RESEARCH ARTICLE

Induction of Metallothionein in the Olfactory Epitheliumof Channa punctatus (Bloch) in Response to Cadmium Exposure:An Immunohistochemical Study

Debraj Roy • Debasree Ghosh • Dipak Kumar Mandal

Received: 11 March 2011 / Revised: 21 April 2012 / Accepted: 8 May 2012 / Published online: 17 June 2012

� Zoological Society, Kolkata, India 2012

Abstract Induction of metallothionein (MT) in the

olfactory epithelium was studied immunohistochemically

in the fish Channa punctatus (Bloch) exposed to sublethal

concentrations of cadmium chloride (2.5 mg/l and 5.0 mg/l).

Results revealed that basal level of MT synthesis occurred

in the olfactory epithelium and cadmium chloride exposure

induced the synthesis of MT. Olfactory receptor cells

(ORCs), supporting cells (SCs), nerve fascicles and

mucous cells exhibit strong immuno-reaction whereas

basal cells were found negative. Results indicated that

ORCs and SCs were the sites of MT synthesis. Mucous

cells accumulated Cd–MT conjugates to eliminate the

metal from the olfactory system.

Keywords Olfactory epithelium � Channa punctatus �Cadmium exposure � Metallothionein induction

Introduction

Olfactory system of fish has an indispensable role in

feeding, reproduction and predator avoidance (Hara 1992).

This sensory system of fish is directly and continuously in

contact with the external environment. Primary neurons of

fish olfactory system provide a direct access for certain

toxicants as well as heavy metals to the central nervous

system (Gottofrey and Tjalve 1991; Hastings and Evans

1991). The ability of the olfactory organ to detoxify and

catabolize the xenobiotic substances is therefore vital for

maintenance of its normal functions. Several reports have

shown that olfactory epithelium expresses different xeno-

biotic metabolizing enzymes such as cytochrome P450,

glutathione S-transferase as in rat (Banger et al. 1994) and

in rainbow trout (Starcevic and Zielinski 1995, 1997).

Cadmium is a well known environmental pollutant and

toxic to some biological tissues. Cadmium produces liver,

lung, renal and testicular injury as well as cancer in rat

(Dudley et al. 1985). Numerous studies have shown that

metallothionein (MT) plays an important role in cadmium

disposition and detoxification. The absence of MT-I and

MT-II in metallothionein knockout mice increases in

organic cadmium induced lethality and hepatotoxicity,

whereas over-expression is associated with protection

(Klaassen and Cagens 1981). MT inductions have been

found in liver, kidney, intestine and pancreas in rat

(Cousins 1985; Bremner and Beattie 1990). Shimada et al.

(2005) localized MT in the olfactory pathway in mercury

exposed mice. However, report on the MT induction in the

fish olfactory organ is not on record. Accordingly, the

present study was designed to examine the impact of

cadmium exposure on the MT induction in the olfactory

epithelium of a common freshwater fish, Channa

punctatus.

Materials and Methods

Experimental Design

Thirty healthy freshwater fish, C. punctatus (20–25 cm in

length) were procured from local freshwater ponds at

Santiniketan, West Bengal, India and acclimatized in lab-

oratory for 15 days. After acclimatization fish were trans-

ferred to glass aquarium of 50 l capacity with ten fish in

each. Each experimental set was constituted with three

D. Roy � D. Ghosh � D. K. Mandal (&)

Department of Zoology (CAS), Visva-Bharati, Santiniketan

731 235, West Bengal, India

e-mail: dkmandal.vb@rediffmail.com

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Proc Zool Soc (Jan-June 2012) 65(1):40–44

DOI 10.1007/s12595-012-0027-2

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aquariums: one aquarium was kept as control and fish of

other two aquariums were exposed to 2.5 and 5.0 mg/l

(1/20th and 1/10th dose of the LC 50 value) of cadmium

chloride respectively for 5 days. Water of the aquariums

was exchanged and doses of cadmium chloride were

renewed in every 2 days interval. Aquariums were kept

under continuous aeration. Two fish from each aquarium

were collected from both control and treated groups on the

day 1 to day 5 at a fixed sampling time to obtain olfactory

tissue. Fish were anaesthetized with MS 222 (Sigma)

solution (100 mg/l) and sacrificed. Olfactory tissues were

dissected out and immediately processed for histology and

immunocytochemical studies.

Histological Study

Olfactory tissues were fixed in aqueous Bouin’s fixative for

18 h. Fixed tissues were rinsed well in 70 % ethanol,

dehydrated through graded ethanol series and embedded in

paraffin of 56–58 �C. Tissues were sectioned serially at

4 lm thickness and stained with eosin and haematoxylin.

Immunohistochemical Study

Olfactory tissues were fixed in Zamboni fixative for 20 h at

room temperature. Fixed tissues were washed in phosphate

buffer (pH 7.2) and distilled water, dehydrated through

graded alcohol and cleared in benzene and embedded in

paraffin of 52–54 �C. Tissues were sectioned at 10 lm

thickness.

The expression of MT in the olfactory system was

detected by using commercially available polyclonal anti-

body from Santa Cruze Biotechnology (SC11377). All

reactions were performed according to avidin–biotin

complex (ABC) method (Hsu et al. 1981). Deparaffinized

and hydrated sections were incubated at 60 �C for 10 min

in 0.1 M citrate buffer (pH 6.0) for antigen retrieval. After

washing in wash buffer the endogenous peroxidase activity

was blocked by treating the slides with 3 % H2O–H2O2

solution for 10 min and treated with 5 % normal goat

serum for 30 min. Then the slides were incubated with

primary antibody, diluted 1:100 in PBS for overnight at

4 �C in moist incubating chamber. After washing in

20 mM Tris–HCl buffer (pH 7.4) containing 0.9 % NaCl,

sections were subsequently incubated at room temperature

with secondary bitinylated antibody (Bangalore Gennei)

diluted 1:200 in PBS for 1 h. Slides were washed twice

with Tris–HCl buffer (pH 7.4) for 10 min and incubated for

30 min with ABC reagents. After washing with wash

buffer slides were incubated for 3 min with DAB (3,3-

diaminobenzidine). As soon as colour developed slides

were rinsed in distilled water and dehydrated through

graded acetone, cleared in xylene and mounted with DPX.

Slides were examined and photographed under BX50

Olympus microscope.

Results

Histological studies revealed that olfactory epithelium

(OE) of C. punctatus was a thick (30–40 lm) sheet of

pseudo-stratified epithelium, folded to form many olfactory

lamellae (OL). In lamellae, OE enclosed a stromal sheet

called central core (CC) which was constituted with con-

nective tissues, blood vessels and nerve fascicles (Fig. 1).

Basal lamina (BL) kept olfactory epithelium separated

from the stromal sheet. Olfactory epithelium could be

distinguished into sensory and non-sensory areas. Sensory

epithelium was composed of columnar ciliated and non-

ciliated olfactory receptor cells (ORCs), supporting cells

(SCs) and round basal cells (BC). BC were situated at the

base of the epithelium and adjacent to the basal lamina. BC

were found in two forms, round globular basal cells (GBC)

and elliptical horizontal basal cells (HBC) (Fig. 1). Non-

sensory epithelium was composed of ciliated columnar

cells. Mucous cell proliferations were found throughout the

epithelium when fish were exposed to CdCl2 (Fig. 2).

Results of the MT-immunohistochemical studies

revealed that the basal level of MT synthesis always

occurred in the sensory epithelium and mucous

cells showed moderate reaction in the control group of

C. punctatus. BC and central core were found negative in

immunoreactions (Fig. 3). However, olfactory epithelium

of the treated fish showed strong MT immuno-reaction

from the day one of exposure to CdCl2 (Fig. 4). ORCs and

SCs were localized as the sites of MT synthesis in the

olfactory epithelium (Fig. 5). BC and basal lamina

Figs. 1–8 Histology and MT immunostaining of the olfactory

epithelium of C. punctatus. 1 Olfactory epithelium (OE) showing

olfactory receptor cells (ORC), supporting cells (SC) and basal cells

(BC) in the form of globular basal cells (GBC) and horizontal basal

cells (HBC). BL indicates the basal lamina in control fish. H&E, bar15 lm. 2 Sensory epithelium showing mucous cells (MC) prolifer-

ation in the fish exposed to 5 mg/l CdCl2 for 5 days. H&E, Bar60 lm. 3 Sensory epithelium showing low level of MT immunore-

actions in ORC, SC and mucous cells (MC) (arrows) and negative in

basal cells and central core (CC) in control fish. Bar 30 lm. 4Olfactory epithelium exposed to 2.50 mg/l CdCl2 for 5 days shows

strong MT immunoreaction in ORC, SC and nerve fascicles (arrows)

but negative in BCs and BL. Bar 30 lm. 5 Intensity of MT

immunostaining increased in ORCs and SCs and nerve fascicles

(arrows) but non-sensory cells (NSC) remain negative in the OE

exposed to 5.0 mg/l CdCl2 for 5 days. Bar 60 lm. 6 ORC and SC and

nerve fascicles (arrows) in the OE exposed to 5.0 mg/l CdCl2 for

5 days exhibit intense MT immunostaining. Bar 30 lm. 7 Mucous

cell (MC) and ORC (arrows) with strong MT immunoreactions in the

OE exposed to 2.50 mg/l CdCl2 for 5 days. Bar 15 lm. 8 Mucous

cells with intense reaction in the OE exposed to 5.0 mg/l CdCl2 for

5 days. Bar 30 lm

b

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exhibited negative in immuno-reaction. Non-sensory cells

(NSC) were also found negative for MT immuno-reaction

(Fig. 5). However, nerve fascicles in the central core

exhibited strong reactions (Fig. 5, 6). Cadmium exposure

induced the proliferation of mucous cells and mucous cells

showed very strong immuno-reaction for MT (Fig. 7, 8).

Discussion

Metallothionein (MT) is a cysteine rich cytosolic protein

essentially found in liver and kidney as means of protective

device against the toxic metals (Klaassen et al. 1999) and it

is the key compound involved in the intracellular handling

of a variety of essential and non-essential metal ions (Liu

et al. 1998). MT mediated hepatoprotection is due to the

high affinity sequestration of cadmium by MT in the

cytosol, thus reducing the amount of cadmium available to

injure other critical organelles (Goering and Klaassen

1984). Shimada et al. (2005) localized the MT in the SCs,

BC, Bowman’s gland cells of olfactory mucosa of rat fol-

lowing exposure to mercury vapour. Tallkvist et al. (2002)

also reported the MT induction in the rat olfactory epi-

thelium due to cadmium exposure. Our investigation pro-

vided the evidences that MT synthesis occurs in the

olfactory epithelium of fish and cadmium induces the

synthesis. MT synthesis was increased with the enhanced

dose and duration of the exposure. Cadmium induced

proliferation of mucous cells and the mucous cells show

strong MT-immunoreactions. Mucous cells were found all

over the epithelium (Mandal et al. 2005) and their popu-

lation increased in the stressed condition. Autometallo-

graphic study by Dang et al. (1999) showed that exposure

with copper led to accumulation of copper in gill mucous

cells of Oreochromis mossambicus. Other autometallo-

graphic study revealed that cadmium ions were localized

mainly in the mucous cells compared to a lesser extent in

other branchial cell types of fish Scophthalmus maximus

exposed to cadmium (Alvarado et al. 2006). These evi-

dences strongly suggest that mucous cells are involved in

chelating and biotransformation of toxic cadmium ion and

provide a device of elimination of the Cd–MT conjugate

from olfactory system through their secretions. The low

level of MT synthesis in the olfactory epithelium in control

group of fish may be related to its general role in protecting

the epithelium from the various types of stress. However,

strong induction of MT in the ORCs and SCs after cad-

mium exposure suggest that metal stress triggers the self

defense mechanism in these cells. Tallkvist et al. (2002)

showed that cadmium administered by intranasal instilla-

tion induced MT in the primary olfactory neurons and the

metal was transported as a Cd–MT conjugate along the

axon of these cells from the olfactory epithelium to the

olfactory bulbs of the brain. The present findings on the

strong MT immuno-reaction in the nerve fascicles in the

central core of the olfactory epithelium support the said

view. Thus, MT induction in the fish olfactory epithelium

seems to be sensitive to cadmium toxicity.

Acknowledgments Authors are grateful to Prof. S. K. Maitra, Head,

Department of Zoology, Visva-Bharati and former Head, Prof.

P. Nath for providing laboratory facilities and Prof. Shelley Bhat-

tacharya for her generous gift of chemicals. Authors are also grateful

to the University Grants Commission, New Delhi, India for financial

support.

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