activity kinetics with arsenic in a cell free system. a GR inhibition profile in response to arsenic trioxide as evidenced by its activity against different GS–SG concentrations. b Double reciprocal plot of GR activity in control and arsenic incubated cytosol. c Concentration and d time dependent ES formation kinetics in arsenic treated cytosol. e Lineweaver–Burk (double reciprocal) plot of GR activity against varied NADPH concentration in control and arsenic incubated cytosol
in rate of ES formation is also evident from the present study. Results clearly demonstrate that maximum substrate binding requires elevated substrate concentration in the reaction system incubated with higher arsenic concentration (Fig. 3c). Not deviating from the fundamental dynamics of ES formation observed
in vitro, arsenic exposed cytosol also results in increased ES complex formation as compared to control accompanied by time dependent inhibition of substrate binding. (Fig. 3d). On the other hand, varying NADPH concentration reduced both Vmax and Km of GR (Fig. 3e).
123
Biometals Fig. 4 Arsenic induced cellular deformation, nuclear breakdown and apoptosis in rat hepatocytes. a Cellular deformation, nuclear damage and phosphatidylserine externalization (apoptosis) is evidenced in eosin– haematoxylin, Hoechst and Annexin V/6CFDA stained cells respectively. b Sequential events of apoptosis progression including cell leakage, mitochondrial damage, apoptosome formation, caspase 3,9 activation, DNA ladder formation and PARP cleavage. c Reduced caspase 3 activation in exogenous GR supplemented cells
Induction of apoptosis as a consequence of GR inhibition Arsenic treatment results in cell damage and nuclear break down as compared to control which is substantially clear from eosin–haematoxylin and Hoechst stained cells (Fig. 4a), Arsenic treated cells demonstrate elevated gamma glutamyl transpeptidase (GGT) released in medium which was reduced in NAC pretreated cells. Western blot analysis reveals elevated cytosolic apoptosis inducing factor (AIF) level, cyt C-caspase 9 conjugation, caspase 3 cleavage, DNA ladder formation and PARP1 cleavage in support of execution of apoptosis (Fig. 4b). Exogenous supplementation of GR system demonstrates reduced caspase 3 activity in arsenic treated cells. Reduction
123
of caspase activity is most prominent in complete GR system assemble with pure GR enzyme and NADPH, the cofactor (Fig. 4c).
Discussion Modulation of GR expression by arsenic induced superoxide anion Eminence of intracellular GR essentially correlated with enhanced intracellular GSH level performing a cytoprotective function (Townsend and Tew 2003; Asmis et al. 2005). Hepatocytes also export GSH through sinusoidal transport into plasma or into bile through canalicular transport. (Rius et al. 2003). The
Biometals
present investigation demonstrates that high GSH level in the cytosol is apparent in arsenic treated cells which correlates positively with the GR expression. Result indicates the essential contribution of GR in maintenance cellular reducing state. Involvement of ROS in induction of GR is clearly demonstrated from subdued GR level in NAC pre-treated cells as compared to arsenic treated cells. On the other hand, the redox regulated transcription factors, Nrf-2 was found to be associated with cyto-protective function regulating expression of glutathione-s-transferase (gst) gene (Alam et al. 1995). Further investigation suggested that Nrf2 regulates GSH synthetic enzymes such as gamma glutamyl cysteine ligase (GCL) and glutathione synthetase (GSS) as well as cysteine/ glutamate exchange transporter that regulates cysteine influx (Erickson et al. 2002; Sasaki et al. 2002). Recently it is also shown in lung and embryonic fibroblast cells that Nrf 2 dependent regulation of GR, independent of its biosynthesis is critical for cell survival (Harvey et al. 2009). Direct correlation between nuclear translocation of Nrf 2 and GR expression is demonstrated here. Keap 1 is the cytoskeletal adaptor protein has been shown to maintain a steady state of cytosolic Nrf2 via Keap1– Nrf2 interaction (Singh et al. 2006). Present investigation demonstrates involvement of Keap 1 in regulation of Nrf-2 in arsenic treated rat hepatocytes. Pretreatment with the GSH synthesis inhibitor, BSO results in profound ROS generation leading to robust expression of gsr (gene of GR) accompanied by enhanced NRF-2 translocation. It is surmised that arsenic exposure heightened the demand of GR expression depending on the requirement of GSH by the cell which is proportional to the magnitude of free radicals generated. However, increase in apoptotic index concomitant with rise in SOA is found to be the signature of inadequate adaptive competency and faulty compensatory mechanism. Inhibitory mechanism of GR by arsenic and arsenic induced free radicals While arsenic mediated free radical induces GR expression the time kinetics study reveals substantial reduction in GR activity. However, NAC pre-treatment demonstrates further augmentation in GR activity. It is known that GR activity follows the ping pong or branched mechanism and product inhibition of GR
by GSH is expected to be non-competitive (Chung et al. 1991). The present investigation clearly elucidates direct inhibition of GR activity manifested either by the metal itself or by end product GSH as a consequence of substantial free radical generation. GR expression level and the GR activity does not correlate linearly, therefore additional investigation has been performed to elucidate the grounds of GR inhibition concomitant with enhanced GR expression level. Kinetics study with GR enriched cytosolic fraction from control and treated cells in presence of different concentrations of GSSG reveals a decrease in Vmax which further confirms that As intoxication inhibition of GR activity. On the basis of existing information of kinetics and biochemical property of GR enzyme (Tandogan and Ulusu 2006, 2010a, b) direct inhibitory mechanism is investigated in arsenic treated cells. In the present study reduction in Vmax is obviously the hallmark of direct catalytic inhibition by arsenic or arsenic generated free radicals. However, increased substrate affinity is also evident from reduced Km for the substrate GSSG, which is apparently accompanied by enhanced GR synthesis. The kinetics of GR activity varies between arsenic intoxicated cells and the cells pretreated with NAC. NAC pre-treated cells demonstrate further increase in Vmax and Km in response to arsenic. This event can directly be correlated with time kinetics indicating that modulation of GR activity is dependent on the magnitude of free radical generation. The scenario is more decisively observed in the activity kinetics of NAC pre-treated cells, where prevention of ROS generation reduces GR expression level although the level remained high as compared to control. Concomitant rise in Vmax is even higher than control which was markedly repressed due to arsenic exposure. Here we hypothesize that other than GSH requirement and availability, activity status of GR is also a key factor for assigning the gsr expression demand and vice-verse. Pattern of arsenic induced free radical mediated GR inhibition appears to be of partially uncompetitive or mixed type where increase in substrate affinity is apparently comparable in enhanced GSSG–GR binding as compared to control. However, a time dependent gradual decrease in substrate binding rate was evident which indicates enzyme inhibition. With varying NADPH concentration the pattern of enzyme inhibition is also found to be partially uncompetitive or mixed where Km and
123
Biometals
Vmax are reduced. In the in vitro assay too the pattern of enzyme inhibition is mixed, especially where the arsenic binding rate with GR is rather less as compared to the arsenic binding rate of GR-NADPH complex. Thus the degree of GR inhibition triggers enhanced enzyme synthesis. Therefore, it is surmised that the cellular adaptive response effects elevated GR expression level by arsenic or arsenic induced free radicals which indirectly leads to partially uncompetitive mode of inhibition. However, time dependent enzyme inhibition is prominent from the reduced rate of substrate binding parallel to induced GSH accumulation either as a result of arsenic interference or due to end product inhibition by GSH. The influence of GR expression or GSH accumulation on enzyme inhibition kinetics was adequately prominent in in vitro experiments. Therefore to understand the direct inhibition mechanism, a cell free system was employed. Results clearly demonstrate direct mixed type of inhibition of GR by arsenic accompanied by a substantial decrease in apparent Km and Vmax. On the other hand the type of inhibition is partially uncompetitive in response to varying NADPH concentration in the cell free system.
detected by released GGT, the membrane bound enzyme involved in metabolic processing of external glutathione. Elevated level of secreted GGT is recently considered as an effective marker of oxidative stress and liver damage often independent of metabolic syndrome (Lee et al. 2004; Lim et al. 2004; Yamada et al. 2006). GGT secretion in the medium was found to be significantly high with promotion of apoptosis which is also confirmed by mitochondrial membrane damage, putative apoptosome formation as well as caspase activation. Apoptosis is eventually reflected in nuclear degradation and Annexin V positive cells. GSH/GSSG maintenance reaction by GR uses NADPH to convert GSSG to GSH. The GSH/GSSG ratio is thus ultimately related to NADPH levels, which is determined by energy status of the cell. (Harvey et al. 2009). To confirm the involvement of GR inhibition in arsenic induced apoptosis further, cells were supplemented with exogenous GR system which significantly reduced caspase 3 activation in arsenic treated cells.
Conclusion Deficiency of GR activity in promotion of apoptosis The worth mentioning reviews by Tandogan and Ulusu (2006), (2010a, b) concluded that during oxidative stress and deficiency of GR, loss of thiol redox balance may cause deleterious consequences for metabolic regulation, cellular integrity, and organ homeostasis due to accumulation of intracellular GSSG. GR inhibition disturbs cellular prooxidant/antioxidant balance and may contribute to the genesis of many diseases. The present investigation demonstrates the apoptotic endpoint due to GR inhibition. We have already reported toxic manifestation of arsenic leading to apoptosis in different cell types, such as differentiated rat hepatocytes and hepatic stem cells (Ray et al. 2008; Agarwal et al. 2009). It is abundantly clear from the sequential events of caspase dependent apoptosis that disruption of mitochondrial membrane integrity occurs due to arsenic. Leakage of AIF in the cytosol is in agreement with the existing report of Holubec et al. (2005). Cellular damage is initially
123
It is concluded that arsenic intoxication exerts remarkable free radical stress leading to induction of GSH. With generation of arsenic induced free radicals, gsr expression is considerably enhanced within the cells leading to increased GR level. However, cellular adaptive proficiency is compromised by inhibited GR activity. Furthermore, from the pattern of inhibition it is concluded that the consequence is much severe as arsenic and arsenic induced free radical acts together in the inhibition process preferably at the site of enzyme–substrate complex both for GSSG as well as NADPH. Consequentially GSH restoration is hindered and caspase dependent apoptosis is implemented. The mechanism is summarized in the graphical abstract. Acknowledgments AR is grateful to Council for Scientific and Industrial Research for a Senior Research Fellowship, SC gratefully acknowledges DST for a SRF (Project No SR/SO/AS22/2008) and SM is grateful to University Grants Commission for financial support and SB acknowledges the National Academy of Sciences, India for the award of Senior Scientist, Platinum Jubilee Fellowship. Conflict of interest
No competing financial interest exists.
Biometals
References Agarwal S, Roy S, Ray A, Mazumder S, Bhattacharya S (2009) Arsenic trioxide and lead acetate induce apoptosis in adult rat hepatic stem cells. Cell Biol Toxicol 25:403–413 Alam J, Camhi S, Choi AMK (1995) Identification of a second region upstream of the mouse heme oxygenase-1 gene that functions as a basal level and induce dependent transcriptional enhancer. J Biol Chem 270:11977–11984 Asmis R, Wang Y, Xu L, Kisgati M, Begley JG, Mieyal JJ (2005) A novel thiol oxidation-based mechanism for adriamycin-induced cell injury in human macrophages. FASEB J 19:1866–1868 Bhattacharya S, Bhattacharya A, Roy S (2007) Arsenic induced responses in freshwater teleosts. Fish Physiol Biochem 33:463–473 Chung PM, Cappel RE, Gilbert HF (1991) Inhibition of glutathione disulfide reductase by glutathione. Arch Biochem Biophys 288:48–53 Datta S, Mazumder S, Ghosh D, Dey S, Bhattacharya S (2009) Low concentration of arsenic could induce caspase 3 mediated head kidney macrophage apoptosis with JNKp38 activation in Clarias batrachus. Toxicol Appl Pharmacol 241:329–338 Erickson AM, Nevarea Z, Gipp JJ, Mulcahy RT (2002) Identification of a variant antioxidant response element in the promoter of the human glutamate–cysteine ligase modifier subunit gene: revision of the ARE consensus sequence. J Biol Chem 277:30730–30737 Estey E, Garcia-Manero G, Ferrajoli A, Faderl S, Verstovsek S, Jones D, Kantarjian H (2006) Use of all-trans retinoic acid plus arsenic trioxide as an alternative to chemotherapy in untreated acute promyelocytic leukemia. Blood 107: 3469–3473 Gore SD, Gojo I, Sekeres MA, Morris L, Devetten M, Jamieson K, Redner RL, Arceci R, Owoeye I, Dauses T, Schachter-Tokarz E, Gallagher RE (2010) Single cycle of arsenic trioxide based consolidation chemotherapy spares anthracycline exposure in the primary management of acute promyelocytic leukemia. J Clin Oncol 28:1047–1053 Harvey CJ, Thimmulappa RK, Singh A, Blake DJ, Ling G, Wakabayashi N, Fujii J, Myers A, Biswal S (2009) Nrf2regulated glutathione recycling independent of biosynthesis is critical for cell survival during oxidative stress. Free Rad Biol Med 46:443–453 Holubec H, Payne CM, Bernstein H, Dvorakova K, Bernstein C, Waltmire CN, Warneke JA, Garewal H (2005) Assessment of apoptosis by immunohistochemical markers compared to cellular morphology in ex vivo-stressed colonic mucosa. J Histochem Cytochem 53:229–235 Inglis JK (1980) Introduction to laboratory animal science and technology. Pergamon Press, Oxford Lee DH, Blomhoff R, Jacobs DJ (2004) Is serum gamma glutamyltransferase a marker of oxidative stress? Free Radic Res 38:535–539 Lim JS, Yang JH, Chun BY, Kam S, Jacobs DR Jr, Lee DH (2004) Is serum c-glutamyltransferase inversely associated with serum antioxidants as a marker of oxidative stress? Free Radic Biol Med 37:1018–1023
Lin CC, Hsu C, Hsu CH, Hsu WL, Cheng AL, Yang CH (2007) Arsenic trioxide in patients with hepatocellular carcinoma: a phase II trial. Invest New Drugs 25:77–84 Loginov AS, Matiushin BN, Tkachev VD (1997) The clinical significance of the liver glutathione system in chronic lesions. Ter Arkh 69:25–27 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Measurement of protein with the Folin phenol reagent. J Biol Chem 193:265–275 Maity S, Roy S, Bhattacharya S, Chowdhury S (2008) Antioxidants in Lampito mauritii are the biomarkers of soil contamination by lead and zinc. Environ Pollut 151:1–7 Martin HL, Teismann P (2009) Glutathione a review on its role and significance in Parkinson’s disease. FASEB J 23: 3263–3272 Mei X, Xu D, Xu S, Zheng Y, Xu S (2012) Novel role of Zn(II)curcumin in enhancing cell proliferation and adjusting proinflammatory cytokine-mediated oxidative damage of ethanol-induced acute gastric ulcers. Chem Biol Interact 197:31–39 Montero J, Morales A, Llacuna L, Lluis JM, Terrones O, Basan˜ez G, Antonsson B, Prieto J, Garcı´a-Ruiz C, Colell A, Ferna´ndez-Checa JC (2008) Mitochondrial cholesterol contributes to chemotherapy resistance in hepatocellular carcinoma. Cancer Res 68:5246–5256 Ray A, Roy S, Agarwal S, Bhattacharya S (2008) As2O3, toxicity in rat hepatocytes: manifestation of caspase-mediated apoptosis. Toxicol Ind Health 34:643–653 Rius M, Nies AT, Hummel-Eisenbeiss J, Jedlitschky G, Keppler D (2003) Cotransport of reduced glutathione with bile salts by MRP4 (ABCC4) localized to the basolateral hepatocyte membrane. Hepatology 38:374–384 Roy S, Bhattacharya S (2006) Arsenic induced histopathology and synthesis of stress proteins in liver and kidney of Channa punctatus. Ecotoxicol Environ Saf 65:218–229 Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, New York Sasaki H, Sato H, Kuriyama-Matsumura K, Sato K, Maebara K, Wang H, Tamba M, Itoh K, Yamamoto M, Bannai S (2002) Electrophile response element-mediated induction of the cystine/glutamate exchange transporter gene expression. J Biol Chem 277:44765–44771 Senft AP, Dalton TP, Shertzer HG (2000) Determining glutathione and glutathione disulfide using the fluorescence probe O-phthalaldehyde. Anal Biochem 280:80–86 Singh A, Misra V, Thimmulappa RK, Lee H, Ames S, Hoque MO, Herman JG, Baylin SB, Sidransky D, Gabrielson E, Brock MV, Biswal S (2006) Dysfunctional KEAP1-NRF2 interaction in non-small-cell lung cancer. PLoS Med 3:e420 Snedecor GW, Cochran WG (1967) Statistical methods. The Iowa State University Press, Iowa S´redzin´ska K, Galicka A, Porowska H, Sredzin´ski Ł, Porowski T, Popko J (2009) Glutathione reductase activity correlates with concentration of extracellular matrix degradation products in synovial fluid from patients with joint diseases. Acta Biochim Pol 56:635–640 Tandogan B, Ulusu NN (2006) A comparative study with colchicine on glutathione reductase. FABAD J Pharm Sci 31:230–237
123
Biometals Tandogan B, Ulusu NN (2010a) Comparative in vitro effects of some metal ions on bovine kidney cortex glutathione reductase. Prep Biochem Biotech 40:405–411 Tandogan B, Ulusu NN (2010b) Kinetic mechanism and molecular properties of glutathione reductase. Protein J 29:380–385 Townsend DM, Tew KD (2003) The role of glutathione-Stransferase in anti-cancer drug resistance. Oncogene 22:7369–7375 Valko M, Rhodes C, Moncol J, Izakovic M, Mazur M (2006) Free radicals, metals and antioxidants in oxidative stressinduced cancer. Chem Biol Interact 160:1–40 Wang X, Zhang J, Zhao L, Hu S, Piao F (2013) Effect of subchronic exposure to arsenic on levels of essential trace
123
elements in mice brain and its gender difference. Biometals 26:123–131 Wiseman DA, Sharma S, Black SM (1999) Elevated zinc induces endothelial apoptosis via disruption of glutathione metabolism: role of the ADP translocator. Biometals 23:19–30 Yamada J, Tomiyama J, Yambe M, Koji Y, Motobe K, Shiina K, Yamamoto Y, Yamashina A (2006) Elevated serum levels of alanine aminotransferase and gamma glutamyltransferase are markers of inflammation and oxidative stress independent of the metabolic syndrome. Atherosclerosis 189:198–205
Arsenic trioxide induced indirect and direct inhibition of glutathione reductase leads to apoptosis in rat hepatocytes Atish Ray • Sarmishtha Chatterjee • Sandip Mukherjee • Shelley Bhattacharya
Received: 4 January 2014 / Accepted: 17 February 2014 Ó Springer Science+Business Media New York 2014
Abstract Glutathione reductase (GR) is an essential enzyme which maintains the reduced state of a cell. Therefore GR malfunction is closely associated with several disorders related to oxidative damage. The present study reports toxic manifestation of arsenic trioxide in respect of GR leading to apoptosis. Isolated rat hepatocytes exposed to arsenic trioxide were analyzed for GR expression and activity. Arsenic resulted in a time dependent inhibition of GR mediated by the superoxide anion. The cellular demand of functional enzyme is achieved by concomitant rise in gene expression. However, direct inhibition of GR by arsenic trioxide was also evident. Furthermore, arsenic induced free radical mediated inhibition of GR was found to be partially uncompetitive and associated with time dependent decrease in the substrate binding rate. Externalization of phosphatidylserine, nuclear degradation, apoptosis inducing factor leakage, apoptosome formation, caspase activation, DNA damage and break down of PARP suggest consequential
A. Ray S. Chatterjee S. Mukherjee S. Bhattacharya (&) Environmental Toxicology Laboratory, Department of Zoology, Centre for Advanced Studies, Visva-Bharati (A Central University), Santiniketan 731235, India e-mail: [email protected] Present Address: A. Ray Immunobiology Group, Department of Zoology, University of Delhi, Delhi, India
induction of apoptosis due to inhibition of GR. The implication of GR was further established from the reduced rate of caspase activation in the arsenic trioxide treated cell, supplemented with complete and incomplete enzyme systems. Keywords Apoptosis Glutathione reductase NAC Free radical Nrf2
Introduction Glutathione reductase (GR) is an enzyme of the flavoprotein disulfide oxidoreductase family that catalyzes the reaction leading to reduction of GSH from GS–SG. The reaction is essentially connected with maintenance of reduced environment in the cells during a toxic exposure. Earlier reports from our research group have already demonstrated that GSH abnormality is associated with the target organ dysfunction and GR is important in the maintenance of redox ratio (Roy and Bhattacharya 2006; Maity et al. 2008; Bhattacharya et al. 2007). The clinical significance of glutathione system is well known and it has been shown that elevation of glutathione dependent enzymes is associated with compensatory process of phase 2 detoxication systems (Loginov et al. 1997). Metal induced free radical generation is of immense importance in cellular damage, DNA breakage and
123
Biometals
cancer (Valko et al. 2006). To demonstrate the significance of GR, it is necessary to consider the series reaction of detoxication process. During formation of excessive amount of reactive oxygen species (ROS) super oxide dismutase (SOD) and catalases produce lipid and hydro peroxides (Mei et al. 2012). Glutathione peroxidase (GPX) detoxifies peroxides with GSH acting as electron donor producing GS–SG as the end product. The restoration reaction of GSSG is catalyzed by GR. Therefore, altered GR expression is associated with a number of pathological conditions, including hepatitis. Different types of cancers and aging are also directly related with altered GR status (Townsend and Tew 2003). Recently, cellular glutathione and GR system is also found to be directly coupled with non hepatic disorder such as Parkinson’s disease and rheumatoid arthritis (Martin and Teismann 2009). Further in joint disease, GR activity protects joint tissue collagen against degradative action of ROS (Sredzinska et al. 2009). Taken together, it is established that GR is of immense importance in disruption of the redox status and consequent disease progression. The present study is therefore focused on arsenic induced alteration in GR expression and activity. Arsenic is of prime importance from the present day perspective because with increasing load of environmental contaminants arsenic poisoning is a serious global issue. Even sub-chronic arsenic exposure is found to affect the levels of trace elements in mice brain, where Fe, Se and Cr levels decreased and that of Cu increased (Wang et al. 2013). Systemic deposition due to slow unavoidable chronic exposure results in severe lesions including certain forms of cancers without exhibiting any immediate effects. On the other hand, arsenic trioxide at a comparatively high dose is often used as a potent chemotherapeutic agent in treating certain forms of cancer including acute promyelocytic leukemia (APL) (Estey et al. 2006; Gore et al. 2010) as well as hepatocellular carcinoma where the weekly accumulative load per adult individual is as high as around 100 mg (Lin et al. 2007). The problems become severe in case of chemo-resistance that may increase the probability of normal cell toxicity without the desired outcome (Montero et al. 2008). It has already been demonstrated with zinc that a bivalent transition metal induces disruption of glutathione metabolism leading to endothelial apoptosis (Wiseman et al. 1999). We have demonstrated manifestation of arsenic toxicity leading
123
to arsenic induced apoptosis in hepatocytes (Ray et al. 2008). The present investigation establishes the association between mechanism of differential GR inhibition and promotion of apoptosis in arsenic treated normal rat hepatocytes. Results depicted here provide a novel comprehensive insight into the significance of altered GR status in arsenic induced oxidative stress/apoptosis of normal differentiated hepatocytes, where arsenic challenge causes loss of GR functionality via bidirectional inhibition.
Materials and methods Chemicals Cell culture medium was procured from Invitrogen Corporation, (Carlsbad, California, USA). All primary antibodies except anti GR antibody were purchased from Santa Cruz Biotechnology Inc., Madison, Wisconsin, USA. Anti GR antibody, mouse and rabbit secondary antibodies, purified GR enzyme, Annexin V-Cy3/CFDA apoptosis detection kit (APO-AC), Hoechst Stain (Bisbenzimide H 33258), other fine chemicals and kits were procured from Sigma Chemical Co. (St Louis, MO, USA). PCR primers were procured from Sigma-Aldrich Corporation. First strand cDNA Synthesis kit and accessory chemicals including Taq DNA polymerase and reverse transcriptase and transfection reagents were procured from Fermentas Life Sciences. All other fine chemicals of analytical grade were purchased from Sisco Research Laboratories (Mumbai, India) and E. Merck (Mumbai, India). Animals Male Swiss albino rats of Sprague–Dawley strain were maintained according to Inglis (1980). All experiments were carried out in accordance with the regulations of the Institutional Animal Ethics Committee. Isolation of hepatocytes Hepatocytes were isolated by collagenase digestion method. Briefly, livers were perfused with 200 mL of Ca??-free Hanks balanced salt solution (HBSS), minced and incubated in 50 mL of Ca??-HBSS containing 0.1 % collagenase type IV for 60 min at
Biometals
37 °C, filtered through 60-lm nylon mesh, and centrifuged at 509g for 1 min. Pellets formed were used as a population of parenchymal cells containing differentiated hepatocytes. Arsenic treatment regimens of isolated hepatocytes Arsenic concentration was selected on the basis of earlier studies which demonstrated that apoptosis in rat hepatocyte is initiated at 10 lM arsenic exposure (Ray et al. 2008). 2 9 106 number of cells per 2 mL of modified basal medium DMEM supplemented with 10 % fetal calf serum (FCS) was plated in 24-well culture plates. Hepatocytes were treated with 10 lM of As2O3 for 0 min 1, 2, 4 and 6 h at 37 °C in a CO2 incubator set at 5 % along with concurrent controls with or without inhibitors. N acetyl cysteine (NAC), exogenous GSH and buthionine sulfoximine (BSO) were used as inhibitors of the de novo GSH synthetic pathway. Exogenous supplementation of glutathione reductase enzyme Glutathione reductase was introduced in the cell population using cationic protein transfection reagent according to the manufacturer’s (Fermentas) instruction. Replicates were used for conventional supplementation by mild permeabilization of the cells with 0.1 % Triton-100 transient shock treatment. Assessment of cytotoxicity and generation of ROS Cell viability was checked by Trypan blue dye exclusion and MTT assays. ROS production was assessed by NBT reduction assay at 0 min, 1, 2, 4 and 6 h in As treated and NAC pre-treated cells (Datta et al. 2009).
Estimation of glutathione reductase activity Cytosol was prepared by ultracentrifugation in a Beckman L 90 K ultracentrifuge. GR activity was measured by monitoring NADPH oxidation rate using NADPH and GSSG as substrate. Reaction mixture was constituted with 0.1 mM NADPH and 1 mM GSSG and the enzyme source (the cytosolic preparation) in 50 mM phosphate buffer (pH 7.6). Decrease in absorbance was recorded at 340 nm for 5 min. Activity was calculated from extinction coefficient of NADPH (6.22 9 103 M-1 cm-1) and expressed in terms of molar fraction of NADPH oxidized min-1 mg protein-1. GR activity was also recorded measuring the rate of GSH production from GSSG by Ellman’s reagent and expressed as lg GSH produced min-1 mg protein-1. For the cell free assay, cytosol was placed in the reaction mixture containing adequate NADPH (100 lM) with varying GSSG concentration. Enzyme kinetics was represented as Lineweaver–Burk Plots. Substrate binding assay and co-immuno precipitation For concentration and time dependent substrate binding assay, cytosol was isolated from arsenic untreated cells. Aliquots of isolated cytosol were incubated with different concentrations of arsenic trioxide (10, 20, 40 lM) for 10 min at 37 °C and formation of enzyme– substrate complex was assessed by Western Blot analysis from co-immuno precipitated (Co-IP) samples. Co-IP was performed using the protocol provided by Abcam plc. Anti GR monoclonal antibody (Sigma) was used to precipitate the enzyme and the bound GSSG fraction was detected using the antibody raised against glutathione (Millipore) by Western blot. Protein was estimated following the method of Lowry et al. (1951) Western blot
Estimation of reduced glutathione GSH was estimated from protein-free clear supernatant, after trichloro-acetic acid (TCA) extraction, using 5,5-dithiobis-2-nitrobenzoic acid (DTNB) at k 405 nm in a Beckman DU 730 spectrophotometer and ophthalaldehyde (OPA) fluorometric method (k excitation = 350, k emission = 430) (Senft et al. 2000).
An aliquot of cytosol containing 100 lg protein was run through 10 % sodium dodecyl sulphate—polyacrylamide gel electrophoresis (SDS PAGE) at a constant voltage (60 V) for 2 h and transferred on a polyvinylidene fluoride (PVDF) membrane (Roy and Bhattacharya 2006). The blotted membranes were incubated in a SNAP i.d. system (Millipore) with
123
Biometals
primary antibodies and with alkaline phosphatase conjugated rabbit IgG as secondary antibody (diluted to 1:2,000). The blots were developed using 5-bromo4-chloro-3-indolylphosphate/nitro blue tetrazolium (BCIP/NBT) as substrate. b actin was considered as loading control.
Immunofluorescence Respective cell populations fixed in 4 % paraformaldehyde were smeared on poly-L-lysine coated cover slips. After permeabilization with ice cold PBS containing 0.25 % Triton X-100 and blocking with PBST with 2.5 % bovine serum albumin (BSA) samples were incubated with primary and secondary antibodies (dilution 1:1,000) for 8 h at 4 °C and 2 h at room temperature respectively and analyzed under a fluorescence microscope (Olympus) with appropriate filters.
Visualization of Annexin-CY3/CFDA stained cells
DNA ladder formation Genomic DNA was isolated from control and treated cells following phenol–chloroform-isoamyl alcohol solvent extraction method. The DNA was precipitated with 3 M sodium acetate and ice cold ethanol (Sambrook et al. 1989). Formation of DNA ladder was investigated in 1.2 % Agarose gel after staining with ethidium bromide. RT-PCR analysis Total RNA from rat hepatocytes was isolated using Tri Reagent (Sigma-Aldrich) as per the manufacturer’s guideline. First strand complementary DNA was synthesized from total RNA as per the protocol provided. PCR was performed according to manufacturer’s instruction for 35 cycles. All test samples were amplified simultaneously from equal quantity of initial template with the particular primer pair using a PCR master mix. PCR reactions were run in a programmable Thermal cycler (Applied Biosystem) with simultaneous NTC (No template control) and GAPDH (internal control). Statistical and image analysis
Freshly harvested cells were incubated in binding buffer and stained with Annexin V-CY3/CFDA according to the manufacturer’s guideline (APO-AC, Sigma). The same fields were studied in green (CFDA) and red fluorescence (Annexin-CY3) using appropriate filter set. Normal cells appeared positive only in green fluorescence and apoptotic cells appeared positive in both green and red fluorescence.
Statistical analysis was done following paired t test (Snedecor and Cochran, 1967). Sigma Plot (SPSS) was used for graphical representation and Image J (available at http://rsbweb.nih.gov/ij/) and Quantity One (Bio Rad) were utilized for image analysis.
Results Cellular deformation and nuclear breakdown Stress imposed on the cell Cellular deformation was studied in routine eosin haematoxylin stained slides. Briefly, smears of 4 % paraformaldehyde fixed cells on poly-L-lysine coated slides were passed through appropriate alcohol gradients and stained with haematoxylin for 15 min and eosin for 1 min. For nuclear degradation studies 1 mg mL-1 Hoechst solution was overlaid on smears prepared from freshly harvested cells. Intact nuclei had regular rounded shape under UV filter in a fluorescence microscope whereas degraded nuclei appeared multi lobed and irregular in shape. The magnitude of nuclear degradation was studied and indexed through random screening.
123
Stress imposed on the cells was investigated by NBT reduction assay. Time dependent superoxide anion (SOA) generation is sufficiently prominent attaining the peak at 1 h (Fig. 1a). Expression profile of glutathione reductase associated with Nrf-2 status GR was found to be induced in arsenic treated cells at 1 h and remained remarkably high as compared to control till the end of the experiment. NAC pre administration exhibits reduction in GR level although
Biometals
Fig. 1 a NBT reduction assay exhibits generation of superoxide anion (SOA) in 10 lM arsenic treated hepatocytes. Magnitude of superoxide anion induction is remarkable at 2–6 h of incubation. (*p \ 0.05). b Elevated level of cellular glutathione reductase (GR) in 10 lM arsenic treated cells. NAC pre-treatment demonstrates reduction in GR level as compared to the cells treated with arsenic alone. c Nuclear translocation of
Nrf2 is associated with decreased cytosolic Keap level in arsenic treated cells. d Generation of superoxide anion, gsr expression, and nuclear translocation of Nrf-2 profile in arsenic treated as well as NAC and BSO pre-treated hepatocytes is concomitant with degree of apoptosis index. Apoptotic index was calculated from magnitude cell death and level of superoxide anion and express as percent of control
it remained higher than control (Fig. 1b). Nuclear translocation of Nrf-2 increased against control in arsenic treated cells concomitantly with an increased GR level and a substantial decrease in Keap1 (Fig. 1c). Toxicity of As to hepatocyte is adequately clear from induced GSH level paralleled to induced SOA generation and apoptotic index which was further investigated in presence of exogenous NAC and BSO. NAC enriched reduced environment prevents induction of intracellular glutathione by scavenging the free radicals along with substantial decrease in apoptotic index. On the other hand, blocking of GSH synthesis with BSO results in GSH depleted cells with profound SOA and augmented apoptotic index. Gsr expression profile demonstrates opposite pattern between NAC and BSO
pre-treated cells. NAC pre-treatment retains gsr expression level lower than arsenic treated cells whereas robust expression is noteworthy in case of BSO pre-treatment. Induction of SOA driven GR level and gsr gene expression was also substantiated from the present study. NAC supplement maintains basal GR level via prevention of gsr over expression. The profile is directly correlated with reduced nuclear Nrf-2 level in GSH pre- administered cells and enhanced NRf-2 translocation in BSO pre-treated cells (Fig. 1d). Inhibition of GR activity Time kinetics study reveals substantial inhibition of GR activity in response to arsenic exposure against
123
Biometals
Fig. 2 Time kinetics of GR inhibition. a Inhibition of GR activity in arsenic treated hepatocytes as detected by NADPH oxidation rate. (*p \ 0.05). b NAC pre-treatment exhibits increased GR activity as compared to NAC untreated cells. (*Significant difference from control, #Significant difference from arsenic treatment; p \ 0.05). c Time dependent substrate
ES (GR–GSSG) complex formation rate in arsenic treated cells. d Lineweaver–Burk (double reciprocal) plot of GR activity against varying substrate (GS–SG) concentration in control, arsenic treated and NAC pre treated cells. e Lineweaver–Burk (double reciprocal) plot of GR activity against varied NADPH concentration in control and arsenic treated cells
untreated cells (Fig. 2a). NAC pre administration maintains the enzyme activity nearer to control (Fig. 2b). Although formation of enzyme–substrate complex (ES) is elevated in treated cells as compared to control time dependent decrease in rate is significant and concomitant with inhibition of GR activity (Fig. 2c). Furthermore, varying concentrations of both the GSSG (Fig. 2d) and NADPH (Fig. 2e) exhibit a
remarkable decrease in the reaction velocity (Vmax) and reduced Km of GR. Direct role of arsenic in GR inhibition is investigated with the cytosol incubated with arsenic trioxide. Result demonstrates reduction both in Vmax and Km in arsenic treated cells as compared to control. Magnitude of decrease in Vmax is significantly higher as compared to decrease in Km (Fig. 3a, b). Concentration (AsIII) dependent decrease
123
Biometals Fig. 3 Modulation of GR activity kinetics with arsenic in a cell free system. a GR inhibition profile in response to arsenic trioxide as evidenced by its activity against different GS–SG concentrations. b Double reciprocal plot of GR activity in control and arsenic incubated cytosol. c Concentration and d time dependent ES formation kinetics in arsenic treated cytosol. e Lineweaver–Burk (double reciprocal) plot of GR activity against varied NADPH concentration in control and arsenic incubated cytosol
in rate of ES formation is also evident from the present study. Results clearly demonstrate that maximum substrate binding requires elevated substrate concentration in the reaction system incubated with higher arsenic concentration (Fig. 3c). Not deviating from the fundamental dynamics of ES formation observed
in vitro, arsenic exposed cytosol also results in increased ES complex formation as compared to control accompanied by time dependent inhibition of substrate binding. (Fig. 3d). On the other hand, varying NADPH concentration reduced both Vmax and Km of GR (Fig. 3e).
123
Biometals Fig. 4 Arsenic induced cellular deformation, nuclear breakdown and apoptosis in rat hepatocytes. a Cellular deformation, nuclear damage and phosphatidylserine externalization (apoptosis) is evidenced in eosin– haematoxylin, Hoechst and Annexin V/6CFDA stained cells respectively. b Sequential events of apoptosis progression including cell leakage, mitochondrial damage, apoptosome formation, caspase 3,9 activation, DNA ladder formation and PARP cleavage. c Reduced caspase 3 activation in exogenous GR supplemented cells
Induction of apoptosis as a consequence of GR inhibition Arsenic treatment results in cell damage and nuclear break down as compared to control which is substantially clear from eosin–haematoxylin and Hoechst stained cells (Fig. 4a), Arsenic treated cells demonstrate elevated gamma glutamyl transpeptidase (GGT) released in medium which was reduced in NAC pretreated cells. Western blot analysis reveals elevated cytosolic apoptosis inducing factor (AIF) level, cyt C-caspase 9 conjugation, caspase 3 cleavage, DNA ladder formation and PARP1 cleavage in support of execution of apoptosis (Fig. 4b). Exogenous supplementation of GR system demonstrates reduced caspase 3 activity in arsenic treated cells. Reduction
123
of caspase activity is most prominent in complete GR system assemble with pure GR enzyme and NADPH, the cofactor (Fig. 4c).
Discussion Modulation of GR expression by arsenic induced superoxide anion Eminence of intracellular GR essentially correlated with enhanced intracellular GSH level performing a cytoprotective function (Townsend and Tew 2003; Asmis et al. 2005). Hepatocytes also export GSH through sinusoidal transport into plasma or into bile through canalicular transport. (Rius et al. 2003). The
Biometals
present investigation demonstrates that high GSH level in the cytosol is apparent in arsenic treated cells which correlates positively with the GR expression. Result indicates the essential contribution of GR in maintenance cellular reducing state. Involvement of ROS in induction of GR is clearly demonstrated from subdued GR level in NAC pre-treated cells as compared to arsenic treated cells. On the other hand, the redox regulated transcription factors, Nrf-2 was found to be associated with cyto-protective function regulating expression of glutathione-s-transferase (gst) gene (Alam et al. 1995). Further investigation suggested that Nrf2 regulates GSH synthetic enzymes such as gamma glutamyl cysteine ligase (GCL) and glutathione synthetase (GSS) as well as cysteine/ glutamate exchange transporter that regulates cysteine influx (Erickson et al. 2002; Sasaki et al. 2002). Recently it is also shown in lung and embryonic fibroblast cells that Nrf 2 dependent regulation of GR, independent of its biosynthesis is critical for cell survival (Harvey et al. 2009). Direct correlation between nuclear translocation of Nrf 2 and GR expression is demonstrated here. Keap 1 is the cytoskeletal adaptor protein has been shown to maintain a steady state of cytosolic Nrf2 via Keap1– Nrf2 interaction (Singh et al. 2006). Present investigation demonstrates involvement of Keap 1 in regulation of Nrf-2 in arsenic treated rat hepatocytes. Pretreatment with the GSH synthesis inhibitor, BSO results in profound ROS generation leading to robust expression of gsr (gene of GR) accompanied by enhanced NRF-2 translocation. It is surmised that arsenic exposure heightened the demand of GR expression depending on the requirement of GSH by the cell which is proportional to the magnitude of free radicals generated. However, increase in apoptotic index concomitant with rise in SOA is found to be the signature of inadequate adaptive competency and faulty compensatory mechanism. Inhibitory mechanism of GR by arsenic and arsenic induced free radicals While arsenic mediated free radical induces GR expression the time kinetics study reveals substantial reduction in GR activity. However, NAC pre-treatment demonstrates further augmentation in GR activity. It is known that GR activity follows the ping pong or branched mechanism and product inhibition of GR
by GSH is expected to be non-competitive (Chung et al. 1991). The present investigation clearly elucidates direct inhibition of GR activity manifested either by the metal itself or by end product GSH as a consequence of substantial free radical generation. GR expression level and the GR activity does not correlate linearly, therefore additional investigation has been performed to elucidate the grounds of GR inhibition concomitant with enhanced GR expression level. Kinetics study with GR enriched cytosolic fraction from control and treated cells in presence of different concentrations of GSSG reveals a decrease in Vmax which further confirms that As intoxication inhibition of GR activity. On the basis of existing information of kinetics and biochemical property of GR enzyme (Tandogan and Ulusu 2006, 2010a, b) direct inhibitory mechanism is investigated in arsenic treated cells. In the present study reduction in Vmax is obviously the hallmark of direct catalytic inhibition by arsenic or arsenic generated free radicals. However, increased substrate affinity is also evident from reduced Km for the substrate GSSG, which is apparently accompanied by enhanced GR synthesis. The kinetics of GR activity varies between arsenic intoxicated cells and the cells pretreated with NAC. NAC pre-treated cells demonstrate further increase in Vmax and Km in response to arsenic. This event can directly be correlated with time kinetics indicating that modulation of GR activity is dependent on the magnitude of free radical generation. The scenario is more decisively observed in the activity kinetics of NAC pre-treated cells, where prevention of ROS generation reduces GR expression level although the level remained high as compared to control. Concomitant rise in Vmax is even higher than control which was markedly repressed due to arsenic exposure. Here we hypothesize that other than GSH requirement and availability, activity status of GR is also a key factor for assigning the gsr expression demand and vice-verse. Pattern of arsenic induced free radical mediated GR inhibition appears to be of partially uncompetitive or mixed type where increase in substrate affinity is apparently comparable in enhanced GSSG–GR binding as compared to control. However, a time dependent gradual decrease in substrate binding rate was evident which indicates enzyme inhibition. With varying NADPH concentration the pattern of enzyme inhibition is also found to be partially uncompetitive or mixed where Km and
123
Biometals
Vmax are reduced. In the in vitro assay too the pattern of enzyme inhibition is mixed, especially where the arsenic binding rate with GR is rather less as compared to the arsenic binding rate of GR-NADPH complex. Thus the degree of GR inhibition triggers enhanced enzyme synthesis. Therefore, it is surmised that the cellular adaptive response effects elevated GR expression level by arsenic or arsenic induced free radicals which indirectly leads to partially uncompetitive mode of inhibition. However, time dependent enzyme inhibition is prominent from the reduced rate of substrate binding parallel to induced GSH accumulation either as a result of arsenic interference or due to end product inhibition by GSH. The influence of GR expression or GSH accumulation on enzyme inhibition kinetics was adequately prominent in in vitro experiments. Therefore to understand the direct inhibition mechanism, a cell free system was employed. Results clearly demonstrate direct mixed type of inhibition of GR by arsenic accompanied by a substantial decrease in apparent Km and Vmax. On the other hand the type of inhibition is partially uncompetitive in response to varying NADPH concentration in the cell free system.
detected by released GGT, the membrane bound enzyme involved in metabolic processing of external glutathione. Elevated level of secreted GGT is recently considered as an effective marker of oxidative stress and liver damage often independent of metabolic syndrome (Lee et al. 2004; Lim et al. 2004; Yamada et al. 2006). GGT secretion in the medium was found to be significantly high with promotion of apoptosis which is also confirmed by mitochondrial membrane damage, putative apoptosome formation as well as caspase activation. Apoptosis is eventually reflected in nuclear degradation and Annexin V positive cells. GSH/GSSG maintenance reaction by GR uses NADPH to convert GSSG to GSH. The GSH/GSSG ratio is thus ultimately related to NADPH levels, which is determined by energy status of the cell. (Harvey et al. 2009). To confirm the involvement of GR inhibition in arsenic induced apoptosis further, cells were supplemented with exogenous GR system which significantly reduced caspase 3 activation in arsenic treated cells.
Conclusion Deficiency of GR activity in promotion of apoptosis The worth mentioning reviews by Tandogan and Ulusu (2006), (2010a, b) concluded that during oxidative stress and deficiency of GR, loss of thiol redox balance may cause deleterious consequences for metabolic regulation, cellular integrity, and organ homeostasis due to accumulation of intracellular GSSG. GR inhibition disturbs cellular prooxidant/antioxidant balance and may contribute to the genesis of many diseases. The present investigation demonstrates the apoptotic endpoint due to GR inhibition. We have already reported toxic manifestation of arsenic leading to apoptosis in different cell types, such as differentiated rat hepatocytes and hepatic stem cells (Ray et al. 2008; Agarwal et al. 2009). It is abundantly clear from the sequential events of caspase dependent apoptosis that disruption of mitochondrial membrane integrity occurs due to arsenic. Leakage of AIF in the cytosol is in agreement with the existing report of Holubec et al. (2005). Cellular damage is initially
123
It is concluded that arsenic intoxication exerts remarkable free radical stress leading to induction of GSH. With generation of arsenic induced free radicals, gsr expression is considerably enhanced within the cells leading to increased GR level. However, cellular adaptive proficiency is compromised by inhibited GR activity. Furthermore, from the pattern of inhibition it is concluded that the consequence is much severe as arsenic and arsenic induced free radical acts together in the inhibition process preferably at the site of enzyme–substrate complex both for GSSG as well as NADPH. Consequentially GSH restoration is hindered and caspase dependent apoptosis is implemented. The mechanism is summarized in the graphical abstract. Acknowledgments AR is grateful to Council for Scientific and Industrial Research for a Senior Research Fellowship, SC gratefully acknowledges DST for a SRF (Project No SR/SO/AS22/2008) and SM is grateful to University Grants Commission for financial support and SB acknowledges the National Academy of Sciences, India for the award of Senior Scientist, Platinum Jubilee Fellowship. Conflict of interest
No competing financial interest exists.
Biometals
References Agarwal S, Roy S, Ray A, Mazumder S, Bhattacharya S (2009) Arsenic trioxide and lead acetate induce apoptosis in adult rat hepatic stem cells. Cell Biol Toxicol 25:403–413 Alam J, Camhi S, Choi AMK (1995) Identification of a second region upstream of the mouse heme oxygenase-1 gene that functions as a basal level and induce dependent transcriptional enhancer. J Biol Chem 270:11977–11984 Asmis R, Wang Y, Xu L, Kisgati M, Begley JG, Mieyal JJ (2005) A novel thiol oxidation-based mechanism for adriamycin-induced cell injury in human macrophages. FASEB J 19:1866–1868 Bhattacharya S, Bhattacharya A, Roy S (2007) Arsenic induced responses in freshwater teleosts. Fish Physiol Biochem 33:463–473 Chung PM, Cappel RE, Gilbert HF (1991) Inhibition of glutathione disulfide reductase by glutathione. Arch Biochem Biophys 288:48–53 Datta S, Mazumder S, Ghosh D, Dey S, Bhattacharya S (2009) Low concentration of arsenic could induce caspase 3 mediated head kidney macrophage apoptosis with JNKp38 activation in Clarias batrachus. Toxicol Appl Pharmacol 241:329–338 Erickson AM, Nevarea Z, Gipp JJ, Mulcahy RT (2002) Identification of a variant antioxidant response element in the promoter of the human glutamate–cysteine ligase modifier subunit gene: revision of the ARE consensus sequence. J Biol Chem 277:30730–30737 Estey E, Garcia-Manero G, Ferrajoli A, Faderl S, Verstovsek S, Jones D, Kantarjian H (2006) Use of all-trans retinoic acid plus arsenic trioxide as an alternative to chemotherapy in untreated acute promyelocytic leukemia. Blood 107: 3469–3473 Gore SD, Gojo I, Sekeres MA, Morris L, Devetten M, Jamieson K, Redner RL, Arceci R, Owoeye I, Dauses T, Schachter-Tokarz E, Gallagher RE (2010) Single cycle of arsenic trioxide based consolidation chemotherapy spares anthracycline exposure in the primary management of acute promyelocytic leukemia. J Clin Oncol 28:1047–1053 Harvey CJ, Thimmulappa RK, Singh A, Blake DJ, Ling G, Wakabayashi N, Fujii J, Myers A, Biswal S (2009) Nrf2regulated glutathione recycling independent of biosynthesis is critical for cell survival during oxidative stress. Free Rad Biol Med 46:443–453 Holubec H, Payne CM, Bernstein H, Dvorakova K, Bernstein C, Waltmire CN, Warneke JA, Garewal H (2005) Assessment of apoptosis by immunohistochemical markers compared to cellular morphology in ex vivo-stressed colonic mucosa. J Histochem Cytochem 53:229–235 Inglis JK (1980) Introduction to laboratory animal science and technology. Pergamon Press, Oxford Lee DH, Blomhoff R, Jacobs DJ (2004) Is serum gamma glutamyltransferase a marker of oxidative stress? Free Radic Res 38:535–539 Lim JS, Yang JH, Chun BY, Kam S, Jacobs DR Jr, Lee DH (2004) Is serum c-glutamyltransferase inversely associated with serum antioxidants as a marker of oxidative stress? Free Radic Biol Med 37:1018–1023
Lin CC, Hsu C, Hsu CH, Hsu WL, Cheng AL, Yang CH (2007) Arsenic trioxide in patients with hepatocellular carcinoma: a phase II trial. Invest New Drugs 25:77–84 Loginov AS, Matiushin BN, Tkachev VD (1997) The clinical significance of the liver glutathione system in chronic lesions. Ter Arkh 69:25–27 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Measurement of protein with the Folin phenol reagent. J Biol Chem 193:265–275 Maity S, Roy S, Bhattacharya S, Chowdhury S (2008) Antioxidants in Lampito mauritii are the biomarkers of soil contamination by lead and zinc. Environ Pollut 151:1–7 Martin HL, Teismann P (2009) Glutathione a review on its role and significance in Parkinson’s disease. FASEB J 23: 3263–3272 Mei X, Xu D, Xu S, Zheng Y, Xu S (2012) Novel role of Zn(II)curcumin in enhancing cell proliferation and adjusting proinflammatory cytokine-mediated oxidative damage of ethanol-induced acute gastric ulcers. Chem Biol Interact 197:31–39 Montero J, Morales A, Llacuna L, Lluis JM, Terrones O, Basan˜ez G, Antonsson B, Prieto J, Garcı´a-Ruiz C, Colell A, Ferna´ndez-Checa JC (2008) Mitochondrial cholesterol contributes to chemotherapy resistance in hepatocellular carcinoma. Cancer Res 68:5246–5256 Ray A, Roy S, Agarwal S, Bhattacharya S (2008) As2O3, toxicity in rat hepatocytes: manifestation of caspase-mediated apoptosis. Toxicol Ind Health 34:643–653 Rius M, Nies AT, Hummel-Eisenbeiss J, Jedlitschky G, Keppler D (2003) Cotransport of reduced glutathione with bile salts by MRP4 (ABCC4) localized to the basolateral hepatocyte membrane. Hepatology 38:374–384 Roy S, Bhattacharya S (2006) Arsenic induced histopathology and synthesis of stress proteins in liver and kidney of Channa punctatus. Ecotoxicol Environ Saf 65:218–229 Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, New York Sasaki H, Sato H, Kuriyama-Matsumura K, Sato K, Maebara K, Wang H, Tamba M, Itoh K, Yamamoto M, Bannai S (2002) Electrophile response element-mediated induction of the cystine/glutamate exchange transporter gene expression. J Biol Chem 277:44765–44771 Senft AP, Dalton TP, Shertzer HG (2000) Determining glutathione and glutathione disulfide using the fluorescence probe O-phthalaldehyde. Anal Biochem 280:80–86 Singh A, Misra V, Thimmulappa RK, Lee H, Ames S, Hoque MO, Herman JG, Baylin SB, Sidransky D, Gabrielson E, Brock MV, Biswal S (2006) Dysfunctional KEAP1-NRF2 interaction in non-small-cell lung cancer. PLoS Med 3:e420 Snedecor GW, Cochran WG (1967) Statistical methods. The Iowa State University Press, Iowa S´redzin´ska K, Galicka A, Porowska H, Sredzin´ski Ł, Porowski T, Popko J (2009) Glutathione reductase activity correlates with concentration of extracellular matrix degradation products in synovial fluid from patients with joint diseases. Acta Biochim Pol 56:635–640 Tandogan B, Ulusu NN (2006) A comparative study with colchicine on glutathione reductase. FABAD J Pharm Sci 31:230–237
123
Biometals Tandogan B, Ulusu NN (2010a) Comparative in vitro effects of some metal ions on bovine kidney cortex glutathione reductase. Prep Biochem Biotech 40:405–411 Tandogan B, Ulusu NN (2010b) Kinetic mechanism and molecular properties of glutathione reductase. Protein J 29:380–385 Townsend DM, Tew KD (2003) The role of glutathione-Stransferase in anti-cancer drug resistance. Oncogene 22:7369–7375 Valko M, Rhodes C, Moncol J, Izakovic M, Mazur M (2006) Free radicals, metals and antioxidants in oxidative stressinduced cancer. Chem Biol Interact 160:1–40 Wang X, Zhang J, Zhao L, Hu S, Piao F (2013) Effect of subchronic exposure to arsenic on levels of essential trace
123
elements in mice brain and its gender difference. Biometals 26:123–131 Wiseman DA, Sharma S, Black SM (1999) Elevated zinc induces endothelial apoptosis via disruption of glutathione metabolism: role of the ADP translocator. Biometals 23:19–30 Yamada J, Tomiyama J, Yambe M, Koji Y, Motobe K, Shiina K, Yamamoto Y, Yamashina A (2006) Elevated serum levels of alanine aminotransferase and gamma glutamyltransferase are markers of inflammation and oxidative stress independent of the metabolic syndrome. Atherosclerosis 189:198–205
