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Thehepatotoxiceffectsof4-nonylphenolonAfricancatfish(Clariasgarepinus):Physiologicalandhistologicalstudy

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The hepatotoxic effects of 4-nonylphenol on African catfish (Clarias garepinus):

Physiological and histological study

Nasser Sayed Abou Khalil

Medical Physiology Department, Faculty of Medicine, Assiut University, Assiut, Egypt.

Mahmoud Abd-Elkareem

Anatomy, Histology and Embryology Department, Faculty of Veterinary Medicine, Assiut University, Assiut,

Egypt.

Abstract 4- Nonylphenol (NP) toxicity in fish attracts much attention due to its ability in targeting several

organs; however, the researches regarding its potential hepatotoxicity are conflict and still require further

investigation. Therefore, the objective of this study is to focus on this issue from the histo-physiological point of

view using NP intoxicated African catfish (Clarias garepinus) as a model of hepatotoxicity. 12 adult fish (6 per

group) were divided into two groups; the first was considered as control and the second was exposed to NP

dissolved in water at a dose of 0.1 mg/kg BW for 3 weeks. A significant reduction in the hepatic alanine

aminotransferase, asparatate aminotransferase and lactate dehydrogenase levels was observed in NP exposed

fish. Concerning the oxidant/antioxidant balance, a significant depletion in superoxide dismutase, catalase and

glutathione peroxidase was found along with a significant elevation in total peroxide and malondialdhyde. The

histopathological examination of the liver tissue revealed that NP had marked hepatotoxic effects including

hepatitis, centrilobular and focal hydropic and fatty degeneration, fatty change (steatosis), apoptosis of

hepatocytes and necrosis of endothelial cells, hepatic coagulative necrosis, and nuclear alterations. Depletion of

the glycogen and increased in pigments (lipofuscin and hemosiderin) content in the hepatocytes were also

recorded. Hemosiderosis and proliferation of the connective tissue around the blood vessels, branches of bile

ducts and in the portal areas were also observed. In the light of these findings, it was concluded that NP has a

well defined hepatotoxic impact in Clarias Gariepinus paving the road towards other studies investigate other

detrimental cyto-physiological influences of this aquatic pollutant.

1. Introduction The causative relationship between endocrine disrupting substance exposure and fish health burden

remains a topic of researches for many years, and represents a scientifically attractive area for investigators due

to gradual rise in ecosystem poisoning risks. The non-biodegradable nature, prolonged persistence in the

environment, and biomagnification through the food chain (Kourouma et al., 2015; Rivero et al., 2008) direct

much attention towards 4-nonylphenol (NP) as a major global hazardous aquatic pollutant. NP is a product

derived from alkylphenol ethoxylates which are used to produce non-ionic surfactants, detergents, emulsifiers

for agrochemicals, antioxidants for rubber manufacture and as lubricant oil additives (Vazquez-duhalt et al.,

2005).

African catfish (Clarias gariepinus) is one of the most important cultured fish in the tropical and

subtropical environments owing to its capacity in satisfaction of aquaculturist and consumer needs, and is

considered a promising candidate model for toxicological studies and monitoring pollutants released in the

aquatic environment (Karami et al., 2016; Shoko et al., 2016).

NP is classified as a highly hepatotoxic substance by inducing adverse changes in the hepatic histoarchitecture

and its metabolizing enzyme activities (Abdulla Bin-Dohaish, 2012; Bhattacharya et al., 2008) most probably

due to upregulation of apoptosis and oxidative stress-related genes, increase in reactive oxygen species

generation and lipid peroxidation, and decrease in antioxidant activities (Kourouma et al., 2015).

Several categories of hepatocellular pathologies are regarded as reliable biomarkers of toxic injury and are

representative of biological endpoints of pollutant exposure (Kumar et al., 2016; Ramalingam and Rajaram,

2016). The mission of this study is to investigate the potential hepatotoxic effect of NP in Clarias garepinus.

2. Materials and methods

2.1. Fish Adult Clarias gareipines were acclimated in aerated recirculating tank containing experimental media

water for two months. Fish were fed with a commercial fish food twice daily, and kept at approximately 28º C

with 12 hr/12 hr light/dark cycle. During the acclimatization period, 20% of the water in each recirculating

system was replaced daily, and fish were fed 5% body weight twice daily with commercial pellets. This

experimental study was done in Fish Lab, Zoology Department, Faculty of Science, Assiut University, Assiut,

Egypt.

2.2. 4-Nonylphenol NP was obtained from Sigma-Aldrich (Schnelldrof, Germany) with purity 99.3%.

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2.3. Experimental design Prior to the experiments, the fishes were determined to be free of external parasites (AFS-FHS, 2003).

To examine the potential hepatotoxic effect of NP, two groups of adult fishes (six per group) were maintained in

100 L glass aquaria; one group was served as control, the second was exposed to NP dissolved in water at a dose

of 0.1 mg/kg BW (Mekkawy et al. 2011; Sayed et al., 2016). During the whole experimental period which

extended for 3 weeks, fish were fed 5% BW twice daily with commercial pellets, and the water was changed

daily.

2.4. Processing of hepatic tissue 1% (w/v) of liver homogenate was prepared in phosphate buffer saline (pH 7.4) with the help of a motor-driven

glass Teflon homogenizer on crushed ice for a minute. The homogenate was centrifuged at 3000 rpm for 15 min

to obtain the supernatant, which is stored at -20 ºC until analysis later on.

2.5. Biochemical measurement Superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) were assayed using

commercially available colorimetric kits according to the manufacturer's protocol (Biodiagnostic, Egypt).

Briefly, SOD was measured based on its ability to inhibit the phenazine methosulphate-mediated reduction of

nitroblue tetrazolium dye to form a red product (Nishikimi et al., 1972). The oxidation of NADPH to NADP+ is

accompanied by a decrease in absorbance at 340 nm providing spectrophotometric means for monitoring GSH-

Px levels (Paglia and Valentine, 1967). CAT was determined based on the fact that 3,5-dichloro -2-

hydroxybenzene sulfonic acid could rapidly terminate the degradation reaction of hydrogen peroxide catalyzed

by CAT and react with the residual hydrogen peroxide to generate a yellow product (Aebi, 1984). Total

peroxide (TPX) was assessed following the procedure of (Harma and Erel, 2005), and calculated from the

standard curve constructed using standard concentrations. Malondialdhyde (MDA) was measured by the

thiobarbituric acid reaction (Ohkawa et al., 1979). Alanine aminotransferase (ALT) and asparatate

aminotransferase (AST) were measured colorimetrically by commercially available kits (Egyptian Company for

Biotechnology, Egypt). Lactate dehydrogenase (LDH) was estimated by colorimetric kit (Stanbio Laboratory,

USA). Total protein was assayed using biuret reagent (Gornall et al., 1949).

2.6. Histological preparation: A-Paraffin sections: Liver were collected from fish and fixed in 10% neutral buffered formalin or Bouin's fluid.

The fixed samples were dehydrated in ascending grades of ethanol, cleared in methyl benzoate and then

embedded in paraffin wax. Paraffin sections at 3-7 µm in thickness were cut and stained with the following

histological stains:-

1- Haematoxylin and Eosin for general histological examination (Harris, 1990).

2- Crossmon's trichrome technique for differentiation of connective tissue and muscle fibers (Crossmon, 1937).

3- Periodic acid Schiff (PAS) technique for demonstration of neutral mucopolysaccharides (McManus, 1946).

4- Perls’ Prussian blue reaction for ferric iron (Stevens, 1986).

5- Nile blue stain for detection of lipofuscin and melanin (Stevens, 1986).

6- Sudan black B for detection of lipofuscin (Stevens, 1986).

B- Semithin sections:

Tissue blocks from the liver of catfish were taken, fixed in 5% gluteraldehyde in cacodylate buffer, washed

several times in phosphate buffer 0.1M, PH 7.3, dehydrated in ascending grades of ethanol, embedded in epoxy

resin of low viscosity (ERL, 4206) as stated by Spurr (1969) and then semithin sections were cut and stained

with toluidine blue.

Paraffin sections and semithin sections were examined by OLYMPUS BX51 microscope and the photos were

taken by OLYMPUS DP72 camera adapted into the microscope.

2.7. Statistical analysis The data were expressed as means ± standard deviation (SD). Statistical differences between groups were

analyzed by independent sample T-test using SPSS program version 16 (SPSS, Richmond, VA, USA).

Differences were considered statistically significant at P < 0.05.

3. Results

3.1. Effect of NP toxicity on the physiological endpoint markers in the liver of

Clarias gareipines As shown in table (1), NP exposed fish were characterized with a significant reduction in the hepatic AST, ALT,

and LDH levels. Marked disturbance in the oxidant/antioxidant profile following NP toxicity was evident by

significant depletion in SOD, CAT, and GSH-Px along with significant elevation in TPX and MDA.

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Table 1: Effects of 4-nonylphenol (NP) on the hepatic enzymes and oxidant/antioxidant parameters of Clarias

garepinus.

Group

Parameter

Control NP P value

ALT (U/mg protein) 23.453 ± 6.719

10.435 ± 2.058

0.000

AST (U/mg protein) 27.225 ± 11.276

8.682 ± 2.166

0.000

LDH (U/mg protein) 63.680 ± 8.601

37.732 ± 2.978

0.000

SOD (U/mg protein) 183.186 ± 11.355

132.568 ± 17.120

0.000

CAT (U/mg protein) 751.688 ± 83.602

606.851 ± 94.397

0.002

GSH-PX (mU/mg protein) 2.075 ± 0.399

1.143 ± 0.138

0.000

TPX (μmol/mg protein) 2499.894 ± 122.542

3292.176 ± 295.808

0.000

MDA (nmol/mg protein) 25.305 ± 8.189

42.669 ± 7.686

0.006

Results are expressed as means ± SD of 6 fish per group.

Statistical differences between groups were analyzed by independent sample T-test.

ALT, alanine aminotransferase; AST, asparatate aminotransferase; LDH, lactate dehydrogenase; SOD;

superoxide dismutase; CAT, catalase; GSH-PX, glutathione peroxidase; TPX, total peroxide; MDA,

malondialdhyde.

3.2. Effect of NP toxicity on the histological endpoint markers in the liver of

Clarias gareipines The histological examination of the liver of the control Clarias Gariepinus revealed that the parenchyma of the

liver was formed of a continuous compact field of branching and anastomosing two cell thick laminae or plates

of hepatocytes. These hepatic plates were separated from each other by hepatic sinusoid. The hepatic sinusoids

were lined with endothelial cells and phagocytic kupffer cells. Hepatocytes were appeared as polyhedral cells

with vacuolated acidophilic cytoplasm contained PAS positive glycogen granules and vesicular rounded central

or eccentric nuclei. There was a far less tendency of the hepatocytes to form distinct lobules as there was scanty

or no amount of connective tissue. The portal areas and veins were scattered through the liver parenchyma

without a well-defined arrangement, and they were surrounded by hepatic parenchyma. The portal areas or

portal triads were appeared as triangular areas of loose connective tissue containing a branch of portal vein,

hepatic artery and bile ductule. (Fig.1).

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Fig. (1): photomicrograph of paraffin sections in the liver of control Clarias Gariepinus illustrating the normal

architecture of the liver. a:Showing central vein (CV), hepatocytes (H), different profiles in venules (V) and the

portal area which appeared as triangular area of loose connective tissue(CT) containing a branch of portal vein

(V), hepatic artery (A), bile ductule (BD) and melanomacrophage centers (MMC).b: Showing hepatocytes (H),

melanomacrophage centers (MMC)and venules (V). Note that there was a far less tendency of the hepatocytes to

form distinct lobules as there was scanty or no amount of connective tissue.c: Showing the hepatic plates (HP)

radiating from central vein (CV) and separated by hepatic sinusoid (arrow).d: Showing the hepatic plates (HP)

composed of branching and anastomosing two cell thick laminae or cords of hepatocytes (H) and separated by

hepatic (blood) sinusoid (BS) which lined by endothelial cells (E) and kupffer cells (K). Note that, the

hepatocytes (H) were polyhedral cells with vacuolated acidophilic cytoplasm and vesicular rounded central and

eccentric nucleus (N).Original magnification, (a& b): 100X, scale bar = 200 µm; c: 400X, scale bar = 50; d:

1000X, scale bar = 20, Hematoxylin and Eosin stain.

The histopathological examination of the liver tissues of intoxicated Clarias Gariepinus revealed that NP had a

hepatotoxic effects represented by hepatitis, centrilobular and focal hydropic and fatty degeneration, fatty

change (steatosis), increased apoptosis of hepatocytes and endothelial cells, hepatic coagulative necrosis,

nuclear alterations, depletion of glycogen in the hepatocytes and increased in pigment (lipofuscin and

hemosiderin) content. Hepatitis was characterized by infiltration of mononuclear leukocytes and congestion in

the blood vessels (Fig. 2 & 3). The centrilobular hydropic degeneration was appeared as swelled lightly stained

hepatocytes surrounding the central vein (Fig. 4).One of the most common hepatotoxic effects of NP intoxicated

Clarias Gariepinus was the fatty degeneration and fatty change (steatosis). There were two types of steatosis,

the first was the microvesicular steatosis which characterized by presence of several small intracytoplasmic fat

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droplets (vacuoles) in the hepatocytes while the second was the marovesicular steatosis which characterized by

presence of large intracytoplasmic lipid vacuole occupying most of the hepatocyte (Fig. 5). The nuclear

alterations in the hepatocytes were including: pyknosis, irregular contour, margination,shrinkage, loss of

chromatin and its basophilia and the absence of the nucleus (Fig. 4 & 5). The apoptotic hepatocytes were

appeared as a round or oval mass with dark eosinophilic cytoplasm and dense deeply stained eccentric nucleus

(Fig. 6a & 5c). Some hepatocytes showed coagulative necrosis and other had pathological pigmentation (Fig. 3

& 6). The hepatic cords were disorganized and degenerated and the hepatocytes had ill clear cell bounders (Fig.

4d & 5). Some blood vessels in the liver of NP intoxicated Clarias Gariepinus were hyalinized and degenerated

and had necrotic endothelial cells (Fig. 6b). Staining of the hepatic tissues with the histochemical stains: PAS,

Nile blue and Suadan black B revealed that the hepatocytes of NP intoxicated Clarias garepinus had numerous,

coarse, PAS positive (magenta or red), Nile blue positive (dark blue) and sudanophilic (black) lipofuscin and /or

ceroid granules (Fig.7 &8). There was depletion in the PAS positive glycogen granules in the hepatocytes of NP

intoxicated Clarias garepinus (Fig. 7). The hepatic sinusoids of NP intoxicated Clarias Gariepinus showed

increased number of kupffer cells (Fig. 4). The hepatocytes of NP intoxicated Clarias garepinus showed

hemosiderosis (Fig. 9). Liver of NP intoxicated Clarias Gariepinus showed increased in the amount of

connective tissue (connective tissue proliferation) around the blood vessels, branches of bile ducts and in the

portal areas (Fig.10).

Fig. (2): Photomicrograph of paraffin sections illustrating the hepatotoxic effects of NP on Clarias garepinus. a:

Showing focal fatty degeneration (F), congestion in blood vessels (C) and increased in the number of

melanomacrophage centers (arrow). b: Showing central vein (CV), melanomacrophage centers (MMC),

centrolobular fatty degeneration (F) and coagulative necrosis (CN). c: Showing central vein (CV) surrounded by

inflammatory leucocytic infiltration (arrow), melanomacrophage centers (MMC), fatty degeneration or change

(F) and congestion in blood vessels (C) d: Showing melanomacrophage centers (MMC), fatty degeneration (F),

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coagulative necrosis (CN) and inflammatory leucocytic infiltration (arrow). Original magnification, a: 40X,

scale bar = 500 µm; b: 100X, scale bar = 200; c: 200X, scale bar = 100 and d: 200X, scale bar = 100.

Hematoxylin and Eosin stain.

Fig. (3): Photomicrograph of paraffin sections illustrating the hepatotoxic effects of NP on Clarias garepinus.

a: Showing congested blood vessels (C), inflammatory leucocytic infiltrations (L) and melanomacrophage

center (MMC). b: Showing congested blood vessels (C), inflammatory leucocytic infiltrations near the

melanomacrophage center (MMC) and coagulative necrosis (CN). Original magnification (a & b) 200X, scale

bar = 100 µm, Hematoxylin and Eosin stain.

Fig. (4): Photomicrograph of semithin sections illustrating the hepatotoxic effects of NP on Clarias garepinus.

a: Showing central vein (CV), centrilobular hydropic degeneration which appeared as swelled lightly stained

hepatocytes (LH), hepatic plates (HP) and healthy hepatocytes (H). b: (Higher magnification of Fig. a) Showing

central vein (CV), kupffer cells (K), lightly stained hepatocytes with hydropic degeneration (LH) and pale

stained nucleus (N), dark stained hepatocytes (DH) with nuclear alteration (arrow head) and intracytoplasmic

lipofuscin pigments (arrow). c: (Higher magnification of Fig. a) Showing central vein (CV) connected with

blood sinusoid (BS) which lined by endothelial cells (E) and kupffer cells (K), lightly stained hepatocytes with

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hydropic degeneration (LH) and pale stained nucleus (N), dark stained hepatocytes (DH) with intracytoplasmic

lipofuscin pigments (arrow). d: Showing hepatocytes with ill clear cell bounders (H), intracytoplasmic

lipofuscin pigments (arrow) and elongated group of deformed red blood corpuscles (DRBC) and macrophages

(M). Original magnification, a: 200X and sale bar = 100 & (b, c and d): 1000X, scale bar = 20 µm, Toluidine

Blue stain.

Fig. (5): Photomicrograph of paraffin sections illustrating the hepatotoxic effects of NP on Clarias garepinus.

a: Showing disorganization of hepatic plates (DHP), necrotic hepatocytes (NH) and fatty degeneration (arrow)

with nuclear alteration (N). b: Showing microvesicular steatosis (MiVS), macrovesicular steatosis (M) with

nuclear alteration; the nucleus became small irregular and eccentric (N), apoptotic hepatocytes (arrow head)

and congested blood vessel with swelled blood cells (RBS). c: Showing microvesicular steatosis (MiVS),

macrovesicular steatosis (M) with nuclear alteration (N), apoptotic hepatocytes (arrow head) and lipofuscin

pigments (arrow). d: Showing disorganization of hepatic plates (DHP), congested central vein (CCV) and

melanomacrophage center (arrow) near the central vein. Original magnification (a, b, c and d) 400X, scale bar =

50µm, Hematoxylin and Eosin stain.

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Fig. (6): Photomicrograph of paraffin sections illustrating the hepatotoxic effects of NP on Clarias garepinus.

a: Showing apoptotic hepatocyte (arrow head) and melanomacrophage centers (MMC) which were formed of

aggregates of macrophages (arrow) filled with pigments (brown colour). b: Showing hyalinized blood vessels

(HBV), endothelial cells of blood vessels (arrow head), degenerated blood vessel (DBV), necrotic endothelial

cells of blood vessels (arrow) and hepatocyte filled with lipofuscin pigments (forked tail arrow). Original

magnification (a & b) 400X, scale bar = 50 µm, Hematoxylin and Eosin stain.

Fig. (7): Photomicrograph of paraffin sections in the liver of Clarias Gariepinus. a: Liver of control Clarias

Gariepinus showing, central vein (CV) connected with blood sinusoid (BS) which lined by endothelial cells (E)

and kupffer cells (K), hepatocytes (H) with few coarse PAS positive lipofuscin granules (arrow head) and many

fine PAS positive glycogen granules around the nucleus (N). b: Liver of NP intoxicated Clarias Gariepinus,

showing an increase in the amount and size of the coarse PAS positive lipofuscin granules (arrow) and

depletion in the PAS positive glycogen granules. Note the hepatocytes with macrovesicular steatosis (fat

change) characterized by presence of large intracytoplasmic lipid vacuole (LV) occupying most of the

hepatocyte (H) and nuclear alteration (N). Original magnification (a & b) 1000X, scale bar = 20 µm, Periodic

Acid Schiff reagent (PAS) and Haematoxylin stain.

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Fig. (8): Photomicrograph of paraffin sections in the liver of Clarias Gariepinus. a: Liver of control Clarias

Gariepinus showing, Nile blue positive melanomacrophage centers (MMC) adjacent to the central vein (CV)

and hepatocytes (H) with few or no Nile blue positive granules. b: Liver of NP intoxicated Clarias Gariepinus,

showing an increase in the amount and size of Nile blue positive melanomacrophage centers (MMC) and

hepatocytes with numerus Nile blue positive lipofuscin granules (arrow). c: Liver of control Clarias

Gariepinus, showing Suadan black B & Hematoxylin positive melanomacrophage centers (MMC) and

hepatocytes (H) with few sudanophilic granules (arrow). d: Liver of NP intoxicated Clarias Gariepinus,

showing an increase in the amount and size of Sudan black positive melanomacrophage centers (MMC) near

the central vein (CV) and hepatocytes with numerus sudanophilic granules (arrow). Original magnification (a &

b) 200X, scale bar = 100 µm, Nile blue stain & (c & d) 1000X, scale bar = 20 µm, Suadan black B and

Hematoxylin stain.

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Fig. (9): Photomicrograph of paraffin sections in the liver of NP intoxicated Clarias garepinus stained by Perls’

histochemical technique showing hemosiderosis; large numerous Prussian blue hemosiderin pigments filling

the hepatocytes (arrow). Original magnification 200X, scale bar = 100 µm, Perls’ Prussian blue reaction.

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Fig. (10): Photomicrograph of paraffin sections in the liver of Clarias Gariepinus. (a & b): Liver of control

Clarias Gariepinus showing, central vein (CV), hepatic plates (HP) and melanomacrophage centers (MMC), a

branch of hepatic artery (A) and portal vein (V). Note the scanty amount of connective tissue around the blood

vessels (arrow). (c & d): Liver of NP intoxicated Clarias Gariepinus showing,fatty degeneration (F),

melanomacrophage centers (MMC) and an increase in the amount of connective tissue (CT) around the blood

vessels, branches of bile ducts (BD) and in the portal areas (PA). Original magnification (a & b):200X, scale

bar = 100 µm;c: 100X, scale bar = 200; d: 400X, scale bar = 50,Crossmon's trichrome stain.

4. Discussion According to the findings of this study, NP induced a profound hepatotoxicity in Clarias garepinus as reflected

on the physiological and histological characters of hepatocyte. These outcome responses could be attributed to

its free radical generating capacity as manifested by shift of oxidant/antioxidant balance towards the oxidant

side. These observations are of outmost significance from the ecotoxicological point of view, and pave the road

towards further studies look for the potential underlying molecular mechanistic pathways in this area of

research.

The clear depletion of hepatic AST, ALT, and LDH levels following NP exposure may be related to the ability

of NP to interact with the cell membranes directly leading to cytolysis with subsequent leakage of these

enzymes into blood stream (Bhattacharya et al., 2008; Cserháti, 1995).As indicated in this study, the obvious

increase in MDA under NP stress reflecting peroxidation of polyunsaturated fatty acids, which construct an

essential building block in the cell membrane, resulting in disruption of membrane fluidity and cell

compartmentalization culminating in cell damage (Stark, 2005). Other factor contributes to explanation of

reduction in the liver function enzymes is the hepatic degeneration and necrosis which are observed on the

histo-pathological examination in this study.

SOD provides the first line of defense against oxygen free radicals by catalyzing the removal of superoxide

radical (O2-) through dismutation and generates hydrogen peroxide, which is consecutively reduced by the

activities of CAT and GSH-PX (Shairibha and Rajadurai, 2014). According to the results of the present study,

NP induced a noticeable reduction in the enzymatic antioxidant levels in the same line with (Kourouma et al.,

2015), possibly attributable to consumption of cellular antioxidant reserve by MDA and TPX, and upregulation

of genes related to reactive oxygen and nitrogen species production, signifying disruption in redox cycling

processes and reflecting inability of the cells to eliminate O2- creating a state of oxidative stress (Matozzo et al.,

2004; Xu et al., 2013). However, conflict data arise from literature depending on the studied species, analyzed

tissue, and exposure duration/concentration (Park, 2015; Wu et al., 2011).

NP intoxicated fish in this study showed a marked increase in the hepatic MDA level in corroboration with the

findings of (Midhila and Chitra, (2015); Park, (2015). This may be attributed to accumulation of fatty acids in

the hepatic structures leading to fatty acid inhibition of ATPase-dependent proton extrusion, binding of tissue

metabolites to cytochromes, and uncoupling of the electron transport chain from monooxygenase activity with

consequent generation of reactive oxygen species which attack unsaturated lipid results in formation of MDA

(Okai et al., 2000; Wu et al., 2011; Ayala et al., 2014; Kourouma et al., 2015) .

The histopathological examination of the hepatic tissues of NP challenged Clarias Gariepinus revealed that NP

had a hepatotoxic effects as manifested by hepatitis, vacuolated cytoplasm, centrilobular and focal hydropic and

fatty degeneration, fatty change (steatosis), increased apoptosis of hepatocytes and endothelial cells, hepatic

coagulative necrosis, nuclear alterations, pigment degeneration, depletion of glycogen in the hepatocytes and

increased pigment (lipofuscin and hemosiderin) content on the same line with previous studies (Khidr et al.,

2012; Van Dyk et al., 2012). Hepatic vacuolated cytoplasm could be attributed to interference with fatty acid

metabolism leading to accumulation of neutral fat that is dissolved by organic solvents during tissue

preparation, leaving unstained empty vacuoles. Fatty infiltration can accompany hydropic degeneration, which

appears as faintly stained vacuoles (Abdulla Bin-Dohaish, 2012). Increased apoptosis may result from

generation of reactive oxygen species and subsequent occurrence of oxidative stress and lipid peroxidation as

evident from the findings of this study and that of Kaptaner and Ünal (2011) and from up-regulation of genes

implicated in apoptosis (Kourouma et al., 2015). Modulation of insulin signaling and enzymes related to

carbohydrate metabolism may be involved as causative factors in the depletion of hepatic glycogen under NP

stress (Jubendradass et al., 2012). Hepatic necrosis could arise from the ability of NP to inhibit calcium pump in

the endoplasmic reticulum (Uguz et al., 2003) as sustained elevations in intracellular calcium concentration

activate several cell death pathways.

The present study indicated that, hepatocytes of NP intoxicated Clarias garepinus had numerous,

coarse, PAS positive (magenta or red), Nile blue positive (dark blue) and sudanophilic (black) lipofuscin and or

ceroid granules. Lipofuscin is a membrane-bound cellular waste formed of non-degradable aggregates of

oxidized proteins, lipids and metals which accumulates inside the lysosomes of cells that do not replicate.

Lipofuscin is insoluble and not degradable by lysosomal enzymes or the proteasomal system nor ejected from

the cell (Jung et al., 2007). The major source of lipofuscin is incomplete lysosomal degradation of damaged

mitochondria (Gray and Woulfe, 2005). Lipofuscin formation appears to depend on the rate of oxidative damage

12

to proteins, the functionality of mitochondrial repair systems, the proteasomal system, and the functionality and

effectiveness of the lysosomes (Jung et al., 2007). It was a yellow-brown material (with H & E) accumulated in

the cytoplasm of hepatocytes of NP intoxicated Clarias Gariepinus. Sudan black B was used as a biomarker to

detect stress-induced cellular senescence in a formalin-fixed paraffin-embedded tissues by detecting lipofuscin

pigments (Georgakopoulou et al., 2013). Stress induced premature senescence is an acute phenomenon in which

the cells stop to proliferate under various stressful conditions (Sikora et al., 2011).

In conclusion, hepatotoxicity is a well marked sign in the NP poisoned Clarias gareipins resulting in depletion

of enzymatic antioxidants increase in free radical-associated peroxidation, and deterioration in the cytological

features of hepatocytes. This study leaves the door open in front of investigating other negative aspects of fish

exposure to this widely spread aquatic contaminant.

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