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INTRODUCTION
Organochlorine pesticides continue to be used in
several developing countries despite concerns regarding
iletiim (Correspondence)
S
El
434
Proliferation andLDHLeakage in ...
435
Cytotoxicity Assay
Statistical Analysis
RESULTS
Cytotoxic effect of endosulfan on cell cultures determined
by proliferation activity is shown in Table 1. Proliferative
activity of mammalian cell cultures was significantly
suppressed in liver WBF344 (10-^-10"'' M) and kidney cells
RK13 (10 '-10'^ M). Insect Sf21 cells were the most sensitive
to this insecticide with significant suppression of their
proliferative activity ranging from 1O'-1O-= M (P<0.01), with
PA = 1.6-65.6% (Table 1).
Cytopathic effect, determined on the basis of microscopical signs of cellular damage and the LDH leakage into
the medium, was observed in WBF344 in the concentrations
Table 1. Cytotoxic effect of insecticide endosuifan on cell cultures tested determined by proiiferation test
Table 1. Insektisit endosulfan'm hiicre ktrieri zerinde proliferasyon testi ile belirlenen sitotoksik etkileri
Concentration of Endosulfan in M (Mean Values)
Cell
Culture
Test
Parameters
101
RK13
ODiSD
%PA+SD
0.040.02
12.1 6*
WBF344
OD45oSD
%PASD
0.02+0.005
7.41.9**
Sf21
ODoSD
%PASD
0.0050.003
1.60.96*
Solvent Control
(DMSO)
10'
10'
10'
0.0203
6.1 9*
0.250.16
75.848.5*
0.370.07
n5.221.2
0.350.04
75.86.0
0.330.05 '^',
0.030.01
o.np.O7
40.725.9**
0.190.03
70.41i:i*
0.32+0.07
118.525.9
0.27.|^^H
11 .i3.7**
0.0050.004
1.61.3**
0.0050.004
1.99.5**
0.130.03
40.6+9.4**
0.21 0.007
65.62.2**
0.320.04
RK13 - Rabbit kidney celi line; WBF344 - epithelial rat liver eeli iine; Sf21 - Spodoptera frugiperda pupai ovarian tissue; DMSO - dimethyisulfoxide;
ODs - opticai density; SD - standard deviation; %PA - percentage of proiiferative activity; M - moiar concentration; ** P<0.01; * P<0.05
Table 2. Cytotoxie effeet of inseetieide endosuifan determined by evaluation of LDH aetivity reieased from eeiis
Table 2. insektisit endosulfan'm hcrelerdenserbestkalan
Cell
Culture
Test
Paramtres
RK13
WBF344
Sf21
LDH aktivitsinindegeriendiriimesi
Solvent Control
(DMSO)
10'
10^
10'
10^
OD,5oSD
% CytotoxicitySD
CPE
1.81 0.18
60.76.0*
1.90.1
66.83.5*
T.230.05
21.60.9
1.20.06
6.0810.1
1.240.46
22.278.3
D45SD
% CytotoxicitySD
CPE
1.860.04
83.61.8**
1.760.03
76.61.3**
1.51 0.13
59.45.1**
0.940.14^
20.03.0
0.940.13 '
23.434.0
0.91 0.03
24.60.8**
0.840.07
20.1 1.7*
0.780.08
16.21.7
ODSD
% CytotoxicitySD
CPE
0.61 0.05
6.00.5
RK13 - Rabbit Kidney ceii iine; WBF344 - epitheiiai rat iiver ceii iine; Sf21 - Spodoptera frugiperda pupal ovarian tissue; DMSO OD^o - opticai density; SD - standard deviation; CPE - cytopathic effect; M - molar coneentration; **P<0.01; * P<0.05
.:0.620.31:|
6.01 3.0''^H
dimethyisuifoxide;
436
Proliferation and LDH Leakage in...
of 10-'-i 0-^ M, in RK 13 cells and the insect cell line Sf21 in the
concentrations of lO-i-lO'^M compared to solvent control
{Table 2). Cytopathic effect and LDH leakage into the
medium were strongly limited in the insect cell line Sf21 and
RK 13 cells in comparison to the inhibition of proliferative
activity.
DISCUSSION
The indiscriminate and injudicious use of pesticides
particularly endosulfan in agriculture and animal husbandry
practices has considerably increased the risk of health hazard.
Traditionally, animal models have been used to examine
the toxic potential of pesticides. For screening purposes,
there is increasing interest in the development of in vitro
methods to replace conventional animal toxicity tests.
Established mammalian and insect cell lines may contribute
to rapid screening of pesticides and discover their modes
of action or may be useful for toxicity prediction of target
organs on chemicals or drug exposure '.
The liver is known to be the main site of xenobiotic
biotransformation, therefore, cell lines of liver origin are
widely used in biomdical research involving xenobiotic
metabolism and hepatotoxicity ". For our study we used
WBF344 established from a single cloned non-parenchymal
epithelial cell isolated from a normal male adult rat liver.
Kidney is the main organ for elimination of xenobiotics
from the body and the use of the cultured cells of kidney
origin to assess specific cytotoxic effects of nephrotoxins
described Li et al.'l Rabbit kidney (RK13) cell line was used
in the present study.
Continuous cell line Sf21 developed from ovarian tissue
of the Spodoptera frugiperda, a moth species that is an
agricultural pest on corn and other grass species, was
chosen for evaluation of direct effect of endosulfan on insect
cells. Cell lines used in this study were also used as model
systems for testing of various xenobiotics, e.g. WBF344
for food and perfume additives ^\ RK13 for mycotoxins '",
and the cell line Sf21 for testing of insect cell extracts ^^
or novel insecticides '^ Manji and Friesen ^^and Tseng et
al.'s studied induction of apoptosis and Takahashi et al.^^
cell cycle arrest induced by radiation in SF21 cell line. The
present work was undertaken to evaluate the direct toxic
effect of endosulfan on model cell cultures mentioned above.
Cytotoxic effects of endosulfan were previously studied
in various cell cultures. Holovsk et al.^" evaluated proliferative
activity of cell lines Madin-Darby Bovine Kidney (MDBK),
Rabbit Kidney (RK13), Porcine Kidney (PK15), and semicontinual line of Bovine Embryonic Pulmonary Cells (BEPC)
after exposure to endosulfan. From these cell cultures
cell proliferative activity was suppressed most intensively
in PK15 culture at the concentrations of 1O'-1O<^ M of
endosulfan. Krovel et al.^^ observed moderate cytotoxicity
437
8. Singh ND, Sharma AK, Dwivedi P, Telang AG, Patil RD, Kumar M:
Ultrastructural pathology of kidney and liver in citrinin and endosulfan
treated pregnant rats. IndJ Vet Pathol, 33 (1 ); 18-24, 2009.
27. Wang J, Yi ChH, Liu XZ, Yuan Y, Bian JCh, Liu ZP: Effects of cadmium
acetate on oxidative stress in primary cultured rat hepatocytes. Chinese
V/efSd, 39,1010-1013, 2009.
10. Davila JC, Rodriguez RJ, Melchert RB, Acosta D Jr: Predictive value
of in vitro model systems in toxicology. Ann Rev Pharmaeol Toxieol, 38, 6396,1998.
11. Mersch-Sundermann V, Knasmuller S, Wu XJ, Darroudi F, Kassie F:
Use of a human-derived liver cell line for the detection of cytoprotective,
antigenotoxic and cogenotoxic agents. Toxicoi, 198, 329-340,2004.
12. Li W, Choy DF, Lam MS, Morgan T, Sullivan ME, Post JM: Use of cultured
cells of kidney origin to assess specific cytotoxic effects of nephrotoxins.
7bx/co/ in Vitro, 17 (1 ): 107-113, 2003.
13. Tang QH, Yang W, Tan YX, Zhou, WP: Sodium butyrate induces rat
liver oval cells WB-F344 differentiating into billiary epithelium cells in vitro.
Annual Internationai Conference of the IEEE Engineering in Medicine and
Bioiogy- Proeeedings, Article number 4030430,576-579,2006.
14. Rumora L, Kovacic S, Rozgaj R, epelak I, Pepeljnjak S, Zanic Grubisic
T: Cytotoxic and genotoxic effects of fumonisin Bl on rabbit kidney RK13
cell line. Areh Toxieol, 76 (1): 55-61, 2002.
15. Staljanssens D, Van Camp J, Herregods G, Dhaenens M, Deforce D,
Van De voorde J, Smagghe G: Antihypertensive effect of insect cells: in
vitro and in vivo evaluation. Peptides, 32 (3): 526-530, 2011.
16. Powell GF, Ward DA, Prescott MC, Spiller DG, White MR, Turner
PC, Earley FG, Phillips J, Rees HH: The molecular action of the novel
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