Arsenic trioxide induced indirect and direct inhibitionof glutathione reductase leads to apoptosis in rat hepatocytes

Atish Ray • Sarmishtha Chatterjee •

Sandip Mukherjee • Shelley Bhattacharya

Received: 4 January 2014 / Accepted: 17 February 2014

� 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, apop-

tosome formation, caspase activation, DNA damage

and break down of PARP suggest consequential

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 cat-

alyzes 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 dys-

function and GR is important in the maintenance of

redox ratio (Roy and Bhattacharya 2006; Maity et al.

2008; Bhattacharya et al. 2007). The clinical signif-

icance 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

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: shelleyb38@gmail.com

Present Address:

A. Ray

Immunobiology Group, Department of Zoology,

University of Delhi, Delhi, India

123

Biometals

DOI 10.1007/s10534-014-9722-y

cancer (Valko et al. 2006). To demonstrate the

significance of GR, it is necessary to consider the

series reaction of detoxication process. During forma-

tion 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 gluta-

thione and GR system is also found to be directly

coupled with non hepatic disorder such as Parkinson’s

disease and rheumatoid arthritis (Martin and Teis-

mann 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 environ-

mental 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 accumula-

tive 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

to arsenic induced apoptosis in hepatocytes (Ray et al.

2008). The present investigation establishes the associ-

ation 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 bidirec-

tional 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, Wis-

consin, 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 Chem-

ical 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 transcrip-

tase 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

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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), exog-

enous 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) instruc-

tion. Replicates were used for conventional supple-

mentation 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 reduced glutathione

GSH was estimated from protein-free clear superna-

tant, 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 o-

phthalaldehyde (OPA) fluorometric method (k excita-

tion = 350, k emission = 430) (Senft et al. 2000).

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. Activ-

ity 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 ade-

quate NADPH (100 lM) with varying GSSG concen-

tration. Enzyme kinetics was represented as

Lineweaver–Burk Plots.

Substrate binding assay and co-immuno

precipitation

For concentration and time dependent substrate bind-

ing 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) sam-

ples. 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

An aliquot of cytosol containing 100 lg protein was

run through 10 % sodium dodecyl sulphate—poly-

acrylamide 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

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123

primary antibodies and with alkaline phosphatase

conjugated rabbit IgG as secondary antibody (diluted

to 1:2,000). The blots were developed using 5-bromo-

4-chloro-3-indolylphosphate/nitro blue tetrazolium

(BCIP/NBT) as substrate. b actin was considered as

loading control.

Immunofluorescence

Respective cell populations fixed in 4 % paraformal-

dehyde 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

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 appropri-

ate filter set. Normal cells appeared positive only in

green fluorescence and apoptotic cells appeared

positive in both green and red fluorescence.

Cellular deformation and nuclear breakdown

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 gradi-

ents 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.

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 syn-

thesized from total RNA as per the protocol provided.

PCR was performed according to manufacturer’s instruc-

tion 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

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

Stress imposed on the cell

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

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123

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 gener-

ation and apoptotic index which was further investi-

gated in presence of exogenous NAC and BSO. NAC

enriched reduced environment prevents induction of

intracellular glutathione by scavenging the free radi-

cals 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

Fig. 1 a NBT reduction assay exhibits generation of superox-

ide 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

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123

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

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

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123

in rate of ES formation is also evident from the present

study. Results clearly demonstrate that maximum

substrate binding requires elevated substrate concen-

tration 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).

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

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123

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 substan-

tially clear from eosin–haematoxylin and Hoechst

stained cells (Fig. 4a), Arsenic treated cells demon-

strate elevated gamma glutamyl transpeptidase (GGT)

released in medium which was reduced in NAC pre-

treated 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 supple-

mentation of GR system demonstrates reduced

caspase 3 activity in arsenic treated cells. Reduction

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

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

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123

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 investi-

gation demonstrates involvement of Keap 1 in regu-

lation of Nrf-2 in arsenic treated rat hepatocytes. Pre-

treatment 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-treat-

ment demonstrates further augmentation in GR activ-

ity. 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 eluci-

dates 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 gener-

ated free radicals. However, increased substrate affin-

ity 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 pre-

treated 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 concentra-

tion the pattern of enzyme inhibition is also found to

be partially uncompetitive or mixed where Km and

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123

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 expres-

sion 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 accumula-

tion either as a result of arsenic interference or due to

end product inhibition by GSH.

The influence of GR expression or GSH accumu-

lation 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 demon-

strate 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.

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 intracel-

lular GSSG. GR inhibition disturbs cellular pro-

oxidant/antioxidant balance and may contribute to

the genesis of many diseases. The present investi-

gation 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

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 apopto-

some formation as well as caspase activation.

Apoptosis is eventually reflected in nuclear degra-

dation 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

It is concluded that arsenic intoxication exerts remark-

able 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/AS-

22/2008) 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.

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123

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