Australian Journal of Basic and Applied Sciences, 5(11): 1062-1068, 2011
ISSN 1991-8178 Histopathological Alterations in The Liver And Lungs of Hoplobatrachus Occipitalis
Exposed to Sub Lethal Concentrations of Cadmium
1
Ezemonye Lawrence Ikechukwu and 2Enuneku Alex Ajeh
1
Department of Animal and Environmental Biology (AEB) University of Benin, Benin Nigeria.
Department of Environmental Science Western Delta University, Oghara Delta State Nigeria.
2
Abstract: The toxic effects of cadmium on the liver and lungs of H. occipitalis after 28 days was
studied. The frog was exposed to 0.25, 0.50, 1.00 and 2.00mg/l cadmium. Cadmium was
bioaccumulated in the liver. Bioaccumulation of cadmium increased significantly (p<0.05) with
increase in cadmium concentration. There was high accumulation of cadmium in the liver which may
alter the levels of various biochemical parameters and cause liver damage. At the end of the study, the
liver and lungs of control frog showed normal structural pattern. The major histopathological changes
observed in the liver were excessive bile secretion and dilation of sinusoids. These changes may be due
to the direct toxic effects of cadmium on the hepatocytes since the liver is the site of detoxification of
all types of toxins and chemicals. Histopathological changes in the lungs were pulmonary haemorrhage
and distortion of lung structure. These changes could lead to loss of lung elasticity and capacity to
carry out gaseous exchange. The results of this study revealed that H. occipitalis manifested
histopathological changes in the liver and lungs when exposed to cadmium concentrations. The
investigation of histological changes in the organs of amphibians is an accurate way to assess the
effects of heavy metals to these amphibian tetrapods in their habitats where they may be exposed
owing to anthropogenic activities.
Key words: H. Occipitalis Cadmium Histopathological Liver Lung.
INTRODUCTION
Environmental contamination from heavy metals is a serious problem of global dimension (Banjerkji et al.,
2003, Van Der Welle et al., 2007). Heavy metals are serious pollutants of the aquatic environment because of
their environmental persistence and ability to be accumulated by aquatic organisms (Maheswaran et al., 2008,
Fargosava 1998). Adult amphibians can acquire heavy metals through their skin or orally by consumption and
respiration. Larvae may also absorb them through their skin (Ezemonye and Enuneku, 2006).
Cadmium is a biotoxic environmental pollutant which accumulates in the body tissues such as the lungs,
liver, kidneys, bones, reproductive organs and the immune system (Egwurugwu et al., 2007, Sofyan et al.,
2007). The toxic effects of cadmium on organisms include nephrotoxicity, carcinoegenicity, teratogenicity and
endocrine disruption (Serafim and Bebianno, 2007). Cadmium may also cause the deterioration of cell
membranes by binding to metalothionein (MT) or glutathione and consequently interfer with the ability of these
proteins to avoid oxidative stress (Cui et al., 2004). Calmium can also replace essential metals such as copper
and zinc in several metalloproteins, altering the protein conformation and affecting their activity because this
element interacts ubiquitously with sulphydryl groups of amino acids, proteins and enzymes (Park et al., 2001,
Giguere et al., 2003). Cadmium can cause oxidative stress through several mechanisms; the Fenton reaction
(Stohs and Bagchi 1995), depletion of cellular glutathione, alterations of mitochondrial electron transfer chain
(Wang et al., 2004) and inhibition of antioxidant enzymes (Hansen et al., 2007).These changes may result in
biochemical and morphological alterations in the affected organs.
Anthropogenic activities such as mining, production and consumption of cadmium and non-ferrous metals
have accelerated the rate of mobilization and distribution of cadmium from non-bioavailable geological matrices
into biologically accessible situations far in excess of natural cycling process (Suru, 2008). These have
predisposed animal and human populations to both subtle and direct exposure pathways with an attendant
increase in cadmium related pathologies (Satarug and Moore, 2004). Bioaccumulation is the building up of a
chemical to a toxic level in an organism’s body. It is the net accumulation of a substance by an organism as a
result of uptake directly from all environmental sources and from all routes of exposure (ASTM 1998).
Primarily, it is the movement of a chemical into the organism from the food or water that is ingested.
Bioaccumulative contaminants are rapidly absorbed out of water-borne ambient environments and concentrated
in the tissues of living aquatic organisms at concentrations that can range from thousands to millions of times
greater than levels in the ambient environment. These absorbed levels are high enough to cause dysfunction in
Corresponding Author: Enuneku Alex Ajeh, Department of Environmental Science Western Delta University, Oghara
Delta State Nigeria. 1062
Aust. J. Basic & Appl. Sci., 5(11): 1062-1068, 2011 the organisms and potential harmful effects to humans. Compounds accumulate in living things any time they
are taken up and stored faster than they are broken down or excreted (Britton, 1998). Bioaccumulation becomes
an environmental problem where chemicals accumulated are toxic, where this will lead to an elevated amount in
the organism’s body.
Given the paucity of information on the ecotoxicology of heavy metals to amphibians, there are basic
research needs on the effects of contaminants including heavy metals to amphibians. Tissue changes in organs
(liver, lungs, kidney, gastrointestinal tract) of test organisms provide histological information on the nature of
toxicants organisms are exposed to in their environments.
Global declines in amphibian populations have caused concern in the scientific community (Houlahan et
al., 2000, Edginton et al., 2004, Ezemonye and Enunueku, 2006) and provided the impetus for this study.
Numerous physical and chemical causes have been postulated (Seburn and Seburn 2000), and in some instances,
interaction of multiple causes have been implicated (Wojtaszek et al., 2004). In many cases, heavy metals from
industrial and agricultural activities have been implicated (Maheswaran et al., 2008). The scientific challenge is
to develop sensitive methodology to detect the specific role of heavy metals in heavy metal toxicity. The
investigation of histological changes in organs of frog is an accurate way to assess the effects of toxic metals in
experimental studies. Hence, this study was undertaken to examine the effect of cadmium at sub lethal
concentrations on histology of the liver and lungs of the adult crowned bullfrog, Hoplobatrachus occipitalis.
MATERIALS AND METHODS
Adults of Hoplobatrachus occipitalis were collected from unpolluted spawning ponds in Oghara
Community in the Niger Delta ecological zone of Nigeria. They were collected using hand nets to prevent injury
to animals during capture since they are active animals. Acclimation to laboratory conditions was done for two
weeks prior to experiments (Goulet and Hontella 2003) in plastic tanks measuring 49cm in length x 29cm in
width x 24cm in height with dechlorinated tap water (2 litres at a slant). The frogs were fed ad libitum daily with
termites. They experienced a natural photoperiod of approximately 10: 14, light/dark period at a laboratory
temperature range of 27-280C.The mean values for the test water quality were as follows; temperature 26±10C;
pH 5.7±0.4; dissolved oxygen 4.7±0.7 ppm and hardness 36±1.24 ppm. The initial mean weight of frogs was
55.23±0.53g. There was no significant difference (p>0.05) between the mean weights of frogs used in the
experiments. Since metabolic activity changes with size and affects the parameters to be measured (Canli and
Furness 1993), individuals of similar weights were used.
Cadmium as CdCl2.H20 was used for the sub lethal tests. Stock solutions of the toxicant (CdCl2) were
prepared by dissolving the toxicant in distilled water to a final volume of 1.0 L. The stock was then diluted
serially into treatment concentrations (Ezemonye and Enuneku 2006). Four sub lethal concentrations (0.25,
0.50, 1.00 and 2.00 mg/l) cadmium were dosed to frogs for 28 days. There were three replicate tanks per
treatment and three individuals per tank including controls. The amphibians were fed with termites.
Twenty-eight days after the exposure of the frogs to cadmium, each frog was sacrificed for the
determination of histopathological alterations in the lungs and liver. For this study, manual tissue processing
method was used. The schedule for medium size tissue blocks (Baker et al., 1998) was adopted. After tissue
processing, tissues were embedded or blocked out using the Leukhand embedding moulds. The L-pieces were
arranged on an aluminium base to form a rectangle. The molten paraffin was then poured into the moulds and
the selected surfaces of the tissues embedded with the aid of a pair of blunt end forceps and allowed to set. The
embedded tissues were separated into different blocks and then attached to wooden blocks with the aid of an
electric spatula. The blocks were then trimmed using a rotary microrome and its knife. At the end of each
trimming, the blocks were knicked to show the direction at which trimming was done. The trimmed tissue
blocks were arranged on ice trays in order to cut thin sections using the rotary microtome at a thickness of 3µ.
Sections were then collected with the help of a camel hair brush and the placed on the slide and then flood
picked with 20% alcohol in order to spread out folds on the sections and then floated out on a water bath with a
temperature of 5-100C below the melting point of the wax used. The sections were picked and floated on a water
bath and then picked with a pre-labelled slide. The slides were dried on a hot plate at a temperature of 5-100C
above the melting point of wax used. These were left on the hot plate for 15 minutes.
The staining methods employed in staining the sections were haematoxylin and eosin method to
demonstrate general tissue structure and Masson’s trichrome method for the demonstration of connective tissue
fibre. Sections were analyzed using the light microscope.
Statistical Analysis:
Data from the experiment was expressed as mean ± standard deviation of 3 replicates. Statistical differences
between treated frogs and control frogs were evaluated using students t-test. The level of significance was set at
p<0.05.
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Aust. J. Basic & Appl. Sci., 5(11): 1062-1068, 2011 Results:
Bioaccumulation of cadmium in the liver increased with increase in concentration of the heavy metal
relative to the control group (Table 1). Bioaccumulation of cadmium in the liver increased (p<0.05) as the
concentration of heavy metal increased.
Table 1: Bioaccumulation of cadmium (ppm) in the liver and kidney of frog H.occipitalis.
Heavy metal
Conc.(mg/L)
Control
0.25
Cadmium
0.50
1.00
2.00
Values significantly different from control groups at p<0.05.
Bioaccumulation
<0.001
0.22±0.02*
0.24±0.01*
0.26±0.02*
1.98±0.13*
Plate 1a shows the normal histological structure of frog liver in untreated media. Histological alterations
observed in the liver of H. occipitalis after exposure to different concentrations of cadmium revealed that the
severity was dose – dependent (Plates 1b-1e).
In the frog exposed to low cadmium concentration (0.25mgll), mild bile secretion was observed in the liver
after 28 days (Plate 1b). In the 0.50mgll concentration, mild bile secretion was still evident with dilation of
sinusoids. With higher concentrations of cadmium (l.00mg/l and 2.00mg/l), severe dilation of sinusoids and
excessive bile secretion were observed.
Plate 1a: Liver of control frog showing the hepatocytes (thin arrowhead) and hepatic sinusoids (thick
arrowheads after28 days (H & E stain x160).
Plate 1b: Frog liver exposed to 0.25 mg/L Cd. Mild bile secretion (B) observed after 28 days. H & E stain x160.
Plate 1c: Frog liver exposed to 0.50mg/l cadmium. Dilation of sinusoids (A) mild bile secretion (B) were
observed after 28 days (H & E x160).
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Aust. J. Basic & Appl. Sci., 5(11): 1062-1068, 2011 Plate 1d: Frog liver exposed to 1.00mg/l Cd. Dilation of sinusoids (A), excess bile (B) were observed (H & E
stain x160).
Plate 1e: Frog liver exposed to 2.00mg/l Cd. severe dilation of sinusoids (A), excess bile secretion observed. H
& E stain x160.
The results of the light microscopic studies showed that morphologic changes were evident in the lungs of
exposed animals and were not observed in the control frogs. Plate 2a showed the normal histological structure of
the lungs. Mild pulmonary haemorrhage was observed in the lung of frog exposed to 0.25mg/l after 28 days as
depicted in Plates 2b and 2c respectively. Haemorrhage was still apparent in lungs exposed to 1.00mg/l
cadmium (Plate 2d). Distortion of lung structure was found in this same concentration. The lungs of frogs
exposed to the highest concentration of cadmium (2.00mg/l) showed severe pulmonary haemorrhage and
distortion of lung structure (Plate 2e).
Plate 2a: Photomicrograph of control frog lung showing the respiratory alveolus (thick arrow Head), capillary
in alveolar wall and inter alveolar Septum (thin arrowhead). H & E x160.
Plate 2b: Lung of frog exposed to 0.25 mg/l Cd. Mild pulmonary haemorrhage (H) was observed after 28 days
(H & E X160).
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Aust. J. Basic & Appl. Sci., 5(11): 1062-1068, 2011 Plate 2c: Lung of frog exposed to 0.50mg /l Cd. Mild pulmonary haemorrhage (H), was observed after 28 days
exposure (H & E X160).
Plate 2d: Lung of frog exposed to 1.00 mg /l Cd. Mild pulmonary haemorrhage(H), and distortion of lung
arcitecture after 28 days. (H & E x 160).
Plate 2e: Lung of frog exposed to 2.00mg/l Cd. Severe haemorrhage and distortion of lung architecture were
observed after 28 days (H & E x160).
Discussion:
In this study, the liver highly bioaccumulated cadmium at the end of the 28 days period of exposure. The
results indicate that the heavy metal accumulation gradually increased during the exposure period. The liver
accumulates relatively higher amounts of heavy metals (Vinidhini and Narayanan 2007). Vinodhini and
Narayanan (2009) who reported that cadmium strongly accumulated in liver and kidney of the common carp
Cyprinus carpio after 32 days exposure. High accumulation of cadmium in liver may alter the levels of various
biochemical parameters. This may also cause severe liver damage (Mayers and Hendricks, 1984).
The use of histopathological responses as important biomarkers for the possible effects of toxic chemicals
on organisms has been reported to be effective (Miller et al., 2007). It is imperative that histological biomarkers
are the indicators of pollutants in the overall health of the entire population in the ecosystem (VelkovaJordanoska and Kostoski, 2005). The exposure of aquatic organisms to sub lethal concentrations of chemical
contaminants in their environment may result in various biochemical, physiological and histological alterations
in vital tissues. The investigation of histological changes in the organs of amphibians is an accurate way to
assess the effects of xenobiotic compounds in experimental studies.
The results of this study revealed that H. occipitalis manifested histopathological changes in the liver and
lungs when exposed to cadmium concentrations.
In the present study, severe bile secretion and dilation of sinusoids were reported in the liver of H.
occipitalis exposed to cadmium. These findings are similar to the reports of Atamanalp et al., (2008) who
observed dilation of sinusoids, congestion of blood vessels and degeneration of hepatocytes in the liver of
Oncorhynkus mykiss exposed to copper sulphate.
The severity of the hepatic alterations observed in this study increased with increase in concentration of
cadmium. These changes may be due to direct toxic effects of the toxicants on hepatocytes since the liver is the
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Aust. J. Basic & Appl. Sci., 5(11): 1062-1068, 2011 site of detoxification of all types of toxins and chemicals (Soufy et al., 2007). The liver is the organ most
associated with the detoxification and biotransformation of foreign compounds that enter the body. However, its
regulating mechanism can be impaired by accumulated toxicants which could result in structural damage
(Camargo and Martinez, 2006). The liver is one of the critical target organs after chronic exposure to cadmium
(Sobha 2007). The liver accumulates substantial amounts of cadmium after both chronic exposures.
The lung is the organ that ensures gaseous exchange and in addition, regulation of immune responses to
inhaled antigens is effected in living systems. In the present study, exposure of H. occipitalis to cadmium
resulted in structural alterations in the lungs. The lung of Hoplobatrachus occipitalis exposed to cadmium
showed haemorrhage and distortion of lung structure. These alterations could lead to loss of lung elasticity and
capacity to carry out gaseous exchange. Toxic substances can injure the lungs thereby disrupting gaseous
exchange and impairing immunological responses. The lungs have been identified as a target organ of heavy
metal toxicity (Roberts, 1999, Egwurugwu et al., 2007).
Conclusion:
Histopathological alterations in the bullfrog exposed to heavy metals can be used as sensitive model to
monitor aquatic heavy metal pollution. The current result indicates that heavy metal contamination definitely
affects the liver causing severe bile secretion, dilation of sinusoids and congestion of blood vessels in the liver
thereby affecting the function of the liver in detoxification and biotransformation of foreign compounds. Heavy
metal exposure to the lungs could cause haemorrhage and distortion of lung structure. These alterations could
lead to loss of lung elasticity and capacity to carry out gaseous exchange. The use of histopathological responses
as important biomarkers for the possible effects of heavy metals on amphibians is an important tool for
monitoring the health of these tetrapods in areas where they have the potential to be exposed.
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