Upload
others
View
3
Download
0
Embed Size (px)
Citation preview
www.JATBAS.com
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)
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
JATBAS,Vol.1,No.2: 198-207,2017 p ISSN:2537-0537,e ISSN:2537-0618 200
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].
JATBAS,Vol.1,No.2: 198-207,2017 p ISSN:2537-0537,e ISSN:2537-0618 201
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.
JATBAS,Vol.1,No.2: 198-207,2017 p ISSN:2537-0537,e ISSN:2537-0618 202
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
JATBAS,Vol.1,No.2: 198-207,2017 p ISSN:2537-0537,e ISSN:2537-0618 203
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)
JATBAS,Vol.1,No.2: 198-207,2017 p ISSN:2537-0537,e ISSN:2537-0618 204
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 *
JATBAS,Vol.1,No.2: 198-207,2017 p ISSN:2537-0537,e ISSN:2537-0618 205
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.
References
1.Glass R (2008): Chronic and long-term effects of
pesticides use in agriculture. Current knowledge
and limits, Toxicol. Lett, 180: p.S21.
2.Battaglin, WA; Thurman, EM; Kalkhoff, SJ; Porter,
SD. (2003):Herbicides and transformation
products in surface waters of the Midwestern
United States. J. Am. Water Res. Assoc, 39: 743–
756.
3.Sengupta A. , Manna K. , Datta S. , Das U. , Biswas
S. , Chakrabarti N. and Dey D. (2017).Herbicide
exposure induces apoptosis, inflammation, immune
modulation and suppression of cell survival
mechanism in murine model . RSC Adv., 7 :
13957.
4.Kawamoto K, Urano K.(1990): Parameters for
predicting fate of organochlorine pesticides in the
JATBAS,Vol.1,No.2: 198-207,2017 p ISSN:2537-0537,e ISSN:2537-0618 206
environment (III) Biodegradation rate constants.
Chemosphere ;21:1141-1152.
5.Watanabe H, Nugyen MHT, Souphasay K, Vu SH,
Phong TK, Tournebize J, Ishihara S.(2007): Effect
of water management practice on pesticide behavior
in paddy water. Agr Water Manag ;88:132-140.
6. Tanetani Y, Kaku K, Ikeda M, Shimizu T.(2013):
Action of a herbicide, thiobencarb. J Pestic Sci
;38:39–43.
7. Eladel HM, Henley WJ, Kobbia IA.(1999): Effects of
thiobencarb on growth and photosynthesis of the
soil alga, Protosiphon botryoides (Chlorophyta). J
Appl Phycol ;10:547–554.
8. Xia J.(2005): Response of growth, photosynthesis and
photoinhibition of the edible cyanobacterium
Nostoc sphaeroides colonies to thiobencarb
herbicide. Chemosphere ;59:561–566.
9. Patrick W. Borthwick James M. Patrick Jr.
Douglas P. Middaugh (1985): Comparative acute
sensitivities of early life stages of atherinid fishes
to chlorpyrifos and thiobencarb. 14(3): 465–473.
10. Ramah,K. (2011): Histopathological study on the
effect of rice herbicides
on grass carp (Ctenopharyngodan idella). African
Journal of Biotechnology Vol. 10(7), pp. 1112-
1116.
11. Fernandez-Vega. C., Sancho, E., Ferrando, M.D.,
Andreu-Molina, E.,( 1999): Thiobencarb toxicity
and plasma AChE inhibition in the European eel. J.
Environ. Sci. Health, B34 (1): 61-73
12. U.S. Environmental Protection Agency, (2000):
Thiobencarb Re-registration Eligibility Decision
(RED).
13. Srinivas N. Pentyala* andC. S. Chetty (1993):
Comparative study on the changes in AChE and
ATPase activities in neonate and adult rat brains
under thiobencarb stress, Journal of Applied
Toxicology,13(1): 39–42.
14. El-Tawil,M F. and Marzouk E. M.A.(2015): Acute
Oral Toxicities of Three Pesticides Used in
Egyptian Rice Farms to Albino Rats. Current
Science International , 4 (2) : 145-154.
15.Kiernan, J. A. (1981): Histological and Histochemical
Methods, Theory and Practice. Pergamon Press.
New York,USA,344 pages.
16- Pearse, A. G. E. (1972): Histochemistry, Theoretical
and Applied, 3rd end.,vol. 2. Churchill Livingstone.
London.
17.Ramos-Vara, J.A. Principles and methods of
immunohistochemistry(2011): Methods Mol Biol.,
691:83-96.
18.Sahai , V. (2013): Carbaryl induced histological
changes in the liver of albino mice . J. Entomology
and Zoology Studies ;1 (4): 145-149 .
19. Hamid S, Mahajan R, Singh H (2012): Carbaryl, A
Pesticide Causes “Toxic Hepatitis” in Albino Rats.
J Cytol Histol 3:149.
20. Munglang M, Nagar M, Prakas R (2009) : Liver in
Carbaryl treated rats morphological & a
monometric stydy. J Anat Soc India, 58: 6-9.
21. Sakr,S.A. Hany A. Abdel Samie, Fatma M. El-
sharkawy (2017): Role of anise oil in ameliorating
methotrexate- induced testicular and hepatotoxicity
in rats. Transylvanian Review Vol XXV, No.
17:4257-4267. 22. Tos-Luty S, Przebirowska D, Latuszynska J,
Tokarska-Rodak M (2001): Histological and
ultrastructural studies of rats exposed to carbaryl.
Ann Agric Environ Med 8: 137-144.
23. Holovska, K., Almasiova,V. ,Cigankova,V. (2014):
Ultrastructural changes in the rabbit liver induced
by carbamate insecticide bendiocarb.
J.Environmental Science and Health, Part B 49(8):
616-628 .
24. Bhushan B, Saxena N, Saxena N.(2013):
Biochemical and Histological Changes in Rat Liver
Caused by Cypermethrin and BetaCyfluthrin.
Arh.Hig.Rada,Toksikol.; 64: 57-67.
25. Sakr SA, El-Messady FA, El-Desouky NI.(2002):
Pyrethroid Inhalation Induced Histochemical
Changes in The Liver of Albino Rats. The Sciences;
2(1): 24-28.
26.Morsy ,F. (2003). Protective Effect of Vitamin C and
Ginseng on Experimental Liver and Kidney Injuries
Induced by Insecticide Profenophos In Male Rats
.The Egyptian Journal of Hospital Medicine Vol.,
10 : 34 – 51 .
27. Sakr, S.A., Hashem, A.M., Nofal,A.E., El-shaer,N.H.
(2017): Protective Effect of Cinnamon Aqueous
Extract on Cypermethrin-Induced Hepatotoxicity in
Albino Rats. World J Pharm. Science.5(5):119-128.
28.Karami-Mohajeri ,S.and Abdollahi,M.(2016). Toxic
influence of organophosphate, carbamate, and
organochlorine pesticides on cellular metabolism of
lipids, proteins, and carbohydrates: A systematic
review. Human and Experimental Toxicology 30(9)
1119–1140.
JATBAS,Vol.1,No.2: 198-207,2017 p ISSN:2537-0537,e ISSN:2537-0618 207
29. Debruyne E (1998): Carbaryl 52-week toxicity study
in the CD1 mouse: Target organs cell cycling
assessment. Study No.: SA 97529. Lab: Rhone-
Poulenc Agro, Centre de Recherche, rue
Dostoievski, Sophia Antipolis, France. Sponsor:
Rhone-Poulenc Agro, rue Pierre Baizet, Lyon,
France.
30. Irisarri E (1996): Carbaryl 52-week toxicity study in
the rat and mouse Target organs cell cycling
assessment Pathology report (post-mortem). Study
No.: SA 95493. Lab: Rhone-Poulenc Agro, Centre
de Recherche, rue Dostoievski, Sophia Antipolis,
France.
31. Marouani N, Hallegue D, Sakly M, Benkhalifa M,
Ben Rhouma K, Tebourbi O. Adverse(2016):
Haemato-Biochemical Effects of Chlorinated
Insecticide in Adult Male Rats. Int. J. Advanced
Res., 4(7): 959-967.
32. Patel, T.; Gores, G.J.; Kaufmann, S.H.(1996): The
role of proteases during apoptosis. FASEB J.,
10:587–597.
33. Li, Q.; Kobayashi, M.; Kawada, T.(2011): Ziram
induces apoptosis and necrosis in human immune
cells. Arch. Toxicol., 85, 355–361.
34. Li, Q.; Kobayashi, M.; Kawada, T.(2015: Carbamate
Pesticide-Induced Apoptosis in Human T
Lymphocytes. Int. J. Environ. Res. Public Health ,
12, 3633-3645.
35. Heikal TM, Mossa ATH, Khalil WKB (2014) :
Protective Effects of Vitamin C against Methomyl-
Induced Injures on the Testicular Antioxidant Status
and Apoptosis-Related Gene Expression in Rat. J
Environ Anal Toxicol 4: 255. doi: 10.4172/2161-
0525.1000255.
36.Sakr SA and Shalaby SY .(2014): Effect of
fenugreek seed extract on carbofuran-inhibited
spermatogenesis and induced apoptosis in albino
rats. Journal of Infertility and Reproductive
Biology, 2 (2) : 36-42.
37. Lari, P.;Abnous, K.;Imenshahidi, M.;Rashedinia,
M.;Razavi, M. and Hosseinzadeh, H. (2013):
Evaluation of diazinon-induced hepatotoxicity and
protective effects of crocin.ToxicolInd Health. Feb
13. [Epub ahead of print].
38.. Farber JL (1994): Mechanisms of cell injury by
activated oxygen species. Environ. Health Perspect
102 Suppl 10: 17-24.
39.. Venkatesh S, Deecaraman M, Kumar R, Shamsi
MB, Dada R (2009): Role of reactive oxygen
species in the pathogenesis of mitochondrial DNA
(mtDNA) mutations in male infertility. Indian J
Med Res.,129: 127-137.
40. D’Souza UJA (2017): Pesticide Toxicity and
Oxidative Stress: A Review. Borneo Journal of
Medical Sciences , 11 (1) : 9 – 19.