heat-shock protein 70 modulates apoptosis signal-regulating kinase 1 in stressed hepatocytes of...

Heat-shock protein 70 modulates apoptosis signal-regulatingkinase 1 in stressed hepatocytes of Mugil cephalus

Ekambaram Padmini • Jayachandran Tharani

Received: 18 March 2014 / Accepted: 19 May 2014

� Springer Science+Business Media Dordrecht 2014

Abstract Oxidative stress causes damage at the

cellular level and activates a number of signaling

pathways. Heat-shock proteins (HSPs) play an impor-

tant role in repair and protective mechanisms under

cell response to stress conditions. HSP70 has been

shown to act as an inhibitor of apoptosis. Apoptosis

signal-regulating kinase-1 (ASK1) activity is regu-

lated at multiple levels, one of which is through

inhibition by cytosolic chaperons HSP70. The current

study was aimed to investigate the alteration in

signaling molecules that allow the fish to survive

under stressed natural field conditions. The study also

investigates the variation in biomolecular composition

of hepatocytes by using Fourier transform infrared

spectroscopy. The impact of stress on hepatocytes was

assessed by measuring the level of lipid peroxides

(LPO), catalase activity (CAT) and assessing the

changes in hepatocytes of Mugil cephalus inhabiting

Kovalam and Ennore estuaries. The expression of

HSP70 and ASK1 were analyzed by immunoblot

analysis and ELISA, respectively. The spectral ana-

lysis showed variations in biomolecular composition

of hepatocytes at a wave number region of

4,000–400 cm-1. There was significant decrease of

CAT activity (p \ 0.01) (25 %) with significant

increase of LPO (p \ 0.001) (35 %) and HSP70

(p \ 0.001) and insignificant increase of ASK1

(p \ 0.05) (16 %) in fish hepatocytes inhabiting

Ennore estuary than Kovalam estuary. In conclusion,

the present study suggests that the survival of fish in

the Ennore estuary under stressed condition may be

due to the upregulation of HSP70 that mediates the

altered signal pathway which promotes cellular resis-

tance against apoptosis.

Keywords Catalase (CAT) � Fourier transform

infrared spectroscopy (FTIR) � Heat-shock

protein (HSP) � Hepatocytes � Lipid peroxides

(LPO) � Mugil cephalus

Introduction

Estuaries, the main contributors of fisheries in India,

suffer from severe damage due to increased industri-

alization and urbanization along the coastal areas. Fish

are the most at threat from aquatic pollution caused by

various internal and external (physicochemical and

biotic) environmental factors and hence considered to

be the bioindicators of water pollution (Adams and

Marshall 1999). Gray mullets, the natural inhabitant of

the estuaries studied, are found in the shallow water of

the intertidal zone that experiences dual fluctuations in

both temperature and salinity. These economically

important fish are under threat because of their

E. Padmini (&) � J. Tharani

P.G. Department of Biochemistry, Bharathi Women’s

College, Affiliated to University of Madras,

Chennai 600108, Tamilnadu, India

e-mail: [email protected]; [email protected]

123

Fish Physiol Biochem

DOI 10.1007/s10695-014-9949-0

continuous exposure to toxic chemical-rich industrial

effluents that are discharged into the estuaries (Pad-

mini and Kavitha 2005). These species have high

economic importance and gastronomic value, which

means that the information provided by our study may

be important both to people using the lagoon resources

and to those responsible for fishery management. Also

they have been demonstrated to be suitable for

biomarker studies (Ferreira et al. 2004; Gorbi et al.

2005; Pacheco et al. 2005).

Oxidative stress reflects a disturbance in the pro-

oxidant and antioxidant systems in favor of the pro-

oxidant. Free radicals are the potent toxic compounds

that are produced continuously in cells during exposure

to environmental toxins and exert a deleterious effect

via their chain reactions (Das and Das 2006), covalent

modification and oxidation of functional groups

thereby causing significant damage to biological

macromolecules and bringing about alterations in the

cellular redox balance (Abele et al. 2002). However,

the harmful effects of free radicals are blocked to a

great extent by antioxidant defense systems.

To maintain the homeostasis under oxidative stress,

the cells apart from the induction of antioxidant

enzymes produce high levels of stress proteins or heat-

shock proteins (HSPs), which protect them against the

damage. HSP70 protein plays appreciable role in

stress tolerance, protein folding, posttranslational

control of the stability and regulation of signal

transduction pathways that control cell growth and

survival (Zugel and Kaufmann 1999). It is expressed

constitutively in most cells and is upregulated by

thermal stress, heat-shock, heavy metal exposure,

oxidative stress and developmental and mitogenic

stimuli. HSPs are potential biomarkers for environ-

mental stress in fish (Iwama et al. 1998). Various

studies demonstrate that Hsp-induced cytoprotection

can be attributed partly to the suppression of apoptosis

(Samali and Orrenius 1998). Apoptosis resistance is

associated with the overexpression of HSPs.

Apoptosis signal-regulating kinase 1 (ASK1) is a

155-kDa ubiquitously expressed protein belonging to

the member of MAPKKK, a serine–threonine protein

kinase. It is activated in response to reactive oxygen

species (ROS), hydrogen peroxide, tumor necrosis

factor (TNF) and other stress stimuli (Morita et al.

2001). It plays a key role in the regulation of signaling

in response to oxidative stress (Goldman et al. 2003).

ASK1 activity is regulated at multiple levels, one of

which is through inhibition by cytosolic chaperons of

the HSP 70 family (Hwang et al. 2005).

Fourier transform infrared (FTIR) spectroscopy is a

non-disturbing technique which provides quantitative

biochemical information about biological samples. It

is a valuable technique due to its high sensitivity in

detecting changes in the molecular constituent of

tissues, such as lipids, proteins and nucleic acids. The

shifts in the peak positions, bandwidths and intensities

of the bands all give valuable structural and functional

information, which may have diagnostic value. With

FTIR spectroscopy, it is possible to monitor changes in

the structure and properties of biomolecules such as

DNA, RNA, proteins, carbohydrates, lipids in biolog-

ical tissues and cell simultaneously (Ci et al. 1999).

The advantages of FTIR method enable it to examine

the initial response to stress with high sensitivity

through spectra from very small amounts of samples.

(Corte et al. 2010) reported that this technique offers a

fast method to fingerprint the global cellular features

under specific conditions (Alvarez-Ordonez et al.

2010). In this context, the current study was aimed

to investigate the level of HSP70 along with ASK1 by

immunoblot and ELISA. An attempt was made to

correlate the FTIR study of HSP70 and ASK1 in

hepatocytes of M. cephalus inhabiting Kovalam

(unpolluted site) and Ennore (polluted site) estuaries.

Materials and methods

Study site

Kovalam and Ennore estuaries were chosen as the two

study sites for the current research work (Fig. 1).

Kovalam estuary (12�47016N, 80�14058E) is situated

on the east coast of India and is about 35 km south of

Chennai. It runs parallel to the sea coast and extends to

a distance of 20 km. It was chosen as the unpolluted

site for the present investigation as it is surrounded by

high vegetation, and it is free from industrial or urban

pollution. Ennore estuary (13�14051N, 80�19031E)

also situated on the east coast of India and is about

15 km north of Chennai. It runs parallel to the sea

coast and extends over a distance of 36 km. This

estuary was chosen as the polluted site as in its

immediate coastal neighborhood are situated a number

of industries, which include petrochemicals, fertiliz-

ers, pesticides, oil refineries, rubber factory and


123

thermal power stations that discharge their effluents

directly into this estuary. Contamination of this

estuary by heavy metals such as lead, cadmium,

mercury, zinc and iron to a significant extent com-

pared to unpolluted estuary has also been confirmed by

previous studies (Raghunathan and Srinivasan 1983;

Padmini and Vijaya Geetha 2007a). It has also been

reported that Ennore estuary significantly differs from

Kovalam estuary in its physical, chemical and biolog-

ical factors (Padmini and Vijaya Geetha 2007b), thus

it has been chosen as the polluted site.

Study animal and sampling

M. cephalus, a natural inhabitant of the estuaries,

identified by the use of Food and Agriculture Organi-

zation (FAO) species identification sheets (Fischer and

Bianchi 1984). M. cephalus with an average length of

30–32 cm were collected from unpolluted and polluted

estuaries using baited minnow traps. Collected fish was

placed immediately into insulated containers filled

with aerated estuarine water at ambient temperature

(25–30 �C) and salinity (24–29 ppt). Fish were main-

tained in the above-specified conditions for 4–5 h until

the start of the experimental procedures. Fish were

killed by severing the spinal cord, and the liver was

removed immediately.

Isolation of hepatocytes

The isolation of hepatocytes was carried out according

to established protocols (Krumschnabel et al. 1994;

Buckley et al. 2004) with slight modification as

described by Padmini and Usha Rani (2008). In brief,

after the fish was anesthetized with a solution of ethyl

m-aminobenzoate (MS-222; 0.5 g l-1 of water), a

INDIA

N

TAMILNADU

CHENNAI

ENNORE

KOVALAM

CHENNAI

BAY

O F

BENGAL

UTHANDI

INJAMBAKKAM

PALAVAKKAM

THIRUVANMIYUR

THIRUVETTIYUR

80°00” 80°06” 80°12” 80°18” 80°24” 80°30”

80°00” 80°06” 80°12” 80°18” 80°24” 80°30”

13°12”

13°06”

13°00”

12°54”

12°48”

13°12”

13°06”

13°00”

12°54”

12°48”

10 0 10 20 km

North Chennai Coast

Central Chennai Coast

South Chennai Coast

Fig. 1 Study area. Geographical locations of the Kovalam (unpolluted) and Ennore (polluted) estuaries


123

midventral incision was made to expose the liver, and

the portal vein was cannulated in the direction of the

liver. The liver was perfused with a perfusion buffer

containing 290 mmol l-1 NaCl, 2 mmol l-1 KCl,

10 mmol l-1 N-2-hydroxyethylpiperazine-N-2-etha-

nesulfonic acid (HEPES), 0.5 mmol l-1 ethylene

glycol-bis (2-aminoethyl)-tetraacetic acid (EGTA)

and 25 mmol l-1 tricine, (pH 7.8), to remove red

blood cells. Liver was then removed, perfused initially

and then incubated in a cell suspension buffer (SB)

(292.5 mmol l-1 NaCl, 5 mmol l-1 KCl,

2.5 mmol l-1 MgCl2, 3 mmol-1 CaCl2, 2 mmol l-1

NaHCO3, 2 mmol l-1 NaHPO4, 5 mmol l-1 glucose

and 50 mmol l-1 Hepes, pH 7.8) that contained

5 units ml-1 type IV collagenase (Sigma, USA) for

1 h, to separate cells. Cells were sieved through 60-

and 200-mm mesh screens and were then pelleted via

centrifugation at 100 g for 10 min. The cells were then

suspended in SB and allowed to recover for 1 h. The

cells were stored at -20 �C prior to assay.

Cell viability assay

The cell viability of hepatocyte preparations was

assessed using trypan blue staining (Strober 2001).

This dye exclusion polluted is used to determine the

number of viable cells present in a cell suspension and

is based on the principle that live cells possess intact

cell membrane that exclude dyes such as trypan blue,

whereas dead cells do not. In brief, suspension cells

were harvested by centrifugation. An equal volume of

0.4 % (w/v) trypan blue was added to a cell suspension

at a concentration of approximately 1 9 106 per ml.

The cells were then incubated for 3 min and loaded

onto a hemocytometer. Nonviable, deep blue cells as

well as viable, clear cells were counted in three

separate fields using bright field optics, and the

viability percentage was calculated by dividing the

number of viable cells by the number of total cells and

multiplying it by 100.

Protein preparation

Hepatocytes were harvested in cell suspension buffer,

centrifuged and resuspended in cell lysis buffer

(20 mM Tris pH 7.5, 1 % Triton X-100, 1 mM

ethylenediamine tetraacetic acid (EDTA), 1 mM eth-

ylene glycol-bis (2-aminoethyl)-tetraacetic acid

(EGTA), 1 mM phenylmethyl sulfonyl fluoride

(PMSF), 5 mM sodium pyrophosphate, 2 mM sodium

orthovanadate and protease inhibitor). The cell sus-

pension was incubated for 30 min at 4 �C, with

occasional shaking, or it was sonicated and centrifuged

at 16,0009g for 10 min in a 4 �C refrigerated micro-

fuge to remove the cellular debris. The supernatant was

the cell lysate, whose protein concentration was

determined by the classical method of Bradford

(1976) with coomassie brilliant blue (CBB) G-250,

using bovine serum albumin as a standard.

Field emission scanning electron microscopy

assay

Hepatocytes were diluted to a concentration of 1:100

(v/v). The cells were then fixed with 4 % glutaralde-

hyde overnight at 4 �C followed by centrifugation at

1009g for 5 min. The supernatant was discarded, and

the pellet was resuspended in 1 % osmium tetroxide

prepared in 0.1 M PBS for 2 h at room temperature.

The centrifugation process was repeated, and the

samples were dehydrated with an ascending ethanol

series (10–100 %). The absolute ethanol was finally

displaced by liquid carbon dioxide which served as the

transitional fluid for critical point drying. Dried

samples were mounted on aluminum stubs and sputter

coated with gold for 60 s (Hitachi, E1010, Europe).

Electron accelerators for FESEM were operated at

10 kV.

Estimation of lipid peroxides (LPO)

The level of lipid peroxides of fish hepatocytes

inhabiting Kovalam and Ennore estuaries was evalu-

ated by the method of Ohkawa et al. (1970). The

results were expressed in terms of nanomoles of MDA/

mg protein.

Estimation of catalase (CAT)

Catalase activity (CAT) of fish hepatocytes inhabiting

Kovalam and Ennore estuaries was evaluated by the

method of Beers and Sizer (1952). The CAT was

expressed as units/mg protein.

Immunoblot analysis of HSP70

Hepatocytes protein aliquots containing 50 lg pro-

teins were ran on 10 % sodium dodecylsulfate-


123

polyacrylamide gel (SDS-PAGE) simultaneously. The

gels were then blotted on to PVDF membranes

(BioTrace PVDF 0.4 lm, Pall Corporation, Germany)

according to the method of Towbin et al. (1979). The

antibodies used were anti-HSP70 (SPA-810) and anti-

b-actin (CSA-400), and followed by goat antimouse

IgG secondary antibody treatment, color development

was done using BCIP-NBT substrate system. The band

intensities were scanned with the Hp Scan Imager and

quantified using the TotalLab software, gels, USA.

The results were confirmed by individually perform-

ing the blotting studies of these proteins.

Quantification of ASK1 using ELISA

The inducible form of ASK1 in unpolluted and

polluted hepatocytes of M. cephalus was quantified

using ASK1 (E91358Hu 96T, Uscn Life Science,

Inc, USA) according to the manufacturer’s

instructions.

Sodium dodecylsulfate-polyacrylamide gel (SDS-

PAGE) Electrophoresis

Sodium dodecylsulfate-polyacrylamide gel electro-

phoresis was carried out in the discontinuous SDS-

PAGE electrophoresis system of Laemmli (1970)

using 10 % (w/v) separating gel and 4 % (w/v)

stacking gel. Protein profile of unpolluted and

polluted hepatocytes of M. cephalus was observed

by SDS-PAGE. The electrophoresis was carried out

using slab type SDS-PAGE with 1.5 % polyacryl-

amide gel. A marker of known molecular weight

(SDS marker, Genei) was also loaded (40 ll) along

with the samples. The apparatus was connected

with constant electric current (30 mA) till the

bromophenol blue (BPB) reached the bottom of

the plate.

Staining and destaining of gel

The gels were put into a container with staining

solution-containing CBB R-250 dissolved in methanol

with acetic acid and double distilled water. Gels were

left in the staining solution for overnight and destained

in methanol, acetic acid and water with shaking until

the bands became visible above the background. Both

staining and destaining steps were carried out while

shaking.

Determination of molecular weight of proteins

Molecular weights of whole cell proteins are analyzed

by SDS-PAGE by using a protein molecular weight

marker. The distance travelled by bands of the marker

was measured. By taking the distance travelled by the

bands of marker (in mm) along x-axis and molecular

weight (in kDa) of the proteins present in the marker

along y-axis, a standard graph was obtained. The

distance covered by each band of the sample was

calculated and with the help of standard graph,

molecular weights of proteins present in the sample

were determined. Based on the graph, bands (MW 70

and 155 kDa) were excised from the gel piece.

Elution of protein from polyacrylamide gel pieces

Place excised gel pieces in clean screw-cap culture or

microcentrifuge tubes. Add 0.5–1 ml of elution buffer

(50 mM Tris–HCl, 150 mM NaCl and 0.1 mM

EDTA; pH 7.5) so that the gel pieces are completely

immersed. Crush the gel pieces using a clean pestle

and incubate in a rotary shaker at 30 �C overnight.

Centrifuge at 5,000–10,0009g for 10 min and care-

fully pipette supernatant into a new microcentrifuge

tube. 70 kDa (HSP70) and 155 kDa (ASK1) proteins

present in an aliquot of the supernatant were quantified

using Western blot analysis and ASK1 ELISA kit

(E91358Hu 96T, Uscn Life Science, Inc, USA),

respectively. The confirmed aliquot of the supernatant

was tested for FTIR analysis.

FTIR spectroscopic analysis

The isolated samples and potassium bromide (all dry

solid state) were lyophilized in order to remove most

bound water, which might interfere with the measure-

ment of amide I, band. Sample was mixed with dried

Kbr and subjected to a pressure of 5 9 106 Pa and

made into a clear pellet of 13 mm diameter and 1 mm

thickness. The spectrometer was continuously purged

with dry nitrogen. The absorption intensity of the peak

was calculated using the baseline method. Each

observation was confirmed by taking at least three

replicates. The spectra were recorded in the range of

4,000–400 cm-1 using FTIR (PerkinElmer FTIR

Spectrometer RX I). In the present study, it is possible

to directly relate the intensities of the absorption bands

of the corresponding functional groups.


123

Statistical analysis

Data were analyzed using statistical software package

version 7.0. Student’s t test was used to ascertain the

significance of variations between unpolluted and

polluted fish hepatocytes. All data were presented as

mean ± SD of 20 fish per estuary. Differences were

considered significant at p \ 0.05, p \ 0.01 and

p \ 0.001.

Results

Cell viability

The liver cells of M. cephalus collected from Ennore

estuary showed decrease in their viability (80 %)

compared with hepatocytes isolated from Kovalam

estuary (92 %) (Fig. 2). As determined by percentage

viability, the results demonstrated here suggest that

estuarine contaminants have toxic effects on aquatic

organisms. Hence, the liver cells of fish from Ennore

estuary were extremely sensitive to such environmen-

tal stress-induced cytotoxicity compared to liver cells

of the counterparts inhabiting Kovalam estuary.

FESEM

Figure 3 describes that the morphological changes of

fish hepatocytes inhabiting Kovalam and Ennore

estuaries. An alteration in membrane structures and

shape of the cells, the signs of swelling and surface

lesions were observed in hepatocytes inhabiting

Ennore estuary than Kovalam estuary.

Lipid peroxides (LPO)

The level of lipid peroxide was showed in fish

hepatocytes inhabiting Kovalam and Ennore estuaries

(Fig. 4). A significant increase in the level of LPO

(p \ 0.001) was observed in hepatocytes inhabiting

Ennore estuary (35 %) compared with hepatocytes of

M. cephalus inhabiting Kovalam estuary.

Catalase activity (CAT)

Catalase activity was illuminated in fish hepatocytes

inhabiting Kovalam and Ennore estuaries (Fig. 5).

There was significant decrease in the level of CAT

(p \ 0.001) was found in hepatocytes inhabiting

Ennore estuary (25 %) compared with hepatocytes

of M. cephalus inhabiting Kovalam estuary.

Immunoblot analysis of HSP70

Blotting analysis showed significant increase in the

expression of HSP70 (p \ 0.001) in hepatocytes of

M. cephalus inhabiting Ennore estuary with

Fig. 2 Cell viability of hepatocytes of M. cephalus inhabiting

Kovalam and Ennore estuaries. Values are expressed as

mean ± SD (n = 20 fish per estuary). *p \ 0.05 when com-

pared with hepatocytes of M. cephalus inhabiting Kovalam

estuary

Fig. 3 FESEM images of

fish hepatocytes inhabiting

Kovalam and Ennore

estuaries. Yellow arrow

indicates the cell size and

morphological changes.

(Color figure online)


123

hepatocytes of M. cephalus inhabiting Kovalam

estuary. The representative blots for proteins are

given in Fig. 6.

ELISA of ASK1

The expression of ASK1 (16 %) was insignificantly

increased in hepatocytes of M. cephalus inhabiting

Ennore estuary compared to Kovalam estuary (Fig. 7).

Expression of HSP70 and ASK1 eluted from SDS-

PAGE

The expression of HSP70 and ASK1 (eluted from

SDS-PAGE) was observed in hepatocytes of M.

cephalus inhabiting Kovalam and Ennore estuaries

(Figs. 8, 9). The expression of HSP70 (p \ 0.001) was

significantly increased along with the expression of

ASK1 (15 %) was insignificantly increased in hepa-

tocytes inhabiting Ennore estuary compared to Kova-

lam estuary.

FTIR analysis

FTIR spectra of M. cephalus inhabiting Kovalam and

Ennore estuaries with structural changes of HSP70 and

ASK1 in the 4,000–400 cm-1 range were demon-

strated in Figs. 10 and 11. The spectrum is quite

complex and contains several bands arising from the

contribution of different functional groups belonging

Fig. 4 Level of lipid peroxides in hepatocytes of M. cephalus

inhabiting Kovalam and Ennore estuaries. Values are expressed

as mean ± SD (n = 20 fish per estuary). #p \ 0.001 when

compared with hepatocytes of M. cephalus inhabiting Kovalam

estuary

Fig. 5 Activity of catalase in hepatocytes of M. cephalus

inhabiting Kovalam and Ennore estuaries. Values are expressed

as mean ± SD (n = 20 fish per estuary). @p \ 0.01 when

compared with hepatocytes of M. cephalus inhabiting Kovalam

estuary

Fig. 6 Immunoblot analysis of HSP70 in hepatocytes of M.

cephalus inhabiting Kovalam and Ennore estuaries. b-actin has

been used as the loading control. a Hepatocytes of M. Cephalus

inhabiting Kovalam estuary. b Hepatocytes of M. Cephalus

inhabiting Ennore estuary

Fig. 7 Level of ASK1 in hepatocytes of M. cephalus inhabiting

Kovalam and Ennore estuaries. Values are expressed as

mean ± SD (n = 20 fish per estuary). *p \ 0.05 when com-

pared with hepatocytes of M. cephalus inhabiting Kovalam

estuary

Fig. 8 Immunoblot analysis of HSP70 (eluted from SDS-

PAGE) in hepatocytes of M. cephalus inhabiting Kovalam and

Ennore estuaries. b-actin has been used as the loading control.

c Hepatocytes of M. Cephalus inhabiting Kovalam estuary.

d Hepatocytes of M. Cephalus inhabiting Ennore estuary


123

to protein, lipids and other biomolecules. The absorp-

tion bands and assignments were shown in Tables 1

and 2.

Discussion

Oxidative stress is a state of unbalanced tissue

oxidation characterized by a disturbance in the free

radical and antioxidant systems (Abele and Puntarulo

2004). The redox cycling of heavy metals as well as

their interactions with organic pollutants is docu-

mented as a major contributor to the oxidative stress

resulting from aquatic pollution (Padmini et al. 2009).

The studies on oxidative stress in fish inhabiting

polluted environment have demonstrated significant

pollution impact on various organs of fish like gill

(Padmini and Sudha 2004), brain (Padmini and

Kavitha 2007), liver (Padmini and Usha Rani 2009)

and erythrocytes (Padmini et al. 2006). Accumulation

of damaged and oxidized macromolecules like lipid,

proteins and DNA in various organs would lead to

decrease in reproduction rate, susceptibility to quick

infection and sudden death of fish in large numbers

(Padmini et al. 2004). The enhancement of oxidative

and nitrative stress status and association of this with

alteration in cell viability are also well documented

(Padmini et al. 2009).

Scanning electron microscopy studies are useful for

the evaluation of toxicant-induced changes in liver

cells (Battle et al. 1997). Hence, comparison of fish

hepatocytes inhabiting Kovalam and Ennore estuaries

using FESEM revealed the structural changes. Regular

surfaces, smooth membranes and characteristic

arrangement of some areas were observed in fish

hepatocytes inhabiting Kovalam estuary (Fig. 3).

However, the signs of swelling, surface lesions and

alteration in membrane structures and shape of the

cells were demonstrated in fish hepatocytes inhabiting

Ennore estuary, indicating the impact of pollutant

stress on the surface morphology of the liver. Consis-

tent with the current findings, profound alterations of

cell surface topography, cytoskeletal disruption and

surface blebs formation have been demonstrated under

Fig. 9 Level of ASK1 (eluted from SDS-PAGE) in hepatocytes

of M. cephalus inhabiting Kovalam and Ennore estuaries.

Values are expressed as mean ± SD (n = 20 fish per estuary).

*p \ 0.05 when compared with hepatocytes of M. cephalus

inhabiting Kovalam estuary

Kovalam Ennore

3733

.37

3669

.08

3388

.91

2993

.00

1774

.20

1666

.35

1456

.59

1355

.48

1067

.42

100015002000250030003500

Wavenumber cm-1

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

Abs

orba

nce

Uni

ts

3778

.13

2889

.12

2828

.51

2396

.44

2351

.04

1834

.92

1773

.40

1681

.80

1648

.83

1539

.76

1515

.45

1364

.62

1339

.68

1244

.00

1056

.93

100015002000250030003500

Wavenumber cm-1

0.00

0.05

0.10

0.15

0.20

0.25

0.30

Abs

orba

nce

Uni

ts

Fig. 10 FTIR spectra of hepatocytes of M. cephalus inhabiting Kovalam and Ennore estuaries with HSP70 expression in the

4,000–400 cm-1 range


123

various stress conditions (Lemasters et al. 1983;

Okanoue et al. 1988).

The present result demonstrates that environmental

pollutants have cytotoxic effects on aquatic organ-

isms, significantly increasing stress, thereby decreas-

ing cell viability in hepatocytes of fish from Ennore

compared to Kovalam. Heavy metals are potent

prooxidants that catalyze lipid peroxidation and may

ultimately lead to oxidative stress (Sampaio et al.

2008). Enhanced free radical production and defective

antioxidant status, a condition usually suggestive of

oxidative/nitrative stress (Tepel et al. 2000), are well

authenticated, and the current results characterized by

increase in LPO along with decrease in CAT levels

confirm both the direction and extent of the previous

observations.

The knowledge of the signaling pathways and

physiological responses to environmental stress con-

dition is essential to understand the mechanism of

adaptation for survival in the stressed condition. HSPs

are a group of inducible proteins some of which are

constitutively expressed and increase in response to

stress, whereas others are expressed only after stress

(Hartl 1996). The induction of increased level of the

stress protein is associated with the development of

resistance to contaminated conditions. It is apparent

that induced stress proteins can act to protect cells

Kovalam Ennore

4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 400.0

0.00

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

1.3

1.4

1.5

1.6

1.71.75

cm-1

A

3916.8

3903.9

3891.2

3853.6

3838.0

3752.3

3456.1

2927.1

2860.0

2367.7

2345.2 1640.2

4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 400.0

0.00

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

1.3

1.4

1.5

1.6

1.70

cm-1

A

3916.4

3902.8

3890.5

3852.5

3840.9

3767.6

3723.8

3688.8

3675.6

3647.4

3627.6

3453.0

3064.8

2925.1

2859.9

2779.2

2679.6

2576.1

2483.1

2364.9

2345.5

2275.4

2202.7

2083.1

1653.0

1577.1

1465.6

1248.5

1108.2

1019.0 670.1

Fig. 11 FTIR spectra of hepatocytes of M. cephalus inhabiting Kovalam and Ennore estuaries with ASK1 expression in the

4,000–400 cm-1 range

Table 1 General band assignments of the FTIR spectra of

hepatocytes of M. cephalus inhabiting Kovalam and Ennore

estuaries

Wave number (cm-1) Functional group

*2,828.51 C–H stretch

*2,396.44 C=O stretch



*1,648.83 C=C stretch

*1,539.76 N–O asymmetric stretch

*1,339.68 N–O symmetric stretch

*1,244 C–N Stretch

Table 2 General band assignments of the FTIR spectra of

hepatocytes of M. cephalus inhabiting Kovalam and Ennore

estuaries

Wave number (cm-1) Functional group

*3,600 to *700 O–H stretch

*3,064.8 C–H stretch

*2,500 to *2,800 C–H stretch

*2,000 to *2,400 C:C and C:N stretch

*1,000 to *1,600 Amide C=O Stretch (1�2� and 3�)

*670 C–H stretch (fingerprint region)


123

from stress-induced damage by preventing protein

denaturation and/or by repairing such damage (Li and

Werb 1982).

Vijayan et al. (1998) have also demonstrated that

significant increase in the inducible HSP70 expression

serves as a sensitive indicator of the cellular stress

response associated with exposure to the contaminant.

Enhanced levels of HSP70 in polluted site fish may

reflect a protective response against environmental

pollutant-related stress. In this study, an increased

expression of HSP70 was observed in fish hepatocytes

inhabiting Ennore estuary (Fig. 6). HSP70 is a pow-

erful chaperon whose expression is induced in

response to a wide variety of physiological and

environmental insults, thus allowing the cell to survive

in lethal conditions. In addition to the protein recovery

functions, HSP70 is also involved in the control of

signaling pathway that control the onset of apoptosis,

as well as protein complexes that are central in the

activation of the apoptotic pathways. HSP70 cytopro-

tective properties may be explained by its anti-

apoptotic function (Rerole et al. 2011). HSP 70

(HSP70) has been shown to act as an inhibitor of

apoptosis (Li et al. 2000).

FTIR spectroscopy is currently used as a detection

tool to study stress-induced changes in the molecular

structure level (Venkataramana et al. 2010). In this

research, FTIR spectra were acquired to detect the

conformational changes and content variations of the

functional groups contributed from protein and lipids of

hepatocytes inhabiting Kovalam and Ennore estuaries.

Figure 10 shows the FTIR spectra of fish hepatocytes

inhabiting Kovalam and Ennore estuaries with structural

changes of HSP70. The bands observed at func-

tional region (*2,828.51, *2,396.44, *2,351.04,

*1,834.92, *1,648.83 and *1,539.76 cm-1) due to

C–H stretch, C=O stretch, C=C stretch and N–O

asymmetric stretching molecules in hepatocytes of M.

cephalus inhabiting Ennore estuaries. The peaks at

*1,339.68 and *1,244 cm-1 were observed in the

fingerprint region due to the presence of deformation and

dipole movement of the molecule in hepatocytes of M.

cephalus inhabiting Ennore estuary.

Apoptosis signal-regulating kinase 1 is a mitogen-

activated protein kinase (MAPK) kinase of the c-Jun

N-terminal kinase (JNK) and p38 MAPK pathways.

ASK1 is preferentially activated by various cytotoxic

stressors and plays pivotal roles in a wide variety of

cellular response to them (Takeda et al. 2008). ASK1

is well-known as a proapoptotic, stress-activated

signaling molecule, and it is under tight regulation at

multiple levels. Molecular chaperons and cochaperons

play a protective role under conditions of cellular

stress by facilitating both refolding and degradation of

misfolded proteins (Gabai et al. 1997; Beere et al.

2000). In addition, HSPs directly modulate stress-

dependent signaling pathways to attenuate cell dam-

age and repress apoptotic events during the response to

stress. ASK1 signaling cascades are regulated by

molecular chaperons. HSP70 affects ASK1 by direct

interactions with JNK (through its peptide-binding

domain) and ASK1 (through the N-terminal ATP-

binding domain). These interactions inhibit JNK and

p38 MAP kinase activities and block ASK1-dependent

apoptosis. The chaperons of the HSP70 family may

inhibit the activity of ASK-1 by having the physical

association with ASK1, thereby inhibiting the homo-

oligomerization of the kinase, and hence act as an

endogenous inhibitor of ASK-1 (Park et al. 2002).

Figure 11 shows the FTIR spectra of fish hepato-

cytes inhabiting Kovalam and Ennore estuaries with

structural changes of ASK1. The ratio of the peak

intensities of the bands observed between *3,600 and

*3,800 cm-1 due to N–H bending and O–H stretch-

ing, respectively, could be used as indicators of the

relative concentration of the protein to water of

biological tissues. The band observed at functional

region *2,000 to *2,400 cm-1 due to C:C and

C:N stretching molecules in hepatocytes of M.

cephalus inhabiting Ennore estuary. The protein

absorption bands mainly located between 1,600 and

1,500 cm-1 contained amide I and amide II bands

(Warnau et al. 1996). The bands between 1,400 and

1,000 cm-1 were of the ‘‘fingerprint’’ region (Pala-

niappan and Renju 2009), amide III and the function

group of nucleic acid and carbohydrates contributed to

these absorption bands in samples. The band at

*670 cm-1 was observed in the fingerprint region

due to the presence of deformation and dipole

movement of the molecule in hepatocytes of M.

cephalus inhabiting Ennore estuary. Overall, the

spectrum of hepatocytes inhabiting Kovalam and

Ennore estuaries with ASK1 protein changes differs

in the shape of absorbance curve, indicating to obvious

changes in structure and contents of biological com-

ponents due to pollution-induced stress.

The analysis of the FTIR spectra collected from

hepatocytes of M. cephalus inhabiting Ennore and


123

Kovalam estuaries revealed that biomolecules were

sensitive to pollution-induced stress. The results of the

current study have provided insight on the stress-

induced conformational changes of biomolecules

including proteins, as well as on the content variation

of these components. The FTIR analysis constructs a

direct link between the functional biomolecules and

the physiological status under heavy metal stress. The

macromolecular characteristics and their contents are

fundamental factors whereby the cell maintains its

normal development and growth. In the present work,

we have clearly demonstrated the functional and

structural changes of protein in the ASK1 signaling

molecule in fish hepatocytes inhabiting Ennore estu-

ary compared to Kovalam estuary. The functional

changes of proteins were also evident from ELISA

study.

The chaperons of the HSP70 family may inhibit the

activity of ASK1 by having physical association

thereby inhibiting the homo-oligomerization of the

kinase and act as an endogenous inhibitor of ASK1

(Park et al. 2002). As HSP70 may inhibit the activity

of ASK1, the expression of ASK1 may be downreg-

ulated and hence there is only 16 % increase in its

expression levels. The evidence presented by the

above study indicates that the overexpression of

HSP70 could reduce ASK1 expression to prevent

stress-induced cellular injury thereby promoting cell

survival in Ennore estuary fish.

Conclusion

The current study results confirm the impact of

environmental pollutant-mediated oxidative stress on

hepatocytes structures. The results also indicated that

the relationship between the degree of antioxidant

deficiency and lipid oxidation could also be used as

biomarkers for toxicity. Upregulation of HSP70, an

early and sensitive biomarker of environmental stress,

downregulates ASK-1 thereby acting as an anti-

apoptotic molecule and promote cell resistance against

contaminating stress-induced apoptosis in the gray

mullet surviving under pollution conditions. In addi-

tion, we demonstrate that HSP70 and ASK-1

expressed under stress conditions show distinct vari-

ation through the FTIR studies which goes to indicate

that tight regulation of signaling proteins can also be

monitored by FTIR. FTIR spectroscopy offers a fast

and efficient tool for detection of qualitative and

quantitative pollutants-induced changes in the context

of molecular structure level in hepatocytes of M.

cephalus. The conformational changes at the biomo-

lecular level indicated by FTIR may be useful tool for

detecting the stress-induced changes.

Acknowledgments The project funded by University Grants

Commission, New Delhi, India. Project referral number-UGC:

41-1281/2012 (SR) is acknowledged.

Conflict of interest The authors report no conflicts of interest.

The authors alone are responsible for the content and writing of

the paper.

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