Abstract: The adverse impacts of heavy metals on fish liver were evident with great variability among organs and species. The present study deals with the histological changes of the hepatocytes of the Nile tilapia, Oreochromis niloticus, following exposure to 2.5, 5, 10 ppm of lead nitrate for 1, 2, 3, 4 weeks. The present results revealed that lead nitrate exerts some histological effects on the hepatic tissue after exposure to the first concentration in the form of dilatation and congestion of the blood vessels, vacuolation of hepatic cells, proliferation of connective tissue and hepatic necrosis. Leucocyte aggregation-mostly lymphatic in nature-was seen infiltrating hepatic tissue. These alterations became more pronounced in liver of fishes exposed to second concentrations indicating more progressive signs of necrosis. The presence of eosinophilic oedematous areas surrounding some blood vessels was also observed. Finally, at the third concentration, in addition to the above alterations, melanomacrophages, which store lipofuscin at the site of necrosis, were observed. These histological results imply that the fish liver may serve as a target organ for the toxicity of sublethal concentrations of lead nitrate.
INTRODUCTION
Lead is widely included in different industrial products such as pipes, battery cases, paints as well as petrol additives in the form of tetra-ethyl lead. Toxicity of lead on fish was considered by many authors including Weir and Hine (1970), Aronson (1971), Davies et al. (1976), Hussein and Mekkawy (2001), Khidr and Mekkawy (2008), Khidr et al. (2008) and Taweel et al. (2012). All life stages of fish are sensitive to the toxic effects of Pb (Mekkawy and Lashein, 2003; Osman et al., 2007a, b, 2008); however, embryos are more sensitive to Pb than are later juvenile stages (Davies et al., 1976; Mekkawy and Lashein, 2003). Pb toxicity can be altered by water quality especially hardness (Weir and Hine, 1970; Davies et al., 1976). Generally, it is postulated that lead (Pb) is one of the most important pollutants in our environment which accumulates in the body due to its low rate of elimination; its biological half-life in bones is about 27 years (Shibamoto and Bjeldanes, 1993).
In Egypt, the river Nile suffers from lead pollution in some areas along its course especially in Assiut Governorate. This contamination could be attributed to several factories: superphosphate, petroleum, cement, soap and oil factories and large number of garbage dump which situated along its course in Assiut (Abdel-Nasser et al., 1996a). Abdel-Nasser et al. (1996b) reported that the lead concentrations in water at different areas of river Nile in Assiut Governorate ranged from 0.200 to 1.940 ppm a value above the permissible limit of WHO (0.10 ppm) or United States Environmental Protection Agency (0.05 ppm). In the same trend, the lead levels in water were 0.18 and 0.257 ppm in Tanta and Helwan areas, respectively (Rizk et al., 1999; Ahmed, 1996).
Histological studies on different fish organs were carried out to evaluate the impacts of different types of stress including diet, heavy metals and pesticides stress (Arnold et al., 2000; Segnini et al., 2000; Jiraungkoorskul et al., 2002; Yang and Chen, 2003; Mekkawy et al., 2007, 2008, 2011a, 2012; Mobarak and Sharaf, 2011; Sayed et al., 2011, 2012a, b; Taweel et al., 2012; Bais and Lokhande, 2012). Most of these studies highlighted on the histopathology of the liver, kidney and other organs. Different degrees of impacts were recorded depending on the dose, age and fish species. According to the previous findings, the present work was suggested and aimed to studying the effect of lead on histological changes of the liver of the Nile fish, Oreochromis niloticus.
MATERIALS AND METHODS
Specimens collection: Forty specimens of the Nile tilapia, Oreochromis niloticus (36.38±0.77 g) in weight and 12.93±0.88 cm in length) were caught in September 2003 from Umm Al-Kossor hatchery about 35 km away from Assiut and transported immediately to the fish laboratory at the Zoology Department, Assiut University.
Experimental design: The experimental fishes were raised in aerated glass aquaria (75x30x30 cm), each of 150 L capacity and acclimatized for two weeks before being used in the experimental study. The experimental fish were fed pellets at a rate of 3% of wet weight twice daily. Feces and residual feed were aspirated regularly. Fishes were weighed and measured at the first day and classified randomly into 4 groups (10 fish each).
Stock solution (1000 ppm) of lead as lead nitrate Analar (BDH) (Pb [NO3]2) was prepared and stored in clean glass bottles and diluted to concentrations of 2.5, 5.0 and 10.0 ppm. Such low sublethal lead concentrations were chosen according to levels monitored by Abdel-Nasser et al. (1996a) and Abdel-Rahman (1997). Lead doses were prepared and added constantly to the aquarium for four weeks. The experimental period (4 weeks lead exposure) was chosen according to Lashein (1999), Rizk et al. (1999) and Hussein and Mekkawy (2001). The test water was replaced daily with the required amount of stock solution to prevent deterioration of water quality and replenish lead levels.
Histopathological preparations: At weekly intervals, representative samples of liver from control and experimental fishes groups (4 surviving fish of each group) were fixed in 10% neutral buffered formalin or aqueous Bouin’s. Fixed tissues were processed routinely for paraffin embedding technique. Embedded tissues were sectioned at 3μ and stained with hematoxylin and eosin (H and E) (Bancroft and Stevens, 1982). For the demonstration of lipofuscin pigment long Ziehl-Neelsen technique was used (Pearse, 1960).
PAS technique (McManus, 1948) was used for demonstration of 1:2 glycol linkage of carbohydrates. Control sections were incubated in human saliva at 37°C for one hour prior to PAS staining. The absence of stained material from such section was taken as an evidence for the presence of glycogen.
For semithin sections, proper sized samples (1 mm3) from the previous tissues were immediately fixed by immersion in 4% glutaraldehyde in 0.1 M cacodylate buffer for 24 h at 4°C and then rinsed in 0.1 M cacodylate buffer. Tissues were post fixed in 1% osmium tetroxide for 1-2 h at 4 °C; the specimens were washed with cacodylate buffer for few minutes several times and subsequently dehydrated in upgraded ethanol series. Embedding of the processed tissues was carried out in Epon 812. Tissues were sectioned at 1 μm (semithin sections) and stained with toluidine blue.
RESULTS
Control liver: As described in most fish species, lobular organization of hepatic tissue is not conspicuous in the liver of Oreochromis niloticus. It rather consists of cord-like structures with hepatic plates emintly visible along blood sinusoids (Fig. 1). The endothelial cells that line these sinusoids as well as their nuclei are flattened and elongated. The blood sinusoids drain into a central vein. Hepatocytes of Oreochromis niloticus appear polygonal in shape; each hepatocyte contains a single spherical nucleus. The nuclei are mostly centrally located within the hepatocytes. Mononuclear leucocytes were sometimes observed in some control specimens. These structures were however, few in number if present. Exocrine pancreatic tissue (hepatopancreas) is a pronounced feature in the liver of Oreochromis niloticus and is clearly visible as darkly stained tissue around the hepatic portal veins. Macrophage centers are found in the normal liver of Oreochromis niloticus, mainly in the vicinity of hepatopancreatic tissue (Fig. 2). The content of these macrophage centers varies in size.
Treatment with 2.5 ppm lead nitrate: Examination of semithin sections of the liver after exposure to 2.5 ppm lead nitrate for 7 to 15 days showed dilatation and wall thickening of some blood vessels, which were congested with blood cells (Fig. 3). Moreover, a considerable number of hepatocytes showed clear cytoplasmic vacuolation with deeply stained nuclei (Fig. 4), while other areas were either still intact or slightly affected.
After 21 days of exposure to the same dose of lead nitrate, besides the proceeding changes, marked proliferation of the connective tissue was seen (Fig. 5). Sometimes patches of leucocytic aggregation, mostly lymphocytic in nature were seen infiltrating hepatic tissue (Fig. 6). By the 28th day of the experiment, areas of hepatic necrosis started to appear in some regions. Other regions showed large patches of haemorrhagic lesions (Fig. 7).
| Fig. 1: | Photomicrograph of a section of control fish’s liver showing CV: central vein, the P: general structure and arrangement of the hepatocytes, diffused exocrine pancreas surrounding the hepatic portal vein. (H and E. X. 100) |
| Fig. 2: | Two photomicrographs of control fish’s liver sections (a) BS: General structure of the liver and the blood sinusoids (BS). (H and E. X. 200) and (b) Exocrine pancreatic tissue (hepatopancreas) surrounding the hepatic portal vein, 8: Macrophage centers were in the vicinity of hepatopancreatic tissue (H and E. X. 400) |
| Fig. 3: | Photomicrograph of a semithin section of fish’s liver exposed to 2.5 ppm of lead nitrate for 7 days showing congested (Cog) and thickened wall of blood vessel. (Toluidine blue. X. 400) |
| Fig. 4: | Photomicrograph of a section of fish’s liver exposed to 2.5 ppm of lead nitrate for 7 days 8: deeply stained nuclei and the vacuolated cytoplasm (arrows). (H and E. X. 1000) |
| Fig. 5: | Photomicrograph of a section of fish’s liver exposed to 2.5 ppm of lead nitrate for 21 days CT: The proliferation of intrahepatic connective tissue and 8: pyknotic nuclei. (H and E. X. 400) |
| Fig. 6: | Photomicrograph of a section of fish’s liver exposed to 2.5 ppm of lead nitrate for 21 days showing inflammatory leucocytic infiltration (arrow). (H and E. X. 1000) |
| Fig. 7: | Photomicrograph of a section of fish’s liver exposed to 2.5 ppm of lead nitrate for 28 days 8: degeneration of hepatic cells with patches of haemorrhages (H). (H and E. X. 400) |
| Fig. 8: | Photomicrograph of a semithin section of fish’s liver exposed to 2.5 ppm of lead nitrate for 28 days 8: lipid droplets in the cytoplasm and a dark areas around the nucleus against lighter stained cytoplasm. (Toluidine blue, X.1000) |
| Fig. 9: | A photomicrograph of a section of fish’s liver exposed
to 5 ppm of lead nitrate for 7 days showing necrotic areas ( |
| Fig. 10: | Photomicrograph of a section of fish’s liver exposed to 5 ppm of lead nitrate for 14 days ED: Faintly stained eosinophilic oedematous area Cog: surrounding congested blood vessel. (H and E. X. 400) |
Examination of the semithin sections revealed numerous lipid droplets in the cytoplasm, which also contained clumped cellular organelles (Fig. 8). The latter appeared as dark areas around the nucleus against the lighter staining cytoplasm.
Treatment with 5 ppm lead nitrate: Examination of liver sections of fish after 7 to 14 days of 5 ppm lead nitrate exposure displayed more progressive signs of necrosis compared to those of the previous group (2.5 ppm lead nitrate) (Fig. 9). The hepatocytes membranes were ruptured with dispersion of cellular contents and loss of stainability (Fig. 9). Other signs of degeneration were manifested including the presence of faintly stained eosinophilic oedematous areas surrounding some blood vessels (Fig. 10). Moreover, the vessels were severely congested with blood and some inflammatory cells.
| Fig. 11: | Photomicrograph of a semithin section of fish’s liver exposed to 5 ppm of lead nitrate for 14 days 8: circular basophilic profiles in the cytoplasm. (Toluidine blue, X. 1000) |
| Fig. 12: | A photomicrograph of a semithin section of fish’s liver exposed to 10 ppm of lead nitrate for 14 days L: Accumulation of lipid droplets in the cytoplasm of hepatocytes. (Toluidine blue, X. 1000) |
Examination of the semithin sections revealed basophilic circular profiles around the nuclei and in the cytoplasm in addition to lipid droplets (Fig. 11). After 21 to 28 days of exposure to the same dose of lead nitrate, a progressive increase in the number of the circular profiles was observed.
Treatment with 10 ppm lead nitrate: Exposure of fish to 10 ppm lead nitrate for 7 to 14 days showed histopathological changes in the liver. Examination of the semithin sections showed numerous lipid droplets in the cytoplasm (Fig. 12). Other hepatocytes showed basophilic granules. After 21 to 28 days of lead exposure, necrotic changes associated with lymphocytic infiltration were detected. Moreover, melanomacrophages, which store lipofuscin at the site of necrosis were also observed in addition to dissociation of hepatic tissue (Fig. 13, 14).
| Fig. 13: | Photomicrograph of a section of fish’s liver exposed
to 10 ppm of lead nitrate for 21 days showing necrotic area ( |
| Fig. 14: | Photomicrograph of a section of fish’s liver exposed to 10 ppm of lead nitrate for 21 days showing the ↑: Melanomacrophage storing lipofuscin pigments. (Long Ziehl. Neelsen technique, X. 1000) |
Histochemical studies: In the hepatocytes of control animals, a considerable amount of polysaccharide material was observed in the cytoplasm as shown by their strong PAS positive reaction (Fig. 15). All these positively stained materials have been proved to be glycogen as verified by PAS staining with and without previous treatment with diastase. In all experimental groups, the hepatocytes displayed a remarkable depletion in the glycogen content. The cytoplasm of different cells exhibited a faint colouration with PAS (Fig. 16).
DISCUSSION
In the present investigation, a remarkable collection of inflammatory invasion of the parenchymal cells of the liver were observed after exposure to lead nitrate.
| Fig. 15: | Photomicrograph of a section of control fish’s liver showing glycogen content in the hepatocytes. (PAS. X. 400) |
| Fig. 16: | Photomicrograph of a section of fish’s liver exposed to 2.5 ppm of lead nitrate for 7 days showing a remarkable depletion in glycogen. (PAS. X. 400) |
Walter and Israel (1974) suggested that such cellular infiltration may due to the presence of necrotic cells which act as an irritant substance attracting the inflammatory cells. Moreover, El-Banhawy et al. (1993) suggested that abundance of leucocytes -in general- and lymphocytes -in particular- are a prominent response of body tissues facing any injurious impact.
In the present study, lipofuscin pigment in melanomacrophage was detected. Melanomacrophages are known to accumulate antigenic materials (Roberts, 1975), store products difficult to eliminate lipofuscin and ceroids (Wood and Yasutake, 1956) and store metals i.e., iron as haemosiderin (Agius, 1979). The number, size and contents of melanomacrophages are highly variable, not only between species but also related to the health status of fish (Matthiessen and Roberts, 1982; Bunton et al., 1987). Infiltration of the necrosed hepatic cells by migrating melanomacrophages in the present study support the suggestion that these cells infiltrate the damaged tissue to engulf lipofuscin pigments. On the other hand, Cheville (1983) suggested that lipofuscin pigments are found in lysosomes of cells undergoing progressive and prolonged oxidation of unsaturated lipids and are a pathognomonic sign of free radical and associated peroxidative injury to polyunsaturated lipids of subcellular membranes (Robbins and Kumar, 1987).
A drastic decrease in glycogen amount in Oreochromis niloticus exposed to lead nitrate was observed in the present investigation. In addition, other studies showed a reduction in hepatic glycogen following exposure to xenobiotic compounds (Gluth and Hanke, 1985, Braunbeck, 1992, Mekkawy et al., 2012) which can be explained by increased glycolytic activity to meet the energy demands imposed by enhanced metabolic activity (Hanke et al., 1983; Gluth and Hanke, 1985). Such activity was confirmed biochemically by Mekkawy et al. (2011b). Moreover, lead acetate intoxication stimulated the adrenals in rats (Wright et al., 1975) via inducing the release of adrenal catecholamines causing glycogenolysis which was also observed in the fish Anabas scandens (Chandravathy and Reddy, 1996). Thus it can be concluded that lead would disturb the carbohydrate metabolism. Other 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; Yuness, 2005) are associated with glycogen depletion; hence, hepatocyte glycogen is non-specific parameter indicating stress of the organism (Sylvie et al., 1996). On the other hand, long-term exposure to contaminants lead to accumulation of glycogen in the fish liver (Kranz and Peters, 1985).
Our results showed that the liver of Oreochromis niloticus exposed to lead nitrate, displayed an increase in the amount of hepatocellular lipid deposits. Fatty infiltration in the liver has been experimentally induced in cat, fish and Atlantic cod which was fed Polychlorinated Biphenyl’s (PCBs) (Hinton et al., 1978; Freeman et al., 1982). A similar increase in lipid droplets was also found in liver cells of Pseudopleuronects americanus after copper poisoning (Baker, 1969) and in Carassius auratus after lead exposure (Franchini et al., 1991). The increase in cytoplasmic lipids may be related to the hypoxic condition induced by lead as observed in other tissues (Fantin et al., 1985). In conclusion, the present study suggested that lead nitrate exerts some toxic effects of the liver of Oreochromis niloticus with regards to the histological changes. Under lead sublethal dose of 45 mg L-1, blood congestion in sinusoids, vaculation of hepatocytes and necroses in the liver of Oreochromis niloticus were observed (Lamchumchang et al., 2007). These authors and Kosai et al. (2011) reported that dietary Ca2+ will be protective in reducing Pb burdens in fish exposed to environments contaminated with waterborne Pb. In a similar way, Hussein and Mekkawy referred to the clay as protective agent in reducing Pb-toxicity. Mekkawy et al. (2011b, 2012) evaluate the dietary tomato paste and vitamin E as a protective agent against toxicity of cadmium.
CONCLUSION
Finally, one can conclude that lead toxicity on fishes could be evaluated by histological characteristics of liver. Such lead-induced histological changes are function of heavy metal doses, time of exposure and fish species. These pathologic alterations reflect the stress-induced biochemical and physiological changes. Further studies are required to evaluate the diet supplementary protective natural antioxidants in counteracting the lead adverse impacts on fishes.