Effects of Endosulfan on Allergic ResponseEffects of Endosulfan on Allergic ResponseTakuya Sugahara,Takuya Sugahara,1,1,* Sogo Nishimoto,* Sogo Nishimoto,22 SatokoSatoko Atobe, Atobe,11 Kosuke Hosoba, Kosuke Hosoba,11 Masaaki Masaaki Okabe,Okabe,22

Yoshimi KakinumaYoshimi Kakinuma11

11Faculty of Agriculture, Ehime University, Matsuyama, Ehime 790-8566, JapanFaculty of Agriculture, Ehime University, Matsuyama, Ehime 790-8566, Japan22Center for Marine Environmental Studies, Ehime University, MatsuyamaCenter for Marine Environmental Studies, Ehime University, Matsuyama, , Ehime Ehime 790-8577, Japan790-8577, Japan*Corresponding author: mars95@agr.ehime-u.ac.jp*Corresponding author: mars95@agr.ehime-u.ac.jp

IntroductionWe focused on endosulfan, one of the agricultural chemicals, because the pesticide was listed as POPs candidates in 2005 (StockholmConvention in 2005 “New POPs”). There are many reports concerning the poisonous effects of endosulfan on reproductive organs. However,there are few reports on evaluation of immune toxicity of endosulfan. Hence, we investigated the effect of β-endosulfan (one of the isomers) onimmune system, especially allergy response.

β-endosulfanα-endosulfan

ExperimentalExperimentalEndosulfanEndosulfan

Endosulfan is an Endosulfan is an organochlorineorganochlorine compound that is used as an compound that is used as aninsecticide and insecticide and acaricideacaricide. This . This colourlesscolourless solid has emerged as a solid has emerged as ahighly controversial agrichemical due to its highly controversial agrichemical due to its acute toxicityacute toxicity,,potential for bioaccumulationpotential for bioaccumulation, and role as , and role as an endocrine disruptoran endocrine disruptor..Banned in more than 62 countries, including the European Union,Banned in more than 62 countries, including the European Union,several Asian and West African nations, and soon in the Unitedseveral Asian and West African nations, and soon in the UnitedStates, it is still used extensively in many other countries includingStates, it is still used extensively in many other countries includingIndia, Brazil, and AustraliaIndia, Brazil, and Australia..

Animal experimentAnimal experiment

0 27 28

・・BloodBlood

・・ SpleenSpleen・・ Thymus Thymus

(d)

ex vivo assayex vivo assay

1) 400 (µ1) 400 (µgg/kg/day) : high dose ( H )/kg/day) : high dose ( H )2) 40 (µ2) 40 (µgg/kg/day) : medium/kg/day) : medium dose ( M ) dose ( M )3) 4 (µ3) 4 (µgg/kg/day) : low dose ( L )/kg/day) : low dose ( L )4) 80% 4) 80% EtOHEtOH : Control ( C ) : Control ( C )

Mouse : LDMouse : LD5050　　6.9-13.5 mg/kg, oral6.9-13.5 mg/kg, oral

Oral Oral administration of administration of ββ-endosulfan-endosulfan

ββ-endosulfan was orally administrated to ovalbumin (OVA)--endosulfan was orally administrated to ovalbumin (OVA)-challenged allergy model mice for 27 days. Blood and spleen werechallenged allergy model mice for 27 days. Blood and spleen werecollected on day 28, and spleen lymphocytes were isolated andcollected on day 28, and spleen lymphocytes were isolated andcultured for 48 cultured for 48 hh. OVA-specific . OVA-specific IgIg levels and cytokine levels in levels and cytokine levels inserum and culture supernatant of spleen lymphocytes wereserum and culture supernatant of spleen lymphocytes weremeasured by enzyme-linked immunosorbent assay (ELISA).measured by enzyme-linked immunosorbent assay (ELISA).

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OVA OVA

ResultsResults

Effect of Effect of ββ-endosulfan on immune system -endosulfan on immune system in vivoin vivo

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β-endosulfan β-endosulfan β-endosulfan

*

ββ-endosulfan was orally administrated mice for 14 days.-endosulfan was orally administrated mice for 14 days.Splenocytes were collected and cultured for 48 Splenocytes were collected and cultured for 48 hh. . IgIg production productionactivity of splenocytes was assessed activity of splenocytes was assessed ex vivoex vivo..

Cont.Cont. β-endβ-end

SpleenSpleen

Oral administration of Oral administration of ββ-endosulfan for 14 -endosulfan for 14 ddinduced atrophy of spleen in mice.induced atrophy of spleen in mice.

Effect of Effect of ββ-endosulfan on allergy response -endosulfan on allergy response in vivoin vivo

β-endosulfan

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Effect ofEffect of ββ-endosulfan on IL-4 level -endosulfan on IL-4 level in vivoin vivo

p < 0.005

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IL-4 production byIL-4 production bysplenocytes splenocytes ex vivoex vivo

HMC L

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C L M H

β-endosulfan

*p < 0.05, **p < 0.01 vs control

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IL-4IL-4

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Th2 type cytokine

TNF-TNF-αα1.2

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Th1 type cytokine

Effect of Effect of ββ-endosulfan on T cell activity in thymus-endosulfan on T cell activity in thymus

*p < 0.05 vs control

IFN-IFN-γγ

TNF-TNF-αα

IL-4IL-4

ThymusThymus

CD4-

CD8-

CD4+

CD8+

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CD4+

Th2

Th1

naïve T cell

ββ-endosulfan-endosulfan

Th2 dominantTh2 dominant

Th2Th2

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AllergyAllergy

Effect of Effect of ββ-endosulfan on degranulation-endosulfan on degranulation

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25 µM25 µM

Cont.Cont.

10 µM10 µM******

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Dose-responseDose-response

*p < 0.05, **p < 0.01, ***p < 0.005 vs control

The effect ofThe effect of ββ-endosulfan on allergen-induced degranulation by rat-endosulfan on allergen-induced degranulation by ratbasophilic leukemia cell line RBL-2H3 cells was examined. As abasophilic leukemia cell line RBL-2H3 cells was examined. As aresult, result, ββ-endosulfan facilitated degranulation by RBL-2H3 cells.-endosulfan facilitated degranulation by RBL-2H3 cells.