Chemico-Biological Interactions 174 (2008) 183192

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Chemico-Biological Interactions
journal homepage: www.elsevier.com/locate/chembioint

Effects of exposure to multiple trace metals on biochemical,

histological and ultrastructural features of gills of a
freshwater sh, Channa punctata Bloch
Suwarna Pandey a , Suhel Parvez b , Rizwan Ahamd Ansari a , Mehboob Ali a ,
Manpreet Kaur a , Faisal Hayat a , Firoz Ahmad a , Sheikh Raisuddin a,,1
a
Ecotoxicology Laboratory, Department of Medical Elementology and Toxicology, Jamia Hamdard (Hamdard University),
New Delhi 110062, India
b
Department of Neurology, University of Magdeburg, Leipziger Str. 44, D-39120 Magdeburg, Germany

a r t i c l e i n f o a b s t r a c t

Article history: The trace metals are frequently encountered as mixtures of essential and non-essential
Received 1 February 2008 elements. Therefore, evaluation of their toxic effects individually does not offer a realis-
Received in revised form 3 May 2008
tic estimate of their impact on biological processes. We studied effects of a mixture of
Accepted 6 May 2008
four essential and toxic metals (Cu, Cd, Fe and Ni) on biochemical and morphological
Available online 18 May 2008
characteristics of the gills of a biomarker freshwater sh Channa punctata (Bloch) using
environmentally relevant concentrations. Fish were exposed to metal mixture through tank
Keywords:
Fish gills water for 7, 15 and 30 days. Biochemical studies as well as light microscopy (LM) and scan-
Metal pollution ning electron microscopy (SEM) revealed signicant metal exposure-induced alterations in
Oxidative stress gills. Besides ultastructural changes, activities of antioxidant enzymes such catalase (CAT),
Morphological alterations glutathione S-transferase (GST) and superoxide dismutase (SOD) were signicantly altered
Adaptation in the gills of exposed sh. The reduced glutathione (GSH) was signicantly (p < 0.001)
decreased, while lipid peroxidation (LPO) was signicantly (p < 0.001) increased. The main
alterations in general morphology of sh gills included spiking and fusion of secondary
lamellae, formation of club-shaped laments, and vacuolization and necrosis of lament
epithelium in the interlamellar regions. SEM studies showed gradual increase of the density
and apical surface area of the chloride cells and transformation of the surface structure of
the pavement cells. The results of this study indicate adaptive as well a toxic responses in
sh gills exposed to mixture of trace metals. Low concentrations of trace metal appear to
compromise the antioxidant defense of gills. Lesions in the gill morphology caused by the
effect of low concentrations of trace metals could lead to functional alterations and inter-
ference with fundamental processes such as maintenance of osmoregulation, gas exchange
and xenobiotic metabolism in the exposed sh populations.
2008 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

The sh gill is a multifunctional organ performing vital

Corresponding author at: Department of Chemistry, College of Natural
Sciences, Hanyang University, Seoul 133-791, South Korea.
Tel.: +82 2 2220 1297; fax: +82 2 2229 9450.
E-mail address: sheikhraisuddin@yahoo.com (S. Raisuddin).
1
Tel.: +91 11 26059688; fax: +91 11 26059663 (in India).
functions such as respiration, osmoregulation, acidbase
balance and nitrogenous waste excretion [1]. Gills are the
rst organs which come in contact with environmental pol-
lutants. Paradoxically, they are highly vulnerable to toxic
chemicals because rstly, their large surface area facilitates
greater toxicant interaction and absorption and secondly,

0009-2797/$ see front matter 2008 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.cbi.2008.05.014
184 S. Pandey et al. / Chemico-Biological Interactions 174 (2008) 183192
their detoxication system is not as robust as that of liver
[2,4]. Additionally, absorption of toxic chemicals through
gills is rapid and therefore toxic response in gills is also
rapid [2,4]. Gills have frequently been used in the assess-
ment of impact of aquatic pollutants in marine as well as
freshwater habitats [3,58]. Metals in particular are one
of the most deleterious environmental toxicants affecting
gills by changing general morphology and ultrastructure of
sh gills [2,4,9]. Heavy metal ions interfere with respiration
and osmoregulation by causing cellular damage to gill cells
[2,10].
The available data indicate that a large majority of stud-
ies on sh gills is based on the evaluation of effects of single
metal exposure mostly for a shorter duration [5,11,12].
Chronic exposure to waterborne heavy metals causes a
variety of physiological and behavioral changes includ-
ing loss of appetite, reduced growth, ion loss, decreased
aerobic scope and mortality in shes [1315]. Metal ions
such as Zn2+ and Cu2+ affect the antioxidants of sh tis-
sues including gills [7,16,17]. Nickel, for instance, induces
histopathological changes in the different tissues of the sil-
ver carp (Hypophthalmichthys molitrix) including fusion of
the gill lamellae and tissue hypertrophy [5].
The trace metals emanating either from natural sources
or from anthropogenic activities interact with aquatic
organisms as mixture of more than one metals or in com-
bination with organic pollutants. Therefore, biochemical
interactions would determine the outcome of the toxic
response. Our previous study on a freshwater sh (Channa
punctata Bloch) revealed that pre-exposure to Cu mod-
ulates toxic effect of chlorinated insecticide, endosulfan
[18]. Subsequently, we showed that Cd pre-exposure mod-
ulates toxicities of deltamethrin [19] and endosulfan [20].
These interacting effects, which were protective in nature,
were more remarkable in kidney and liver, whereas, gill
showed enhanced toxicity. It was suggested that met-
allothioneins (MTs), which have antioxidant and radical
scavenging activities, were not strongly induced in gills
by Cd [19,20]. C. punctata, a common Indian freshwater
sh has shown potential as a biomarker species in eval-
uation of xenobiotically induced oxidative stress [2124].
There is paucity of information on effect of multiple
trace metal exposure at environmentally relevant con-
centrations on sh gills. Also, it is not known whether
such an exposure scenario would compromise the antiox-
idant defense of gills or not. In order to generate this
information we studied effects of four essential and non-
essential metals viz., Cu, Cd, Fe and Ni on C. punctata gills
focusing on histological and morphological changes and
alteration of enzymatic and non-enzymatic antioxidant
prole. The selected metals have been reported in most
of the Indian rivers [25,26] and the concentrations used
in this study are very close to environmentally relevant
levels.
2. Materials and methods
2.1. Chemicals
Bovine serum albumin (BSA), butylated hydroxy toluene
(BHT), 1-chloro-2,4-dinitrobenzene (CDNB), dithio-bis-2-
nitrobenzoic acid (DTNB), ethylenediamine tetraacetic
acid (EDTA) disodium salt, Folin reagent, glutarade-
hyde, glutathione reductase (GR), nicotinamide adenine
dinucleotide phosphate (NADP), nicotinamide adenine
dinucleotide phosphate reduced (NADPH), nitroblue tetra-
zolium (NBT), oxidized glutathione (GSSG), reduced
glutathione (GSH), phenazonium methosulfate and sul-
fosalicylic acid were purchased from SigmaAldrich, Inc.
(St. Louis, MO, USA). Hydrogen peroxide (H2 O2 ) and
ortho-phosphoric acid (OPA) were procured from CDH
Chemicals (Mumbai, India). Cadmium chloride (CdCl2 ),
copper chloride (CuCl2 ), ferrous chloride (FeCl2 ), nickel
chloride (NiCl2 ), osmium tetroxide, 2-thiobarbituric acid
(TBA), Trishydrochloride were from Hi-Media Labs (Mum-
bai, India).
2.2. Fish
C. punctata (Perciformes: Channidae; weight range
30 2 g; length range 14 2 cm) obtained from a local
hatchery were maintained in glass aquaria (60-l capac-
ity; 70 cm 35 cm 40 cm) following standard proce-
dure [27]. Water was kept oxygen saturated by aer-
ation and aquarium temperature was maintained at
25 1 C with a photoperiod of 12 h light and 12 h
darkness. Physico-chemical characteristics of aquarium
water analyzed by the procedures of APHA [27] were
as follows: dissolved oxygen (DO) = 4.6 0.4 mg/l, bio-
logical oxygen demand (BOD) = 11.7 0.3 mg/l, chemical
oxygen demand (COD) = 24.9 1.3 mg/l, total suspended
solid (TSS) = 3.0 0.8 mg/l and turbidity (in NTU) = 4 0.4.
Aquarium water was replaced every 24 h to minimize con-
tamination from metabolic wastes.
2.3. Exposure
After acclimatization for 2 weeks sh were divided into
four groups (n = 15). One group served as control and the
other three as exposed groups for three different time dura-
tions of 7, 15 and 30 days. Fish were exposed to a mixture
of metals containing 50 g/l CuCl2 , 80 g/l CdCl2 , 750 g/l
FeCl2 and 150 g/l NiCl2 prepared in ultrapure water. The
concentrations of metals used in this study are compara-
ble to concentrations reported in Yamuna river water in
a recent study conducted by the Central Pollution Control
Board (CPCB) of India [28]. The experiment was planned in
such a way that sh from all the groups were sacriced on
the same day.
2.4. Preparation of post-mitochondrial supernatant
(PMS)
On completion of exposure, sh were briey immersed
in MS222 (tricaine methanesulfonate, Sigma) and dis-
sected to excise out gill tissues. Tissues were washed in
ice-cold saline (0.85% NaCl) and homogenized 10% (w/v)
in cold phosphate buffer (0.1 M, pH 7.4) containing KCl
(1.17%) to prepare post-mitochondrial supernatant (PMS)
using the method of Parvez et al. [29]. The homogenates
were ltered through a muslin cloth and subsequently
centrifuged at 800 g for 5 min at 4 C to separate
 ID Title Pages

Effects of exposure to multiple trace metals on biochemical, histological and ultrastructural features of gills
2581698 10
of a freshwater fish, Channa punctata Bloch

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