**1. Introduction**

162 Zoology

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plants for oviposition by the tobacco hornworm, *Manduca sexta*. *Entomologia Experimentalis et Applicata*, Vol. 12, No. 5, (December 1969), pp. 504-508. ISSN 0013Endocrine-disrupting chemicals (EDCs) include synthetic and naturally occurring chemicals that affect the balance of normal functions in animals (Razia et al. 2006). It has been found that exposure to natural and synthetic estrogenic chemicals may adversely affect wildlife and human health (Colborn et al. 1993). In vitro exposures (Soto et al. 1992; Soto et al. 1994; Toomey et al. 1999) have confirmed the effects of EDCs on tissue structure and cellular processes. Nonylphenol ethoxylates (NPEs) are EDCs which are used globally in the production of plastics, pesticides, and cleaning products and are present in sewage effluents around the world (Talmage, 1994). It has been reported that NP is the most important degradation product of NPEs because of its enhanced resistance towards biodegradation, toxicity, ability to bioaccumulate in aquatic organisms, and estrogenicity (Ahel et al. 1994). NP is found in surface waters, aquatic sediments, and ground water (Bennie, 1999; Talmage, 1994) and it is estrogenic in various aquatic animals (Nimrod and Benson, 1996; Talmage, 1994; Servos, 1999).

The application of environmental toxicological studies on non-mammalian vertebrates is rapidly expanding; and for aquatic system, fish have become valuable indicator for the evaluation of the effects of noxious compounds (Khidr and Mekkawy, 2008). Histology and histopathology can be used as biomonitoring tools for health in toxicity studies (Meyers and Hendricks, 1985). Histoplathological alterations are biomarkers of effect exposure to environmental stressors, revealing alterations in physiological and biochemical function (Hinton et al. 1992). Histopathology, the study of lesions or abnormalities on cellular and tissue levels is useful tool for assessing the degree of pollution, particularly for sublethal and chronic effects (Bernet et al. 1999). More than one tissue may be studied for assessment of the biological effects of a toxicant on localized portions of certain organs and also for assessment of subsequent derangements (degradations) in tissues or cells in other locations and this allows for diagnoses of the observed changes (Adeyemo, 2008). NP has been shown

<sup>\*</sup> Corresponding Author

Histopathological Alterations in some Body Organs

experimental setup.

**2.2 4-nonylphenol** 

**preparations** 

**2.6 Ethical statement** 

**3. Results** 

**2.3 Experimental setup** 

of Adult *Clarias gariepinus* (Burchell, 1822) Exposed to 4-Nonylphenol 165

(conductivity 2000 ls/cm; pH 7.5; oxygen 88–95% saturation; temperature 27-28 ºC; photoperiod 12:12 light: dark). After 2 week acclimatization, fishes were used for the

The adapted adult fish classified into four groups (10 fish per each): control, 4-nonylphenoltreated group (for15 day/ for 0.05mg/l day), 4-nonylphenol-treated group (for 15 day/for 0.08mg/l day), and 4-nonylphenol-treated group (for 15 day/for 0.1 h/ day). In the present study, the range of NP exposures was 0.05-0.1 mg/l and the exposure concentrations are environmentally relevant. The conditions of the experiment were as that of acclimatization

For microscopic preparations, after 15 days, 3 surviving fish of each group were removed and dissected. Small pieces of the liver, kidneys, gills, and skin were taken and immediately fixed in 10% neutral buffered formalin. Fixed tissues were processed routinely for paraffin embedding technique. Embedded tissues were sectioned at 5-7μ in thickness and then stained with Harris' hematoxylin and eosin stain (H & E) and Masson's Trichrome (TRI) stain according to Bancroft and Steven, (1982). Sections were visualized and studied using

Small pieces of liver of newly scarified fish were fixed in 2 % glutaraldehyde, washed in cacodylate buffer and post-fixed in 1% osmium tetroxide. Dehydration was carried out in ascending grads of alcohol and then embedded in epon-araldite mixture. Semithin sections of liver were cut at 1µm and stained with toluidine blue for examination under a light microscope. Ultrathin sections were stained with uranyl acetate followed by lead nitrate (Johannessen, 1978). Electron micrographs were obtained using a Jeol JEM 1200 EX Transmission Electron Microscope at Electron microscope center of Assiut University.

All experiments were carried out in accordance with the Egyptian laws and University guidelines for the care of experimental animals. All procedures of the current experiment have been approved by the Committee of the Faculty of Science, Assiut University, Egypt.

Throughout the duration of the experiment, the number of fish that died was 1.2, 3.75, 5.5, and 8 % for control, 0.05, 0.08 and 0.1 mg/l 4-nonylphenol respectively. Lesions were observed in the gills, skin, kidneys, and liver of sampled fish for all 4-nonylphenol at all

4-Nonylphenol was obtained from Sigma- Aldrich (Schnelldrof, Germany)

with changing all the tap water and concentrations of 4-nonylphenol every day.

**2.4 Hematoxylin-Eosin (HE) and Masson's Trichrome (TRI) histopathological** 

OLYMPUS microscope model BX50F4 (Olympus optical Co., LTP. Japan).

**2.5 Transmission electron microscope (TEM)** 

to cause histopathological changes in the germ and Sertoli cells of the male eelpot (Christiansen et al. 1998). The skin of fish is continuously exposed to and in direct contact with the environment pollutants such as NP. Histological changes in skin of rainbow trout with mucosomes in goblet cells were recorded after exposure to 10 µg/l of 4-nonylphenol. Several studies demonstrated the high susceptibility of skin to environmental pollutant impacts (Burkhardt-Holm et al. 1997; Iger et al. 1995; Shephard, 1994). Burkhardt-Holm et al. (1997) hypothesized that in trout, xenobiotic estrogens might affect the skin, like natural estrogens, via the steroid receptor.

In trout species, nonylphenol was found to accumulate in the liver, gill, skin, gut, fat, and kidney tissue (Ahel et al. 1993; Coldham et al. 1998; Lewis and Lech, 1996). So that, 4 nonylphenol may affects those organs in corresponding with its impacts on reproductive ones. Most of NP studies revealed sever effects on the liver and gonads of fish tissues (Christiansen et al. 1998; Jobling et al. 1996; Lech et al. 1996) and the corresponding metabolism. The liver is important in many aspects of nutrition, including lipid and carbohydrates storage and alterations in liver structure may be useful as biomarker that indicate prior exposure to environmental stressors (Hinton and laurén, 1990). Stressorsassociated alterations of hepatocytes may be found in the nucleus or cytoplasm or both (Marchand et al. 2008). Malik and Hodgson, (2002) reported that the liver plays a major role in complex enzymatic processes of thyroid hormones conversion. So, liver dysfunction and disease affects thyroid hormone metabolism. Although gills are not only the prime organs for gaseous exchange, they perform several other physiological functions including osmoregulation and excretion. Parashar and Banerjee, (2002) reported that changes in environmental parameters often damage this delicate vital organ which has direct contact with aquatic environment. Many studies demonstrated that increased concentrations of different pollutants including several heavy metals seriously damage the gills of teleostean fish (Dutta et al. 1996; Wendelaar Bonga, 1997)).

African catfish (*Clarias gariepinus*), an omnivore freshwater fish, is a popular delicacy relished throughout tropical Africa (Nguyen and Janssen, 2002) due to fast growth rate, high stocking-density capacities, high consumer acceptability and high resistance to poor water quality and oxygen depletion (Adewolu et al. 2008; Akinwole and Faturoti, 2007; Karami et al. 2010). Because it is a prominent culture species (Adeyemo, 2008), the African catfish has been used in many fundamental experimental researches (Mahmoud et al., 2009).

The present work is an extension of previous studies of the present authors (Mekkawy et al., 2011; Mahmoud et al., 2011; Sayed et al., 2011) to determine to what extent the histopathological variations in some organs of the adult catfish, *Clarias gareipinus* (Burchell, 1822) are simultaneously correlated with biochemical and physiological NP-induced changes especially in respect with endocrine disruption.
