Abstract: The present study was carried out to investigate the potential protective effects of tomato paste (9 mg lycopene kg-1 b.wt.) in comparison with vitamin E (50 mg kg-1 b.wt.) against the impacts of cadmium (Cd) toxicity (4.64 mg L-1, ¼ of 96 h- LC50) Oreochromis niloticus exposed for 15 and 30 days. Cd impacts were evaluated in terms of histopathological and histochemical characteristics of the liver and hepatopancreas. Various changes have been observed in the liver tissues of the fish exposure to Cd in the two periods. These changes included liver cord disarray, enlargement of ballooning degeneration, dilatation and congestion of some blood vessels with blood cells. A considerable number of hepatocytes showed cytoplasmic vacuolation. Küpffer cells were evident in the dilated hepatic sinusoids. A gradual increase in the damage was noticed by longer period of exposure. The pathologic effect of cadmium on hepatopancreas included a decrease in the size of the acinar cells with an increase in their number. Histochemically, glycogen content decreased in the liver and hepatopancreas with the increase of the period of exposure. These Cd-induced parameters were significantly improved with supplementation of vitamin E and/or tomato paste. These findings were emphasized by liver enzymes ones demonstrating the beneficial supplementation of vitamin E and/or tomato paste in reducing the harmful effects of Cd on the normal enzyme levels and structures of the liver and hepatopancreas.
INTRODUCTION
The health of aquatic ecosystems is best evaluated by fish physiological and biochemical characteristics which serve as biomarkers of environmental pollution (Kock et al., 1996). In this concern Oreochromis niloticus, is a common appropriate model in toxicological research (Figueiredo-Fernandes et al., 2006a, b; Garcia-Santos et al., 2006) and in biomonitoring programmers (Gadagbui et al., 1996).
Heavy metals were recorded naturally in varying levels in all ground and surface waters (Martin and Coughtrey, 1982). According to Mason (1991), these metals represent one of the five major types of toxic pollutants commonly present in surface waters. Some heavy metals are nonessential and play no significant biological roles whereas others are essential elements for the normal metabolism of organisms (Cross and Sunda, 1985; Rainbow, 1985; Rainbow and White, 1989; Sanders, 1997). Anthropogenic activities cause an increased discharge of various concentrations of both essential and nonessential metals into natural aquatic ecosystems. Before significant changes can be identified in fish behavior or external appearance, the initial effects of heavy metal pollution may be evident at cellular or tissue levels (Bais and Lokhande, 2012; Van Dyk et al., 2007).
The nonessential heavy metal, Cadmium (Cd) is has an accumulative toxicity effect to aquatic organisms even in minute concentrations (Cicik and Engin, 2005). It has been demonstrated to stimulate free radical production in concern with the lipid, protein and DNA oxidative deterioration and initiating various pathological conditions in humans and animals (Hussein and Mekkawy, 2001; Manca et al., 1991; Sarkar et al., 1997; Shaikh et al., 1999; Shen and Sangiah, 1995).
Variability in the Cd-induced toxic effects on fish is evident especially in interrupting development and growth (Lemaire-Gony and Lemaire, 1992; Mekkawy and Lashein, 2003), anemia (Abdel-Rahman, 1997; Houston et al., 1993; Larsson et al., 1985), preventing Ca+2 uptake through the gills (Verbost et al., 1987), disturbing liver functions (Soengas et al., 1996), skeletal deformations (Muramoto, 1981) and pathological changes in some tissues and organs (Karlsson-Norrgren et al., 1985). Cadmium fish toxicity and factors controlling such toxicity were discussed and reviewed by Alabaster and Lloyd (1982) and Lloyd (1992) and Heath (1995).
The study of pollutant effects on the histology of different organs of fish is an important basic task leading to understanding the impact of such pollutants on a given ecosystem (Meyers et al., 1992; Young et al., 1994). The liver of fish is an important organ sharing in many metabolic processes and is the major site of detoxification (Hogan and Knowles, 1968; Verity and Reigth, 1967). According to Hinton and Lauren (1990), the liver is essential for both the metabolism and the excretion of toxic substances and so it serves as a model for studying the interactions between environmental factors and hepatic structures and functions (Brusle and Anadon, 1996). Heavy metals-induced hepatic histological changes could be considered as biomarkers. The estimation of Cd-free radical generations and the antioxidant defense has become an important aspect of investigation in mammals and animals especially with the natural antioxidants like tomato carotenoids including β-carotene, γ-carotene and lycopene (Bramley, 2000; Clinton, 1998; Krinsky, 2001; Tapiero et al., 2004; Visioli et al., 2003) and vitamin E (α-tocopherol). In animal health, these naturally occurring antioxidants inactivate harmful free radicals produced through normal cellular activity and from various pollutants and stressors (El-Demerdash et al., 2004a). These micronutrients could, at least enhance immunity by maintaining the functional and structural integrity of important immune components (Chew, 1995; El-Demerdash et al., 2004b; Pregiosi et al., 1998; Yousef et al., 2003). Therefore, the assessment of the relative antioxidant potency of vitamin E and carotenoids has received particular attention (El-Demerdash et al., 2004a; Heber and Lu, 2002; Wertz et al., 2004) with emphasize on the conclusion that lycopene has a higher antioxidant potential than α-tocopherol and β-carotene (Di Mascio et al., 1989; Rao and Agarwal, 1999; Woodall et al., 1997). There is no interpretation of the mechanism controlling the antioxidant property of carotenoids (Aust et al., 2001; Woodall et al., 1997).
Human consumption of lycopene-rich foods was found to be related to the lower levels of some health problems including cancer and cardiovascular diseases (Mekkawy et al., 2010). According to some authors, it is difficult to determine what proportions of their health-protective effects are attributable to lycopene because lycopene-rich foods contain a number of other beneficial phytonutrients (Hadley et al., 2002). Since tomatoes and tomato products are the major dietary source of lycopene (Zhang et al., 2008), lycopene could be used as indicator of the additive or synergistic anti-carcinogenic effects of their phytonutrients (Mekkawy et al., 2010). Emphasizing on such observation, Boileau et al. (2003) stated that consumption of tomato powder but not lycopene inhibited prostate carcinogenesis. Tomatoes develop sets of interacting compounds (carotenoids) to accomplish their antioxidant functions rather than relying on single compounds (Gann and Khachik, 2003). These compounds behave in similar way in animal body. The antioxidant role of these phytonutrients depends on the ability of animal species to achieve their biologically relevant tissue concentrations.
According to the aforementioned findings and low price of tomatoes in a definite season in Egypt, the present work was suggested as extension to previous work (Mekkawy et al., 2010) and aimed to study the effect of cadmium on the light of diet supplementation of tomato paste and/or vitamin E on histological and histochemical characteristics of the Nile fish, Oreochromis niloticus (Linnaeus 1758). Do the current results have applicable role in human liver stressed by heavy metal toxicity is a question to be answered.
MATERIALS AND METHODS
Sample collection and treatment manipulation: One hundred and twelve healthy fish of the Nile tilapia, Oreochromis niloticus (120±17.8 g in weight, 19±1.04 cm in length) were caught from the fish farm of Faculty of Agriculture, Assiut University, Egypt. Fishes immediately were transported to the fish laboratory in the Department of Zoology, Faculty of Science, Assiut University. The experimental fishes were reared in aerated glass tanks (100 L capacity) and acclimatized for two weeks before being used in the experimental study. The experimental fish fed pellets at a rate of 4.5% of fish body weight twice daily. Feces and residual food were aspirated regularly. The water temperature, pH and Dissolved Oxygen (DO) concentrations were measured daily (24.2±.08 °C, 6.8±.11 pH and 6.5±.89 mg L-1 DO). Light cycle was 12 light and 12 h dark.
Preparation of tomato paste to adjust the lycopene dose: Tomatoes used for the experiment were obtained from the local market. Fresh peeled, deseeded tomatoes were pulped well to a smooth consistency in a warring blender. The lycopene content in tomato paste was estimated spectrophotometrically according to the methods of Ranganna (1976) and Choudhari and Ananthanarayan (2007). The lycopene concentration in the tomato paste was 30.028 mg/100 g (Okajima et al., 1998). Based on the review of Xianquan et al. (2005), such concentration could not be affected by current conditions of diet preparation and storage of a short time (37°C for 4 weeks). In addition to lycopene, tomato paste composition include water, proteins, carbohydrates, fibers, calcium, potassium, zinc, copper, manganese, iron, vitamin C, vitamin E, β-carotenoids and other phytonutrients.
Experimental design: Fishes were weighed, measured and classified randomly into 8 groups (14 fish/tank) according to dose of cadmium, tomato paste in terms of lycopene, vitamin E and their combinations (Table 1). The diets (maize and soy bean, 15 mg kg-1 fish) were pellet after addition of vitamin E and tomato paste doses for the treated groups and the addition of suitable amounts of molasses and water. The diets were dried at room temperature and stored in small bags for fish feeding.
Stock solution (1000 ppm) of cadmium as cadmium chloride (CdCl2 2.5H2O) was prepared and stored in clean glass bottles and diluted to concentration of 4.64 mg L-1. Such low sublethal cadmium concentration (¼ of 96 h- LC50) was chosen according to levels monitored by Almeida et al. (2002). Cadmium doses were prepared and added constantly to the aquarium for four weeks.
| Table 1: | The fish groups exposed to cadmium (4.64 mg L-1) and tomato paste (9 mg lycopene/kg body weight) and vitamin E (50 mg kg-1 body weight) and their combinations |
| C: Control, VE: Vitamin E, Tp: Tomato paste and Cd: Cadmium | |
The test water was replaced daily with the required amount of stock solution to prevent deterioration of water quality and replenish cadmium levels. Tomato paste was added to the diet in concentration of 30 mg kg-1 b.wt. (9 mg lycopene kg-1 b.wt.). Dose response of lycopene was described previously by Rodriguez et al. (2004). Also, vitamin E (α-tocopherol) was supplemented in 50 mg kg-1 b.wt. Such vitamin E concentration was chosen according to levels monitored by Ortuno et al. (2001). It is worthy to mention that vitamin E (α-tocopherol) in tomato paste was estimated to be 38.67±2.29 mg/100 g tomato paste dry weight with no effect by industrial processing (Capanoglu et al., 2008).
Histological and histopathological examination: For microscopic preparations, after intervals of 15 and 30 days, 3 surviving fish of each group were removed and sacrificed. Small pieces of the liver were taken and immediately fixed in 10% neutral buffered formalin. Fixed tissues were processed routinely for paraffin embedding technique. Embedded tissues were sectioned at 7 μ in thickness and then stained by following stains: Harris's hematoxylin and eosin stain (HE) (Bancroft and Stevens, 1982) and Masson's Trichrome stain for the collagenous fibers (Humason, 1979). This method is preceded by hematoxylin staining for a brilliant permanent nuclear stain.
Histochemical preparation: Estimation of general carbohydrates represents the important parameter among the histochemical ones. For the demonstration of the polysaccharides status, periodic acid Schiff's (PAS) technique was applied (McManus, 1946). In this regard, carbohydrates were first oxidized with 0.1% periodic acid; aldehyde groups (-HCO-HCO), were liberated from the glycol reagent, producing a compound of magenta coloration. Alcian blue (AB-PH 2.5) and periodic acid-Schiff (PAS) method visualized by Mowry (1956) was indicated by appearance of blue color for acid mucin, magenta for neutral mucin and mixture of the two colors for carbohydrate. The nuclei colored pale blue.
RESULTS
Control liver: In control liver of Oreochromis niloticus the cords of hepatocytes tend to have a regular radial pattern enclosing the sinusoidal network for a short distance into the perivenular areas (Fig. 1a-c). These areas become less regular outside the perivenular zone. The individual hepatocyte is polygonal in shape and has a single spherical nucleus. The nuclei are mostly centrally located within the hepatocytes with some nuclei tending to occur closer to the cell periphery bordering the sinusoids. The endothelial cells that line these sinusoids as well as their nuclei are flattened and elongated (Fig. 1b-d).
The exocrine pancreatic tissue (hepatopancreas) is a pronounced feature in the liver of Oreochromis niloticus. It consists of a large number of acini (Fig. 1b). Each acinus is made up of conical glandular cells with an eccentric, deeply stained nucleus and prominent nucleolus.
| Fig. 1(a-b): | Sections of control fish liver and hepatopancreas. (a) Fish liver showing the general structure, Blood sinusoids (BS), Central vein (CV), hepatocytes (H) and Küpffer cell (K) (H and E X 400). (b) Fish hepatopancreas (P) showing the portal vein (PV) and the basophilic portion with nucleus and the acidophilic cytoplasm of the acinar cells. (H and E X 400) |
| Fig. 2(a-b): | Sections of control fish liver and hepatopancreas. (PAS X 400). (a) Control fish liver showing the great amount of glycogen in the cytoplasm of hepatocytes. (b) Control fish hepatopancreas (P) showing glycogen content in hepatopancreatic tissue |
The cytoplasm is homogenous and basophilic in the nuclear portion of the cell; the remaining part of the cell is acidophil (Fig. 1b).
PAS-technique exhibited distinct distribution of the polysaccharide materials in the hepatocytes and pancreatic cells of the control fish for 15 and 30 days. The positively stained materials have been proved to be glycogen as verified by PAS-staining with and without pre-treatment with diastase. Such reaction appeared comparatively higher in the hepatocytes around the central vein areas than at the peripherals (Fig. 2a, b).
Treatment with tomato paste or vitamin E and their combinations: Histological examination revealed that the treatment of fish with tomato paste and/or vitamin E for 15 days had no effect on the appearance of the liver structure and hepatopancreas which had essentially normal appearance. An improvement in the histological structure of the hepatocytes and pancreatic tissues was noticed in fish administrated lycopene and/or vitamin E for 30 days as compared to the control ones. The boundary between cells was noticed more or less clearly and the nuclei of the hepatic cells were seen to be enlarged and hyperchromatic (Fig. 3a-c, Fig. 4a-c).
PAS-technique exhibited distinct distribution of the polysaccharide materials appeared as massive red colored patches located exclusively in the cytoplasm of the hepatocytes and pancreatic cells in the fish administrated lycopene and/or vitamin E (Fig. 5a-c, 6a-c) for 15 and 30 days. The positively stained materials have been proved to be glycogen as verified by PAS-staining with and without pre-treatment with diastase. Like the control, PAS-reaction appeared comparatively higher in the hepatocytes around the vein areas than at the peripherals.
Treatment with cadmium chloride: In comparison with the control, variable alterations had indicated in the liver sections of Cd-treated fish for 15 days. These alterations were mainly represented by liver nonhomogenous architecture (Fig. 7a-c). Sinusoidal lumen was collapsed and few Küpffer cells were observed. Hydropic and vacuolar degenerations were evident in some specimens. Various degrees of vacuolar degeneration, varying from mild to complete replacement of the hepatic cells by vacuoles (Fig. 7a) were noticed in some places with the rupture of hepatocyte membranes. Dilatation of some blood vessels congested with blood cells was revealed (Fig. 7a).
| Fig. 3(a-c): | Sections of treated fish liver for 30 days showing the general structure of the liver. Blood sinusoids (BS), central vein (CV), hepatocytes (H) and Küpffer cell (K). (H and E.X 400). (a) Tomato paste treatment, (b) Vitamin E treatment and (c) Vitamin E and tomato paste simultaneous treatment |
| Fig. 4(a-c): | Sections of treated fish hepatopancreas for 30 days showing small aggregation of melanomacrophage (Mm), portal vein (PV) and acinar cells with basophilic and acidophilic cytoplasm. (H and E. X 400). (a) Tomato paste treatment, (b) Vitamin E treatment and (c) Vitamin E and tomato paste simultaneous treatment |
| Fig. 5(a-c): | Sections of treated fish liver for 30 days showing, great amount of glycogen in the cytoplasm of hepatocytes. (PAS X 400). (a) Tomato paste treatment, (b) Vitamin E treatment and (c) Vitamin E and tomato paste simultaneous treatment |
Figure 2b-c show proliferation of the hepatic cells with a decrease in cell size. Accordingly, the hepatocytes lost their normal polygonal shape and boundary between cells became invisible. Also, infiltration of the inflammatory cells inside the central vein and between the hepatocytes and extravasated erythrocytes (haemorrhage) were observed (Fig. 7b). Areas of hepatic necrosis started to appear in some regions and rupture of the wall of the central vein were noticed (Fig. 7c).
In addition to preceding changes, more conspicuous signs of irregularly shaped advanced necrotic areas were recorded for Cd-treated fishes for 30 days. The hepatocytes are delimited by ruptured cell membranes in some areas and dispersion of cell contents and loss of stainability in others (Fig. 8a). Individual cells were necrotic with condensed granules, some of them were characterized by the absence of nuclei while the others were having pyknotic nuclei and extravasated erythrocytes (haemorrhage) were observed (Fig. 8a). Moreover, more hydropic and little vacuolar degeneration of the hepatocytes were observed more than the previous period with infiltration of inflammatory cells, dilatation in blood sinusoid and granulated cytoplasm (Fig. 8b).
Cd-exposed fish for 15 days showed an aggregation of melanomacrophage and inflammatory cells around the hepatopancreatic acini. Extravasated erythrocytes (haemorrhage) and dilatation in portal vein of hepatopancreas was revealed (Fig. 9a). In addition to the previous alterations, Cd-exposed fish for 30 days revealed proliferation of the acinar cells, Absence of the acidic portion of these cells, rupture and peripheral fibrosis in the portal vein and aggregation of melanomacrophage cells were observed (Fig. 9b).
Examination of liver sections stained with Masson's trichrome stain produced an accumulation of connective tissue fibers around the central vein and hepatic sinusoids and infiltration of inflammatory cell with necrotic area after 15 days (Fig. 10a). Such accumulation of connective tissue increases with an inflammatory area after 30 days of Cd-exposure (Fig. 10b).
| Fig. 6(a-c): | Sections of treated fish hepatopancreas for 30 days showing, good PAS-reactivity in the acinar cell. (PAS X 400). (a) Tomato paste treatment, (b) Vitamin E treatment and (c) Vitamin E and tomato paste simultaneous treatment |
| Fig. 7(a-c): | Sections of treated fish liver exposed to (4.46 mg L¯1) cadmium chloride for 15 days showing histological altrations in liver tissues. (H andE X 400). (a) Showing dilatation of blood vessel (DBV), congested with blood (Cog), Küpffer cells (K) and fatty degeneration (lipid droplets) (L), (b) Showing proliferation of hepatocytes (crowded of nuclei), haemorrhage (Hr), infiltrations of inflammatory cell (IF) and large necrotic areas (N) and (c) Showing vaculation of hepatic cells and large necrotic areas (N). Rupture of wall of the central vein (RCV) |
| Fig. 8(a-b): | Sections of treated fish liver exposed to (4.46 mg L¯1) cadmium chloride for 30 days. (H and E X 400), (a) Showing ruptured central vein lining (CV), haemorrhage (Hr), necrotic zone (N) with condensed granules and pyknotic nuclei (Pk) and (b) Showing congested blood sinusoids (BS), inflammatory area (IF), fatty degeneration (lipid droplets) (L) and necrosis (N) |
| Fig. 9(a-b): | Sections of treated fish hepatopancreas exposed to (4.46 mg L-1) cadmium chloride, (a) for 15 and (b) for 30 days. (H and E. X 400). (a) Showing haemorrhage (Hr), infiltration of inflammatory cells (IF) between the hepatopancreatic tissues and aggregation of melanomacrophage cells (Mm) around the pancreatic acini. Dilatation in portal vein (PV) and (b) Showing proliferation of the pancreatic acini (crowded pancreatic cells), notice the absence of the acidophilic portion of the pancreatic cells, aggregation of melanomacrophage cells (Mm) between the acini, necrotic area (N), also, peripheral fibrosis (PF) of the portal vein |
| Fig. 10(a-d): | Sections of treated fish liver (a and b) and hepatopancreas (c and d), exposed to (4.46 mg L-1) cadmium chloride for 15 and 30 days. (Masson's trichrome X 400). (a) Showing an accumulation of connective tissue fiber (CT), inflammatory (IF) and necrotic (N) areas, (b) Showing a huge amount of connective tissue fiber (CT) around the blood vessel, inflammatory (IF) and necrotic (N) areas, (c) Showing a large amount of connective tissue fibers (CT) located around the portal vein (PV), an aggregation of lymphocytes (LY) between the hepatopancreatic tissues and dilatation of portal vein (DPV) and (d) Showing peripheral fibrosis (connective tissue) (CT) of the portal veins, congestion (Cog) of the portal vein |
Moreover, examination of hepatopancreas sections revealed a marked increase in the collagenous fibers in the wall of the portal vein (peripheral fibrosis) and between the tissues with an aggregation of lymphocytes between the hepatopancreatic tissues after 15 and 30 days (Fig. 10c-d). Also, dilatation of the portal vein and blood congestion inside the portal vein were noticed.
In the liver of Cd-administered fish, histochemical investigation revealed that there was a remarkable depletion in the glycogen content in hepatocyte and hepatopancreatic tissues after 15 day (Fig. 11a-b). The cytoplasm of the majority of hepatocytes exhibited a faint coloration with PAS reaction compared to those of control ones. After 30 days of exposure, more depletion in the glycogen content in both organs was observed (Fig. 11c-d).
Treatment with cadmium chloride plus tomato paste: Cd-exposed fish dietary supplemented with lycopene for 15 days retained their normal appearance of the hepatic tissues reflecting the protective potential effect of lycopene (Tomato paste). Each hepatocyte has its own nucleus. The number of Küpffer cells increased with a marked decrease in the number of the melanomacrophage cells. However, some cells still suffering from vacuolar degeneration in some areas and blood congestion inside the central vein (Fig. 12a). After 30 days of exposure and supplementation of tomato paste, the hepatic tissues retained its normal structure with tiny aggregations of melanomacrophage cells and infiltration of inflammatory cell (Fig. 12b).
| Fig. 11(a-d): | Sections of fish liver (a) and hepatopancreas(b) exposed to (4.46 mg L-1) cadmium chloride for 15 days, and fish liver (c) and hepatopancreas (d) exposed to the same dose for 30 days. (PAS, X 400). (a) Showing a remarkable depletion of glycogen in the liver cells, (b) Showing depletion in glycogen content of hepatopancreas, (c) Showing moderate amount of glycogen in the liver cells after 30 days and (d) Showing more depletion of glycogen content in the hepatopancreas after 30 days |
| Fig. 12(a-d): | Sections of treated fish liver (a and b) and hepatopancreas (c and d) exposed to lycopene+(4.46 mg L-1) cadmium for 15 and 30 days. (H and E X 400), (a) Fish liver Showing normal structure of hepatic tissue with blood congestion (Cog) in central vein (CV) and in blood sinusoids and clear cytoplasmic vacuolation. The number of Küpffer cells increased (K), (b) Fish liver Showing the general structure and arrangement of the hepatocytes, central vein (CV) and infiltration of inflammatory cell (IF) and small aggregation of melanomacrophage (Mm) after 30 day of exposure, (c) Fish hepatopancreas showing proliferation of the pancreatic acini and blood congestion (Cog) in portal vein (PV) and (d) Fish hepatopancreas showing the normal structure of the hepatic and hepatopancreatic tissues after 30 day of exposure |
The hepatopancreatic tissue shows normal structure with presence of its acidic portion, few aggregation of melanomacrophage (Mm) and blood congestion in portal vein were detected after 15 days (Fig. 12c). After 30 days of exposure and supplementation of tomato paste, the hepatopancreatic tissues retained its normal structure (Fig. 12d). So time of dietary supplementation of tomato paste may affect the process of repairing hepatocytes, sinusoids, blood vessels and hepatopancreatic tissue.
PAS-technique revealed distinct accumulation of carbohydrate materials in the hepatocytes and pancreatic acini in comparison to those of Cd-treated fish after 15 days (Fig. 13a-b). More accumulation of carbohydrate materials was observed in liver of fish treated for 30 days (Fig. 13c-d). These results referred to the role of tomato paste in preservation of carbohydrate materials.
Treatment with cadmium chloride plus vitamin E: Administration of vitamin E (α-tochopherol) plus cadmium to fishes for 15 days, produced histopathological alterations in the liver tissue.
| Fig. 13(a-d): | Sections of treated fish liver (a) and hepatopancreas(b) exposed to tomato paste with (4.46 mg L-1) cadmium for 15 days and fish liver (c) and hepatopancreas (d) exposed to the same dose for 30 days. (PAS, X 400). (a) Fish liver showing a remarkable good amount of glycogen in the liver cells. (b) Fish hepatopancreas showing moderate amount of glycogen content in the hepatopancreas. (c) Fish liver Showing moderate amount of glycogen in the liver cells after 30 days and (d) Fish hepatopancreas showing more depletion of glycogen content in the hepatopancreas after 30 days |
These alterations included proliferation of hepatocytes in some fishes as indicated by the crowded of the nuclei and the limits between cells not clear. Some of hepatocytes showed vacuole degeneration (Fig. 14a). In others, the majority of the hepatocytes and sinusoids presented normal appearance.
After 30 days of exposure to the same dose of cadmium and vitamin E, improvement of the hepatic tissue was noticed when compared to those of Cd-treated fish. The majority of the hepatocytes and sinusoids showed normal appearance with small infiltration of the lymphocyte cells beside the central vein (Fig. 14b). However, some samples still suffering from a great reduction in the size of the hepatocytes. An aggregation of these cells in clumps was noticed as well the boundaries between cells are not clear. The nuclei of these aggregated cells were seen to be enlarged and hyperchromatic.
After 15 days of exposure, the hepatopancreatic tissue display peripheral fibrosis and dilatation in portal vein of hepatopancreas. Tiny aggregations of melanomacrophage cells were observed (Fig. 14c).
| Fig. 14(a-d): | Sections of treated fish liver (a and b) and hepatopancreas (c and d) exposed to vitamin E plus (4.46 mg L-1) cadmium for 15 and 30 days. (H and E. X 400). (a) Fish liver showing proliferation of some hepatocytes and vacuole degeneration. (b) Fish liver showing the general structure and arrangement of the hepatocytes with lymphatic infiltration (LY) beside the central vein (CV) after 30 days (c) Fish hepatopancreas showing small aggregation of melanomacrophage cells (Mm), peripheral fibrosis (PF) and dilatation in portal vein (PV) of hepatopancreas and (d) Fish hepatopancreas showing normal structure of hepatopancreatic tissue with few number of melanomacrophage (Mm) cells after 30 days |
| Fig. 15(a-d): | Sections of treated fish liver (a and b) and hepatopancreas (c and d) exposed to vitamin E with (4.46 mg L-1) cadmium for 15 and 30 days. (PAS X 400). (a) Fish liver Showing a moderate amount of glycogen in the liver cells. (b) Fish liver showing good amount of glycogen content in the liver cells after 30 days. (c) Fish hepatopancreas Showing depletion in glycogen content in the hepatopancreatic tissue and (d) Fish hepatopancreas showing increase in glycogen content in the hepatopancreas after 30 days |
After 30 days of exposure, the pancreatic cells of the examined sections retained its normal structure with a few number of melanomacrophage cells (Fig. 14d). One can conclude that tomato paste has a better protective role than vitamin E in this concern.
Histochemically, PAS-technique revealed a good preservation of carbohydrate materials (glycogen) in hepatocytes by vitamin E after 15 and 30 days (Fig. 15a-b). However, the hepatopancreatic tissue revealed slight accumulation of glycogen in the acinar cells compared to those of Cd-treated fish after 15 days, such accumulation increased after 30 days (Fig. 15c-d). These results reflect the protective role of vitamin E in preservation of glycogen in a way similar to tomato paste.
Treatment with cadmium chloride, vitamin E and tomato paste: Apart from mild hydropic degeneration and proliferation of hepatic cells, an improvement in the hepatic tissue with slight cytoplasmic vacuolation was noticed when compared to those of Cd-treated fish or Cd- treated fish plus vitamin E for 15 days (Fig. 16a). After 30 days, beneficial improvements were noticed in the hepatic tissue of the exposed fishes. The majority of the hepatocytes and sinusoids had normal appearance with few proliferation of hepatic cell (Fig. 16b).
The hepatopancreatic tissue shows proliferation of the pancreatic acini, peripheral fibrosis and large aggregation of melanomacrophage cells around the pancreatic acini after 15 days of exposure (Fig. 16c). After 30 days of exposure to the same doses of cadmium, tomato paste and vitamin E, the pancreatic tissue of the examined sections retained its normal structure with small aggregation of melanomacrophage cells around the pancreatic acini (Fig. 16d).
| Fig. 16(a-d): | Sections of treated fish liver (a and b) and hepatopancreas (c and d) exposed to tomato paste, vitamin E plus (4.46) cadmium for 15 and 30 days. (H and E. X 400). (a) Fish liver showing more or less normal structure of the liver (CV) central vein, (b) Fish liver showing proliferation and normal structure and arrangement of the hepatocytes, central vein (CV). after 30 days, (c) Fish hepatopancreas showing proliferation of the pancreatic acini, large aggregation of melanomacrophage (Mm) around the pancreatic acini and peripheral fibrosis (PF) in portal vein and (d) Fish hepatopancreas showing normal structure of pancreatic tissue with small aggregation of melanomacrophage cell (Mm) after 30 days |
| Fig. 17(a-d): | Sections of treated fish liver (a and b) and hepatopancreas (c and d) exposed to tomato paste, vitamin E with (4.46 mg L-1) cadmium for 15 and 30 days. (PAS.X 400). (a) Fish liver showing a moderate amount of glycogen in the liver cells, (b) Fish liver showing good amount of glycogen content in the liver cells after 30 days, (c) Fish hepatopancreas showing depletion in glycogen content in the hepatopancreatic tissue and (d) Fish hepatopancreas showing depletion in glycogen content in the hepatopancreatic tissue after 30 days |
By using PAS- technique, a good restoration of glycogen content was noticed in the liver cells and hepatopancreatic tissue after 15 days with increased accumulation of glycogen after 30 days of exposure of fish to cadmium chloride, tomato paste and vitamin E synchronal (Fig. 17a-d).
DISCUSSION
Heavy metals disturb different living processes at the molecular and subcellular levels of biological organization leading to cell injury and in turn to degenerative and neoplastic diseases in target organs (Folmar, 1993; Cicik and Engin, 2005; Jacobson-Kram and Keller, 2001; Pacheco and Santos, 2002). Accordingly, changes in histopathological and biochemical characteristics as bioindicators are reliable evident of toxicity in fish organs (Schwaiger et al., 1996; Teh et al., 1997). There are several reports on the impact of environmental toxicants on fish revealed by histopathological, histochemical and biochemical studies of vital organs such as gills, liver and kidney (Barlas, 1999; Cengiz and Unlu, 2002; Cengiz et al., 2001; Erkmen et al., 2000; Gill et al., 1988; Richmonds and Dutta, 1989). To what extent this situation was revealed in Cd-stressed O. niloticus fed supplemented diet with tomato paste or vitamin E is considered.
Gill et al. (1990), Cengiz et al. (2001), Fanta et al. (2003), Cengiz and Unlu (2006), Velmurugan et al. (2007) and Benli et al. (2008) reported hepatic lesions including hypertrophy, vacuolization, nuclear pyknosis, karyolysis, karyohexis, fatty degeneration, hydropic degenerations, sinusoids enlargement, hemorrhage, infiltration of mononuclear lymphocyte, Cloudy swelling, focal necrosis, increase of Küpffer cells, circulatory disturbance, narrowing of sinusoids and congestion.
The miniaturization of histological changes in fish liver is a highly sensitive and accurate way to assess the effects of xenobiotic compounds in field and experimental studies (Figueiredo-Fernandes et al., 2007). In the present investigation, some of pathological features noticed in the liver of fish exposed to 4.64 mg L-1 of cadmium chloride at both periods of exposure were the cytoplasmic vacuolation and fatty degeneration associated with lipid accumulation. Hinton and Lauren (1990) reported that vacuolation of hepatocytes are associated with the inhibition of protein synthesis, energy depletion, disaggregation of microtubules, or shifts in substrate utilization. A similar result was reported by Van Dyk et al. (2007) on Oreochromis mossambicus after treatment with cadmium and zinc. Such vacuolation was regarded by El-Banhawy et al. (1986) as a cellular defense mechanism against injurious substances to prevent their interfering with cellular metabolism. The cytoplasmic vacuolization may be also attributed to the increased number and activities of lysosomes and their contents El-Banhawy et al. (1993). Desai et al. (1984) and Gabr (1986) confirmed these conclusions on Oreochromis mossambica and Oreochromis niloticus after treatment with monocrotophos and diasinon insecticides, respectively.
Cellular degeneration followed by nuclear pyknosis and necrosis was observed in the present investigation. These alternations are in agreement with those of Wassif et al. (2000), Elezabi et al. (2001), Fathalla et al. (2001), Mohamed et al. (2005) and Van Dyk et al. (2007). Such cellular degeneration occurs directly by denaturation of volume-regulating ATPases or indirectly by disruption of the cellular energy transfer processes required for ionic regulation (Hinton and Lauren (1990). El-Banhawy et al. (1993) mentioned that there is a relationship between pathological alternations (liver damage) and reduction in activities of oxidative enzymes such as cytochrome oxidase, succinic dehydrogenase, coenzyme I, diphosphopyridine nucleotidase DPN, cytochrome p-450 and β-glucuronidase under various pathological conditions.
Desai et al. (1984) suggested that the necrosis could be either due to direct effect of the compound on the cells or to an accumulation of acetylcholine in the tissues.
Venous congestion was recorded in the present study may be due to the decline in the haematological parameters which observed in the present study. Such observation was reported by Mekkawy et al. (1996), since the haemoglobin contents are insufficient for the respiration of the tissue. This results was in agreement with Fathalla et al. (2001), Khidr et al. (2001) and Mohamed et al. (2005).
The remarkable abundance of the lymphocytic infiltration in the liver tissues was another pathological sign recorded in the present work. Such pathological sign was confirmed by many authors as a response of heavy metal stress (El-Banhawy et al., 1993; Khidr et al., 2001; Mohamed, 2009; Wassif et al., 2000). The Küpffer cells were increased in size and number as a defense mechanism against stress (Kadry, 1989; Mahmoud, 1999; Popper and Schaffner, 1957) and against any foreign material in the circulating blood (Mahmoud, 1999).
In the present investigation, a remarkable collection of inflammatory cells adjacent to some blood vessels and invasion of the parenchyma cells were observed. Such cellular infiltration might be due to the presence of necrotic cells which act as an irritant substance attracting the inflammatory cells (Walter and Israel, 1974). In general, the abundance of leukocytes and lymphocytes in particular is a prominent response of body tissues facing any injurious impacts (El-Banhawy et al., 1993).
The number, size and contents of melanomacrophages are highly variable, not only between species but also related to the health status of fish (Bunton et al., 1987; Matthiessen and Roberts, 1982). Migration of such melanomacrophages into the liver parenchyma after exposure to cadmium was observed in the present study. A similar result has been reported by Braunbeck et al. (1990) in Zebra fish (Brachydanio rerio) and trout (Salmo gairdneri) following exposure to 4-chloroaniline (Braunbeck et al., 1992) in Oryzias latipes after exposure to diethylnitrosamine and by Oulmi et al. (1995a) in rainbow trout (Oncorhynchus mykiss) exposed to sublethal concentration of linuron. The migration of macrophages and removal of damaged cell components as well as necrotic hepatocytes represented adaptive rather than degenerative features (Oulmi et al., 1995a).
Chronic liver diseases are characterized by progressive accumulation of connective tissue undergoing fibrotic degeneration (Popper and Kent, 1975). Under Cd-stress, a remarkable collection of connective tissue fiber adjacent to some blood vessels in the liver tissue was observed in the present work. The common link between chronic liver damage and hepatic fibrosis representing severe oxidative stress, has been found to be associated with the activation of Hepatic Stellate Cells (HSC) (Svegtiati Baroni et al., 1998). Such oxidative stress seems to occur at a prefibrotic stage suggesting a causative relation between oxidative events and liver fibrosis (Vendemiale et al., 2001). These findings were confirmed by many authors (El-Sokkary et al., 2005; Gualdi et al., 1994; Hernandez-Munoz et al., 1997; Muriel et al., 1994; Niemela et al., 1995).
The disruption or dissolution of normal arrangement of the hepatocytes observed in the present study has been frequently reported by Couch (1975), Smith and Piper (1975) and Hacking et al. (1978).
The aggregation of lymphocytes around the hepatopancreatic acini, proliferation of the hepatopancreatic acini, crowdness of cells and depletion in the acidity of the apical portion of pancreatic acinar cells were observed in the hepatopancreas of fish exposed to 4.64 mg L-1 of cadmium chloride at both periods of exposure. Wassif et al. (2000) and Hussein and Mekkawy (2001) recorded similar results when the first authors exposed Oreochromis niloticus to different concentrations of atrazine and the others exposed Oreochromis zillii to sublethal concentration of lead.
Depletion in the acidity of the apical portion of pancreatic acinar cells after exposure to cadmium was observed in the present study. A similar result has been reported by Hara et al. (1994) who found that bis (tributylin) oxide (TBTO) inhibited both the synthesis and secretion of zymogen granules in the rat exocrine pancreas. They attributed this inhibition to mitochondria dysfunction due to the toxic effects of TBTO. Oulmi et al. (1995b) reported that the exposure of rainbow trout to atrazine caused disorganization of Golgi fields in the distal segment cells of the renal tubules.
In the present investigation, histochemical results showed a remarkable decrease in glycogen amount in the liver of fish exposed to cadmium chloride. This finding is consistent with other studies, which showed a reduction in hepatic glycogen following exposure to xenobiotic compounds (Biagianti-Risbourg, 1996; Gluth and Hanke, 1985) and exposure to herbicides (Mekkawy et al., 1996; Wassif et al., 2000).
This observation might be due to increased glycolytic activity to meet the energy demands imposed by enhanced metabolic activity, hormone-mediated stress phenomenon (Gluth and Hanke, 1985; Hanke et al., 1983). Heath (1995) explained the loss of glycogen as a depressed feeding and/or elevated levels of the stress hormones, cortisol and adrenaline. Glycogen can also be depleted in response to some physiological processes such as sexual maturation (Yamamoto and Egami, 1974), or nonchemical stresses, such as temperature (Braunbeck et al., 1987) and hypoxia (De Zwaan and Zandee, 1972; Mekkawy et al., 1996; Yuness, 2005).
In the present study, administration of antioxidants tomato paste (tomato paste) as dietary supplementation remarkable protective effects and restored the general structure and the carbohydrate contents of the liver. Matos et al. (2001) reported minimum histopathological and no macro- morphological changes in the liver treated with tomato paste against Ferric Nitrilotriacetate toxicity in Rats. These results postulated that carotenoids (lycopene) are well known as highly efficient scavengers of singlet-oxygen (1O2) and other excited species. During 1O2 quenching, energy is transferred from 1O2 to the lycopene molecule, converting it to the energy-rich triplet state and thus preventing their damage (Atessahin et al., 2005).
In the present study, administration of antioxidant vitamin E to fish exposed to cadmium chloride display remarkable protective effects and restored the general structure and carbohydrate contents in the liver. The present results are in agreement with those observed in liver of Clarias gariepinus exposed to vitamin E against Lead -induced oxidative stress (Mohamed, 2006) and in liver of rats exposed to vitamin E against cisplatin -induced oxidative stress (Naziro et al., 2004).
The histopathological and histochemical changes resulted from cadmium oxidative stress was nearly disappeared in tissues of the studied organs by administration of lycopene and Vitamin E.