proliferation and ldh leakage in cell cultures of animal and insect origin exposed to insecticide...

8/9/2019 Proliferation and LDH Leakage in Cell Cultures of Animal and Insect Origin Exposed to Insecticide Endosulfan

1/6

Kafkas Univ Vet Fak Derg

19

(3):

433-437, 2013

DOI:10.9775/kvfd.2di 2.7982

: http: //vetdergi.kafkas.edu.tr

: http://vetdergikafkas.org

Proliferation and LDH Leakage in Cell Cultures of Anim al and

Insect Origin Exposed to Insecticide Endosulfan

Natal ia KOVALKOVICOV

*

Jura j PISTL

^

Toms OSANK

**

Jana POLLKOV** Piotr DZIADCZYK*** Jaro slav LEGTH*

[1] This research

v\/as

supported by

VEGA

Grants No. 1/0287/11,

1/0855/12

and the National Reference Laboratory for

Pesticides,

University of Veterinary Medicine and Pharmacy in

Kosice

Department of Pharmacology and Toxicology, University of Veterinary Medicine and Pharmacy, Komenskho 73,

041 81 Kosice-SLOVAK REPUBLIC

* * Department of Microbiology and Immunology, University of Veterinary Medicine and Pharmacy, Komenskho 73,

041 81 Kosice-SLOVAK REPUBLIC

* * * Rzeszw University of Technology, Department of Biochemistry and Biotechnology, Powstacw Warszawy 6, 35-

959 Rzeszw - POLAND

^ Makale Kodu ArticleCode):KVFD-2012-798

Summary

In thepresent study three di f ferent cel l cul tures, der ived f ro m ra bbi t k idne y (RKl3) ,rat liver (WBF344)andinsect o rigin (Sf21),

were usedtoexamine the cy totoxic ef fectofthe insect ic ide en dosul fan. Cytotoxic ity was d etermine don thebasisofcel l prol i fer ation

act iv i ty ;thecel lular da ma ge was assessedbyevaluat ionof cytopathic effect and lactate dehydrogenase (LDH) leakage. Endosulfan

treatm ent suppressed prol i ferat ive ac t iv i ty

in

cell cultures as follows: insect Sf21 cells (lO-'-lO

M;

P

WBF344 ( lO-^ - lO

M >

RKl 3 cel ls (10'-1 0-^

M .

LDH leakage into

the

me d i u m

was

increased

in

WBF344 cells

at

1 0 ' - 1 0 ^

M

(P


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Proliferation andLDHL eakage in

sol id has emerged as a highly controversial agrichemical

due to i ts acute toxicity ' , persistence in the environment,

potent ia l for bioaccumulat ion, and role as an endocrine

disru pto r I Banned in m ore tha n 63 countries, it is stil l used

extensively to control insect pests inc luding whi tef lys,

aphids, leafhoppers, Colorado potato beetles and cabbage

worms in vegetab les , co t ton, and f ru i ts in some other

countries including India and Brasi l . Because of i ts unique

mode of act ion, i t is useful in res is tance management ^

On the other hand, endosul fan is one of the most tox ic

pesticides on the market today, responsible for many fatal

pes t i c ide po ison ing inc idents in humans and an imals

around the w orld. It is absorbed throu gh the intestinal tract,

the lungs, and the

skin.

Toxicokinetics of ^ C-endosulfan in

rats was described by Chan et al. .Monitoring of the residue

levels of insecticide revealed i ts accumulation in various

t issues and f lu ids ^^ The commonest mani festat ions of

endosu l fan in tox icat ion are neuro log ica l a l thoug h o ther

organ dysfunction also occurs \ Hepatotoxicity and nephro-

toxicity in humans a nd animals exposed to endosulfan have

been documented in many studies ^

The aim of our s tudy was to compare th e di rect ef fect

o f d i f fe rent endosu l fan concent ra t ions on pro l i fe ra t i ve

act iv i ty and cel lu lar damage of mammal ian and insect

cel l cultures.

MATERIAL and METHODS

The Insecticide

Tested

Chemical data on en dosulfan are depicted in Fig /\ Endo-

sulfan was dissolved in dimethylsulfoxide (DMSO, Lachema,

Brno, Czech Republ ic), of which the f inal concentrat ion

in the main tenance medium was

1 % .

The basic molar

Fig1 .Chemical data on endosulfan '

CAS No. 115-29-7; Molecular formula: C^HjCleOjS; Mol, wt.: 406.9;

concentrations of insecticide, freshly prepared before eac

experiment, were 10

' -10 ' ^

M and added to cel l cultures a

the rate of 1 %of to tal cell volum e; i.e. the a ctual doses wer

lOOx lower than the basic ones. After endosulfan exposure

cel l prol i feration, lactate dehydrogenase release and ce

dsintgration were evaluated in cell cultures of mammalia

and insect or ig in . ,

Cell

ultures

Cel l l ines (k indly provided from Virological Inst i tute

Bratislava, Slovakia) RKl

3

(rabb it kidn ey), WBF344 (rat liver

and th e IPLBSF-21 (the pup al ovarian cel ls of the fal l army

worm, podopterafrugiperda Sf21) were used in the stud

RKl 3

cells were cultured in minima l essential m edium (MEM

sup plem ente d w ith 10% (v/v) foetal calf serum (FCS) and

1 % (v/v) antib iotic solution of P enicill in G Sodium Salt (PNC

and Streptomycin Sulphate (STM) (Gibco, Invitrogen, Corp.

USA) at 37C. WBF344 cel ls we re cu lture d in Du lbecco 's

mo dified Eagle's mediu m (D-MEM) wit h 1 0% (v/v) FCS, 1 %

(v/v) PNC and STM in h um id ifie d 5% CO^ at 37C. Sf-900 I

SFM m ediu m was used for insect cell l ine (Gibco, Invitrogen

Corp.,

USA) supplemen ted with

1 %

(v/v) PNC and STM. Sf2

cells were cultured at 27C.

ell

Density and Cytopathic Effect

Cell density and cytopathic effect dete rmined on the basis

of microscopical signs of cel lular damage (granulation and

vacuol isat ion of cytoplasm, rounding of f and detachment

of cel ls f rom the bottom of cul t ivat ion vessel , rupture o

ce l l s ) were eva luated by s tandard count ing techn ique

using an inverted microscope (Carl Zeiss, Germany) a

mag ni f icat ions of 400 x af ter 24 h exposure to e ndosu l fan

Proliferation Test PT)

Cells were seeded in 100 ml of cel l culture medium in

96-multiwel l culture plate (Corning, Inc., USA) at a density

o f

2x

OVml . A f ter 24 h incubat ion d i f fe rent endosu l fan

concentrat ions were then added and treated cel ls were

incu ba ted for 48 h. There were f ive repl icates of each

treatment. After the exposure period a colorimetric imm uno-

assay was used to quantify cell proliferation (Cell Proliferation

ELISA Ki t , BrdU-color imetr ic , Roche Diagnost ics, GmbH,

Germany). This was based on the measurement of 5-brom o-

2'-deoxyuridine (BrdU) incorporation during DNA synthesis.

Twenty four hours before the end of the cul t ivat ion, 10

HM BrdU was added and the cells were reincubated . After

removing the cul ture medium, denaturat ion of DNA and

f i xa t ion o f the ce l l s on the bot tom of we l l s , 100 ml o f

ant i -BrdU-peroxidase label led conjugate was added and

allowed t o react for 90 m in at 25C. The im mu ne complexes


3/6

435

KOVALKOVICOV PISTL CSANK

POLLKOV DZIADCZYK LEGTH

residual cell viability expressed as percentage of proliferative

act iv it y (% PA): %PA = [OD^ 'f /

OD ' ' ^ ^

x 100; where

QQendosuifan 5 |.f^g mean value of OD of cells treated with

insecticide and 0D^^ is the mean value of OD of cel ls

treated w i th the solvent con trol , measured at 450 nm .

Cytotoxicity A ssay

Cells were seeded in 100 ml of complete medium in

96-multiwell culture plate (Corning, Inc., USA) at

a

density

of 2x10Vml and incubated for 24 h. Growth medium was

changed to maintenance medium with 1%(v/v) FCS and

different endosulfan concentrations were then added and

cells were incubated for additional 24 h. There were five

replicates of each treatment. After the exposure period

a colorimetric assay was used to quantify cytotoxicity/

cytolysis by measuring LDH activity released from damaged

cells (Cytotoxicity Detection Kit'''- ^ Roche Diagnostics,

GmbH,Germany).

To

each well on th e 96-we ll plate 100

reaction mixture was added and plate was incubated

for 30 min at room temperature. After incubation 50 ul

of stop solution was added to each well.Optical density

(OD) was measured in an ELISA-multiwell reader (BIO-RAD

Laboratories, Inc., USA) at 450 nm (OD^jj,). To calculate

percent cytotoxicity in each plate low control (LC) and high

control (HC) were set up and the percentage of cytotoxicity

was calculated acco rding to the form ula: Cytotoxicity (%)

iosuifan_ QQi-c/ o Q H : - O D ^ S X 1 0 0 ; w h e r e

proliferation and ldh leakage in cell cultures of animal and insect origin exposed to insecticide...

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