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A Dissertation

for the Degree of Doctor of Philosophy

Toxicity mechanisms of

chlorpyrifos and trifloxystrobin

pesticides in human keratinocytes

클로르피리포스와 트리플록시스트로빈 농약의

피부세포 내 독성 기전 연구

February 2017

By

Yoonjeong Jang, D.V.M.

Veterinary Pathobiology and Preventive Medicine

College of Veterinary Medicine

Graduate School of Seoul National University

i

ABSTRACT

Toxicity mechanisms of

chlorpyrifos and trifloxystrobin

pesticides in human keratinocytes

Yoonjeong Jang, D.V.M.

Veterinary Pathobiology and Preventive Medicine

College of Veterinary Medicine

Graduate School of Seoul National University

Pesticides have provided significant benefits including plant

disease control and increased crop yields since they were

developed and utilized. However, pesticide causes many adverse

effects on non-target organisms including human, which

necessitate precise toxicity tests and risk assessment.

Integumentary system is a main exposure site of pesticide

exposure. Therefore, it is important to investigate toxicity

mechanism of pesticides especially in skin. Chlorpyrifos, and

trifloxystrobin are widely-used pesticides for both agricultural

and residential application. Although various studies have reported

ii

toxicity and health-related effects by their exposure, the toxicity

mechanism in skin cells has not been well-characterized. The

present study determined the potential mechanism involved in

skin toxicity of chlorpyrifos and trifloxystrobin using HaCaT

human skin keratinocyte cell line.

First, after treating chlorpyrifos to HaCaT cells, we examined

changes in the cells. Chlorpyrifos triggered reactive oxygen

species (ROS) generation and mitochondrial oxidative stress. We

focused on NLRP3 inflammasome, known to induce innate

immune response. We used mitochondrial ROS (mROS) scavenger

mitoTEMPO to demonstrate a role for mROS in NLRP3

inflammasome and programmed cell death induced by

chlorpyrifos. These results showed that chlorpyrifos provoked

NLRP3 inflammasome and pyroptosis/apoptosis via an increase of

mROS in HaCaT cells, and could propose that chlorpyrifos

induces innate immune response and skin inflammation through

activating the NLRP3 inflammasome in skin epithelial cells.

Second, skin toxicity mechanism of trifloxystrobin was explored

using HaCaT cells. Following treatment of trifloxystrobin, cell

viability, and subsequent Annexin V-FITC/propidium iodide

assay, TUNEL assay and Western blotting were performed to

investigate the cell death mechanism of trifloxystrobin. Exposure

to trifloxystrobin resulted in diminished viability of HaCaT cells

in both a time- and concentration-dependent manner. The cell

death was derived through apoptotic pathways in the HaCaT

iii

cells. Furthermore, we explored the effect of trifloxystrobin on

TRAIL-mediated extrinsic apoptosis using siRNA transfection.

Knockdown of death receptor 5 suppressed

trifloxystrobin-provoked apoptosis. These results indicate that

trifloxystrobin induces TRAIL-mediated apoptosis and has an

inhibitory effect in keratinocytes that can interfere with the

barrier function and integrity of the skin.

Third, we investigated the impact of trifloxystrobin on exposed

skin at the cellular organelle level in skin keratinocytes. HaCaT

cells were treated with trifloxystrobin for 48 h and trifloxystrobin

showed detrimental effects on mitochondria evidenced by altered

mitochondrial membrane potential and morphology. To identify

autophagic degradation of the damaged mitochondria, confocal

imaging and Western blotting were performed. Trifloxystrobin

induced autophagy and mitophagy-related proteins in HaCaT

cells. The mitoTEMPO was applied to further explore the

mechanism of trifloxystrobin-mediated mitophagy, and it

alleviated the effects on mitophagy induction. Our findings

indicated that mitochondrial damage and mitophagy may play a

role in trifloxystrobin-induced toxicity in human keratinocytes

and this could be suggested as a mechanism of cutaneous

diseases developed by exposure.

Taken together, these findings could be suggested as

mechanisms of cutaneous diseases developed by the pesticide

exposure. Furthermore, our studies can contribute to the

iv

assessment of pesticide-induced diseases and be considered in the

management of pesticide use and its adverse effects.

Keywords: Pesticides, Pesticide exposure, Toxicity mechanism,

Reactive oxygen species, Human skin keratinocyte

Student number: 2014 - 21932

v

TABLE OF CONTENTS

Abstract..................................................................................i

Table of Contents................................................................v

List of Figures...................................................................vii

List of Abbreviations.........................................................ix

General Introduction............................................................1

Chapter I. Chlorpyrifos induces NLRP3 inflammasome

and pyroptosis/apoptosis via mitochondrial oxidative

stress in human keratinocyte HaCaT cells......................7

Abstract.................................................................................8

1.1. Introduction....................................................................9

1.2. Materials and Methods...............................................13

1.3. Results..........................................................................21

1.4. Discussion....................................................................44

Chapter Ⅱ. Trifloxystrobin induces tumor necrosis

factor-related apoptosis-inducing ligand (TRAIL)-

mediated apoptosis in HaCaT, human keratinocyte

cells......................................................................................49

Abstract...............................................................................50

vi

2.1. Introduction..................................................................51

2.2. Materials and Methods...............................................54

2.3. Results..........................................................................59

2.4. Discussion....................................................................72

Chapter Ⅲ. Trifloxystrobin-induced mitophagy through

mitochondrial damage in human skin keratinocytes.....76

Abstract...............................................................................77

3.1. Introduction..................................................................78

3.2. Materials and Methods...............................................81

3.3. Results..........................................................................85

3.4. Discussion....................................................................95

References...........................................................................99

Abstract in Korean...........................................................118

vii

LIST OF FIGURES

General Introduction

Figure 1. Chemical structures of chlorpyrifos (A) and

trifloxystrobin (B).

Chapter I. Chlorpyrifos induces NLRP3 inflammasome and

pyroptosis/apoptosis via mitochondrial oxidative stress in

human keratinocyte HaCaT cells

Figure 1.1. Effects of CPF on cell viability and oxidative stress

of HaCaT cells.

Figure 1.2. Increase of mitochondria damage and mitochondrial

ROS induced by CPF.

Figure 1.3. The induction of NLRP3 inflammasome in CPF

treated HaCaT cells and the reversal by ROS scavenger NAC.

Figure 1.4. Diminished CPF induced-NLRP3 inflammasome in

response to mROS scavenger, mitoTEMPO.

Figure 1.5. The effects of CPF on pyroptosis and apoptosis.

Figure 1.6. Changes of pyroptosis and apoptosis in the presence

of caspase-1 inhibitor and mitoTEMPO.

Chapter Ⅱ. Trifloxystrobin induces tumor necrosis factor-

related apoptosis-inducing ligand (TRAIL)-mediated

apoptosis in HaCaT, human keratinocyte cells

viii

Figure 2.1. The effect of trifloxystrobin on cell viability.

Figure 2.2. Increase in apoptosis induced by TRF exposure.

Figure 2.3. The effect of TRF exposure on pro-apoptotic protein

expression.

Figure 2.4. Change in apoptosis by siRNA transfection.

Figure 2.5. Changes in protein expression following the

transfection of DR5 siRNA.

Chapter Ⅲ. Trifloxystrobin-induced mitophagy through

mitochondrial damage in human skin keratinocytes

Figure 3.1. The effect of TRF on cytotoxicity in HaCaT cells

using the CCK-8 assay.

Figure 3.2. The effects of TRF on mitochondrial damage.

Figure 3.3. The effect of TRF on autophagy induction.

Figure 3.4. Induction of mitophagy by TRF and the effects

alleviated by mROS scavenger.

ix

LIST OF ABBREVIATIONS

ALR: AIM2-like receptor

Apaf-1: apoptotic protease activating factor 1

ASC: apoptosis-associated speck-like protein containing a CARD

Bad: Bcl-2-associated death promoter

BECN1: beclin 1

BSA: bovine serum albumin

CCK-8: cell counting kit-8

CLSM: confocal laser scanning microscope

CPF: chlorpyrifos

Cyt c: cytochrome c

DCF: 2,7-dichlorofluorescein

DMSO: dimethyl sulfoxide

DR: death receptor

ELISA: enzyme–linked immunosorbent assay

GSH: glutathione

GSSG: glutathione disulfide

H2DCFDA: 2′,7′-dichlorodihydrofluorescein diacetate

IL-1β: interleukin-1β

JC-1: 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazol

-carbocyanine iodide

LAMP2: lysosome-associated membrane protein 2

LC3B: microtubule associated protein 1 light chain beta

x

LDH: lactate dehydrogenase

MMP: mitochondrial membrane potential

mROS: mitochondrial reactive oxygen species

MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

NAC: N-acetyl-cysteine

NLR: NOD-like receptor

NLRP3: NOD-like receptor family pyrin domain-containing 3

PARP: poly (ADP-ribose) polymerase

PBS: phosphate-buffered saline

pDRP1: phosphorylated-dynamin related protein 1

PI: propidium iodide

PINK1: PTEN-induced putative kinase 1

PRRs: pattern recognition receptors

PS: phosphatidylserine

ROS: reactive oxygen species

SEM: standard error of the mean

SRM: super resolution microscope

TRAIL: tumor necrosis factor-related apoptosis-inducing ligand

TRF: trifloxystrobin

TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labeling

1

GENERAL INTRODUCTION

Pesticides are essential to control agricultural diseases and pests

to increase food production and improve plant breeds on limited

farmland (Jang et al., 2014). Nowadays, the use of pesticides has

become much more common due to increased pest problem

caused by climate change. However, these agrochemicals are not

species-specific and non-target organisms including humans can

also be affected by exposure (Sooresh et al., 2015). In case of

Korea, it has been documented many different pesticides

including insecticides and fungicides have been applied in

orchards and farmlands, and some widely used pesticides were

detected in Korean rivers (Shim et al., 2007). It is important

because pesticide contamination in the water environments may

reflect the pesticide use, and have an impact on non-target

organisms. Thus, there has been a great public concern regarding

occupational and non-occupational pesticide exposure and its

adverse effects such as skin inflammation since their introduction.

A notable example is pesticide residue. Concern for public health

has been raised because of remaining pesticides in foods and

environmental contamination caused by the spraying of pesticides.

When pesticides contaminate soil, rivers, and oceans, they may

be able to produce adverse effects on non-target ecosystems.

Non-occupational exposure, which is occurred through the use of

2

improper hazardous pesticides in cities, public parks, golf club,

and other environments such as swimming pools, is a growing

issue and also has a potential harmful influence on public health.

Therefore, it is necessary to identify the toxicity by pesticide

exposure.

To assess pesticide toxicity, a majority of laboratory animals

have been employed. Recently, people show ethical concerns and

severe opposition to traditional in vivo toxicity tests. Technical

development of in vitro tests using cultured cells has progressed

satisfactorily to allow these tests to be considered as alternatives

to animal experiments. In vitro tests using human-derived cell

lines offer several advantages over in vivo methods, including

controlled testing conditions, a high level of standardization, a

reduction in variability between experiments, low cost, a small

amount of required material, a limited amount of toxic waste, and

reduced need for animals (Araújo et al., 2014).

The integumentary system is a major direct site of exposure

during pesticide use. Multiple studies have reported that skin

inflammation such as allergic contact dermatitis was triggered in

farm workers (Koch, 1996). However, there is lack of knowledge

concerning the mechanism by which skin disease develops in

response to pesticide exposure. An understanding of pesticide

toxicity is important for safe use and to prevent excessive

exposure. Therefore, it is important to investigate underlying

toxicity mechanisms of pesticides in human skin cells and assess

3

the risks of pesticide use.

Pesticides include insecticides, fungicide and herbicides etc.

Chlorpyrifos (Fig. 1A) is an organophosphate insecticide that has

been broadly used for agricultural pest control and managing

residential indoor pests since its registration (Gomez, 2009).

However, chlorpyrifos has shown non-target toxicity to exposed

people. In particular, skin can easily come in contact with

chlorpyrifos products both in occupational and non-occupational

settings. Through epidemiological and experimental studies, it has

been suggested that exposure to chlorpyrifos through skin may

cause dermatitis and skin sensitization (Penagos et al., 2004;

ArystaLifeScience, 2010; Wang et al., 2014). Among the important

fungicides, trifloxystrobin (Fig. 1B) is a widely used, systemic,

broad-spectrum strobilurin-derived fungicide that plays an

important role in the disease management in agriculture around

the world. The mode of action in fungi is to bind to the quinol

oxidation site of cytochrome b in the mitochondrial electron

transport chain and thereby block electron transfer, which leads

to the inhibition of energy (ATP) production and then the fungi

ultimately die (Balba, 2007). As such, trifloxystrobin could be

toxic to non-target species including eukaryotes, and it has been

reported to induce allergic reaction and dermal sensitization (US

EPA, 1999; Paranjape et al., 2015). Furthermore, chlorpyrifos and

trifloxystrobin have been used even at many golf courses in

Korea, and detected in the soils (NIER, 2014). However, there is

4

a lack of studies on their toxicity mechanisms in the skin. Thus,

it is necessary to explore how they have effects on skin after

the exposure.

Fig. 1. Chemical structures of chlorpyrifos (A) and trifloxystrobin

(B) (From Sigma-Aldrich, Inc.).

One of representative adverse effects induced by pesticide

exposure is inflammation such as dermatitis. Various mechanisms

and factors can provoke the inflammation pathway. First,

induction of oxidative stress linking pesticide exposure to health

effects has been reported in farmworkers who have used

pesticides, and biomarkers of the oxidative stress may provide

the link for pesticide contact to a number of health issues (Muniz

et al., 2008). The oxidative stress and abnormal increase of ROS

can cause a cascade of subsequent toxic pathways that may lead

to diseases including inflammation and immune responses (Reuter

et al., 2010).

In response to stimuli, the innate arm of the immune system

precedes and primes the more advanced adaptive immune system

5

(Lamkanfi and Dixit, 2012). The innate immune system can

recognize activators through receptors called pattern recognition

receptors (PRRs) leading to the induction of an immune response

(Medzhitov and Janeway, 2002). NOD-like receptor (NLR)

proteins in PRR families expressed by epithelial cells trigger a

defense mechanism leading to assembly of a cytosolic protein

complex called inflammasome (Lamkanfi and Dixit, 2014).

NOD-like receptor family pyrin domain-containing 3 (NLRP3)

inflammasome is induced by various physical and chemical

stimuli (Gross et al., 2011). The formation of NLRP3

inflammasome modulates innate immunity and inflammation via

two pathways that include processing of cytokine precursors and

pyroptosis.

Cell death could be initiated by a range of physiological and

pharmacological stimuli and play a role in the disturbed

homeostasis and function of the skin (Henseleit et al., 1996,

Lippens et al., 2009). The extrinsic apoptosis, one of important

cell death mechanisms, in the epidermis has been reported to be

involved with several skin diseases such as atopic dermatitis and

contact hypersensitivity (Wehrli et al., 2000). Also, it has been

shown that regulation of autophagy is important to the human

physical condition and may contribute to the pathogenesis of

several disease processes (Todde et al., 2009).

As the frontline of pesticide exposure, diverse adverse reactions

could be developed if the pesticide has detrimental effects on the

6

skin. Therefore, it is significant to identify the toxicity

mechanisms of pesticides using human cells. In the present

study, we investigated potential mechanisms involved in skin

toxicity of chlorpyrifos and trifloxystrobin using HaCaT human

skin keratinocyte cell line. The elucidated mechanisms could

provide better understanding of the toxicity process in skin cells

by pesticide exposure.

7

Chapter Ⅰ

Chlorpyrifos induces NLRP3

inflammasome and pyroptosis/apoptosis

via mitochondrial oxidative stress in

human keratinocyte HaCaT cells

(Published in 2015, Toxicology)

8

ABSTRACT

Chlorpyrifos (CPF) has been widely used around the world as a

pesticide for both agricultural and residential application. Although

various studies have reported toxicity and health-related effects

from CPF exposure, the molecular mechanism of CPF toxicity to

skin has not been well-characterized. The present study

determined the potential mechanism involved in skin toxicity of

CPF using the HaCaT human skin keratinocyte cell line. After

treating to HaCaT cells, CPF triggered reactive oxygen species

(ROS) generation and mitochondrial oxidative stress. We focused

on NLRP3 inflammasome, known to induce innate immune

response. We used mitochondrial ROS (mROS) scavenger

mitoTEMPO to demonstrate a role for mROS in NLRP3

inflammasome and programmed cell death induced by CPF. Our

results showed that CPF provoked NLRP3 inflammasome and

pyroptosis/apoptosis via an increase of mROS in HaCaT cells.

This study proposes that CPF induces innate immune response

and skin inflammation through activating the NLRP3

inflammasome in skin epithelial cells. CPF may lead to cutaneous

disease conditions and antioxidants could be proposed for therapy

against skin exposure to CPF.

9

1.1. INTRODUCTION

Chlorpyrifos (CPF) is an organophosphate pesticide that has

been broadly used for agricultural pest control and managing

residential indoor pests since its registration (Gomez, 2009).

However, CPF has shown non-target toxicity to exposed people.

Main exposure sites of the pesticide are integumentary and

respiratory systems. In particular, skin can easily come in contact

with CPF products both in occupational and non-occupational

settings. Through epidemiological and experimental studies, it has

been suggested that exposure to CPF through skin may cause

dermatitis and skin sensitization (Penagos et al., 2004;

ArystaLifeScience, 2010; Wang et al., 2014). Due to the

preponderance of direct skin exposure to CPF, it is necessary to

explore the mechanism of skin inflammation induced by CPF.

Several studies have reported that CPF induced oxidative stress

in neuronal cells and animal studies (Giordano et al., 2007;

Saulsbury et al., 2009). The mechanism of oxidative stress from

CPF may be via production of reactive oxygen species (ROS)

from mitochondria. ROS can cause damages to important

biomolecules or cells. If unchecked, it leads to systemic diseases

including inflammation and immune responses (Reuter et al.,

2010). Among the mechanisms of inflammation induced by

oxidative stress, innate immune process can be modulated by

10

dysfunction of mitochondria, the main source of ROS

(López-Armada et al., 2013).

In response to stimuli, the innate arm of the immune system

precedes and primes the more advanced adaptive immune system

(Lamkanfi and Dixit, 2012). The innate immune system can

recognize activators through receptors called pattern recognition

receptors (PRRs) leading to the induction of an immune response

(Medzhitov and Janeway, 2002). NOD-like receptor (NLR)

proteins and AIM2-like receptor (ALR) proteins in PRR families

expressed by epithelial cells and macrophages trigger a defense

mechanism leading to assembly of a cytosolic protein complex

called inflammasome (Lamkanfi and Dixit, 2014).

The inflammasome is a multiprotein complex that consists of a

sensor protein such as NLRs, an adaptor protein ASC

(apoptosis-associated speck-like protein containing a CARD), and

caspase-1 cleaving cytokines. Unique from other inflammasomes

that are activated by specific stimuli, NLRP3 (NOD-like receptor

family pyrin domain-containing 3) inflammasome is triggered by

various physical and chemical stimuli (Gross et al., 2011). NLRP3

inflammasome is promoted by not only pathogens but also

danger-associated molecular patterns including crystals, ATP, and

oxidative stress. Mitochondria can orchestrate innate immunity

and the release of mitochondria-derived ROS (mROS) is reported

as a crucial activator of NLRP3 inflammasome (Kepp et al., 2011;

Zhou et al., 2011).

11

The formation of NLRP3 inflammasome in host defense

modulates innate immunity via two pathways that include

processing of cytokine precursors and pyroptosis. This complex

modulates the secretion of proinflammatory cytokines including

interleukin-1β (IL-1β) by cleaving its pro-form under the

presence of IL-1β converting enzyme caspase-1 (Martinon and

Tschopp, 2004). After the mature IL-1β is secreted to the

extracellular space, it is able to bind to receptors and activate

cells as an important mediator molecule in cytokine network. It

recruits inflammatory cells and causes immune related diseases

(Arend et al., 2008). IL-1β activated by the inflammasome takes

part in regulating systemic and cutaneous inflammatory reactions

and contributes to inflammatory and auto-inflammatory disease of

skin (Contassot et al., 2012). Pyroptosis is a form of programmed

cell death requiring caspase-1 activated by the inflammasome.

The role of pyroptosis is lysis of affected cells which could be

deleterious to the organism. Thus, pyroptosis is characterized by

cell swelling, release of cytosolic elements, and inflammation

(Miao et al., 2011). The ensuing ROS production and

mitochondrial damage are responsible for intrinsic pathway of

apoptosis (Simon et al., 2000). The cell damage involves the

formation of apoptosome containing apoptotic protease activating

factor 1 (Apaf-1), and caspase-9 that initiate an apoptotic

cascade pathway. These programmed cell deaths remove damaged

cells to maintain the homeostasis.

12

As the frontline of pesticide exposure, diverse adverse reactions

could be developed if the pesticide has detrimental effects on the

skin. Therefore, it is important to identify potential mechanisms

of CPF on the induction of skin inflammation. The objective of

this study was to determine: (1) whether CPF could cause

mitochondrial oxidative damage, and (2) whether NLRP3

inflammasome and programmed cell death were induced by CPF

through mitochondrial ROS generation in human keratinocyte

representative HaCaT cells.

13

1.2. MATERIALS AND METHODS

1.2.1. Cell culture and treatment

HaCaT cells (immortalized human keratinocyte cell line) were

maintained in high-glucose Dulbecco’s modified Eagle’s medium

(DMEM, GE Healthcare Life Sciences, Logan, UT, USA) at 37

°C in a humidified incubator containing 5% CO2. The complete

medium was supplemented with 10% fetal bovine serum (FBS,

Thermo Scientific, Logan, UT, USA) and 1%

penicillin/streptomycin (GE Healthcare Life Sciences). Chlorpyrifos

(CPF), dimethyl sulfoxide (DMSO), N-acetyl-cysteine (NAC), and

mitoTEMPO were purchased from Sigma–Aldrich (St. Louis,

MO, USA). Caspase-1 inhibitor

(Ac-Tyr-Val-Ala-Asp-chloromethylketone, Ac-YVAD-CMK) was

purchased from Bachem (Bubendorf, Switzerland). CPF was

dissolved in DMSO to obtain stock solutions of 100 and 500 mM.

NAC and mitoTEMPO were dissolved in autoclaved distilled

water to make stock solutions of 500 mM and 10 mM,

respectively. Caspase-1 inhibitor was dissolved in DMSO to

obtain stock solution of 100 mM. The stock solutions were

diluted to acquire treating concentrations. In this study, cells

were treated with 100 μM CPF for 24 h. NAC 500 μM,

mitoTEMPO 2.5 μM, and caspase-1 inhibitor 25 and 100 μM

were pre-treated for 1 h before CPF was added. Non-treated

14

(only media) cells were used as control. Vehicle control was 0.4%

DMSO for MTT assay or 0.1% DMSO for other assays.

1.2.2. Cell viability assay

The cell viability assay was performed using

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

(MTT, Biosesang, Seongnam, Korea) assay. HaCaT cells were

seeded onto 96-well plate. The cells were treated with CPF from

31.3 μM to 2000 μM (serial dilution) for 24 h. After 20 μL of

MTT solution (5 mg/ml) was added to each well, the plate was

additionally incubated at 37 °C for 2 h with 5% CO2 atmosphere

in the dark. After removing the supernatant, DMSO was added

to each well to solubilize formazan crystals formed by the cells.

The absorbance of each well was measured at 540 nm using a

microplate spectrophotometer reader (Bio-Rad, Richmond, CA,

USA). Result was expressed as a percentage of response in the

control wells.

1.2.3. Measurement of reactive oxygen species

For determining intracellular ROS production, 2′,7′

-dichlorodihydrofluorescein diacetate (H2DCFDA) assay was

performed after treatment with 100 μM of CPF for 24 h.

H2DCFDA (Molecular Probes, Eugene, OR, USA) at 25 μM was

loaded into each samples for 30 min at 37 °C. The cells were

washed with phosphate-buffered saline (PBS), trypsinized and

15

resuspended with 500 μL PBS. The number of fluorescent cells

(cell counts) was analyzed by FACS Aria II flow cytometer (BD,

Franklin Lakes, NJ, USA) (10,000 events per sample) and FlowJo

software (Tree Star Inc., Ashland, OR, USA). Mitochondrial ROS

was measured by MitoSOX™ Red (Molecular Probes). After

treatment, the trypsinized cells were incubated with 5 μM of

MitoSOX at 37 °C for 10 min. Following washing with PBS, the

fluorescent product was resuspended in PBS. The number of

fluorescent cells was analyzed by FACS Aria II flow cytometer

(10,000 events per sample) and FlowJo software. Data were

graphed relative to control.

1.2.4. Glutathione assay

To measure glutathione (GSH)/glutathione disulfide (GSSG)

ratio, luminescence-based GSH/GSSG-Glo™ Assay (Promega)

was proceeded according to the manufacturer’s instructions after

24 h of treatment with 100 μM CPF. The luminescence (RLU) of

GSH and GSSG level was detected by luminometer (Berthold).

GSH/GSSG ratios were calculated from the equation [RLU of

GSH—RLU of GSSG]/[RLU of GSSG/2].

1.2.5. Mitochondrial membrane potential

The change of mitochondrial membrane potential (MMP, ΔΨm)

caused by CPF was assayed by using a membrane-permeant

JC-1 dye (5,5′,6,6′-tetrachloro-1,1′,3,3′

16

-tetraethylbenzimidazol-carbocyanine iodide, Molecular Probes).

JC-1 dye accumulates in mitochondria in a potential-dependent

manner. In healthy cells with a high ΔΨm, JC-1 accumulates in

the mitochondria as J-aggregates with red fluorescence. As the

MMP declines in apoptotic or unhealthy cells, JC-1 cannot

accumulate in the mitochondria and stays in the cytosol as

monomeric form which has a green fluorescence (Koybasi et al.,

2004). After 24 h of 100 μM CPF treatment, 5 μg/ml of JC-1 dye

was added to cells followed by incubation at 37 °C for 30 min.

The cells were washed with PBS and stained with NucBlue Live

ReadyProbes Reagent (Molecular Probes) for nuclei staining. The

alteration in live cells was observed with a confocal laser

scanning microscope (CLSM, LSM710, Carl Zeiss Microscopy

GmbH, Oberkochen, Germany).

1.2.6. Ca2+ measurement with Fluo-4, AM

HaCaT cells were seeded onto 8 well chamber slide. Following

treatment for 24 h, they were stained with MitoTracker Deep

Red at 100 nM for 15 min and Fluo-4, AM at 2 μM (Molecular

Probes) followed by additional incubation at 37 °C for 30 min.

Cells were then washed with PBS and NucBlue Live

ReadyProbes Reagent was added for nuclei staining in live cells.

Images presenting the level of Ca2+ (green fluorescence) were

obtained using the CLSM (LSM710, Carl Zeiss Microscopy

GmbH).

17

1.2.7. Immunofluorescence

Untreated and treated HaCaT cells were washed with PBS and

fixed in 4% paraformaldehyde at room temperature (RT) for 15

min. They were washed again with PBS and permeabilized with

cold methanol/acetone solution (1:1) at RT for 15 min. The cells

were blocked with 3% bovine serum albumin (BSA) in

Tris-Buffered Saline and Tween 20 (TTBS) at RT for 1 h and

incubated at 4 °C overnight with anti-NLRP3 primary antibody

(1/200, AdipoGen, San Diego, CA, USA). Following three washes

with TTBS, a secondary antibody Alexa Fluor 488 goat

anti-mouse IgG (Molecular Probes) was added at RT for 2 h.

The cells were mounted with Fluoroshield™ with DAPI mounting

medium (Sigma–Aldrich). Samples were imaged using confocal

microscopy LSM710.

1.2.8. Western blot analyses

After measuring the protein concentration of each cell lysate

using Pierce™ BCA Protein Assay Kit (Thermo Scientific,

Waltham, MA, USA), equal amounts (25 μg) of each sample

were loaded and separated by 10–12% sodium dodecyl

sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Then,

the proteins were transferred to nitrocellulose membranes by

using iBlot system (Life Technologies, Carlsbad, CA, USA).

Membranes were blocked with 5% skim milk in TTBS at RT for

18

1 h and incubated 4 °C overnight with primary antibodies against

caspase-1 (Casp-1), ASC, Apaf-1, Bcl-2-associated death

promoter (Bad), cytochrome c (Cyt c), actin (Santa Cruz

Biotechnology, Santa Cruz, CA, USA), caspase-9 (Casp-9), or

caspase-3 (Casp-3) (Cell Signaling, Danvers, MA, USA). After

three washes with TTBS, the membranes were incubated with

secondary antibodies conjugated with horseradish peroxide (HRP)

at RT for 2 h. After washing, the bands were detected by using

Image analyzer ATTO EZ Capture MG (ATTO Corp., Tokyo,

Japan). Densitometric analysis was performed by using CS

analyzer 3.0 program (ATTO Corp.) and the band intensities

were expressed relative to control.

1.2.9. Enzyme—linked immunosorbent assay (ELISA)

Secretion of IL-1β into cell culture media was measured by

using ELISA kit (R&D Systems, Minneapolis, MN, USA)

according to the manufacturer's protocol. Briefly, after 24 h of

treatment of each experiments in serum free media, the

supernatants were concentrated by using Amicon® Ultra

centrifugal filter devices (Millipore, Billerica, MA, USA). The

samples and the standard solution were added for 2 h, human

IL-1β conjugate for 1 h, and substrate solution for 20 min to the

wells. The wells were washed three times between each step.

After adding the Stop Solution, the absorbance of each well was

detected by a microplate spectrophotometer reader (Bio-Rad). The

19

amount of IL-1β (in pg/ml) was calculated based on the

standard curve obtained from the same experiment.

1.2.10. AnnexinV-FITC/PI apoptosis assay

Pyroptosis and apoptosis were measured with EzWay Annexin

V-FITC Apoptosis Detection kit (Koma Biotech, Seoul, Korea).

After each treatment, cells were detached and stained with

Annexin V-FITC for 15 min, and then propidium iodide (PI) was

added before analyzing by flow cytometry. The fluorescent

intensity of the cells (10,000 events/sample) was measured by

using FACS Aria II. Percent of the stained cells were graphed

according to PI positivity as pyroptosis and Annexin V-FITC

positivity as a measure of total apoptosis.

1.2.11. Terminal deoxynucleotidyl transferase dUTP nick end

labeling (TUNEL) assay

Apoptotic cells were visualized by DeadEnd™ Colorimetric

TUNEL System (Promega). TUNEL assay was based on the

ability of terminal deoxynucleotidyl transferase (TdT) to label the

ends of broken double-stranded DNA to detect apoptotic cells

with DNA degradation during apoptosis (Kyrylkova et al., 2012).

After 24 h of CPF treatment, TUNEL assay was performed

according to the manufacturer’s protocol. Images were obtained

by using a microscope (Nikon Eclipse Ti-S, Melville, NY, USA).

The degree of apoptosis was evaluated as ratio of the number of

20

apoptotic nuclei (TUNEL positive) to the total nuclei.

1.2.12. LDH assay

The level of lactate dehydrogenase (LDH) was measured by

using CytoTox 96® NonRadioactive Cytotoxicity Assay

(Promega). Cells were pretreated with 25, 100 μM caspase-1

inhibitor (Ac-YVAD-CMK) or 2.5 μM mitoTEMPO for 1 h

followed by treatment with 100 μM CPF for 24 h. The

supernatants (100 μL/well) were transferred into a new 96 well

plate and CytoTox 96® Reagent (LDH reaction solution) was

added to each well. After incubating the plate at RT for 30 min

in the dark, Stop Solution was added and the absorbance was

then recorded at 490 nm by using a microplate spectrophotometer

(Bio-Rad). The result was expressed as a percentage of control.

1.2.13. Statistical analysis

The statistical significance of the results was evaluated by

Student’s t-test and one-way ANOVA with Tukey’s multiple

comparison test using GraphPad Prism 5 Software (San Diego,

CA, USA). p < 0.05 was considered statistically significant

compared to the respective control group (*p < 0.05, **p < 0.01,

and ***p < 0.001).

21

1.3. RESULTS

1.3.1. Cytotoxicity and oxidative stress induced by CPF in

HaCaT cells

Based on MTT assay, the cell viability declined in a

d.ose-dependent manner after exposure to CPF for 24 h (Fig.

1.1A). This result showed that CPF had an inhibitory effect on

HaCaT cells. The IC50 value of CPF was estimated to be 820 μM.

For mechanism studies, 100 μM, approximately 10% of IC50, was

selected for the subsequent experiments so that the viability of

HaCaT cells would not be directly impacted by CPF significantly.

To identify whether CPF could induce oxidative stress to skin

keratinocyte HaCaT, we measured intracellular ROS generation

with H2DCFDA. H2DCFDA is known for detecting general ROS

including hydroxyl radical, hydrogen peroxide, peroxynitrate, and

peroxyl radical (Dinnen et al., 2013). CPF augmented the number

of ROS-generated cells based on fluorescent intensity changes in

flow cytometry compared to the vehicle control (Fig. 1.1B,C). In

addition, glutathione (GSH) and glutathione disulfide (GSSG)

levels were quantified to confirm the effect of CPF on ROS

generation. Because GSH is reversibly oxidized to GSSG under

oxidative conditions, the GSH/GSSG ratio is used to measure

ongoing oxidative stress (Campochiaro et al., 2015). The

glutathione to oxidized glutathione ratio was significantly

22

decreased in HaCaT cells after CPF treatment (Fig. 1.1D). These

data indicated that CPF could affect ROS generation and alter

the redox status of HaCaT cells.

23

24

Fig. 1.1. Effects of CPF on cell viability and oxidative stress of

HaCaT cells. (A) The cell viability was determined with MTT

assay and presented as percent of control. The cell viability was

decreased in a concentration-dependent manner at 24 h. (B)

Intracellular ROS was assessed by H2DCFDA using flow

cytometry. The ROS level was enhanced after treating cells with

100 μM CPF for 24 h. (C) Histogram represents DCF

fluorescence level relative to control. (D) The redox status

change by 100 μM CPF was determined by the GSH/GSSG

assay. Each result is represented as mean ± standard error of

the mean (SEM) (n ≧ 3, *p < 0.05, **p < 0.01 and ***p <

0.001 versus vehicle control (Veh. Cont.)).

25

1.3.2. Impact on mitochondrial damage and mitochondrial

ROS

In order to identify mitochondrial damage by CPF, ΔΨm loss of

HaCaT cells was detected by JC-1 dye staining. JC-1 exhibits

red fluorescence in healthy cells (high ΔΨm) and green

fluorescence in MMP-disrupted cells (low ΔΨm) (Castedo et al.,

2002). After treatment with CPF, the fluorescence shown by

confocal images was shifted from red to green indicating

alteration of ΔΨm in HaCaT cells (Fig. 1.2A). Oxidative stress

causes Ca2+ influx to the cytoplasm and mitochondria (Ermak and

Davies, 2002) and mitochondrial ROS could induce Ca2+ increase

in cells (Waypa et al., 2002). To examine the change of

intracellular and mitochondrial Ca2+, cells were stained with

MitoTracker deep red and Fluo-4, AM. As shown by CLSM,

Fluo-4, AM green fluorescence was increased in the cytosol of

CPF treated cells compared to that in the controls. Merged form

of green and MitoTracker deep red was also detected in CPF

treated cells (Fig. 1.2B). Therefore, to confirm whether CPF

induced mitochondrial ROS, mitoSOX was used as a

mitochondrial ROS indicator. Based on flow cytometry, mROS

generation was significantly increased in HaCaT cells after

exposure to 100 μM CPF for 24 h compared to that in the

controls (Fig. 1.2C,D). These results demonstrated that CPF could

damage mitochondria and induce mitochondria-related ROS.

26

27

Fig. 1.2. Increase of mitochondria damage and mitochondrial ROS

induced by CPF. (A) JC-1 dye staining showed alteration of

MMP induced by 100 μM CPF for 24 h. Each scale bar of the

confocal images represents 10 μm. (B) To detect intracellular Ca2+

level after treatment, the cells were stained with Fluo-4, AM

(green) and MitoTracker deep red (red) and then imaged by

CLSM. Ca2+ levels in the cytosol and mitochondria were

increased by CPF (scale bar = 10 μm). (C) Increased

mitochondrial ROS generation in CPF-treated HaCaT cells was

identified using MitoSOX and analyzed by flow cytometry. (D)

Histogram represents the altered fluorescence relative to control.

Each result is represented as mean ± SEM (n = 3, ***p < 0.001

vs. Veh. Cont.).

28

1.3.3. Increase of NLRP3 inflammasome in CPF treated

HaCaT cells

We next examined whether CPF could induce NLRP3

inflammasome in HaCaT cells through immunofluorescence

analysis. The intensity of NLRP3-green fluorescence was

conspicuously enhanced in CPF-treated cells compared to that in

the controls. And the cells exposed to CPF and NAC 500 μM,

general ROS scavenger, showed down-regulated effect (Fig.

1.3A). Furthermore, changed protein expression for components of

NLRP3 inflammasome was analyzed. Caspase-1 and ASC were

up-regulated in CPF-treated cells, which was negated when

pre-treated NAC, and was in agreement with the confocal

microscopy results (Fig. 1.3B). The Western blot analyses were

quantified by densitometry (Fig. 1.3C). It has been reported that

pro IL-1β is cleaved and IL-1β is secreted from cells as a

proinflammatory cytokine after NLRP3 inflammasome is activated

(Caicedo et al., 2009). Therefore, we measured the amount of

IL-1β secreted into the cell culture media using ELISA. Secreted

IL-1β was significantly increased by CPF treatment and

diminished in presence of NAC (Fig. 1.3D). These data suggested

that NLRP3 inflammasome could be stimulated by CPF in HaCaT

cells and that the inhibition of ROS could protect cells against

the damage.

29

30

Fig. 1.3. The induction of NLRP3 inflammasome in CPF treated

HaCaT cells and the reversal by ROS scavenger NAC. (A)

Immunofluorescence analysis was performed with NLRP3

antibody (green) and DAPI (blue) for nuclei staining. The

confocal images showed increased expression of NLRP3 caused

by 100 μM CPF and it was reduced by 500 μM NAC (scale bar

= 10 μm). (B) Western blot was conducted with each cell lysate

to determine increased expression of caspase-1 and ASC. Actin

was used as a loading control. (C) Densitometric analysis of

three independent experiments. (D) The change of secreted IL-1β

in media was detected using ELISA. The level of IL-1β was

increased after treatment with 100 μM CPF for 24 h and it was

reversible by pre-treatment with 500 μM NAC. Each result is

represented as mean ± SEM (n ≧ 3, *p < 0.05, **p < 0.01 vs.

Veh. Cont. and #p < 0.05 vs. CPF)

31

1.3.4. Reversed induction of NLRP3 inflammasome by mROS

scavenger, mitoTEMPO

To demonstrate the effect of mROS on NLRP3 inflammasome

caused by CPF, we used mitoTEMPO, a mitochondrial-targeted

antioxidant, to treat cells. MitoTEMPO is a highly positively

charged TEMPO derivative that is concentrated in the

mitochondria matrix where it acts as a superoxide scavenger (Liu

et al., 2010). After 24 h treatment, MitoSOX staining revealed

noticeable decrease of mROS generation in mitoTEMPO 2.5 μM

pre-treated cells compared to cells with CPF treatment alone

(Fig. 1.4A,B). Based on immunofluorescence analysis, the

activation of NLRP3 by CPF was profoundly inhibited in

response to mitoTEMPO (Fig. 1.4C). Concordantly, we determined

the changed protein levels with Western blot. Our results

revealed that mitoTEMPO treatment caused down-regulation of

caspase-1 and ASC induced by CPF (Fig. 1.4D,E). To confirm

the effect of mitoTEMPO on IL-1β production, IL-1β ELISA

was performed. Significant inhibition of IL-1β secretion was

detected from mitoTEMPO pre-treated media and this further

demonstrated that CPF-induced activation of NLRP3

inflammasome was reversed by the reduction of mROS (Fig.

1.4F). Overall, the NLRP3 inflammasome could be triggered as a

result of CPF-induced mROS. Our results indicate that

CPF-induced mROS plays an important role in triggering NLRP3

inflammasome and secreting inflammatory cytokine. In addition,

32

mitoTEMPO could act as a protectant against CPF-induced

mROS.

33

34

Fig. 1.4. Diminished CPF induced-NLRP3 inflammasome in

response to mROS scavenger, mitoTEMPO. (A) HaCaT cells

were incubated with 100 μM CPF and 2.5 μM mitoTEMPO. After

24 h, the change in mROS generation was examined using

MitoSOX staining, and analyzed by flow cytometry. (B)

Histogram represents the altered fluorescence relative to control.

(C) Immunofluorescence analysis was performed with NLRP3

antibody (green) and DAPI (blue) for nuclei staining and imaged

with confocal microscope. The increased expression of NLRP3 by

CPF was reversed in the presence of 2.5 μM mitoTEMPO (scale

bar = 10 μm). (D) The protein levels of caspase-1 and ASC in

each cell lysates were determined by Western blot. The

decreased levels of each were detected after mitoTEMPO

treatment. Actin was used as a loading control. (E) Densitometric

analysis of three independent experiments. (F) From IL-1β

ELISA on each culture media, mitoTEMPO treatment showed

inhibition of IL-1β secretion compared to the CPF only-treated.

Each result is represented as mean ± SEM (n ≧ 3, *p < 0.05,

**p < 0.01 and ***p < 0.001 vs. Veh. Cont. and ##p < 0.01 and

###p < 0.001 vs. CPF).

35

1.3.5. Pyroptosis and apoptosis in CPF-treated HaCaT cells

We next investigated the programmed cell death of HaCaT cells

that could be mediated by mROS and the inflammasome resulting

from CPF exposure. In programmed cell death, pyroptosis and

apoptosis could be brought about by an increase of ROS and

oxidative stress (Fleury et al., 2002; Chung et al., 2012). To

determine whether pyroptosis and apoptosis could be induced by

CPF in HaCaT cells, Annexin V-FITC/PI assay was performed

and analyzed by FACS. Pyroptosis is associated with rapid loss

of cell membrane integrity, while apoptosis is characterized by

maintenance of membrane integrity and externalization of

phosphatidylserine onto the cell surface (Sagulenko et al., 2013).

Therefore, cells that stained positive to PI indicates pyroptosis.

Cells undergoing apoptosis stain positively with Annexin V-FITC

and remain negative to PI staining. However, in the end stage of

apoptosis, cells stain positively to both Annexin V-FITC and PI.

Both pyroptosis assessed by increase of PI-positive cells (upper

panel) and apoptosis assessed by increase of Annexin

V-FITC-positive cells (right panel) were detected in CPF-treated

HaCaT cells compared to the controls (Fig. 1.5A,B). Next,

changes in expression levels of pro-apoptotic proteins were

determined with Western blot analyses to confirm apoptosis. In

the presence of CPF, Apaf-1, Casp-9, Casp-3, Bad, and Cyt c

related in the apoptotic pathways showed increased expression

(Fig. 1.5C,D). In order to confirm that pyroptosis and apoptosis of

36

HaCaT cells were caused by CPF, we performed TUNEL assay

that has been used to determine the localization of apoptotic

DNA fragmentation and chromatin condensation in situ (Gavrieli

et al., 1992). TUNEL assay can visualize nuclear DNA cleavage

during pyroptosis and apoptosis (Bergsbaken and Cookson, 2007).

In CPF treated cells, the amount of apoptotic nuclei (dark brown)

was noticeably increased compared to that in the controls (Fig.

1.5E). The ratio of apoptotic nuclei to total nuclei was quantified

from each of the three fields (Fig. 1.5F). These results signified

that HaCaT cells may undergo programmed cell death including

pyroptosis activated by NLRP3 inflammasome and apoptosis

mediated by mitochondrial intrinsic pathway after treatment with

CPF.

37

38

Fig. 1.5. The effects of CPF on pyroptosis and apoptosis. (A)

Levels of pyroptosis and apoptosis were measured by Annexin

V-FITC/PI assay using flow cytometry. Pyroptotic (PI positive)

and apoptotic (Annexin V-FITC positive) cells were increased

after CPF exposure for 24 h. (B) Histograms represent percent of

pyroptotic cells (upper panel) and percent of total apoptotic cells

(right panel). (C) Western blot was conducted using Apaf-1,

Casp-9, Casp-3, Bad, and Cyt c antibodies. Actin was used as a

loading control. The pro-apoptotic proteins were up-regulated by

100 μM CPF. (D) Densitometric analysis of three independent

experiments. (E) Apoptosis induced by CPF was visualized by

the TUNEL assay. The arrows indicate apoptotic nuclei. (F)

Increased amount of apoptotic staining in CPF treated cells was

represented as the ratio of apoptotic nuclei to total nuclei. Each

result is represented as mean ± SEM (n = 3, *p < 0.05, **p <

0.01 and ***p < 0.001 vs. Veh. Cont.).

39

1.3.6. Effect of caspase-1 inhibitor and mitoTEMPO on

CPF-induced pyroptosis and apoptosis

To further identify how CPF provoked pyroptosis and apoptosis,

we analyzed the effect of CPF in presence of caspase-1 inhibitor

and mitoTEMPO. Pyroptosis, which is a form of inflammatory

cell death, can trigger cell lysis and lead to release of LDH

(Wellington et al., 2014). CPF treated cells resulted in increased

release of LDH, while in the presence of caspase-1 inhibitor 25,

100 μM or mitoTEMPO 2.5 μM, the cells had reversed effect

detected by the diminished release of LDH to cell culture media.

From this data, we ascertained that CPF could trigger pyroptosis

via caspase-1 pathway and mROS generation (Fig. 1.6A).

Annexin V-FITC/PI assay was measured by flow cytometry to

verify whether pyroptosis and apoptosis occurred through

CPF-induced mROS. Populations of both pyroptotic (PI positive,

upper panel) and total apoptotic cells (AnnexinV-FITC positive,

right panel) were reduced in the existence of mitoTEMPO

compared to CPF-only treated cells (Fig. 1.6B,C). To reinforce

the impact of mitoTEMPO on antiapoptotic effect, we

demonstrate the changed protein expression from Western blot

analysis. In agreement with above data, the expression levels of

pro-apoptotic proteins, Apaf-1, Casp-9, Casp-3, and Bad that

were induced by CPF, were down-regulated in response to

mitoTEMPO (Fig. 1.6D,E). Overall, the roles of casp-1 and

mROS in pyroptosis and apoptosis induced by CPF were

40

confirmed by these experiments. Moreover, caspase-1 inhibitors

or mROS scavengers were identified for preventing inflammation

and cell death in CPF-mediated damage.

41

42

43

Fig. 1.6. Changes of pyroptosis and apoptosis in the presence of

caspase-1 inhibitor and mitoTEMPO. (A) Pyroptosis is

characterized by cell lysis and LDH release. Increased LDH

release was measured from the supernatant of CPF treated cells

and the reversed effect was detected in the presence of

caspase-1 inhibitor (Cas-1 inh.) or mitoTEMPO. (B) Annexin

V-FITC/PI assay was performed to identify the decrease of

pyroptosis and apoptosis after treatment in the presence of 2.5 μ

M mitoTEMPO. (C) Histograms represent percent of pyroptotic

cells (PI positive) and percent of total apoptotic cells (Annexin

V-FITC positive). (D) To support the anti-apoptotic effect of

mitoTEMPO, the changed expression of pro-apoptotic proteins

was determined by Western blot. The induced proteins by CPF

were down-regulated by mitoTEMPO treatment. (E)

Densitometric analysis of three independent experiments. Each

result is represented as mean ± SEM (n ≧ 3, *p < 0.05, **p <

0.01 and ***p < 0.001 vs. Veh. Cont., #p < 0.05, ##p < 0.01 and

###p < 0.001 vs. CPF).

44

1.4. DISCUSSION

With increased industrialization and urban growth and an

increased demand from agriculture, harmful chemicals (especially

pesticides) are released into the environment that endanger public

health (Sharpe and Irvine, 2004). Humans have been exposed to

pesticides and experience negative effects of pesticides such as

dermatitis, respiratory disease, etc. Induction of oxidative stress

linking pesticide exposure to health effects has been reported in

farmworkers who have used organophosphates including

chlorpyrifos and biomarkers of the oxidative stress may provide

the link for pesticide contact to a number of health issues (Muniz

et al., 2008). The oxidative stress and abnormal increase of ROS

can trigger a cascade of subsequent toxic pathways associated

with disease development.

Skin exposure accounts for about 90% of the exposure from

pesticide users (Abhishek et al., 2014). In addition to its role as a

physical barrier, skin takes a part in immune defense where the

innate immune system plays an important role. The epithelial

cells of the skin can secrete various inflammatory cytokines to

stimulate immune cells (Kupper and Fuhlbrigge, 2004). In

particular, epidermal keratinocytes as part of the outermost layer

of the skin are directly exposed to pesticides and may cause side

effects. Thus, it is valuable to study dermal toxicity of pesticide

45

at the main exposure site. However, pesticides toxicity towards

keratinocytes has been inadequately studied particularly with

regard to innate immune response related to the inflammasome

pathways. This study was designed to identify innate immune

activation via mitochondrial oxidative stress occurred by

chlorpyrifos, a widely-used and common pesticide. To study skin

exposure, we used the HaCaT skin keratinocyte cell line. CPF

induced ROS with activation of NLRP3 inflammasome and

resulted it in programmed cell death. Additionally, attenuated

effects of mROS production on this pathway was demonstrated

by using a mitochondria-targeted antioxidant, mitoTEMPO. Our

results suggest that the induction of inflammasome is a possible

mechanism of dermatitis when CPF has contact the skin.

Several studies have reported that CPF is able to induce

oxidative stress and altered GSH/GSSG ratio as part of a

defense process (Crumpton et al., 2000; Verma et al., 2007). In

this study, we found that CPF could cause ROS production in

keratinocytes. Intracellular ROS generation was increased and

GSH/GSSG ratio was decreased in response to CPF. These

results indicate that CPF has an impact on oxidative stress and

cellular redox status in HaCaT cells. Since mitochondria play an

important role in ROS generation and apoptosis induction

(Kasahara et al., 2011) and mitochondria are vulnerable to

xenobiotics, we determined whether CPF could lead to

mitochondrial damage and increase mitochondrial ROS. Altered

46

MMP and increased Ca2+ levels by CPF raised the possibility of

mROS generation. Our results showed that CPF induced mROS

in HaCaT cells and imply that CPF-induced toxicity can be

mediated by mitochondrial oxidative stress.

Increased ROS influences various physiological and pathological

processes including inflammation. An important mechanism of

inflammation is the induction of inflammasome that activates

caspase-1 and processes inflammatory cytokines (Green et al.,

2011). Innate immune response to cellular damage and

pathological insult is interrelated with mitochondria and mROS, a

common activator of the NLRP3 inflammasome (West et al.,

2011). In addition, a recent study suggests that mitochondria

targeting agents can induce mROS and enhance NLRP3

inflammasome associated with cellular redox status (Jabaut et al.,

2013). In this study, we investigated the effect of CPF on NLRP3

inflammsome. CPF increased the expression level of NLRP3,

caspase-1 and ASC in HaCaT cells. The activated NLRP3

inflammasome caused secretion of IL-1β detected. To evaluate

the role of mROS induced by CPF on NLRP3 inflammasome, we

used mitochondria-targeted ROS scavenger mitoTEMPO.

MitoTEMPO reduced the mROS produced by CPF and decreased

the formation of NLRP3 inflammasome. A previous study

reported that activation and secretion of IL-1β processed by

inflammasome can mediate innate immunity and skin contact

hypersensitivity by skin sensitizers (Watanabe et al., 2007).

47

Therefore, our results suggest that CPF can induce the

inflammasome and subsequent innate immune response through

mitochondrial oxidative stress and promoting immune response

such as hypersensitivity and dermatitis.

Inflammasome can also promote pro-inflammatory or pro-death

functions of caspase-1. The activated caspase-1 can trigger

inflammatory type of programmed cell death called pyroptosis

(Tait et al., 2014). Besides, mROS is to contribute to programmed

cell death (Fleury et al., 2002). Mitochondria-generated ROS may

play a crucial role in increase of pro-apoptotic proteins, which

can trigger the activation of caspase cascade and apoptosis (Ott

et al., 2007). It has been reported that CPF induced apoptosis in

PC 12 cell line (Lee et al., 2012) and mouse retina in vivo (Yu et

al., 2008) through ROS generation and that it was attenuated by

antioxidants. In this study, we showed that CPF could induce

pyroptosis and apoptosis in HaCaT cells. The cell lytic pyroptosis

was inhibited by caspase-1 inhibitor. And mitoTEMPO-treated

cells were able to reverse the impact of CPF on cell death. This

is in agreement with a previous study showing that treatment of

mitoTEMPO could attenuate mitochondrial apoptosis (Liang et al.,

2010). Increased expression of Apaf-1 and caspase-9 revealed

that CPF could induce the intrinsic pathway of apoptosis through

apoptosome activation. The apoptosome has been reported to be

triggered by cytochrome c, thus promoting the activation of

caspase-9 through Apaf-1 (Zou et al., 1999). Interestingly,

48

inflammasome and apoptosome have similar characteristics.

Similar to inflammasome, apoptosome is a macromolecular

complex interacting through CARD domains. It serves as an

activator of caspases as a response to cellular damage (Gross et

al., 2011). Taken together, these results indicate that caspase-1

processed by the inflammasome may be involved in another

pathway of programed cell death and that mitochondrial-produced

ROS plays an important role in pyroptosis and apoptosis

triggered by CPF.

In conclusion, our research first demonstrated that the exposure

of CPF could provoke NLRP3 inflammasome and programmed cell

death through the induction of mitochondrial oxidative stress in

human skin keratinocyte. Through this pathway, innate immune

response may be activated and subsequent cutaneous

inflammation could develop after skin exposure to CPF pesticide.

More detailed in vivo studies may be necessary to explore the

mechanisms of innate immunity and dermatitis induced by CPF.

Furthermore, our results revealed that agents including caspase

inhibitors or ROS scavengers might be used as a new class of

anti-inflammatory therapeutics to repair damages caused by

pesticides.

49

Chapter Ⅱ

Trifloxystrobin induces tumor necrosis

factor-related apoptosis-inducing ligand

(TRAIL)-mediated apoptosis in HaCaT,

human keratinocyte cells

(Published in 2016, Drug and Chemical Toxicology)

50

ABSTRACT

As the outermost layer of the body, the skin plays an important

role in exposure to pesticides, which could have negative impacts

on human health. Trifloxystrobin is a widely used fungicide of

the strobilurin class, however, there is little information regarding

the skin contact-associated toxic mechanism. Therefore, the

present study was performed in order to identify the skin toxicity

mechanism of trifloxystrobin using HaCaT (keratinocyte of

human skin) cells. Following 24 or 48 h treatment, cell viability,

and subsequent Annexin V-FITC/propidium iodide assay, TUNEL

assay and Western blotting were performed to investigate the

cell death mechanism of trifloxystrobin. Exposure to

trifloxystrobin resulted in diminished viability of HaCaT cells in

both a time- and concentration-dependent manner. The cell death

was derived through apoptotic pathways in the HaCaT cells.

Furthermore, we explored the effect of trifloxystrobin on

TRAIL-mediated extrinsic apoptosis using siRNA transfection.

Knockdown of death receptor 5 suppressed trifloxystrobin-

provoked apoptosis. These results indicate that trifloxystrobin

induces TRAIL-mediated apoptosis and has an inhibitory effect in

keratinocytes that can interfere with the barrier function and

integrity of the skin. This could be proposed as a mechanism of

skin toxicity by trifloxystrobin and considered in the management

of pesticide exposure.

51

2.1. INTRODUCTION

Pesticides have been beneficially utilized to control agricultural

and residential pests in order to improve crop yields and public

health. However, these substances are not species-specific and

non-target organisms including humans can also be affected by

exposure (Sooresh et al., 2015). A number of adverse effects

from using pesticides can hinder public health. For instance,

multiple studies have reported that skin inflammation such as

allergic contact dermatitis was triggered in farm workers (Koch,

1996). Thus, it is important to perform toxicological tests and to

assess the risks of pesticide use.

Many different fungicides are applied worldwide every year.

Numerous cases causing skin irritation and cutaneous diseases

were more closely related to the exposure to fungicides rather

than insecticides, herbicides, fumigants (Lisi, 1992; Penagos, 2002;

Roberts and Reigart, 2013). Among the important fungicides,

trifloxystrobin (TRF) is a widely used, systemic, broad-spectrum

strobilurin-derived fungicide that plays an important role in the

disease management in agriculture around the world. As such,

trifloxystrobin could be toxic to non-target species including

eukaryotes, and it has been reported to induce allergic reaction

and dermal sensitization (Paranjape et al., 2015; US-EPA, 1999).

However, there is a lack of studies on the mechanism of

52

fungicides toxicity on the skin. Therefore, it is necessary to

study how fungicides have effects on skin after the exposure.

The epidermis of the integumentary system is the most direct

and important organ in the exposure of pesticides. Although

keratinocytes, as the outermost and major cells of the epidermis,

provide the initial barrier and protect the body from the external

environment (Denecker et al., 2007), they are frequently damaged

by exposure to toxicants including pesticides. Apoptosis could be

initiated by a range of physiological and pharmacological stimuli

and play a role in the disturbed homeostasis and function of the

skin (Henseleit et al., 1996; Lippens et al., 2009).

Among the cell death mechanism, apoptosis is regulated by two

classes of pathways. One is an extrinsic pathway that initiates a

signal cascade by binding extracellular ligands to pro-apoptotic

death receptors (DRs) located at the cell membrane and the other

is an intrinsic pathway that is evoked by the intracellular

environment depending on the presence of mitochondrial

pro-apoptotic factors (Gonzalvez and Ashkenazi, 2010). In the

extrinsic pathways, tumor necrosis factor-related

apoptosis-inducing ligand (TRAIL)-mediated apoptosis has

recently been cast in the limelight. TRAIL is a member of the

tumor necrosis factor (TNF) family and is produced by a wide

range of normal cells. TRAIL-mediated apoptosis is derived by

interactions between its death receptors, which are composed of

TRAIL-R1 (DR4), TRAIL-R2 (DR5), decoy receptor (DcR) 1,

53

DcR2, and a soluble receptor named osteoprotegerin. Among

these receptors, only DR4 and DR5 contain the death domain on

the intracellular face. After binding with the ligand, they lead to

the apoptotic signaling pathway via the formation of a

death-inducing signaling complex (Wang and El-Deiry, 2003).

Subsequently, caspase-8 is recruited and autoactivated which

initiates apoptosis through the activation of downstream effector

caspases such as caspase-3, -6, and -7 (Wehrli et al., 2000). The

extrinsic apoptosis in the epidermis has been reported to be

involved with several skin diseases such as atopic dermatitis and

contact hypersensitivity (Wehrli et al., 2000).

The toxicity mechanism of trifloxystobin on the skin has not

yet been sufficiently elucidated. Therefore, the objective of the

present study was to investigate the manner in which

trifloxystrobin affects skin cells following exposure. We examined

the impact of trifloxystrobin on TRAIL-mediated apoptosis using

DR5 siRNA in HaCaT, human skin keratinocyte cell line. Our

data suggest that the fungicide trifloxystrobin sensitizes

keratinocytes to TRAIL-mediated apoptosis, which could trigger

cutaneous diseases via a pathophysiological response.

54

2.2. MATERIALS AND METHODS

2.2.1. Cell culture and treatment

HaCaT is the human immortalized skin keratinocyte cell line.

The cells were cultured in high-glucose Dulbecco's modified

Eagle's medium (DMEM) complete medium (GE Healthcare Life

Sciences, Logan, UT) containing 10% fetal bovine serum

(Thermo Scientific, Logan, UT) and 1% penicillin/streptomycin

(GE Health Care Science,) in a 5% CO2 incubator at 37 °C.

Trifloxystrobin (TRF) and dimethyl sulfoxide (DMSO) were

purchased from Sigma-Aldrich (St. Louis, MO). TRF was

dissolved in DMSO to produce stock solutions of 1 and 500 

mM. The stock solutions were diluted to obtain treatment

concentrations. In this study, the cells were exposed to 0.05%

DMSO as a vehicle control (Veh. Cont.) and 0.5 μM TRF for 24

or 48 h.

2.2.2. Cell viability assay

Cell viability assay was measured using

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

(MTT, Biosesang, Seongnam, Korea) assay. About 4000 cells per

well were seeded on 96-well plates. Cells were treated with

medium as a control and 0.1% DMSO as a vehicle control. TRF

was serially diluted from 500 μM to 2 μM. After 24 or 48 h, 20

55

μl of MTT solution (5 mg/ml) was added to each well. Following

additional incubation in the dark for 2 h at 37 °C, the

supernatants were removed and DMSO was added to dissolve

the formed formazan crystals. The absorbance of the samples

was detected at 540 nm using a microplate spectrophotometer

(Bio-Rad, Richimond, CA). The values were calculated as a

percentage of the control.

2.2.3. Annexin V-FITC/PI apoptosis assay

Detection of apoptosis was performed using the EzWay Annexin

V-FITC apoptosis detection kit (Koma Biotech, Seoul, South

Korea). For flow cytometry analysis, following 48 h of each

treatment, cells were trypsinized and stained with both Annexin

V-FITC and propidium iodide (PI) or Annexin V-FITC only. The

fluorescence staining of the cells was measured using a BD

FACS Calibur (BD Biosciences, San Jose, CA) (10,000 events per

sample). For confocal imaging, after 48 h of the treatment, the

cells were washed and stained with Annexin V-FITC and PI.

The nuclei were stained with NucBlue® Live ReadyProbes™

Reagent (Molecular Probes, Eugene, OR) containing Hoechst

33342 which binds to DNA and is used to identify the condensed

nuclei in apoptotic cells. The images were obtained using a

confocal laser scanning microscope (CLSM, LSM710, Carl Zeiss

Microscopy GmbH, Oberkochen, Germany).

56

2.2.4. Terminal deoxynucleotidyl transferase dUTP nick end

labeling (TUNEL) assay

Apoptotic cells were detected by the TUNEL assay using a

DeadEnd™ Colorimetric TUNEL System kit (Promega, Madison,

WI). After 48 h of treatment with 0.5 μM TRF, the TUNEL

assay was performed according to the protocol of the

manufacturer. The TUNEL assay can detect apoptotic cells in

situ based on the measurement of nuclear DNA fragmentation

and chromatin condensation using biotinylated nucleotide and

terminal deoxynucleotidyl transferase (TdT) to label the 3′-OH

DNA ends of double-stranded DNA breaks. The images were

obtained using a microscope (Nikon Eclipse Ti-S, Melville, NY).

2.2.5. Analysis of protein expression

The protein concentration of each cell lysates was measured

using a PierceTM BCA protein assay kit (Thermo Scientific,

Waltham, MA). Equal amounts of proteins were loaded onto 10–

12% sodium dodecyl sulfate-polyacrylamide gels and separated by

electrophoresis. Subsequently, using an iBlot system (Life

Technologies, Carlsbad, CA), the proteins were transferred to a

nitrocellulose membrane. The membranes were blocked in 5%

skimmed milk for 1 h at room temperature (RT) and incubated

overnight at 4 °C with primary antibodies against apoptotic

protease activating factor 1 (Apaf-1), cytochrome c (Cyt c), actin

(Santa Cruz Biotechnology, Santa Cruz, CA), Procaspase-9

57

(Procasp-9), Procaspase-8 (Procasp-8), cleaved casp-8,

Procaspase-3 (Procasp-3), cleaved casp-3, poly (ADP-ribose)

polymerase (PARP), cleaved PARP (Cell Signaling, Danvers,

MA), and DR5 (Enzo Life Sciences, Farmingdale, NY). After

washing the membranes with Tris-buffered saline containing

Tween 20 (TTBS), the membranes were incubated with the

corresponding horseradish peroxide (HRP)-conjugated secondary

antibodies for 2 h at RT. The protein bands were developed

using LuminataTM Forte Western HRP substrate (Millipore,

Billerica, MA) and an ATTO EZ Capture MG (ATTO Corp.,

Tokyo, Japan) image analyzer. The densitometric analysis of each

band was conducted using the CS analyzer 3.0 software (ATTO

Corp., Tokyo, Japan).

2.2.6. Immunofluorescence

HaCaT cells were seeded on an 8-well chamber. Following 48 h

of the treatment, cells were incubated with MitoTracker red

(Invitrogen, Waltham, MA) at 37 °C for 30 min. The cells were

then washed with PBS, fixed in 4% paraformaldehyde at RT for

15 min and permeabilized with methanol/acetone 1:1 solution at

RT for 10 min. Subsequently, the cells were blocked with 3%

bovine serum albumin (BSA) at RT for 1 h, and then incubated

with cyt c antibody at 4 °C overnight. After washing with

TTBS, Alexa Fluor-488 (green) goat anti-mouse IgG (Molecular

Probes, Eugene, OR) was used as a secondary antibody at RT

58

for 2 h. The sample was mounted with Fluoroshield™ mounting

medium containing DAPI (Sigma-Aldrich, St. Louis, MO). The

images were obtained using CLSM (LSM710).

2.2.7. siRNA transfection

HaCaT cells were suspended and transfected with 500 nM

siRNA per 2.5×105 cells using a Neon® transfection system

(Invitrogen, Waltham, MA) based on electroporation technology.

The following day, the cells were treated with 0.05% DMSO or

0.5 μM TRF. Scrambled siRNA (siScr) was purchased from

Genolution Pharmaceuticals (Seoul, South Korea) and DR5 siRNA

was purchased from Bioneer (Daejeon, South Korea). The DR5

siRNA sequence was sense 5′-CAGACUUGGUGCCCUUUGA-

3′ and antisense 5′-UCAAAGGGCACCAAGUCUG-3′.

2.2.8. Statistical analysis

The statistical significance of the data was determined by

Student’s t-test and one-way ANOVA followed by the Newman

–Keuls post-hoc test, using the Graphpad Prism 5 Software

(San Diego, CA). p < 0.05 was considered statistically significant

to the compared group.

59

2.3. RESULTS

2.3.1. Trifloxystrobin reduces the viability of HaCaT cells

The cytotoxic effect of TRF on HaCaT cells was assessed

using MTT assay. The cell viability decreased in a

concentration-dependent manner. In the exposure for 48 h, the

cells were drastically inhibited by TRF (Fig. 2.1). The IC50 value

at 24 h was calculated to be 22.9 μM, and that at 48 h was 5.14

μM. With respect to toxicological mechanism study, based on

preliminary study and the IC50 value, 0.5 μM TRF was

administered in subsequent experiments. The 0.1% DMSO did not

affect the viability of cells compared with control. Thus, in this

study, 0.05% DMSO was used as a vehicle control. These data

indicate that TRF induces cell death and inhibitory effect on

HaCaT cells, and thus, we focused on the type of TRF-induced

cell death.

60

Fig. 2.1. The effect of trifloxystrobin on cell viability. HaCaT

cells were treated with TRF from 500 to 2 μM for 24 or 48 h.

The viability was subsequently measured using an MTT assay.

TRF showed both concentration- and time-dependent inhibitory

effects. The IC50 values at 24 and 48 h were 22.9 and 5.14 μM,

respectively. The results were processed as a percentage of the

control, which is indicated to be 100%. Each value is represented

as the mean ± standard error of the mean (SEM).

61

2.3.2. Apoptosis is induced in HaCaT cells upon exposure to

TRF

To investigate the mechanism by which TRF promotes cell

death, an Annexin V-FITC/PI assay using flow cytometry was

performed with TRF-treated cells. Phosphatidylserine (PS) in the

plasma membrane of apoptotic cells is translocated from the

interior to the exterior of the membrane. The externalized PS

binds to Annexin V, which acts as a probe for undergoing

apoptosis, and PI penetrates late stage apoptotic cells through the

damaged membrane (Hwang et al., 2011). This process indicates

that Annexin V-FITC-positive and PI-negative cells are

considered to be undergoing early apoptosis and that Annexin

V-FITC- and PI-positive cells are considered to be in the late

stages of apoptosis. Increased apoptosis, as a result of TRF

exposure, was detected by flow cytometry (Fig. 2.2A,B). To

clarify TRF-induced apoptotic cells, only the Annexin V-FITC

stain was used, and the positive cells were increased compared

with the vehicle control (Fig. 2.2C). Furthermore, a TUNEL

assay was conducted to visualize the apoptotic state that was

triggered by TRF. Based on the TUNEL system, apoptotic nuclei

are stained dark brown by the chromogen, diaminobenzidine

(DAB) which detects horseradish peroxidase-labeled streptavidin

bound to biotinylated nucleotides of fragmented nuclear DNA.

The numbers of apoptotic nuclei and total nuclei in each image

were counted and calculated as a ratio of apoptotic nuclei to total

62

nuclei in order to show the increase in TRF-induced apoptosis.

The dark brown positive cells increased in response to TRF,

whereas few positive cells were detected in the vehicle control

(Fig. 2.2D,E). To confirm the apoptotic state, the expression of

pro-apoptotic proteins was detected using Western blotting

analysis after 0.5 μM of TRF treatment for 24 or 48 h. The

pro-apoptotic proteins, apaf-1, procasp-9, procasp-8, and cyt c

were induced by TRF in a time-dependent manner (Fig. 2.3A,B).

Moreover, cyt c released from mitochondria into the cytosol was

visualized by immunofluorescence (Fig. 2.3C). Therefore, these

experiments demonstrate that TRF caused apoptosis in HaCaT

cells through either intrinsic or extrinsic pathways. In particular,

casp-8 belongs to an extrinsic pathway that includes

TRAIL-mediated apoptosis, upon which we focused.

63

64

Fig. 2.2. Increase in apoptosis induced by TRF exposure. (A)

Apoptosis was measured using an Annexin V-FITC/PI assay by

flow cytometry. The lower right panel shows early apoptosis

(Annexin V-FITC-positive, PI-negative) and the upper right

panel shows late apoptosis (Annexin V-FITC-positive,

PI-positive). Early and late apoptotic cells were increased by

exposure to 0.5 μM TRF for 48 h. (B) Histograms present the

percentage of total apoptotic cells. (C) Histograms show increased

Annexin V-FITC single positive cells upon TRF exposure. (D) A

TUNEL assay was used to visualize TRF-induced apoptosis. The

yellow arrows indicate apoptotic nuclei. (E) The stained apoptotic

nuclei were counted and presented as the apoptotic nuclei/total

nuclei ratio. The amount of apoptotic staining increased after 0.5

μM TRF treatment. The result is represented as the mean ±

SEM (n = 3, ***p < 0.001 versus Veh. Cont.).

65

Fig. 2.3. The effect of TRF exposure on pro-apoptotic protein

expression. (A) Western blotting analysis was performed using

apaf-1, procasp-9, procasp-8, and cyt c antibodies after treatment

with 0.5 μM TRF for 24 or 48 h. Actin indicates the loading

control. The pro-apoptotic proteins were induced by TRF and

time-dependent manner. (B) Densitometric analysis was

conducted in three independent experiments. (C)

Immunofluorescence images were obtained by CLSM to visualize

cyt c in the cells. The amount of cyt c (green) and release from

mitochondria (red) were increased by TRF exposure. The results

are expressed as the mean ± SEM (n = 3, *p < 0.05, **p < 0.01

and ***p < 0.001 versus Veh. Cont.).

66

2.3.3. TRF triggers TRAIL-mediated apoptosis of HaCaT

cells

To identify whether TRF triggered TRAIL-mediated apoptosis

in HaCaT cells, we observed the effect of DR5 knockdown of

TRAIL receptors using siRNA transfection to inhibit

TRAIL-mediated apoptosis. Scrambled siRNA was used as a

transfection control. After 24 h of siDR5 transfection, DR5

expression was effectively inhibited, and number 1 siDR5

down-regulated the expression to 50% compared with the control

(Fig. 2.4A,B). Thus, number 1 siDR5 was used in subsequent

experiments. Following siRNA transfection and subsequent

treatment with 0.5 μM TRF for 48 h, Annexin V-FITC/PI

apoptosis assay using fluorescence microscopy was performed to

determine how knockdown of DR5 in TRAIL-mediated apoptosis

has impact on TRF-induced apoptosis. In TRF-treated HaCaT

cells and TRF-treated siScr-transfected cells, both only Annexin

V-FITC (green)-positive cells and Annexin V-FITC (green)/PI

(red)- positive cells were observed, and the nuclei of the

apoptotic cells showed stronger staining with the NucBlue Live

ReadyProbe Reagent (blue), indicating nuclear condensation in

apoptosis. In contrast, the knockdown of DR5 led to a

pronounced reduction in TRF-induced apoptosis, and the nuclei

staining pattern was similar to that of the vehicle control. This

result indicates that there was a correlation between DR5

knockdown and the inhibition of TRAIL-mediated apoptosis by

67

TRF (Fig. 2.4C). Moreover, to support TRF-induced

TRAIL-mediated apoptosis, the changes in protein expression

after siRNA transfection were measured using Western blot. DR5,

procasp-8, procasp-3, and PARP in the TRAIL-mediated pathway

were down-regulated in DR5-knockdown cells by siDR5 in

comparison with only TRF-treated and TRF-treated

siScr-transfected cells. In addition, cleaved forms of casp-8,

casp-3 and PARP indicating activation through their proteolytic

processing were reduced by DR5 knockdown (Fig. 2.5A,B). Taken

together, these data confirm that TRF induces apoptosis via the

TRAIL-mediated pathway.

68

69

Fig. 2.4. Change in apoptosis by siRNA transfection. (A)

Scrambled siRNA (siScr) and DR5 siRNA (siDR5) were

transfected using the Neon transfection system. Following DR5

knockdown using two different siRNAs, number 1 siDR5

effectively inhibited the protein expression by 50%. (B)

Densitometric analysis was conducted in three independent

experiments. The error bars represent the mean ± SEM (n = 3,

*p < 0.05 versus Cont. or siScr). (C) Cells were transfected with

number 1 siDR5 for the AnnexinV-FITC/PI apoptosis assay.

After siRNA transfection and 48 h of treatment with TRF, the

change in fluorescence was obtained by CLSM. Only

TRF-treated HaCaT cells and siScr-transfected TRF-treated cells

were stained with Annexin V-FITC (green), indicating early

apoptosis, or with both Annexin V-FITC (green) and PI (red),

indicating late apoptosis. However, in the DR5 knocked down

cells, TRF treatment resulted in the staining being hardly

observed, and few cells were exhibited Annexin V-FITC- or

PI-positive. In addition, the nuclear condensation recognized by

Hoechst (blue) was decreased in DR5 siRNA-transfected cells.

The images show that there is a correlation between DR5

knockdown and reduced apoptosis induced by TRF.

70

71

Fig. 2.5. Changes in protein expression following the transfection

of DR5 siRNA. (A) After transfection of siScr and siDR5 using

the Neon transfection system, 0.05% DMSO and 0.5 μM TRF

were administered to HaCaT cells for 48 h. Western blot was

performed using DR5, procasp-8, cleaved casp-8, procasp-3,

cleaved casp-3, PARP, and cleaved PARP antibodies. Actin

indicates the loading control. The DR5-knocked down cells did

not show overexpression and activation of the apoptotic proteins

that were induced by TRF. (B) Densitometric analysis was

conducted in three independent experiments. The results are

expressed as the mean ± SEM (n = 3, *p < 0.05, **p < 0.01

and ***p < 0.001 versus TRF or siScr+TRF).

72

2.4. DISCUSSION

Although pesticides were developed to control pests and improve

quality of life, many pesticides could present potential risks to

the environment and to human health. Concerns related to

pesticide use have been raised due to the detrimental effect on

people exposed to them. In particular, the exposure to pesticides

has shown an association with the development of skin diseases

(Guo et al., 1996; Spiewak, 2001). Since the skin receives the

most exposure to pesticides, it is important to identify pesticide

toxicity specifically in the skin. However, little is known

regarding the toxicological mechanism of the widely-used

fungicide strobilurins. Therefore, the objective of this study was

to investigate the cytotoxic mechanism of trifloxystrobin using

the representative human skin keratinocyte cell line, HaCaT

which has been used as an in vitro skin model (Di Caprio et al.,

2015).

It has been reported that several fungicides that are used to

control soybean rust inhibited the cell growth or disrupted the

cell cycle in Chinese hamster ovarian cells, and that

trifloxystrobin displayed a more significant toxic response (Daniel

et al., 2007). Thus, here, we examined the cytotoxicity of

trifloxystrobin on skin keratinocyte HaCaT cells. The cell

viability was drastically decreased in a concentration- and

73

time-dependent manner demonstrating that trifloxystrobin can

trigger cell death of keratinocytes (skin epithelial cells).

There are various mechanisms of cell death including apoptosis,

necrosis, mitotic catastrophe, necroptosis, and pyroptosis

(Kroemer et al., 2009). Numerous studies have demonstrated that

pesticides can induce apoptosis of cells resulting in dysregulation

of the related function (Ahmadi et al., 2003; Calviello et al., 2006;

Fukuyama et al., 2010). Apoptosis is a programmed cell death to

maintain normal cellular homeostasis, and the induction of

apoptosis can be a primary pathological event that causes tissue

remodeling and diseases (MacFarlane and Williams, 2004).

Therefore, in the present study, we evaluated whether

trifloxystrobin induced apoptosis as a mechanism of toxicity. The

Annexin V-FITC/PI assay showed an increase in early and late

apoptosis compared with the control, upon the addition of

trifloxystrobin, and the exposed cells had DNA fragmentation, a

characteristic of apoptosis. Furthermore, the expression of

proteins involved in apoptotic pathways was up-regulated

following trifloxystrobin treatment. Apaf-1 and casp-9 form

apoptosomes from the mitochondrial factor, cyt c, and induce

intrinsic pathways of apoptosis (Zou et al., 1999). Whereas

casp-8 is activated by the death domains of receptors associated

with extrinsic pathways such as TRAIL-induced apoptosis

(Falschlehner et al., 2007). These results indicate that

trifloxystrobin derives apoptotic pathways which can be a mode

74

of cell death in HaCaT cells.

A previous study showed that the contact allergen nickel could

increase the susceptibility of keratinocytes to TRAIL-mediated

apoptosis as a crucial factor in sustaining epidermal integrity

(Schmidt et al., 2010). Thus, we investigated

trifloxystrobin-induced TRAIL-mediated apoptosis in HaCaT

cells. The extrinsic apoptosis is activated by cell surface death

receptors. Cell surface expression of the death receptors DR4 or

DR5 is demanded for induction of extrinsic pathway of apoptosis

(Zhang and Zhang, 2008). To assess TRAIL-mediated apoptosis

as a result of trifloxystrobin, we introduced DR5 siRNA into cells

to knockdown DR5, and thus inhibit the induction of

TRAIL-mediated apoptosis. The transfection of siDR5 effectively

down-regulates DR5 in HaCaT cells. As DR5 was knocked down,

apoptosis induced by trifloxystrobin was suppressed, similar to

the vehicle control. This was in accordance with a previous

study in which DR5 siRNA significantly reduced apoptosis

compared with control siRNA (Koyama et al., 2010). In addition,

trifloxystrobin-treated cells with siDR5 transfection revealed the

changed expression of DR5 and reduced activation of its

downstream proteins in the TRAIL-mediated pathway. The

diminished proteins reinforced the idea that trifloxystrobin played

a role in the induction of extrinsic apoptosis. Taken together, our

data demonstrate that DR5 is involved in trifloxystrobin-induced

apoptosis, and that exposure to trifloxystrobin causes apoptosis

75

via the TRAIL-mediated pathway in skin epithelial cells. It has

been suggested that epithelial cell apoptosis could amplify or

maintain inflammatory processes that are associated with the

phenomenon of allergic diseases such as eczematous dermatitis

(Simon, 2009; Trautmann et al., 2000). Thus, these findings imply

that skin exposure to fungicides can disrupt skin functions

related to barrier and integrity maintenance, and subsequently

lead to the development of cutaneous diseases.

In conclusion, this study identified that the fungicide

trifloxystrobin sensitizes keratinocytes to TRAIL-mediated

apoptosis, which may trigger skin diseases. Epidermal

keratinocytes are the outermost layer of the body, and could have

harmful impacts on the barrier function and coherence of skin

through exposure to environmental chemicals, especially

pesticides. Therefore, understanding of the mechanism of

trifloxystrobin-induced toxicity in keratinocytes may help the

development of therapeutic strategies against pesticide exposure.

Additional studies that analyze mechanisms and pathophysiology

of pesticides in more detail would be necessary in order to

identify and control their impact on human health.

76

Chapter Ⅲ

Trifloxystrobin-induced mitophagy

through mitochondrial damage in human

skin keratinocytes

(Published in 2016, The J ournal of

Toxicological Sciences)

77

ABSTRACT

Trifloxystrobin is a strobilurin class fungicide, the mode of action

of which is to block the mitochondrial electron transport chain

and inhibit energy production in fungi. Although adverse effects

have been reported by occupational or environmental exposure of

fungicides, the pathophysiological mechanism in human cells

remains poorly understood. In the present study, we investigated

the impact of trifloxystrobin on exposed skin at the cellular

organelle level using HaCaT, the human skin keratinocyte cell

line. Cells were treated with trifloxystrobin for 48 h and

trifloxystrobin showed detrimental effects on mitochondria

evidenced by altered mitochondrial membrane potential and

morphology. To identify autophagic degradation of the damaged

mitochondria, confocal imaging and Western blotting were

performed. Trifloxystrobin induced autophagy-related proteins in

HaCaT cells. The mitochondrial reactive oxygen species

scavenger mitoTEMPO was applied to further explore the

mechanism of trifloxystrobin-mediated mitophagy in human skin

cells. PINK1 and Parkin were overexpressed by trifloxystrobin,

and mitoTEMPO alleviated the effects on mitophagy induction.

Taken together, our findings indicated that mitochondrial damage

and mitophagy may play a role in trifloxystrobin-induced toxicity

in human keratinocytes and this could be suggested as a

mechanism of cutaneous diseases developed by exposure.

78

3.1. INTRODUCTION

Various fungicides have been applied worldwide for agricultural

and public pest control. However, fungicide exposure has the

potential to cause adverse effects in humans, particularly at the

major exposure site, the skin. Therefore, an understanding of

fungicide toxicity is important for safe use and to prevent

excessive exposure. There are many epidemiological and

toxicological studies on fungicides that were developed a long

time ago and used continuously. However, since strobilurin class

fungicides are a relatively new class of fungicides, discovered in

the 1990s and introduced to the market in the late 1990s and

early 2000s (Bartlett et al., 2002), toxicological data are relatively

lacking.

The strobilurins, which are produced from strobilurin A of

Strobilurus tenecellus, are an important class of fungicides. They

have played a role in the disease management of various

agricultural products and are widely used, accounting for over

10% of the fungicide market (Bartlett et al., 2002). The mode of

action in fungi is to bind to the quinol oxidation site of

cytochrome b in the mitochondrial electron transport chain and

thereby block electron transfer, which leads to the inhibition of

energy (ATP) production and then the fungi ultimately die

(Balba, 2007). Among the strobilurin class, trifloxystrobin (TRF)

79

is a broad-spectrum, widely-used fungicide. As such,

trifloxystrobin could be toxic to non-target species, similar to

nontarget eukaryote species. Thus, it is necessary to study the

manner in which trifloxystrobin affects human skin cells

following exposure with regard to mitochondria.

The mitochondrion is an essential cellular organelle that

generates cellular energy in the form of ATP from the citric acid

cycle and electron transport chain. In addition to this,

mitochondria perform many important functions, such as

regulation of membrane potential, metabolism, reactive oxygen

species (ROS), and cell signaling (McBride et al. 2006).

Therefore, damaged mitochondria show collapse of the membrane

potential, increased ROS production, disturbance of fusion and

fission dynamics, and altered other cell signaling pathways (Chen

and Chan, 2009; Lefeuvre et al., 2005). These have detrimental

effects on the cell and ultimately impaired and irreparable

mitochondria are removed or lead to cell death and disease

development.

In response to foreign substances and harmful influence, cells

induce the autophagic degradation process of cellular components.

Autophagy is characterized by a double-membrane

autophagosome, which is made by a multiprotein complex

including beclin 1 and elongation by the LC3 conjugation system.

Then, the autophagosome is fused with the lysosome and the

components are degraded by lysosomal hydrolytic enzymes (Glick

80

et al., 2010; Tanida, 2011). Selective autophagic degradation of

mitochondria is called mitophagy. Triggered by lethal stimuli

causing mitochondrial malfunction, PTEN-induced putative kinase

1 (PINK1) accumulates in the membrane of depolarized

mitochondria, and initiates a mitophagic process by recruiting

Parkin. Once recruited to the mitochondria, Parkin promotes

autophagic degradation through the recruitment of LC3-containing

autophagosomes to depolarized mitochondria (Narendra et al.,

2010; Sica et al., 2015).

Because the mechanism of damage by fungicide exposure in

view of cellular organelles in human skin remains to be

elucidated, it is significant to identify the toxicological mechanism

in vitro using human cells. Therefore, in the present study, we

reported that trifloxystrobin causes mitophagy through

mitochondrial damage and alleviated effects by antioxidant in

HaCaT human skin keratinocytes. This could be proposed as a

plausible pathophysiological mechanism of fungicide exposure in

the skin epithelium.

81

3.2. MATERIALS AND METHODS

3.2.1. Cell culture and treatment

HaCaT (immortalized human keratinocyte cell line;

nontumorigenic) cells were maintained in high-glucose Dulbecco's

modified Eagle's medium (DMEM, GE Healthcare Life Sciences,

Logan, UT, USA) with 10% fetal bovine serum (FBS, Thermo

Scientific, Logan, UT, USA) and 1% penicillin/streptomycin (GE

Health Care Science) at 37 °C in a 5% CO2 humidified incubator.

Trifloxystrobin (TRF; CAS number 141517-21-7, purity (HPLC

area %) 99.5%), mitoTEMPO, and dimethyl sulfoxide (DMSO)

were purchased from Sigma-Aldrich (St. Louis, MO, USA). TRF

was dissolved in DMSO for 1 and 100 mM stock solution.

mitoTEMPO was dissolved in autoclaved distilled water for a 10

mM stock solution. The stock solutions were diluted to acquire

treating concentrations. In this study, cells were treated with

0.05% DMSO as vehicle control or 0.5 μM TRF for 48 h. 2.5 μM

mitoTEMPO was preloaded for 1 h before TRF treatment.

3.2.2. Cell cytotoxicity assay

Cell cytotoxicity was assayed by the Cell Counting Kit-8

(CCK-8, Dojindo Molecular Technologies, Kumamoto, Japan).

HaCaT cells were seeded on a 96-well plate. 0.2% DMSO was

used as a vehicle control. TRF was serial-diluted from 200 μM

82

to 0.39 μM. After 48 h, 10 μL of CCK-8 solution/100 μL medium

was added to each well. The cells were further incubated in the

dark for 2 h at 37°C. The absorbance was measured at 450 nm

using a microplate spectrophotometer (Bio-Rad, Richmond, CA,

USA). The absorbance values were calculated as percentages of

the control.

3.2.3. Detection of mitochondrial damage

The change in mitochondrial membrane potential (MMP, ΔΨm)

was analyzed by using a JC-1 dye and MitoTracker Red

CMXRos (Molecular Probes). After 48 h of TRF treatment, 5 μ

g/mL of JC-1 dye, which accumulates in a potential-dependent

manner in mitochondria was loaded onto cells and incubated at

37°C for 30 min. The samples were trypsinized and resuspended

in PBS. Then flow cytometric analysis of JC-1 was performed

using a FACS AriaII flow cytometer (BD, Franklin Lakes, NJ,

USA) (10,000 events/sample). 100 μM of MitoTracker Red

CMXRos, which accumulates in mitochondria by membrane

potential, was added to cells and incubated at 37 °C for 30 min.

Cells were fixed in 4% paraformaldehyde and mounted with

Fluoroshield™ with DAPI mounting medium (Sigma-Aldrich). The

images were obtained with a confocal laser scanning microscope

(CLSM, LSM710, Carl Zeiss Microscopy GmbH, Oberkochen,

Germany) to detect the change in MMP and a super resolution

83

microscope (SRM, ELYRA PS.1, Carl Zeiss Microscopy GmbH)

to observe mitochondrial morphology.

3.2.4. Immunofluorescence

Cells were fixed in 4% paraformaldehyde at room temperature

(RT) for 15 min and permeabilized with cold methanol/acetone

solution (1:1) at RT for 10 min. The cells were blocked with 3%

bovine serum albumin (BSA) and incubated with anti- beta

(LC3B) primary antibody (Cell Signaling, Danvers, MA, USA).

Followed by incubating with a secondary antibody, Alexa Fluor

555 goat anti-rabbit IgG (Molecular Probes), the cells were

mounted with Fluoroshield™ with DAPI mounting medium. The

confocal images were detected by using CLSM, LSM710.

3.2.5. Measurement of protein expression

Proteins in each samples were collected using lysis buffer and

the protein concentration was measured with PierceTM BCA

Protein Assay Kit (Thermo Scientific, Waltham, MA, USA). For

Western blotting, 25μg of protein from each sample was loaded

onto 10%-12% sodium dodecyl sulfate-polyacrylamide gel

electrophoresis (SDS-PAGE). Next, the transferred nitrocellulose

membranes were blocked with 5% skim milk and then incubated

with primary antibodies of lysosome-associated membrane protein

2 (LAMP2), beclin 1 (BECN1), PTEN-induced putative kinase 1

84

(PINK1), Parkin (Abcam, Cambridge, UK), LC3B, and

phosphorylated-dynamin related protein 1 (pDRP1/Ser616) (Cell

Signaling). After washing with TTBS, the membranes were

incubated with secondary antibodies conjugated with horseradish

peroxide (HRP). The detection of protein bands and densitometric

analysis were performed by using Image analyzer ATTO EZ

Capture MG and CS analyzer 3.0 program (ATTO Corp., Tokyo,

Japan).

3.2.6. Detection of mitophagy

Following treatment, cells were stained with 2 µL Cyto-ID®

Green Detection Reagent (Cyto-ID® Autophagy detection kit,

Enzo Life Sciences, Farmingdale, NY, USA)/1mL media at 37 °C

for 3 h and then 100 nM MitoTracker at 37 °C for 20 min. The

stained cells were fixed in 4% paraformaldehyde for 15 min and

mounted with Fluoroshield™ with DAPI mounting medium. The

images were detected by using CLSM, LSM710.

3.2.7. Statistical analysis

The statistical significance of data was determined by Student’s

t-test and one-way ANOVA with Tukey’s post-hoc test using

Graphpad Prism 5 Software (San Diego, CA, USA). Results were

considered statistically significant at p<0.05 with the compared

group.

85

3.3. RESULTS

3.3.1. Trifloxystrobin induces cytotoxicity and mitochondrial

damage in HaCaT skin keratinocyte

To determine the concentration for this mechanistic study, the

cytotoxicity assay of TRF on HaCaT cells was performed using

the CCK-8 assay. After 48 h of exposure, cytotoxicity occurred

in a dose-dependent manner (Fig. 3.1). The IC50 value at 48 h

was calculated as 3.32 μM. For the mechanistic study, HaCaT

cells were treated with 0.5 μM of TRF in subsequent

experiments based on the IC50 value and preliminary study.

Because 0.2% DMSO as a vehicle control did not affect

cytotoxicity compared with the control, 0.05% DMSO was applied

to other assays as a vehicle control.

To identify the effect of TRF on mitochondria considering the

mode of action of TRF, we used MMP dependent dyes, JC-1 and

MitoTracker Red CMXRos. JC-1 is a parameter of mitochondrial

health in cells and the red/green fluorescence ratio using flow

cytometry provides information on comparative MMP between

cell groups. As shown in Fig. 3.2A, the red/green ratio was

decreased in TRF-treated cells. Furthermore, using

MMP-dependently stained MitoTracker Red CMXRos, reduced

red fluorescence was observed by confocal imaging in TRF

treatment compared to in 0.05% DMSO treatment (Veh. Cont.)

86

(Fig. 3.2B). These results indicated that HaCaT cells showed a

decrease in MMP when treated with 0.5 μM TRF for 48 hr

(Pendergrass et al., 2004). In addition, morphological changes in

mitochondria were observed using a super resolution microscope.

Mitochondrial fragmentation and shape changes were detected in

cells with TRF exposure (Fig. 3.2C). These data demonstrated

that TRF causes inhibition and damage to the mitochondria of

HaCaT cells.

87

Fig. 3.1. The effect of TRF on cytotoxicity in HaCaT cells using

the CCK-8 assay. Cells were treated with TRF from 0.39 to 200

μM for 48 h (n=4). Cells showed concentration-dependent

cytotoxic effects. The IC50 value was measured as 3.32 μM. The

results were presented as percentages of the control indicating an

average of 100%. The graph is represented as mean ± standard

error of the mean (SEM).

88

Fig. 3.2. The effects of TRF on mitochondrial damage. (A) Flow

cytometric analysis using the JC-1 assay after TRF treatment.

The Red/Green fluorescence ratio was decreased in cells treated

with 0.5 μM TRF for 48 h indicating reduced MMP in

comparison with vehicle control (Veh. Cont.). (B) Membrane

potential-dependent MitoTracker Red CMXRos staining observed

by CLSM (scale bar = 5 and 10 μm), and (C) by SRM for

morphological change in mitochondria (scale bar = 5 μm).

89

3.3.2. Autophagy detection provoked by TRF in HaCaT cells

To investigate the effect of damage by TRF in HaCaT cells, we

examined autophagy using immunofluorescence and Western blot.

After TRF treatment, increased LC3B dots (red) representing

autophagosome formation were observed compared to Veh. Cont.

using immunofluorescence analysis imaged by CLSM (Fig. 3.3A).

To further confirm TRF-induced autophagy, Western blot

detecting the autophagosome marker proteins, BECN1 and

LC3BII, and the lysosome marker protein LAMP2 was performed.

These autophagy-related proteins were significantly up-regulated

in cells with 0.5 μM TRF treatment for 48 h in agreement with

the immunofluorescent results (Fig. 3.3B,C). These data clearly

indicated that TRF exposure induced autophagy in HaCaT cells.

90

91

Fig. 3.3. The effect of TRF on autophagy induction. (A) After 0.5

μM TRF treatment for 48 h, immunofluorescence analysis was

performed with LC3B antibody (red) and mounted using DAPI

(blue) containing mounting solution. LC3B expression was

visualized by CLSM. Increased LC3B dots were observed in

TRF-treated cells compared with Veh. Cont. (scale bar = 5 and

10 μm). (B) Western blot analysis detecting autophagy marker

proteins, LAMP2, BECN1 and LC3B. All autophagy-related

proteins were increased in cells incubated with TRF for 48 h.

Actin expression was used as a loading control. The assay was

repeated in triplicate and showed similar results. (C)

Densitometric analysis of Western blot bands. *p < 0.05, **p <

0.01 and ***p < 0.001 versus Veh. Cont.

92

3.3.3. TRF-induced mitophagy and the alleviated effect by

mitochondrial ROS scavenger

To determine autophagic degradation of damaged mitochondria

by TRF exposure in HaCaT cells, mitophagy induction was

evaluated using confocal imaging and analysis of protein

expression change. In TRF-treated cells, autophagic dots (green)

stained with Cyto-ID® autophagy detection dye were increased

and merged form with mitochondria (red) was observed by

CLSM imaging (Fig. 3.4A). Then Western blot was performed to

examine changes in mitophagy-related proteins. Phosphorylation

of DRP1 at Ser616 stimulating mitochondrial fission was

increased in TRF treated cells. Consistent with the data,

mitophagy marker proteins, PINK1 and Parkin were also

upregulated (Fig. 3.4B,C). To further identify the damaging

mechanism of TRF, HaCaT cells were treated with mROS

scavenger mitoTEMPO before TRF exposure. Pretreatment of

cells with mitoTEMPO alleviated the effects of TRF on

mitophagy induction. Taken together, these results suggested that

mitophagy was provoked by TRF in HaCaT cells and increased

mROS could be one of mechanisms of TRF-induced mitophagy.

93

94

Fig. 3.4. Induction of mitophagy by TRF and the effects

alleviated by mROS scavenger. HaCaT cells were treated with or

without mROS scavenger, 2.5 μM mitoTEMPO for 1 h and then

treated with 0.05% DMSO (Veh. Cont.) or 0.5 μM TRF for 48 h.

(A) After treatment, cells were stained with Cyto-ID® autophagy

detection reagent (autophagy, green) and MitoTracker

(mitochondria, red). The change in fluorescence was visualized by

CLSM. Increased autophagy and merged form with mitochondria

were observed in TRF-treated cells compared with Veh. Cont.

and reduced by mitoTEMPO pretreatment (scale bar = 5 μm).

(B) Western blot analysis with mitochondrial fission protein

pDRP1 and mitophagy marker proteins PINK1 and Parkin. Actin

expression was used as a loading control. The assay was

repeated in triplicate and showed similar results. (C)

Densitometric analysis of Western blot bands. **p < 0.01 versus

Veh. Cont., #p < 0.05, and ##p < 0.01 versus TRF.

95

3.4. DISCUSSION

Pesticide use is well known to cause adverse effects, and some

fungicides have been epidemiologically reported to cause diseases

of the skin, which is the major exposure route (Ginkel and

Sabapathy, 1995; Saunders and Watkins, 2001; Spiewak, 2001).

However, the pathogenesis and toxicological mechanism in skin

cells are not yet clearly identified. Although human exposure to

pesticides occurs at a low level, hazardous health effects and

toxicity at the molecular and cellular levels have been constantly

documented after exposure to various pesticides (Androutsopoulos

et al., 2013). Therefore, it is necessary to investigate the

response of skin cells to pesticides under conditions of low

concentration and relatively long-term exposure similar to

occupational or environmental exposures to pesticide. In this

study, we explored the toxicological mechanism of a fungicide,

trifloxystrobin (TRF) in human skin cells, establishing such

treatment conditions (0.5 μM, 48 h)

It has been reported that mitochondria-targeted drugs altered

mitochondria-related cell status, including reductions in membrane

potential and ATP production and increase in mROS production

(Jabaut et al., 2013). As such, we assumed that the

mitochondria-targeted fungicide TRF, the mode of action of

which is to block the mitochondrial electron transport chain, may

96

also influence the mitochondria in human cells, same eukaryotes.

Our data show that TRF causes representative mitochondrial

damage such as altered membrane potential and morphology in

skin keratinocytes. Since mitochondria are essential to regulate

healthy skin physiology, the progression of skin diseases may be

associated with aberrant mitochondrial status and functions

(Feichtinger et al., 2014). Thus, these could suggest that TRF

exposure to skin resulting in a detrimental influence on

mitochondria could be a cause of developed cutaneous disease by

the exposure.

Autophagy is a degenerative mechanism that enables cells to

control homeostasis and remove defective cellular components.

The autophagic process can be triggered by starvation, damaged

cytoplasmic components, and oxidative stress. Several pesticides

have been reported to induce autophagy by exposure

(González-Polo et al., 2007; Park et al., 2013) Thus, autophagy

induction was assessed after treatment of TRF in HaCaT cells.

We observed increased expression of an important early

autophagosme component, BECN1 and increased lapidated LC3B,

LC3BII in a key autophagic process following exposure to TRF.

Lysosomal transmembrane protein LAMP2 was also

overexpressed indicating increased lysosome fused with

autophagosome.

When these results of damaged mitochondria and induced

autophagy were combined, mitophagy could be induced. Then, we

97

visualized TRF-mediated mitophagy and confirmed with changed

protein expression of PINK1 and Parkin in the mitophagy

process. To further examine the mechanism of mitophagy

induction by TRF in skin keratinocytes, we identified

mitochondrial fission protein and applied the special antioxidant of

mROS, mitoTEMPO. Mitochondria undergo fission and fusion to

maintain mitochondrial dynamics and functions and increased

fission facilitates mitophagy. A central player in mitochondrial

fission is DRP1, which is regulated by phosphorylation such as

phosphorylation at Ser616 that activates fragmentation activity

(Chan, 2012; Gomez-Lazaro et al., 2008). In TRF-treated cells,

pDRP1 (Ser616) was increased in agreement with mitochondrial

morphological change. mitoTEMPO-treated cells showed

ameliorated effects on the phosphorylation induced by TRF.

Furthermore, mitoTEMPO reduced mitophagy induction. These

findings indicated that mitochondrial oxidative stress through

mitochondrial damage induced by TRF could be considered as a

step of TRF-mediated mitophagy.

Previous studies have reported that regulation of autophagy is

important to the human physical condition and may contribute to

the pathogenesis of several disease processes (Todde et al., 2009).

Taken together, the evidence indicates that mitochondrial damage

and mitophagy induced by TRF may result in cutaneous toxicity,

and this proposes the possible cellular mechanism of

dermatological diseases developed by skin exposure of the

98

fungicide.

In conclusion, we investigated for the first time the toxicological

mechanism of TRF in human skin keratinocytes at the cellular

organelle level. Our study demonstrated that TRF exposure to

skin induced mitochondrial damage with respect to the mode of

action to fungi of the strobilurin fungicide class. Subsequent

autophagic degradation of the damaged mitochondria was

protected by an mROS-target antioxidant, which could be

suggested as preventive and therapeutic strategy in preparation

for adverse effects of TRF exposure. Further in vivo studies are

needed to explore detailed pathophysiological mechanism in the

skin exposed to TRF. Moreover, we propose that our findings be

applied to research on the toxicity and pathological mechanism of

other fungicides.

99

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국문 초록

클로르피리포스와 트리플록시스트로빈

농약의 피부세포 내 독성 기전 연구

장 윤 정

수의학과

수의병인생물학 및 예방수의학 전공

서울대학교 대학원

농약을 개발하고 사용한 이래로, 농약은 식물 질병 컨트롤, 농작물

수확 증대 등 많은 이점을 제공하였다. 그러나 농약 사용은 인간 등

비표적 (non-target) 생물체에도 다양한 부작용을 나타내어, 정확한

농약 독성시험과 위해성평가가 필요하다. 농약의 주 노출 장소는

피부이므로, 특히 이러한 피부에서 농약의 독성 기전 연구가

중요하다. 클로르피리포스와 트리플록시스트로빈은 농업에서 뿐

아니라 주거 환경에서도 널리 쓰이는 농약이다. 비록 농약 노출에

의한 독성 및 건강 영향에 관한 다양한 연구들이 보고된 바 있으나,

피부세포에서 독성 기전은 정확히 밝혀지지 않았다. 본 연구에서는

인간 피부 각질세포주인 HaCaT을 사용하여 클로르피리포스와

트리플록시스트로빈 농약의 피부 독성 기전을 연구하였다.

첫번째로, HaCaT 세포에 클로르피리포스를 처치 후, 세포 내

119

변화를 관찰하였다. 클로르피리포스는 활성산소종 (ROS) 생성 및

미토콘드리아의 산화적 스트레스를 유발하였다. 선천성 면역 반응을

유도한다고 알려진 NLRP3 인플라마좀에 초점을 두었다.

미토콘드리아 활성산소종 (mROS) 제거제인 mitoTEMPO를

사용하여, 클로르피리포스에 의해 유도되는 NLRP3 인플라마좀과

예정세포사에서 mROS의 역할을 증명하였다. 이러한 결과들은

클로르피리포스가 HaCaT 세포 내에서 mROS의 유도를 통해

인플라마좀과 pyroptosis/apoptosis를 일으킨다는 것을 보여준다.

이는 클로르피리포스가 피부 상피세포에서 NLRP3 인플라마좀의

활성화를 통해, 선천성 면역 반응과 피부 염증을 유도함을 제안할

수 있다.

두번째로, HaCaT 세포를 사용해 트리플록시스트로빈의 피부 독성

기전을 연구하였다. 트리플록시스트로빈 처치 후,

트리플록시스트로빈에 의한 세포 사멸 기전을 조사하기 위해

세포생존율 및 Annexin V-FITC/propidium iodide 실험, TUNEL

실험, Western blot을 수행하였다. 트리플록시스트로빈 노출은 시간,

농도에 따라 HaCaT 세포의 생존율을 감소시켰다. 이러한 세포

사멸은 apoptosis 경로를 통해 유도되었다. 더 나아가, siRNA를

사용해 TRAIL-매개 외인성 apoptosis에서 트리플록시스트로빈의

영향을 탐색하였다. death receptor 5의 발현 감소는

트리플록시스트로빈에 의한 apoptosis를 억제하였다. 이러한 결과는

트리플록시스트로빈이 TRAIL-매개 apoptosis를 유도하여 피부

각질세포에 억제 효과를 가지며, 이는 피부의 장벽 기능과

통합성(integrity)을 저해할 수 있음을 지시한다.

세번째로, HaCaT 세포 내 세포소기관 수준에서

120

트리플록시스트로빈의 영향을 연구하였다. 세포에

트리플록시스트로빈의 48시간 노출은 미토콘드리아 막전위 및 모양

변화 등 미토콘드리아에 해로운 영향을 끼쳤다. 이러한 손상된

미토콘드리아의 자가소화작용 (autophagy)을 확인하기 위해

공초점현미경과 Western blot을 수행하였다. 트리플록시스트로빈은

세포 내 autophagy 및 mitophagy 관련 단백질의 발현을

유도하였다. 트리플록시스트로빈 매개 mitophagy의 기전을

탐색하기 위해 mitoTEMPO를 적용하였고, 이는 mitophagy 유도

효과를 완화시켰다. 이러한 발견들은 트리플록시스트로빈에 의한

인간 피부세포 독성에서 미토콘드리아 손상과 mitophagy가 역할을

할 수 있으며, 이는 농약 노출에 의해 유도된 피부병의 기전으로

제안될 수 있음을 지시한다.

결론적으로, 이러한 발견들은 농약 노출에 의해 유발되는 피부

질병의 기전으로 제안될 수 있다. 더 나아가, 본 연구 결과는

농약에 의한 질병 평가와 농약 사용 및 그 부작용의 관리 등에

기여할 수 있을 것이다.

주요어 : 농약, 농약 노출, 독성 기전, 활성산소종, 인간 피부

각질세포

학 번 : 2014 – 21932