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published online 2 October 2012Toxicol Ind HealthSarmishtha Chatterjee, Atish Ray, Sandip Mukherjee, Soumik Agarwal, Rakesh Kundu and Shelley Bhattacharya

Low concentration of mercury induces autophagic cell death in rat hepatocytes  

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Article

Low concentration of mercuryinduces autophagic cell death inrat hepatocytes

Sarmishtha Chatterjee1, Atish Ray1, Sandip Mukherjee1,Soumik Agarwal1, Rakesh Kundu2 andShelley Bhattacharya1

AbstractIn the present study, we attempted to elucidate the induction of autophagy in rat hepatocytes by a lowconcentration of mercury. Hepatocytes treated with different doses of mercuric chloride (HgCl2; 1, 2.5, 5 and10 mM) and at different time intervals (0 min, 30 min, 1 h, 2 h and 4 h) show autophagic cell death only at 5 mMHgCl2 within 30 min of incubation. At 1 and 2.5 mM HgCl2, no cell death is recorded, while apoptosis is found at10 mM HgCl2, as evidenced by the activation of caspase 3. Autophagic cell death is confirmed by the presence ofmonodansylcadaverine (MDC) positive hepatocytes which is found to be highest at 1 h. Atg5-Atg12 covalent-conjugation triggers the autophagic pathway within 30 min of 5 mM HgCl2 treatment and continues till 4 h ofincubation. In addition, damage-regulated autophagy modulator (DRAM) expression gradually increases from30 min to 4 h of treatment with mercury and a corresponding linear decrease in p53 has been observed. It isconcluded that a low concentration (5 mM HgCl2) of mercury induces autophagy or nonapoptotic programmedcell death following an Atg5-Atg12 covalent-conjugation pathway, which is modulated by DRAM in ap53-dependent manner.

KeywordsRat liver, autophagy, mercury, Atg 5-12, MDC, DRAM

Introduction

Mercury is an all pervasive environmental contami-

nant, which gets fixed and stored in tissues of organ-

isms directly affecting the individual’s health (Streit,

1992). It interacts with the sulfhydryl group of pro-

teins by displacing other metals from their natural-

binding sites (Flora et al., 2008). Constant exposure

of liver to heavy metals causes programmed cell death

by apoptosis, autophagy or necrosis. Although pro-

grammed cell death occurs at a negligible rate under

normal conditions (Bursch et al., 1985; James and

Muskhelishvili, 1994), autophagy has been observed

under pathological conditions (Martinet et al., 2006;

Wang et al., 2010). Although autophagy, the type II

programmed cell death, is considered a genetically

programmed mechanism by which cellular proteins

and organelles are degraded nonselectively (Kopitz

et al., 1990; Levine and Klionsky, 2004), there is an

evidence from several independent groups, indicating

a selective autophagic elimination of depolarized

mitochondria, endoplasmic reticulum and peroxi-

somes (Elmore et al., 2001; Hamasaki et al., 2005;

Iwata et al., 2006; Kissova et al., 2004). Yu et al.

(2006) showed that catalase, a key enzyme of the

antioxidant defense mechanism, was selectively

1 Environmental Toxicology Laboratory, Department of Zoology,Centre for Advanced Studies, Visva-Bharati University, Santinike-tan, West Bengal, India2 Molecular Endocrinology Laboratory, Department of Zoology,Centre for Advanced Studies, Visva-Bharati University, Santinike-tan, West Bengal, India

Corresponding author:Shelley Bhattacharya, Environmental Toxicology Laboratory,Department of Zoology, Centre for Advanced Studies,Visva-Bharati University, Santiniketan 731235, West Bengal,IndiaEmail: shelleyb38@gmail.com

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eliminated during autophagic cell death. Therefore,

an alternative death induction mechanism by autop-

hagy may consist of selective elimination of vital

organelles and/or proteins may be involved in cell

survival and homeostasis, although the mechanism

regulating this selectivity still remains obscure. This

process has been implicated in a range of disorders

and therefore is of major interest. Formation of the

double membrane cytosolic vesicle, the autophago-

some, is the hallmark of this evolutionary-conserved

process.

During the early formation of autophagosome, the

membranes enlarge in magnitude by altering the

shape to form a cup-fashioned structure called phago-

pores (Chan et al., 2006). Phagopores are formed

either by the isolation of original membrane or by the

assimilation of additional lipids or tubulation of the

existing compartments. Sequestration of cytosolic

content finally takes place in these vesicles (Klionsky

et al., 2007; Mizushima et al., 2008) and the mature

autophagosome is formed by the fusion of autophagic

vesicle and lysosome (Martinet et al., 2006). Mono-

dansylcadaverine (MDC) is a potential marker to

detect these autophagic vesicles (Biederbick et al.,

1995). However, some specific molecular markers are

also known to express during this complex process.

Several proteins that are involved in the formation

of autophagosomes are identified. Atg6/Beclin-1

(BCN1, homolog of yeast Atg6), a lipid kinase

Bcl-2 interacting protein, mediates the autophagy

induction (Cao and Klionsky, 2007; Klionsky and

Emr, 2000). Atg5-Atg12 conjugation is also a partici-

pant in the initial autophagosome formation. Further-

more, damage-regulated autophagy modulator

(DRAM), composed of 238 amino acid proteins and

transactivated by p53, is a modulator of autophagic

regulation (Crighton et al., 2007).

We have reported earlier (Ray et al., 2008) that the

higher concentration of metals drives the rat hepato-

cytes toward caspase-dependent pathway with an

indication of vacuolization in the cell before the onset

of apoptosis. However, a few recent reports demon-

strated that the lower concentration of metals causes

the induction of autophagic cell death in different cell

types; arsenic was reported to induce autophagy at a

concentration of 2 mM in malignant glioma cell lines

(Kanzawa et al., 2003) and at 6 mM in lymphoblastoid

cell lines (Bolt et al., 2010); Dong et al. (2009)

recorded autophagy in vascular endothelial cells at

less than 10 mM cadmium (Cd); while more recently,

Chargui et al. (2011) reported Cd-induced autophagy

in rat kidney at an in vivo dose of 0.3 mg Cd/kg body

mass. Mercury is a ubiquitous hepatotoxicant that

affects all the organisms deleteriously. Considering

the dearth of information on the nature of cytotoxicity

of low concentration of mercury, we have attempted

here to elucidate autophagic cell death in rat hepato-

cytes at low levels of mercury exposure.

Materials and methods

Chemicals and reagents

Collagenase type IV, MDC, Hoechst-33258, Annexin

V-Cy3™ (Apoptotic detection kit; Cat. no. APO-AC),

poly-L-lysine, primary antibodies for anti-Atg5 (anti-

rabbit; Cat. no. A0856), anti-Atg12 (anti-rabbit; Cat.

no. PRS4421), anti-Atg6 (anti-rabbit; Cat. no.

B6061) and alkaline phosphatase (ALP)-conjugated

anti-rabbit secondary antibody were procured from

Sigma-Aldrich Chemicals Private Ltd (St Louis,

Missouri, USA). Other primary antibodies such as,

anti-TIGAR (Tp53-induced glycolysis and apoptosis

regulator) (sc-67273), anti-DRAM (sc-98654), anti-

caspase 3 (sc-7148), anti-p53 (sc-6243) and anti-Bcl-

2 (sc-492) were purchased from Santa Cruz Biotech-

nology Inc. (Madison, Wisconsin, USA). Dulbecco’s

Modified Eagle Medium (DMEM) and other tissue

culture materials were obtained from Gibco-BRL (Life

Technologies Inc., Gaithersburg, Maryland, USA). All

other chemicals used were of analytical grade, pur-

chased from Sisco Research Laboratories (Mumbai,

India) and E. Merck (Darmstadt, Germany).

Animals and their maintenance

Adult male albino rats of the Sprague–Dawley strain

weighing 180–220 g were maintained under standard

laboratory conditions in clean polypropylene cages

(two animals per cage) under 12 h of light/dark cycle

at 25 + 2�C, with food (rat chow; Hindustan Lever

Ltd, Bombay, India) and water provided ad libitum

(Inglis, 1980). Animals were acclimated for about a

week prior to use. Rats showing any abnormal

behavior were removed from the cage immediately.

All the experiments were performed following the

guidelines prescribed by the Institutional Animal

Ethics Committee.

Isolation of hepatocytes

A two-step collagenase digestion method (Shimano

et al., 2003) with some modifications was used to isolate

hepatocytes. In brief, livers were perfused at 37�C with

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200 ml of Ca2þ free Hanks’-balanced salt solution

(HBSS), containing 0.05% collagenase type IV. The

livers were promptly removed and minced with a razor.

For the isolation of parenchymal cells, the minced livers

were incubated in 50 ml of Ca2þ-HBSS containing

0.1% of collagenase type IV for 60 min at 38�C, filtered

through a 60-mm nylon mesh and centrifuged at 50g for

2 min. Pellets were used as a sample of parenchymal

cells containing differentiated hepatocytes.

Mercuric chloride treatment of isolatedhepatocytes

An aliquot of 1 � 106 cells were cultured in 24-well

plates in DMEM and nonessential amino acids

supplemented with penicillin (100 U/ml) and strepto-

mycin (100 mg/ml) in a humidified 95% O2/5% CO2

atmosphere at 37�C. The cells were treated with mer-

curic chloride (HgCl2) at different doses (1, 2.5 , 5 and

10 mM) and different time intervals (30 min, 1 h, 2 h

and 4 h) or as per the design of the experiment keep-

ing a concurrent control.

Assessment of cell viability

Aliquots of 5 mL of control and HgCl2-treated cell

suspensions were taken for Trypan blue dye exclu-

sion assay to check the viability of the cells using a

hemocytometer slide and are represented as percent-

age viability. The rate of cytotoxicity of HgCl2was determined in a 96-well plate by estimating

the formation of 3-(4,5-dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromide (MTT) formazan using

the MTT Cell Growth Assay kit (Millipore, Chemi-

con, Cat. No. CT02, Bedford, Massachusetts, USA)

following the manufacturer’s instructions. The

absorbance was measured at 570 nm (test wave-

length) and 630 nm (reference wavelength) in a

Thermo Multiskan Ascent 96/384 Plate Reader

(Thermo Labsystems, Massachusetts, USA). Results

of all the three individual experiments are calculated

as percentage cell death and are expressed as

mean + SEM.

Detection of autophagolysosome by MDC

Control and HgCl2-treated cells were fixed in ice-cold

methanol and allowed to adhere on poly-L-lysine coated

slides. Cells were incubated with 0.05 mM MDC dis-

solved in phosphate-buffered saline (PBS) at 37�C for

10 min to label the autophagic vesicles by following a

previously described method (Biederbick et al., 1995).

After incubation, cells were thoroughly washed with

PBS (four times) and images were captured in fluores-

cence microscope (Carl Zeiss, Goettingen, Germany)

using Jenoptik software (ProgRes1 CapturePro 2.7).

Nuclear staining by Hoechst-33258 and EtBr

Nuclear DNA was visualized in liver cells by staining

with the DNA-specific dye Hoechst-33258 (Bisbenzi-

mide H). Ice-cold methanol-fixed liver cells were resus-

pended in 200ml of PBS and 2 ml of Hoechst-33258 was

added to a final concentration of 1 mg/ml and incubated

for 10 min. Excess stain was washed with PBS, and the

cells were observed immediately under the fluorescence

microscope (Carl Zeiss) using blue filter. Ethidium bro-

mide (EtBr at 1 mg/ml) staining was also carried out in a

similar manner and visualized under the microscope

with an ultraviolet filter.

AnnexinV-Cy3 and 6-CFDA staining ofhepatocytes

AnnexinV-Cy3 and 6-CFDA (6-carboxyfluorescein-

diacetate) staining was performed following the man-

ufacturer’s protocol (Sigma-Aldrich Chemicals

Private Ltd). Briefly, liver cells were allowed to

adhere on poly-L-lysine-coated slide and then stained

with AnnexinV-Cy3 and 6-CFDA reagents in 1X

binding buffer at room temperature for 10 min in the

dark. Excess stain was removed by blotting the slides

with 1X binding buffer and images were captured in

fluorescence microscope (Carl Zeiss) using Jenoptik

software (ProgRes1 CapturePro 2.7). The cells

could be visualized as green (live cells), red (necro-

tic) and green plus red (apoptotic).

Electrophoresis and immunoblotting

Control and HgCl2-treated hepatocytes were lysed and

the protein content was measured according to Lowry

et al. (1951). A total of 60 mg of protein was resolved

on 10% SDS-PAGE (Sodium Dodecyl Sulfate Poly

Acrylamide Gel Electrophoresis) and transferred to

PVDF (Poly Vinylidene Fluoride) membranes (Milli-

pore) with the help of semidry trans blot apparatus

(Bio-Rad Trans-Blot1 SD Cell, Hercules, California

USA) using transfer buffer (25 mM Tris, 193 mM gly-

cine, 20% methanol, pH 8.5). The membranes were first

incubated with primary antibodies of Atg5 (rabbit poly-

clonal), DRAM (rabbit polyclonal), p53 (rabbit polyclo-

nal), Atg12 (rabbit polyclonal) and caspase 3 (rabbit

polyclonal) followed by ALP-conjugated anti-rabbit

Chatterjee et al. 3

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secondary antibody at 1:1000 dilution following a pre-

viously described method from our laboratory (Roy and

Bhattacharya, 2006). The protein bands were detected

using 5-bromo 4-chloro 3-indolyl phosphate/nitroblue

tetrazolium.

Results

Assessment of cell viability of rat hepatocytes

There was no mortality of cells exposed to 1 and

2.5 mM of mercury. Cell viability decreased from

95% at 30 min to 89.7% and 89.4% at 4 h when

exposed to 5 and 10 mM of HgCl2, respectively

(Table 1), indicating gradual increase in the percentage

cell death for up to 4 h of treatment with mercury.

Reduction in viability (% control) was 5.17% and

5.49% at 5 and 10 mM, respectively, after 4 h of treat-

ment with mercury. Since treatment with mercury

results in cytotoxicity, we performed MTT assay to

check the effect of low concentration (5 mM) of treat-

ment with mercury on hepatocytes. A minimal increase

in cell death upon treatment with mercury was

recorded and compared with the corresponding con-

trols, and the significant cell death was noted only

between 1 h (2.4% death) and 2 h (5.4% death) of incu-

bation when compared with the 0 h control (Figure 1).

Low concentration of mercury-induced formationof autophagosomes

MDC is a well known indicator of autophagosome for-

mation and the rat hepatocytes treated with 5 mM HgCl2clearly demonstrated an appearance of autophagic vesi-

cles from 30 min to 4 h of incubation, whereas highest

number of MDC-positive hepatocytes was observed at

1 h (Figure 2). However, at 10 mM of HgCl2, no autop-

hagic cell death was seen (result not shown). Therefore,

5 mM of HgCl2 was selected for further investigations.

Treatment with mercury did not alter nuclear andcellular morphology

It was abundantly clear from Figures 3(a) and (b) that

no apoptotic degradation of nucleus occurred until 4 h

of 5 mM HgCl2 treatment. Change in cellular mor-

phology was also assessed in treated rat hepatocytes

by labeling them with AnnexinV-Cy3 and 6-CFDA

double stain, which would indicate apoptotic (green

plus red) or necrotic (red) cell death. In the present

experiment, the majority of cells were AnnexinV-

Cy3 negative, indicating the absence of any type of

programmed cell death (Figure 4).

Expression of the potential autophagic markerson exposure to mercury

Autophagosome signal is initiated by Atg5-Atg12 cova-

lent conjugation, which is evidently demonstrated.

Table 1. Determination of hepatocyte viability (%) by Trypan blue dye exclusion

Time ofincubation

Hepatocyte viability (%)Reduction in hepatocyteviability (% of control)

ControlTreated with5 mM HgCl2

Treated with10 mM HgCl2

Treated with5 mM HgCl2

Treated with10 mM HgCl2

30 min 96.10 + 0.6 95.50 + 0.5 95.60 + 0.5 0.62 0.521 h 95.50 + 0.5 94.20 + 0.4 94.40 + 0.6 1.36 1.152 h 94.80 + 0.4 91.30 + 0.5 91.20 + 0.7 3.69 3.794 h 94.60 + 0.4 89.70 + 0.6 89.40 + 0.3 5.17 5.49

HgCl2: mercuric chloride.

Figure 1. Assessment of cell death by MTT assay. Isolatedrat hepatocytes were incubated with 5 mM HgCl2 from 0 to4 h; white columns correspond to every time period. Lysateswere prepared and subjected to MTT analysis. Each value isthe mean + SEM of five independent experiments; *p < 0.05(versus control). HgCl2: mercuric chloride; MTT: (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide).

4 Toxicology and Industrial Health

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Figure 2. Detection of autophagosomes by MDC. Isolated hepatocytes were incubated without (control) or with 5 mMHgCl2 for different time periods. On termination, incubations cells were subjected to MDC staining. Arrows indicateMDC positive hepatocytes. Scale ¼ 20 mm. MDC: monodansylcadaverine; HgCl2: mercuric chloride.

Figure 3. Detection of change in nuclear morphology by Hoechst-33258 and EtBr. Control and 5 mM HgCl2-treatedhepatocytes were subjected to Hoechst-33258 (a) and EtBr (b) staining and changes in nuclear morphology wereobserved. Arrows indicate Hoechst-33258 and EtBr positive nucleus. Scale¼ 20 mm. EtBr: ethidium bromide; HgCl2: mer-curic chloride.

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Treatment of hepatocytes with 5 mM HgCl2 induced

the expression of Atg5-Atg12 complex from 30 min

to 4 h of incubation. However, at 1 and 2 h, expres-

sion of Atg5-Atg12 complex remained remarkably

high, which decreased in 4 h indicating autophago-

some formation within 30 min and continuing up

to 4 h of treatment with mercury (Figure 5(a)).

Similar result was observed for Atg12 expression.

As Atg5 and Atg12 proteins exist in the form of

Atg5-Atg12 complex, it is difficult to detect the

monomeric forms of these proteins. Therefore, when

the blots were probed to detect free Atg5 (*32 kDa)

or Atg12 (*21 kDa), both of them showed a single

band for Atg5-Atg12 dimer at 55 kDa (Figure 5(a)).

However, TIGAR was not expressed at any time

point of the experimental incubations (Figure not

shown). Therefore, to confirm the phenomenon of

autophagy in hepatocytes at 5 mM HgCl2, we

checked the expression levels of DRAM and p53,

which are the modulators of this process. Interest-

ingly, the expression of DRAM linearly increased

from 30 min to 4 h of treatment with mercury

(Figure 5(b)) in contrast to the level of p53, which

gradually decreased throughout the incubation period

(Figure 5(c)). Moreover, there was no change in the

expression of cytosolic Atg6, Bcl2 and caspase 3

indicating that 5 mM HgCl2 did not trigger apoptosis

(data not shown). To check whether a higher concen-

tration of HgCl2 induced apoptosis, we performed

the Western blot analysis for caspase 3 expression.

A linear increase in the expression of both pro-

caspase and caspase 3 from 30 min to 4 h of incuba-

tion of hepatocytes with 10 mM HgCl2 substantiated

the induction of apoptosis (Figure 6).

Discussion

Mercury is a well-established environmental toxicant

that affects the immune system, damages the nervous

system and gastrointestinal tract and also activates

numerous intracellular signal transduction pathways,

resulting in the induction of programmed cell death

due to apoptosis (Shenker et al., 2000). Induction of

autophagy at a low concentration of heavy metals has

been reported earlier in cell types other than hepato-

cytes (Bolt et al., 2010; Chargui et al., 2011; Dong

et al., 2009; Kanzawa et al., 2003). However, there

is no report on autophagic cell death at low

Figure 4. AnnexinV-Cy3 staining of live cells. Hepatocytes were treated with or without 5 mM HgCl2 for 4 h and weresubjected to AnnexinV-Cy3 and CFDA (6-carboxyfluorescein diacetate) double staining as described in the Materials andMethods section. Viable cells are indicated (arrow) as CFDA positive (green). AnnexinV-Cy3 positive and dual positivecells were absent. Scale ¼ 20 mm. HgCl2: mercuric chloride.

6 Toxicology and Industrial Health

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concentration of HgCl2. Herein, we report for the first

time that hepatocytes exposed to low concentration

(5 mM) of HgCl2 lead to autophagic cell death. The

time kinetics of the type II programmed cell death

correlates well with the cellular deformation, where pro-

nounced autophagosome formation at 5 mM HgCl2 was

visualized under the microscope. Thus, it is abundantly

clear that autophagy initiates before apoptosis dur-

ing concentration-dependent metal exposures corro-

borating earlier observations (Dong et al., 2009;

Gonzalez-Polo et al., 2005).

In the present investigation, hepatocytes exposed

to low concentration of HgCl2 resulted in minimal

cell death from 30 min to 4 h of incubation. To con-

firm whether this cell death was due to autophagy or

apoptosis, we used a specific autophagic marker

(MDC) and found autophagic vesicles within 30 min

of treatment continuing until 4 h of incubation. How-

ever, nuclear staining by Hoechst 33258 and EtBr

confirmed no nuclear deformation, indicating nona-

poptotic cell death. Externalization of phosphatidyl-

serine and phosphatidylethanolamine (PE) is a

hallmark of the changes in the cell surface during

apoptosis (Koopman et al., 1994; Lecoeur et al.,

1997). Negative staining by AnnexinV-Cy3 con-

firmed the absence of apoptosis in 5 mM HgCl2-

treated hepatocytes. Since all the microscopic obser-

vations indicated the induction of autophagic cell

death, a study with specific protein markers of autop-

hagy was worth pursuing to confirm these

observations.

There are several isoforms of Atg proteins that are

indicative of cell death following the autophagic

pathway. The autophagic protein Atg5 remains con-

jugated to Atg12, an ubiquitin-like modifier, and two

other proteins Atg7 and Atg10 for the formation of

autophagosome (Wang and Klionsky, 2003). The

conjugation of Atg5 to Atg12 normally occurs soon

after the individual proteins have been synthesized

(Mehrpour et al., 2010). The Atg5-Atg12 conjugate

localizes to the preautophagosomal structure, a puta-

tive center for autophagosome formation in mam-

mals (Suzuki et al., 2001). Almost all the

conjugates of Atg5 and Atg12 exist in the form of

Atg5-Atg12 dimer, and therefore, it is difficult to

detect the monomeric form of these proteins. Atg5-

Atg12 conjugate is translocated from the cytosol to

the membranes of autophagosome during nutrient

deprivation and immediately upon the completion

of autophagic vacuole formation (Mizushima et al.,

2001). These two autophagic proteins are known to

participate in elongation of the autophagosome

membrane during vacuole formation (Hanada

et al., 2007). Atg5-Atg12 complex facilitates LC3-

PE mediated autophagy (Eisenberg-Lerner et al.,

2009). However, in the present study, we have not

yet been able to clearly establish the role of LC3-

PE; our preliminary data suggest that there is an

Figure 5. Immunoblotting of autophagic markers. Thehepatocytes treated with 5 mM HgCl2 were incubated fordifferent time periods and lysates were subjected to immu-noblot analyses with anti-Atg5 and anti-Atg12 (a), anti-DRAM (b) and anti-p53 (c) antibodies. b-Actin served asan internal control. Figures are representative of one of thefive independent experiments. DRAM: damage-regulatedautophagy modulator; HgCl2: mercuric chloride.

Figure 6. Induction of apoptosis by 10 mM HgCl2. Hepato-cytes treated with 10 mM HgCl2 were subjected to immu-noblot analysis with anti-caspase 3 antibody. Two bands,one of pro-caspase 3 (34 kDa) and another of caspase 3(17 kDa) are observed. b-Actin served as an internal con-trol. Figures are the representatives of one of the threeindependent experiments. HgCl2: mercuric chloride.

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involvement of LC3 in mercury-induced autophagy

(data not shown). Thus, our model indicates that

Atg5-12 complex is playing an important role in

mercury-induced autophagic pathway. In autophagy,

there is a membrane-trafficking process that serves

to deliver cytoplasmic proteins and organelles to the

lysosome for degradation. The major modulators of

the events are TIGAR, DRAM and p53 (Bensaad

et al., 2009; Crighton et al., 2007). The p53-

inducible TIGAR (Tp53-induced glycolysis and

apoptosis regulator) protein has a role in the deple-

tion of reactive oxygen species (Bensaad et al.,

2009). Thus, TIGAR can limit autophagy and drive

the cells toward apoptosis. We checked the expres-

sion level of TIGAR and no notable change was

observed, which clearly indicates that the exposure to

low levels of mercury leads to autophagy. Recent

reports suggested that p53 inhibits mTOR (mammalian

target of rapamycin) (a cell growth regulator) via acti-

vation of AMP (adenosine mono phosphate)-

responsive protein kinase (AMPK) and regulates its

downstream targets including autophagy (Budanov and

Karin, 2008; Feng et al., 2005). Autophagosome for-

mation is promoted by p53 (Crighton et al., 2006) and

DRAM, a lysosomal membrane-spanning protein, acts

at the final stage of autophagy to form autophagolyso-

some (Green and Chipuk, 2006). In the present study,

we have amply demonstrated the inverse relationship

of p53 and DRAM expressions in the event of autop-

hagy induced by a low concentration of mercury.

Conclusion

It is concluded that a low concentration (5 mM) of

HgCl2 has a direct autophagic effect on hepatocytes

as evidenced by the formation of autophagosomes,

where Atg5-Atg12 covalent-conjugation has a direct

role and DRAM has a distinct p53-dependent regula-

tory role in nonapoptotic programmed cell death.

Acknowledgements

SC and SB are grateful to the Department of Science and

Technology, Ministry of Science and Technology; AR and

SA are grateful to Council of Scientific and Industrial

Research (CSIR) for providing the Senior Research

Fellowship and SM acknowledges University Grants

Commission for providing the Research Fellowship in

Sciences to Meritorious Students. RK is thankful to CSIR

for his Research Associateship. All the authors gratefully

acknowledge Prof Samir Bhattacharya, Molecular Endocri-

nology Laboratory for his critical suggestions.

Funding

Financial support for this research work was from the

Department of Science and Technology, Ministry of Sci-

ence and Technology, Government of India (Project No.

SR/SO/AS-22/2008). Authors also appreciate the Univer-

sity Grants Commission–Centre for Advanced Studies

(UGC-CAS) grants (UGC No. F 5-3/2007 (SAP II) to the

department, which enabled the present study.

Conflict of interest

The authors declared no conflicts of interest.

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