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Evaluation of the hepatotoxicity of thiobencarb herbicide in albino rats

Ferial A. El-messady

Zoology, Department, Faculty of Science,Menoufia University, Egypt

E.mail. [email protected]

Abstract

Thiobencarb is a thiocarbamate herbicide extensively used for weed control in paddy fields. The present work studied the

hepatotoxic effect of thiobencarb using histological, histochemical and immunohistochemical methods. Two groups of

male albino rats were used. G1: These animals (10 rats) were served as normal controls, G 2: This group (15 rats) was

administrated with thiobencarb at dose level (44.78 mg/kg/ orally / 3 days a week (equivalent to 1/20 of LD50) for 6

weeks. Treating rats with thiobencarb induced congestion of blood vessels, leucocytic infiltration, cytoplasmic

vacuolation of the hepatocytes and fatty infiltrations. Histochemical results revealed reduction of carbohydrates and total

proteins. Moreover, the hepatocytes of thiobencarb-treated rats showed increase expression of PCNA and caspase-3.It is

concluded from these results that the hepatotoxicity induced by thiobencarb could be possibly explained by its direct

cytotoxic effect and/or indirectly via the increased level of ROS and apoptosis-mediated genesin liver of rats. .

Keywords: Thiobencarb, Hepatotoxicity, Carbohydrates, Proteins, PCNA, Caspase-3

Introduction

Accidental exposure with pesticides in human

and animals result from advertent use. Populations at

highest risk of high dose exposure are producers,

hygienic and pesticide workers. The toxicity of

pesticides to mammals has received much attention in

recent years because animals exposed to these

insecticides showed changes in their physiological

activities besides other pathological features [1].The use

of herbicides to control weeds has been recognized as a

part of agricultural practices throughout the world.

Unfortunately, the indiscriminate use of these

herbicides to improve agricultural production and yield

may have impacts on non-target organisms, especially

aquatic life forms and their environment[2].The use of

herbicides can cause deleterious effects on organisms

and human health including neurotoxicity , lung damage,

birth defects, cancer, immunomodulation, disruption of

reproductive functions and histopathological alterations

in vital organs [3].

Thiobencarb S-4-chlorobenzyl diethyl

(thiocarbamate), a thiocarbamate herbicide, is

extensively used for weed control in paddy fields in

many countries. It is used as a weed killer in rice

cultivation in Egypt and other countries. The half-life of

thiobencarb in water is 12 days under aerobic conditions

and more than 40 days under anaerobic conditions at

200C [4]; in soil, it is 2–3 weeks under aerobic

conditions and 6–8 months under anaerobic conditions

[5]. Thiobencarb was reported to inhibit the synthesis of

fatty acids and proteins through an antagonistic effect

on auxins in higher plants[6]. It also inhibited the growth

of algae [7] and cyanobacteria [8]. Thiobencarb was

found to have deleterious effects on fish. Patrick et al.

Journal of Advanced Trends in

Basic and Applied Science Vol.1, No.2:198-207, 2017

(Print ISSN: 2537-0537, Online ISSN:2537-0618)

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JATBAS,Vol.1,No.2: 198-207,2017 p ISSN:2537-0537,e ISSN:2537-0618 199

[9] reported that thiobencarb was toxic in early life

stages of atherinid fishes, Leuresthes tenais,Menidia

menidia and Menidia peninsulae. Exposing grass carp

(Ctenopharyngodan idella) to thiobencarb induced

histopathological alterations in gills, muscle and liver

[10]. Thiobencarb was found to induce significant

inhibitory effects on plasma AChE activity on European

Eel [11]. Concerning the effect on mammals, it caused

decreased body weight gains, food consumption and

food efficiency, as well as increased blood urea nitrogen

in rats [12]. Srinivas et al.[13] reported that

Thiobencarb affected AChE and ATPase activities in

neonate and adult rat brains. The aim of the present

study was to assess the toxicity of thiobencarb on the

liver of albino rats.

Material and Methods

Animals and treatments

Adult male albino rats (Rattus norvegicus)

weighing 130 ± 10 g were used. They were obtained

from the breeding center of experimental animals,

Helwan, Egypt. Animals were kept in the laboratory

under constant temperature (24±2°C) for at least one

week before and throughout the experimental work.

They were maintained on a standard diet composed of

55% corn starch, 20% casein, 15% corn oil, 5% salt

mixture and 5% vitaminzed starch (Egyptian Company

of Oils and Soap Kafr-El Zayat, Egypt). Water was

available ad libitum. All the experiments were done in

compliance with the guide for the care and use of

laboratory animals approved by Faculty of Science,

Menoufia University.Animals were divided into two

groups:

Group 1: These animals (10 rats) were served as normal

controls

Group 2: This group included 15 rats and was

administrated with thiobencarb at dose level (44.78

mg/kg/ orally / 3 days a week (equivalent to 1/20 of

LD50 , for 6 weeks) [14 ].

Histological and histochemical investigation

For histological study, animals were sacrificed

after 6 weeks, liver specimens were immediately

removed and fixed in 10% neutral formalin for 24

hours. After fixation, specimens were dehydrated in

ascending series of ethyl alcohol, cleared into two

changes of xylene, infiltrated in three changes of molten

paraffin wax with melting point of 58- 60 oC and then

embedded in molten paraffin blocks. Sections of 5

microns thickness were cut by using rotary microtome

and mounted on clean slides. Sections were stained with

Ehrlich's haematoxylin and counter stained with Eosin,

and examined under light microscope. For histochemical

demonstration of total carbohydrates periodic acid

Schiff’s technique (PAS) was used [15]. Total proteins

were detected using the mercury bromophenol blue

method [16].

Immunohistochemical investigation

For immunohistochemical localization of PCNA,

and caspase3 fixed wax sections were stained using the

avidin-biotin peroxidase method [17].Formalin-fixed

paraffin-embedded tissue sections were deparaffinized,

endogenous peroxidase activity was blocked with H2O2

in methanol and the sections were heated in 0.01 mol/l

citrate buffer in a microwave pressure cooker for 20

minutes. The slides were allowed to cool to room

temperature, and nonspecific binding was blocked with

normal horse serum for 20 minutes at room temperature.

The MIB-1 monoclonal antibody was used for detection

of nuclear PCNA, a marker of proliferating cells (1:40,

code No. M7187, Dako, Cambridge, UK). Anti-caspase-

3 (Dako) monoclonal antibodies were used for detection

of caspase-3. Counterstaining was performed using

Mayer's hematoxylin (Cat. No. 94585, BioGenex,

Menarini Diagnostics, Antony, France).

Image analysis

Digital images were analyzed by a semi-

quantitative scoring system (Image J software, Java

based application for analyzing images). The

immunohistochemical stained sections were analyzed in

10 microscopic fields under high-power field (×400)

microscope. In each field, percentage of positive stained

area was calculated as mean of 10 fields / slide.

Statistical analysis

The data were expressed as mean ± standard error.

Data were analyzed using Student’s t-test and

homogeneity of variances (Levene test) using statistical

program of social science (SPSS) software for windows.

P < 0.05 value was used.

Results

Histopathology

Examination of the liver of control rats showed

the typical features of normal hepatic architecture (Fig.1


A).Examination of Liver sections of rats treated with

thiobencarb for six weeks showed dilated and congested

central and portal veins Fig.1 B,C) and bile ductular

proliferation (Fig.1D. Leukocytic infiltration was

evident (Fig.2A). Cytoplasmic vacuolation was appeared

in the majority of hepatocytes with pyknotic nuclei

(Fig.2B). Fatty infiltration of different fat droplets was

noticed in the cytoplasm of the hepatocytes (Fig.2C).

Histochemical observations

Examination of liver section of a control rat

showed that the hepatocytes contain pink granules of a

strong PAS reaction in the pole of cytoplasm of

hepatocytes (glycogen flight) while nuclei are exhibited

negative stain (Fig.3A). Examination of liver of rats

treated with thiobencarb for 6 weeks showed a

depletion of the carbohydrates content (Fig. 3B).

Liver of a control rat showed normal protein

content in the hepatocytes as dense blue granules in

cytoplasm, cell membrane, nuclear membrane,

chromatin bodies, nucleoli and Kupffer cells (Fig. 4A).

Liver sections of rats treated with thiobencarb for 6

weeks showed a marked reduction of the protein

contentin the cytoplasm, while the nuclei were pyknotic

(Fig. 4B)

Immunohistochemical results

The expression of PCNA appeared in a few

nuclei of the hepatocytes of control rats (Fig. 5A). After

six weeks of treatment with thiobencarb, rats showed a

strong expression of PCNA in many nuclei of the

hepatocytes (Fig.5B). The results in table 1 showed the

area percentage of PCNA positive nuclei of the

hepatocytes in the control and treated groups. The area

percentage of PCNA positive nuclei of the hepatocytes

showed highly significant elevation (P˂0.05) in rats

treated with thiobencarb for six compared with control

group.

Caspase-3 was expressed in cytoplasm of few

hepatic cells as brown color (Fig.6A). After six weeks of

treatment with thiobencarb, an increase in expression of

caspase-3 immunoreactivity was observed in the

cytoplasm of large number of hepatocytes (Fig.6B).

The Data in table 1 showed that the percentage area of

caspase-3 expression in cytoplasm of hepatocytes

showed a significant increase (P<0.05) in rats treated

with thiobencarb for six weeks when compared with

control group.

Discussion

Since liver is the organ where most of the

substances undergo first pass metabolism, it becomes an

organ of extreme importance to study the effect of

various substances. The present results showed that

thiobencarb administratin induced histopathological

alterations in the liver of rats. Many histological

alterations were recorded in liver of rats orally given

thiobencarb for six weeks. The normal structure of

hepatic lobules was lost, congested and dilated veins,

bile duct proliferation, leukocytic infiltration, activated

Kupffer cells within wide sinusoids, cytoplasmic

vacuolation, pyknotic nuclei and fatty infiltration in the

hepatocytes were detected. These changes seemed to

follow the same pattern as that previously enumerated by

many investigators under the stress of different

carbamates. Sahai [18] reported that carbaryl caused

different histological changes in liver of mice such as

fibrosis and inflammatory infiltrate around the portal

triads along with the dilatation and congestion of the

blood vessels and proliferation of bile ductules and areas

of hemorrhage . Hamid et al.[19] reported that treating

rats with carbaryl caused hepatocyte degeneration with

pyknotic nuclei, cytoplasmic vacuolation and fibrosis.

Munglang et al. [20] noted that carbarylcaused

hepatocellular degeneration, necrosis and

morphometrical changes in liver of rats.

Cytoplasmic vacuolation was observed in the

cytoplasm of hepatocytes of thiobencarb treated rats.

Sakr et al. [21] attributed cytoplasmic vacuolation to the

oxidative stress that generate superoxide anions which

cause lipid peroxidation. Lipid accumulation leads to

alteration and damage of cellular lipid membranes with

paralysis of Na-K pump and hepatocytes edema.

Congestion in the blood vessels might be due to loss of

fluid from the blood and vessels engorged with red

blood corpuscles [22]. Fatty infiltration was observed in

the liver of the treated rats. It was reported that fatty

infiltration resulted by the inhibition of lipogenesis that

directly inhibited the expression and activity of fatty acid

synthase (FAS) in liver. Inhibition of FAS results in

accumulation of malonyl-Coenzyme A (CoA), an

important inhibitor of mitochondrial fatty acid oxidation

(FAO). The target for malonyl- CoA as an inhibitor of

FAO is carnitine palmitoyltransferase 1, the enzyme

catalyzing the first step for transporting fatty acids into

the mitochondria. Short and long-term administration of

bendiocarb affects the liver ultrastructure of rabbits. It

caused increase in the number of peroxisomes, dilatation

of rough endoplasmic reticulum and proliferation of

smooth endoplasmic reticulum and increase of lipid

droplets [23].


Fig.1. A. Section in the liver of a control rat showing central vein (CV),

Sinusoidal space (S) with kupffer cell (K) and hepatocyte (H), X400

B. Section in the liver of a rat treated with thiobencarb showing congested

and enlarged central vein(CG) X200.

C. Congested and enlarged portal vein (PV) X200.

D. Liver section of treated rat showing bile ductular proliferation (arrows)

H&E,X400.


Fig.2. A. Liver section of a treated rat showing leucocytic infiltrations (LI), X400.

B.Cytoplasmic vacuolation of the hepatocytes (arrows).

C. Fatty infiltrations, (arrows), H&E X400


Fig.3. A. Liver section of a control rat showing PAS positive materials in the cytoplasm of the cells.

The nuclei gave a negative reaction.

B. Section of liver of a treated rat showing weak PAS reaction in most of hepatocytes,

( PAS X 400).

Fig.4. A. Normal protein content in the hepatocytes of a control rat appears as dense bluish

bodies in the cytoplasm.

B. Reduction of the protein content in the hepatic cells of a treated rat.

(Mercury bromophenol blueX 400)


Fig.5. A. Expression of PCNA in nuclei of hepatocytes of a control rat (arrow)

B.Increase in expression of PCNA in hepatocytes of a treated rat,

(immunohistochemical stain, X400).

Fig.6. A. Slight expression of caspase-3 in cytoplasm of hepatocytes of a control rat.

B. Strong expression of caspase-3 in hepatocytes of a treated rat,

(Immunohistochemical stain, X400).

Table (1).Mean staining area percentage of PCNA and caspase-3 in control and

thiobencarb-treated group.

(*) Significant at P<0.05 against control group

Groups PCNA

(mean ±SD)

Caspase-3

(mean± SD)

Control 0.2 ± 0.01 3.4 ± 0.5

Thiobencarb 44.5 ± 2.3* 51.3 ± 3.5 *


In this study, thiobencarb treatment caused

reduction of carbohydrates in the hepatocytes. Similar

results were observed under the effect of different

pesticides. Bhushan [24] reported that hepatic proteins

and glycogen decreased in liver of rats treated with

cypermethrin. Sakr et al. [25] showed that inhalation of

tetramethrin caused decrease of glycogen and proteins. It

was suggested that the depletion of carbohydrates is due

to the release of hydrolytic enzymes from ruptured

lysosomes under the toxic effect of toxic agents [26].

Reduction of total proteins was observed in liver of

trated rats. Similarly, Sakr et al. [27] reported that

cypermethrin administration induced decrease in total

proteins in hepatic cells of rats. This decrease in proteins

was attributed to that carbamates impair the enzymatic

pathways involved in metabolism of carbohydrates, fats

and protein within cytoplasm, mitochondria, and

proxisomes [28].

Immunohistochemical results indicated that

thiobencarb increased the expression of PCNA in

hepatic tissue of rats. Cabamates were found to affect

PCNA expression. Debruyne [29] reported that mean

number of PCNA-positive increase in hepatocytes and

renal cortical tubular cells of rats exposed to carbaryl.

Irisarri [30] measured cellular proliferation by PCNA

staining in the liver, urinary bladder and thyroid gland of

rats that had been exposed to carbaryl for 52 week in a

dietary study . There was a small increase in cell cycling

activity in the male thyroid and female liver at

7500 ppm. The authors added that the increase in PCNA

expression may be associated with an increase in the

number of cells that accumulate in the S phase of the cell

cycle. Also, the increase in hepatocyte proliferation may

be at least related to regenerative liver response to

pesticide, since during liver growth, histological signs of

necrosis and vacuolated cytoplasm were present [31].

Expression of caspase-3 was increased in

hepatocytes of thiobencarb-treared animals. Caspase-3 is

a key protease activated during the early stages of

apoptosis and, like other members of the caspase family,

is synthesized as an inactive proenzyme that is processed

in cells undergoing apoptosis by self-proteolysis and/or

cleavage by another protease such as caspase 8 or 9 [32].

Different pesticides was found to cause apoptosis. ziram,

a carbamate pesticide, induced significant increase in

apoptosis in human T cells, which contributed to the

inhibition of CTL activity [33]. The three carbamate

pesticides,carbary, maneb and thiram induced apoptosis

human T cells in a dose- and time-dependent manner at

higher concentrations via the caspase-3 pathway [34].

Subchronic exposureof rat to methomyl for 28 days was

associated with a significant upregulation of the

expression of apoptosis-related genes, Tp53, Bcl-2,

CASP3 and CASP9 in testes of rats [35]. Sakr and

Shalaby [36] reported that carbofuran induced testicular

apoptosis as indicated by increase of caspase-3 and bax

in germ cells. Lari et al.[37] reported that diazinon

induced hepatic apoptosis through activation of

caspases-9 and -3 and increasing Bax/Bcl-2 ratio.

The reactive oxygen species (ROS) are

continuously generated inside the body as a

consequences of exposure to a lot of exogenous

chemicals in our ambient environment including

pesticides. Under normal circumstances, the ROS

generated are eliminated by enzymatic and non-

enzymatic antioxidant systems present in the body.

Harmful effects caused by ROS occur as a consequence

of an imbalance between the formation and elimination

of these species culminates in an oxidative stress [38].

ROS readily attack and induce oxidative damage to

various bio-molecules including proteins, lipids,

mitochondria, lipoproteins and DNA which alter the

pathways of these bio-molecules [39]. Carbamates were

known to generate reactive oxygen species (ROS) ,

results in oxidative stress and increase lipid

peroxidation with a reduction in CAT, SOD and GST

levels in experimental models in different organs [40].

The hepatotoxicity induced by thiobencarb in this study

could be possibly explained by its direct cytotoxic effect

and/or indirectly via the increased level of ROS and

apoptosis-mediated genes.

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