International Journal of Zoological Research

Year: 2011 | Volume: 7 | Issue: 1 | Page No.: 1-18
DOI: 10.3923/ijzr.2011.1.18

Lead Acetate-induced Histopathological Changes in the Gills and Digestive System of Silver Sailfin Molly (Poecilia latipinna)

Yomn Mohamed Shahat Mobarak and Mariam Mahmoud Sharaf

Abstract: This study aims to investigate the effects of a single sublethal concentration of lead acetate on the gills and digestive system of the silver mollies. After LC₅₀ determination, twenty five mollies were randomly chosen and divided into two groups. The first one served as control; while the second group exposed to 0.8 mg lead acetate/liter of H₂O for 96 h. Afterwards fish were anesthetized, dissected out and the gills, liver, pancreas, stomach and intestine were processed for paraffin embedding, stained with haematoxylin and eosin and examined by light microscopy. Lead acetate-exposed fish exhibited a decrease of swimming activity and brilliant silvery body color, accumulation of lead acetate on ovarian surface and an increased secretion of mucus from gills and skin. The gills showed hyperplasia, hypertrophy and destruction of the lamellar architecture, fusion of lamellae and lamellar clubbing. The livers showed disarrangement of hepatic cords, shrinkage of hepatocytes and dilatation of liver sinusoids and extravasation of blood. Hepatopancreas damage included loss of contact between hepatocytes and pancreocytes, lysis of pancreocyte membranes and appearance of pyknotic/apoptotic nuclei. The exocrine pancreas revealed necrosis, increased adipocytes and atrophy of pancreatic acini. The stomach exhibited irregularity, shrinkage and fusion of its microvilli, pyknotic/apoptotic nuclei of microvilli epithelium and atrophy of submucosal zone. The intestinal damage included fusion of intestinal microvilli, necrosis and irregularities of the microvilli cells, microvilli loss, flattening and hypertrophy. The study concluded that lead acetate exposure resulted in severe histopathological changes in the gills and in the selected digestive organs of silver mollies.

Keywords: atrophy, adipocytes, necrosis, Hyperplasia and microvilli loss

INTRODUCTION

Lead (Pb) is an immunotoxicant which through human exposure results in immune function changes and has the potential to adversely affect human health. It has many uses in industry including pipes, paints, enamels, glazes, motor industry and others. The major hazard in industry arises from the inhalation of dust and fume but the organic compounds may also be absorbed through the skin. It induces a broad range of physiological, biochemical and neurological dysfunctions in humans (Nordberg et al., 2007). Developing individuals (embryos, fetuses and children) are the most susceptible populations to Pb. Exposure to low levels of Pb during early development was found to produce long-lasting cognitive and neurobehavioral deficits, persistent immune changes, reduced fertility, a delay in sexual maturity, irregular estrus and reduced numbers of corpora lutea in human and experimental animals (Mobarak, 2008). Several reports have indicated that Pb can cause neurological, hematological, gastrointestinal, reproductive, circulatory, immunological, histopathological and histochemical changes all of them related to the dose and time of exposure to Pb (Ademuyiwa et al., 2007; Park et al., 2006; Vega-Dienstmaier et al., 2006; Patrick, 2006; Berrahal et al., 2007; Farrag et al., 2007; Reglero et al., 2009; Abdallah et al., 2010; El-Neweshy and El-Sayed, 2010; Mirhashemi et al., 2010). The liver plays a major role in lead’s metabolism (lead poisoning cause adverse effects to hepatic cells) because after Pb exposure liver is one of the major organs involved in the storage, biotransformation and detoxification (Sivaprasad et al., 2004). In fish Pb is accumulated mostly in gill, liver, kidney and bone. Fish eggs show increasing Pb levels with increased exposure concentration and there are indications that Pb is present on the egg surface but not accumulated in the embryo (Birge et al., 1979). The toxicity of Pb and other heavy metals, their accumulation in edible tissues, their effects on growth and metabolic processes of carb (Cyprinus carpio) and fish were previously studied (Adam and Basset, 1990; Al-Akel, 1994; Allen, 1995; Alam and Maughan, 1995; Ay et al., 1999; Andreji et al., 2006; Ashraf et al., 2006; Erdogrul and Erbilir, 2007; Has-Schon et al., 2006; Agah et al., 2009). Lead at a concentration of 0.48 ppm caused inhibition of egg hatching in several fish species including rainbow and lake trout, channel catfish, bluegill and white sucker fish (Weber et al., 1997). However, unique to fish, Pb exposure may cause excessive mucus secretion; this may interfere with the role of the gill in diffusion of gases - the uptake of oxygen from water and expulsion of carbon dioxide (Weber et al., 1997). Lead acetate (LA) and mercuric chloride have genotoxic and cytotoxic damage in gill and fin epithelial cells of Carassius fish Auratus auratus (Cavas, 2007).

The silver sailfin molly (Poecilia latipinna) are fresh water ornamental fish usually found in brackish canals or gently flowing warm water of small creeks, in ponds, lakes, marshes and, often vegetated, backwaters and pools of streams and drains (Allen et al., 2002). It is a popular ornamental fish traded globally by the aquarium industry and much of the demand for P. latipinna throughout the world is satisfied by its captive culture in India and elsewhere (Gosh et al., 2003). They consume animal material: rotifers, small crustaceans (such as copepods and ostracods) and aquatic insects. They also heavily predate mosquito larvae and pupae; larger fish eat more plant material. The sailfin molly is a live-bearing poeciliid fish without parental care that lives in mix-sexed shoals of 10-20 individuals (Witte and Noltemeier, 2002; Aspbury, 2007). There is a positive relationship between female size and fecundity (Travis et al., 1990) and male preferentially choose larger females as mates (Ptacek and Travis, 1997; Gabor, 1999) and produce more sperm in response to these females (Aspbury and Gabor, 2004). Females also copy the mate choice of Amazon mollies (Poecilia formosa), a heterospecific gynogen that requires sperm from sailfin males to induce embryogenesis (Schlupp et al., 1994). No information is available on the toxicity of Pb or other heavy metals in the P. latipinna. On the other hand, a good deal of information is now available on the accumulation of Pb in muscle and other body compartments of fish. Therefore, the present study was undertaken to find out the possible histopathological effects of Pb as lead acetate on the gills, liver, pancreas, stomach and intestine of sailfin silver molly (P. latipinna).

MATERIALS AND METHODS

This study was conducted in the Zoology Department, Faculty of Science, Suez Canal University, Ismailia, Egypt from February-2007 to June-2007. Healthy silver mollies, used in the study were purchased from a fish breeder in Ismailia, Egypt. In the laboratory they were acclimated, for two week inside transparent rectangular glass tanks (60x30x30 cm). Fish rearing and maintenance procedures were as previously described by Briggs et al. (1996). Briefly, the water in the aquaria was obtained from a header tank containing constantly aerated water to remove any residual chlorine and in order to keep the amount of oxygen not less than 4 mg L^-1. The water composed of deionised water mixed (5:1) with local tap water and NaCl (450 mg L^-1), resulting in a conductivity of 1.2 ms cm^-1. The water temperature was kept between 21 and 23°C, average pH was 8.8, average salinity during the experiments was 0.189-0.290%. The fish were fed twice daily with commercial dry flake food (Tetra Guppy, Tetra Werke, Melle, Germany) supplemented with a dry fish meal twice a week. Faeces accumulating at the bottom of the aquaria were siphoned off weekly. The LA toxicity test was based on the standard method of US Environmental Protection Agency (1995) in aquaria with 30 L capacity.

Fish exposures to lead acetate: To estimate the mortality rate and histopathological effects of LA on gills, liver, pancreas, stomach and intestine of silver mollies, a total of 89 fish were divided into three groups as follows.

Group I. 96 h LC₅₀ determination: To evaluate the fish viability and LC₅₀ of LA, eight subgroups of mollies were exposed to different concentrations (0.2, 0.4, 0.6, 0.8, 0.9, 01, 1.1, 1.2 mg L^-1) of LA in dechlorenated and filtrated tap water at ones. The parameters of water quality (temperature, dissolved oxygen, salinity and pH) in the aquaria were determined before and periodically during the experiments; 8 fish were used per concentration. Stock solutions of the test compound (CH₃COO)₂ 3H₂O_, Merck) and their dilutions were made according to the guidelines given in the standard methods (Organization for Economic Co-operation and Development, 1993). The mortality rate was determined from 24-96 h following exposure. Then the LC₅₀ was calculated by regression analysis. The estimated LC₅₀ of LA was about 1.1 mg L^-1. The concentration selected for the study was 0.8 mg L^-1. The data were also evaluated according to the following formula (Klassen, 1991).

where, LC₅₀ and LC₁₀₀ indicate the lethal doses for the 50 and 100% of the samples. Value a gives the difference between the two consecutive doses, b the arithmetic mean of the mortality caused by two consecutive doses and n the number of samples in each group.

Group II and III: Control and LA-exposed fish: Group II served as control (10 fish) was kept in LA- free experimental water, keeping all other conditions constant. The third LA-exposed (15 fish) group was exposed to 0.8 mg LA/L. The aquaria were not aerated at the time of dosing with LA. No food was given to both control and LA-exposed groups during the experiments. The amount of (0.8 mg L^-1) LA to be added to the aquarium was calculated after accurate determination of the aquarium volume. The parameters of water quality (temperature, dissolved oxygen, salinity and pH) in the aquaria were determined before and periodically during the experiments. The experiments were carried out for a period of 96 h. During this period, LA exposure waters were renewed daily. After 96 hours of exposure to the above mentioned LA concentration, the mollies (control and LA exposed) were anesthetized, dissected then any change in the general viscera was recorded and photographed. Then the gills, liver, pancreas, hepatopancreas, stomach and intestine were removed, rinsed in physiological saline (0.9% NaCl) and fixed in Bouin's solution for 48 h. Afterwards, they were washed in 70% ethanol, dehydrated, cleared in xylene and infiltrated with the embedding medium (a mixture of equal volumes of parablast and paraffin wax), serially sectioned, stained with haematoxylin and eosin, cleared in xylene and mounted in neutral Canada balsam (DPX). Then the gills, liver, pancreas, stomach and intestine of controls as well as LA exposed fish were carefully examined with the light microscope, described and photographed using Leitz microscope fitted with a camera and connected to a computer.

RESULTS

Mortality rate of LA- exposed mollies: A concentration-dependent mortality rate was observed in mollies exposed once to different concentrations (0.2, 0.4, 0.6, 0.8, 0.9, 01, 1.1, 1.2 mg L^-1) of LA. No mortality was observed up to 24-96 h from 0.2 or 0.4 or 0.6 or 0.8 mg L^-1 of LA. The fish exposed to the concentrations of 0.9 or 01 or 1.1 mg L^-1 of LA showed 12.5, 37.5 and 50% mortality after 96 h of LA exposure, respectively. The calculated number of the above mentioned equation was about 1.13. At the concentration of 1.2 mg LA/L, 100% mortality was recorded within a period of 24 h following exposure. On the basis of above data, the estimated LC₅₀ of LA was 1.1 mg L^-1 (Table 1). No mortality was observed in the control or LA- exposed group during the experiment.

The external morphology of silver molly of control and LA-exposed fish were illustrated in Fig. 1a and b, respectively. When LA-exposed fish were dissected out there was no sings of LA accumulation on organs except ovaries in which minute white colored particles appeared accumulated on its surface (Fig. 1d). However, these particles were not found on any of the control ovaries (Fig. 1c). In the control and experimental group no visible changes in behavior or external appearance were observed, except for a decrease of the swimming activity and brilliant silvery body color as well as an increased secretion of mucus from gills and skin of LA-exposed fish compared with the control ones (Fig. 1b). On the other hand, in all control fish no histopathological lesions were observed in any of the observed organs, while all fish exposed to 0.8 mg LA/L exhibited histopathological changes in the gills, liver, hepatopancreas, pancreas, stomach and intestine. Histological changes in the gill, liver, pancreas, hepatopancreas, stomach and intestinal tissues of control and LA-exposed silver mollies were described and presented in Fig. 2-8.

Histopathological changes of gills and digestive system
Gills: Each gill filament of control fish covered externally by squamous epithelial cells and supported internally by a supporting cartilage located just internal to the afferent arteriole (Fig. 3a, b).

The gill lamellae composed of thin epithelium and separated by inter lamellar space, chloride cells (faintly stained acidophilic cells) were located near the base of the lamellae and between them (Fig. 3b). The distinct histopathological change in gills of fish exposed to 0.8 mg LA/L was represented by hypertrophy and destruction of the lamellar architecture, hyperplasia that resulted in the fusion of many lamellae (Fig. 3c).

The epithelial covering of the gill filaments was hyperplastic and edematous with vacuolated epithelial covering of the gill rakers. The interlamellar spaces were lost and became one large space (Fig. 3d). At higher magnifications, the lamellar blood vessels showed damage ranging from reduction of endothelial cell numbers to wall destruction, many lamellae became short and clubbing with swollen tips and complete degeneration of gill arch and filaments were also observed (Fig. 3d, f).

Liver: The normal histological structure of liver sections in control mollies was illustrated in Fig. 3a and b. The hepatic parenchyma was homogeneous, often weakly basophilic. It had no distinct lobules and composed of branching two-cell thick laminae of hepatocytes bordered by sinusoids. Between the neighboring sinusoids, the hepatocytes were arranged as plates of hexagonal cells. The cell membrane of individual hepatocytes was clearly visible through light microscopy analysis (Fig. 3b). Among hepatocyte plates, variable sized pancreatic tissues were embedded (Fig. 3a). In the LA-exposed group degenerative effects were evident among hepatocytes such as disarrangement of hepatic plates, shrinkages of hepatocyte, condensed cytoplasm and contact loss between hepatocytes, faded hepatic cells, dilatation of bile canaliculi and extravasation of blood of the liver sinusoids, frequent necrosis evidenced by pyknosis and vacuole formation. Also, the presence of a hemorrhagic spot was detected in the hepatic parenchyma with a large amount of erythrocytes (Fig. 3d).

Pancreas and hepatopancreas: In the liver of control mollies there were areas of endocrine pancreas (hepatopancreas) that located near the small veins of the hepatic portal vein (Fig. 3a). The exocrine pancreas was found as islet of Langerhans and was associated with the endocrine pancreas and scattered in the mesentery, primarily near the pylorus and associated with bile ducts and anterior hepatic portal vein (Fig. 4c). The veins represented endothelial cells with a few erythrocytes in their interior. The pancreocytes were seen as islets with their own lamellar lining in an acinar pattern surrounding a branch of the portal vein. Each acinus consisted of a single layer of pyramidal broad based cells that rested on a basal lamina. The nucleus of the acinar cell located towards the base of the cell and the basophilic cytoplasm towards the lumen of the acinus and contained acidophilic zymogen granules (Fig. 4a, c). These components of the pancreas were enclosed within a thin capsule that consisted of poorly stained cords of fusiform cells with large distinct nuclei, interspersed with, blood sinuses. In the liver sections of mollies exposed to 0.8 mg LA/L hepatopancreas damage was characterized by widened extracellular spaces and loss of contact between hepatocytes and pancreocytes, altered pancreocytes with apparent volume reduction and disassociation, an apparent lysis of pancreocyte membranes (necrotic cells) was also observed (Fig. 4b). In many cases this was associated with the appearance of pyknotic/apoptotic nuclei. The histopathological changes noted in the exocrine pancreas were represented by acinar tissue damage of varying severity, focal or diffuse pancreatic cell necrosis, increased adipocytes, pancreatic atrophy and dramatic atrophy of the pancreatic acini (Fig. 4d, e).

Stomach: In cross sections of control mollies, the stomach was covered externally by a serosa of a simple squamous epithelium and lined internally by tubular-shaped microvilli consisted of a short ciliated columnar epithelium that lacking gastric glands. The serosa was followed by a triple layer of non-striated muscularis. The outer layer was longitudinal and the inner layer was circular with the middle layer running at an oblique angle to the two these layers. The follow up of the cardiac and pyloric parts of the stomach sections revealed that the layer of musculature surrounding the stomach was thin in the cardiac region but became thicker in the pyloric portion. This muscular layer was followed by a dense connective tissue layer or the submucosa (Fig. 5). The stomach of LA-exposed mollies exhibited irregularity, shrinkage and fusion of stomach microvilli as well as atrophy of the submucosal zone (Fig. 6a). In such cases the microvilli lost their normal appearance and became highly folded, sometimes appeared inverted (Fig. 6b). The changes of submucosa were represented by vacuole formations, shrinkage and complete absence in some parts (Fig. 6c). Also, a large number of these microvilli displayed pyknotic/apoptotic nuclei of their epithelium (Fig. 6d).

Intestine: The intestine of control mollies was a short thin tube with a thin double layer musculature, the outer layer was longitudinal and the inner layer was circular. The mucosa of the anterior and intermediate intestine was similar with the exception of the number and size of mucosal microvilli, which were more numerous and shorter in the anterior region. These microvilli appeared as small pocketlike evaginations of the intestinal wall, lined by a simple layer of columnar ciliated cells with eosinophilic goblet cells (Fig. 7a). The intestinal microphotographs of LA-exposed mollies were illustrated (Fig. 7b, c, 8a-d). Fusion of intestinal microvilli and wide intercellular spaces among epithelial cells of microvilli, an increased rate of cell death, necrosis and irregularities of the microvilli cells were observed (Fig. 7b, c). Furthermore, fusion of all intestinal microvilli and absence of some of them, inflammation at the site of the microvillus loss and adjacent parts, complete damage, flattening and hypertrophy of the intestinal microvilli were also observed (Fig. 8c, d).

DISCUSSION

The results presented here revealed that LA exposure resulted in various histopathological changes in the gills, liver, hepatopancreas, pancreas, stomach and intestine of silver mollies and no one of the exposed fish could escape the toxic effect of LA. This result could be explained by the fact that lead accumulates selectively in the gills (the principal site of gaseous exchange, body fluid pH regulation and nitrogenous waste excretion) of freshwater teleost fish that may leads to impairment of gill functions as respiration, osmotic and ionic regulation, acid-base regulation and excretion of nitrogenous wastes. Impairment of these functions will certainly affect structure and function of different fish organs and eventually may lead to the observed histopathological changes. In the present study it was observed that LA-exposure caused an increased secretion of mucus from gills and skin. This result is in accordance with Weber et al. (1997) who declared that unique to fish, lead exposure may cause excessive mucus secretion and this may interfere with the role of the gill in diffusion of gases - the uptake of oxygen from water and expulsion of carbon dioxide. The observed LA accumulations on the ovarian surface of the present study corroborate results of earlier studies of LA toxicity recorded by Birge et al. (1979) that fish eggs show increasing lead levels with increased exposure concentration and there were indications that lead was present on the egg surface but not accumulated in the embryo.

The present histopathological change in gill cells (the cells most directly exposed to LA) included hypertrophy and deformation of gill filaments, hyperplasia of epithelial cells that resulted in the fusion of many lamellae and curling at the tips of gill lamellae. Similar observations were reported due to exposure of other freshwater fish to lead (Abd-El-Gawad, 1999; Martinez et al., 2004; Palaniappan et al., 2008) and copper (Park and Heo, 2009; Kosai et al., 2009). Also, Grosell et al. (2006) and Spokas et al. (2008) declared that gills accumulate the highest lead concentration which may cause disturbance of ion regulation and respiratory gas exchange. Besides, massive lamellar hyperplasia that led to fusion and shortness of many lamellae, lamellar clubbing with swollen tips and complete degenerations of gill arch and filaments were observed. Similarly, Cruz et al. (1988) suggested that hyperplasia resulted in the fusion of many gill lamellae markedly reducing the respiratory surface area of some filaments.

The livers of LA-exposed fish in the present study showed disarrangement of hepatic cords, shrinkages of hepatocyte, condensed cytoplasm and contact loss between hepatocytes. These histopathological changes are in accordance to studies conducted on other freshwater fish exposed to different heavy metals such as exposure of Tilapia mossambica to cadmium (Usha and Ramamurthi, 1989; Suresh, 2009) and Hypophthalmichthys molitrix (Valenciennes) to nickel (Athikesavan et al., 2006). Furthermore, the observed dilatation of bile canaliculi and liver sinusoids, extravasation of blood and necrosis/pyknosis cell nuclei in liver of LA-exposed mollies are in agreement with Abd-El-Gawad (1999) who declared similar liver histopathological changes in LA-exposed Oreochromis niloticus. According to Wolf and Wolf (2005) fish liver exposure to toxic agents may result in the accumulation of lipid droplets or glycogen. In the present study the hepatopancreas and pancreas of LA-exposed mollies exhibited increased incidence of vacuoles and/or adiopcytes, probably due to depletion of glycogen and/or lipid in the hepatocytes. Similar results were described by Coimbra et al. (2007) in Nile tilapias exposed to endosulphan insecticide in their feed, where hepatic lesions and an increase in hepatic vacuolization were noted. Also similar findings were found by Fiuza et al. (2009a) in O. niloticus orally administered crude ethanol extract and choloroform fractions with their feed. Furthermore, the exocrine pancreas of LA-exposed fish showed acinar tissue damage of varying severity, focal or diffuse pancreatic cell necrosis, increased adipocytes, pancreatic atrophy and dramatic atrophy of the pancreatic acini. These alterations were also noted in zebrafish pancreas that were exposed to 2,3,7,8 tetrachlorodibenzo-p-dioxin (Henry et al., 1997) and in the exocrine pancreas of tilapias that received crude ethanol extract and chloroform fraction (Fiuza et al., 2009b).

The atrophy of the submucosal zone and the microvilli loss and inflammation observed in stomach of LA-exposed fish of the present study suggests a possible irritant and/or toxic activity by LA, which probably led to the inflammatory response in the stomach submucosa. It was suggested that lead increases the formation of gastric ulcers by interfering with the oxidative metabolism in the stomach that increased the incidence of gastric ulcer (Olaleye et al., 2007). The implication of this is that lead causes an increase in the formation of free radicals, which, if not mopped up by free radical scavengers, will expose the stomach to inflammation and gastric mucosal damage. These adverse effects of lead as well as its inhibition of enzyme activities (Dai et al., 2009; Abdallah et al., 2010) might be the main inducer of the obtained intestinal histopathological damage of the exposed mollies. The study concluded that LA exposure resulted in an increased secretion of mucus from gills and skin and no sings of LA accumulation on organs except ovaries in which minute white colored particles appeared accumulated on its surface. Also, in all control fish no histopathological lesions were observed in any of the observed organs, while all fish exposed to 0.8 mg LA/L exhibited histopathological changes in the gills, liver, hepatopancreas, pancreas, stomach and intestine.

REFERENCES