PM -Induced Imbalance of Intracellular Reactive Oxygen ...Nov 26, 2018 · D B C Time (h) 0. 0.25 0.5 0.75 1. 1.25 0 2 4 8 12 24 DEP ent DEP+NAC 0. 17.5 35. 52.5 70. 87.5 0 2 4 8

PM2.5-Induced Imbalance of Intracellular Reactive Oxygen Species Cause Vascular

Dysfunction and Developmental Disorder

招名威博士/美國毒理學家

Ming-Wei Chao, PhD, DABT

Chung Yuan Christian University/BioScience Technology

[email protected]

http://ctlab501.com

2018.11@SOT-AACT Chao LabFB Page

mailto:[email protected]

http://ctbal501.com/

Air quality in Taiwan

(Taiwan)

(Chung Yuan Christian University)

Taipei

PM @ 2018/11/14

http://aqicn.org/map/

Air pollutants contain Particulate Matter

PM2.5 cause cardiovascular and systemic effects

Modified from Brook, Clinical Sci, 115, 175-187, 2008

Permeability Vasoconstriction

Hypertension

Top 10 cause of death relation to PM2.5

(WHO 2015)

Top 10 causes of death

Ischemic heart disease

Trachea, bronchus, lung cancers

Lower respiratory infections

Chronic obstructive pulmonary disease

Stroke

Diabetes mellitus

Alzheimer disease and other dementias

Diarrheal diseases

Tuberculosis

Road injury

http://aqicn.org/map/

Air quality in the world

Beijing PM2.5

PM in Beijing @ 2018/11/14

The smog caused 12,000 people dead, 200,000 injured

Fang et al, Sci Total Environ, 2016, 569-570:1545-52

The smog caused people dead in China (2013)

• Cardiovascular issue is the main disorder that PM2.5 might cause

10 years Air quality change in Taiwan

2017

2016

2015

2014

2013

2012

2011

2010

2009

2008

Adapted from Environ Health Perspect, 2006,114:6,810–817

Type II

Pneumocyte

Type I

Pneumocyte

Capillaries

Alveolus

Alveolar Wall: Blood-air barrier

Basement

membranes

Nemmar et al, Brief Rapid Comm, 2002

PM2.5 translocate into the circulation and cause damages

By what mechanism that PM2.5 translocate into the circulation?

D. 1 µg/ml

B. 0 µg/ml

10 µg/ml 100 µg/ml

1 µg/ml

The endothelial in vitro model experiment:

A. The PM2.5 particle B. The model treated with PM2.5

30 μm 5 μm

A

Chao et al. Toxicology. 2011

Antioxidative mechanism: Nrf-2 and HO-1 activation in PM2.5 treated capillary-tube cells

Chao et al. Toxicology. 2012

Nrf2 MergeNuclei

0.

100.

200.

300.

400.

1 10 100

Intr

acellu

lar

RO

S (

% o

f contr

ol)

DEP

DEP+NAC

A B

Concentration of DEP (µg/mL)

C

E

0 µg/ml

D

TNF-α

IL-6

IL-1β

Cytoplas

mic

Extracts

Nuclear

Extracts

0.

3.

6.

9.

12.

1 2 3 4 5 6

Control

1 µg/mL DEP

10 µg/mL DEP

100 µg/mL DEP

Imm

unoblo

t quantita

tion

(fold

of contr

ol)

HO-1 TNF-α IL-6 IL-1β IκB-α Nuclear

p65

*

**

***

**

**

**

*

*

**

100 µg/ml

10 µg/ml

1 µg/ml

F

Bright-field ROS

Tseng et al, PLoS One, 2015

Chao et al, Toxicology, 2012

PM2.5 disrupt our vascular junction with going paracellular route

0 mg/ml 1 mg/ml 10 mg/ml

Chao et al, Toxicology, 2011Tseng et al, PLoS One, 2015

1. Para-cellular

(hydrophilic)2. Trans-cellular

(hydrophobic)

The mechanism that PM2.5 translocate into the circulation and

cause damages

Transcellular mechanism - Vesiculo-vacuolar organelle (VVO)

D

B

C

Time (h)

0.

0.25

0.5

0.75

1.

1.25

0 2 4 8 12 24

DEP

DEP+NAC

LC

3 p

rese

nt

0.

17.5

35.

52.5

70.

87.5

0 2 4 8 12 24

LC

3 p

unct

a/ce

ll

Phase

Contrast

β-actin

Nuclei

Merge

Carbon Black

ParticlesPMA + Carbon Black

Particles

0h 2h 0h 2h

PM2.5 cause autophagy in endothelial capillary

Wang et al, ToxSci, 2017

A

DEP DEP+NACDEP+N3a

Time (h)

LC3-ILC3-II

ATG12-ATG5

p62

0 2 4 8 12 24 0 2 4 8 12 24

α-tubulin

p53

Mdm2

14 kDa

12 kDa

55 kDa

62 kDa

55 kDa

53 kDa

50 kDa

242

Lysosome

Nuclei

Merge

0h 2h 4h

DEP treatment time points

8h 12h 24h

The

cell

s w

hic

h D

EP

accu

mula

ted

in l

yso

som

es

(# p

er 1

00

cel

ls)

0.

12.5

25.

37.5

50.

0 2 4 8 12 24

***

***

***

******

Time (h)

F

EA

PM2.5-caused endothelial apoptosis might lead to atherosclerosis

D

Tseng et al, Cardiovascular Toxicology, 2015

Rap

α-tubulin

Bcl-2

DEP DEP+NAC

Time (h)

Bax

Cytochrome c

CQ

Bad

0 2 4 8 12 24 0 2 4 8 12 2424

50 kDa

26 kDa

20 kDa

23 kDa

14 kDa

2

DEP + N3a

100 mg/mL0 mg/mL

100 mg/mL+NAC10 mg/mL

B

C

0

10

20

2 4 8 24

TNF-α

Cyto

kin

es l

evel

(fo

ld o

f co

ntr

ol)

Time (h)

ICAM-1

IL-6 MCP-1

E-SelectinVCAM-1

Anch

ori

ng m

ole

cule

s

level

(fo

ld o

f co

ntr

ol)

0

2.5

5

2 4 8 24

0

2.5

5

2 4 8 24

0

2.5

5

2 4 8 24

0

5

10

2 4 8 24

0

2.5

5

2 4 8 24

**

* ***

**

* ***

*

□ Control

25 μg/mL DEP

50 μg/mL DEP


AB

DC

G

E

H

0 h

4 h

8 h

12 h

24 h

H2O2

CQ

Rap

DEP

ATG12-KO

F

Time (h)

0.

7.5

15.

22.5

30.

0 2 4 8 12 24

Flu

ore

scen

t ce

lls

(cel

l n

um

ber

per

100

cell

s)

□ MitoSOX

■ H2DCFDA

*** ***

***

***

**

Cel

l n

um

ber

(% o

f to

tal

cell

s)

Time (h)

0.

3.

6.

9.

12.

0 4 8 12 24

Necrotic cell deathEarly apoptosisLate apoptosis

0.

30.

60.

90.

120.

150.

Tai

l D

NA

(% )

Tai

l le

ngth

(p

ixel

)

Tai

l

mo

men

t

□ DEP

■ DEP + ATG12-KO

0.

17.5

35.

52.5

70.

0.

20.

40.

60.

80.

0 4 8 12 24

Time (h)

0 4 8 12 24

Time (h)0 4 8 12 24

Time (h)

□ DEP

■ DEP + ATG12-KO

□ DEP

■ DEP + ATG12-KO

*********

***

******

******

************

0h

12h

4h 8h

24h Bleach

Annexin VP

I

0 2 4 8 12 24

DEP + ATG12-KO

Time(h) Sham

Cytochrome c

Bax

ATG12-ATG5

LC3-I

LC3-II

Bcl-2

α-tubulin

p62

Bad

12 kDa

14 kDa

55 kDa

62 kDa

26 kDa

23 kDa

20 kDa

14 kDa

50 kDa

p53

Mdm2

53 kDa

55 kDa 0.

0.75

1.5

2.25

3.

0 2 4 8 12 24

Pro

tein

exp

ress

ion

(fo

ld o

f co

ntr

ol)

Time (h)

0.

0.7

1.4

2.1

2.8

0 2 4 8 12 240.

0.75

1.5

2.25

3.

0 2 4 8 12 24

ATG12-ATG5 p62 LC3-II* * * *

0.

0.75

1.5

2.25

3.

0 2 4 8 12 24

Mdm2

0.

0.75

1.5

2.25

3.

0 2 4 8 12 24

p53

0.

27.5

55.

82.5

110.

137.5

0 2 4 8 12 24

Cel

l via

bil

ity

(% o

f co

ntr

ol)

□ DEP

■ DEP + ATG12-KO§ § §

§ †

Time (h)

** *

**

** **

Blockage of autophagy might stop PM2.5-induced endothelial apoptosis


Cytotoxicity

Summary

PM2.5

p53/mdm2 ApoptosisAutophagy


(Kaohsiung)

Maternal Exposure Embryo Fetus Adult

Embryonic ToxicityImmune ResponseReproductive Toxicity

Neuron DevelopmentMicroarray

Offspring inflammatoryNeuron morphologyBehavior Test

PM2.5 Exposure

In vivo model studies

0

50

100

150

0 1 5 10 50 1000 1 10 100 1000 10000

Concentration of PM2.5 (mg/mL)

Cel

lv

iab

ilit

y(%

of

contr

ol)

Ex

trac

ellu

lar

RO

S g

ener

atio

n

(fold

of

contr

ol)

0

5

10

15

20

1 10 100 1000 10000

A B

D

0

20

40

60

80

100

120

100 1000

Inte

nsi

ty (

G/d

)

Diameter (nm)

C

The PM2.5 size is about 400 nm

Tseng et al, Am J Chin Med, 2016

Control 100 μg 400 μg

A B C

D E F

G H I

**

**

**

**

PM2.5 cause Embryonic Toxicity

Yu et al, in manuscript

PM2.5 cause Fetal Brain Malformation

0

1

2

3

4

0 2.5

Neu

trop

hil

s

Nu

mb

er(1

06/m

l)

Concentration of PM2.5 (mg/m3)

Neutrophils

GMI 0 μg/kg

GMI 0.33 μg/kg

GMI 3.3 μg/kg

A

C

B

D

E F

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Per

cen

tage o

f

Infl

am

mato

ry C

ells

Basophils

Eosinophils

Neutrophils

Monocytes

Lymphocytes

GMI 0 0 0.33 0.33 3.3 3.3

PM2.5 0 2.5 0 2.5 0 2.5

0

1

2

3

4

5

6

7

0 2.5

WB

C N

um

ber

(10

6/m

l)


GMI 0 μg/kg

GMI 0.33 μg/kg

GMI 3.3 μg/kg

0

1

2

3

4

0 2.5

Baso

ph

ils

Nu

mb

er(1

06/m

l)


Basophils

GMI 0 μg/kg

GMI 0.33 μg/kg

GMI 3.3 μg/kg0

1

2

3

4

0 2.5E

osi

nop

hil

s

Nu

mb

er(1

06/m

l)Concentration of PM2.5 (mg/m3)

Eosinophils

GMI 0 μg/kg

GMI 0.33 μg/kg

GMI 3.3 μg/kg

0

1

2

3

4

0 2.5

Mo

nocy

tes

Nu

mb

er(1

06/m

l)


Monocytes

GMI 0 μg/kg

GMI 0.33 μg/kg

GMI 3.3 μg/kg

PM2.5 induce maternal inflammation responses

Yu et al, under revision

PM2.5 induce maternal inflammation responses

050

100150

百分比

(%)

懷孕率

050

100150

百分比

(%)

出生率

0

0.5

1

1.5

重量

(g)

胎鼠體重

**

Chao et al, Environmental Toxicology, 2017

PM2.5 affect miRNAs in fetal brain

Chao et al, Environmental Toxicology, 2017

rno-miR-9a-3p↓

rno-miR-489-3p↓

rno-miR-30d-5p↓

rno-miR-346↓

rno-miR-339-5p↓

rno-miR-191a-5p↓

rno-miR-433-3p↓

rno-miR-151-3p↓

rno-miR-107-3p↓

rno-miR-181a-5p↓

rno-miR-139-5p↓

Control

Tbr1 SATB2 Merge DAPI

PM2.5: 0.25 mg


PM2.5: 2.5 mg


PM2.5 cased fetal brain cortex development delay

PM2.5: 0.025 mg


0

0.5

1

1.5

Control 0.02mg 0.2mg 2mg

TBR1

0

0.5

1

1.5

2

2.5

3

Control 0.02mg 0.2mg 2mg

SATB2

Rat

io o

f T

BR

1+/D

AP

I ce

lls

(Fo

ld o

f C

on

tro

l)

Rat

io o

f S

AT

B2

+/D

AP

I ce

lls

(Fo

ld o

f C

on

tro

l)

***

RL

50µm

0 0.025 0.25 2.5 0 0.025 0.25 2.5

PM2.5 increase brain inflammation in the offspring

0

50

100

150

200

250

0 2.5 25

Exp

ress

ion

of

CD

68

(Fold

of

Con

trol)


A

C

B

D

0%

20%

40%

60%

80%

100%

0 2.5

Per

cen

tag

e o

f

Infl

am

ma

tory

Cel

ls


Basophils

Neutrophils

Monocytes

Lymphocytes

0

0.5

1

1.5

2

2.5

3

3.5

0 2.5

Neu

tro

ph

ils

Nu

mb

er(1

06/m

l)



A B C

0

2

4

6

8

10

12

0 2.5

WB

C N

um

ber

(1

06)


GMI 0 μg/kg

GMI 0.33 μg/kg

GMI 3.3 μg/kg

0

0.5

1

1.5

2

2.5

3

0 2.5

IgE

Exp

ress

ion

(Fo

ld o

f C

on

tro

l)


GMI 0 μg/kg

GMI 0.33 μg/kg

GMI 3.3 μg/kg

0

0.2

0.4

0.6

0.8

1

1.2

0 2.5

His

tam

ine

Exp

ress

ion

(Fo

ld o

f C

on

tro

l)


GMI 0 μg/kg

GMI 0.33 μg/kg

GMI 3.3 μg/kg

PM2.5 increase inflammatory & asthma ratio in offspring


Weng et al, Biochemical Pharmacology, 2018

PM increases the amounts of blood vessels in the offspring dentate gyrus

Control 0.025mg

0.25mg 2.5 mg

Control PM2.5

Neural cell morphology: spine spots decrease(MATLab Bonfire software analysis)


Behavior Test: 對照組 Behavior Test: PM2.5組

Max

Min

GMI (µg/kg)

0 0.33 3.3

0

2.5

25

PM

2.5

(m

g/m

3)

GMI (µg/kg)

0 0.33 3.3

0

2.5

25

PM

2.5

(m

g/m

3)

GMI (µg/kg)

0 0.33 3.3

0

2.5

25

PM

2.5

(m

g/m

3)

PM2.5 (mg/m3)

0 2.5 25

0

10

20

30

40

50

0 2.5 25

Late

ncy

(se

c)

Concentration of PM2.5 (mg/m3) Yu et al, under revision

Cell damage Molecule damage

How to protect ?

Is that going through by anti-oxidative stress?

What is the prevention against PM2.5??

Upregulation of endogenous GSH arises protection against ROS

Intr

ace

llula

r G

SH

(U

/mg)

&

GS

SG

(U

/mg)

leve

l

Control 7 days 14 days 21 days 28 days

Treatment period of GTDE

10

14

8

2

4

6

12

0

GSH

GSSG

C D E


A B

Concentration of PM2.5 (μg/mL)

0.

2.

4.

6.

8.

0 500 1000

Super

ox

ide

anio

n.le

vel

(fold

of

contr

ol)

RO

S i

nte

nsi

ty

0.

2.

4.

6.

8.

0 500 1000

MD

A l

evel

(fo

ld o

f

contr

ol)

0

2

4

6

8

0 500 1000

Control

GTDE (One week)

GTDE (Two weeks)

Control

GTDE (One week)

GTDE (Two weeks)

Control

GTDE (One week)

GTDE (Two weeks)

B

CControl GTDE (Two Weeks)GTDE (One Week)

500 mg/mL

1000 mg/mL

Control

G. tsugae blocks ROS generation and its sequential DNA damages

H2O2

0.

50.

100.

150.

200.

0 500 1000

0.

50.

100.

150.

200.

0 500 1000

Tail % Tail Length (% of control)

GTDE

A

0.

1.5

3.

4.5

6.

Control 1 Week

GLE

2 Week

GLE

Fo

ld o

f co

ntr

ol

0.

0.375

0.75

1.125

1.5

Control 1 Week

GLE

2 Week

GLE

0.

0.375

0.75

1.125

1.5

Control 1 Week

GLE

2 Week

GLE

SOD-1 Catalase

Control

PM2.5 0 mg/mL

PM2.5 500 mg/mL

PM2.5 1000 mg/mL

One week Two weeks Control One week Two weeks Control One week Two weeks

HO-1ControlGTDE (One week)GTDE (Two weeks)

D

0.

2.5

5.

7.5

10.

12.5

15.

0 500 1000

Cas

pas

e 3

/7 a

ctiv

ity

(Fo

ld o

f C

ontr

ol)

#

***

§# #


Control

GTDE (One week)

GTDE (Two weeks)


0.

1.25

2.5

3.75

5.

1 2 3 4 5 6 7

GSH/GSSG ratio

− 500 1000 500 1000 500 1000

− − − + + + +One week Two weeks

**

**

0.

0.55

1.1

1.65

2.2

0 500 1000

Vas

cula

r P

erm

eabil

ity (

Fold

of

contr

ol)

A B C

0.

0.75

1.5

2.25

0 500 1000

VE

GFA

rel

ease

(fo

ld o

f

contr

ol)


GTDE restores PM2.5–induced vascular permeability

Vascular Permeability VEGF-A release

PM2.5

GTDE

PM2.5 (mg/m3) Body Weight (g) Hemoglobin (g/dL) White Blood Cell (cells/mL) Red Blood Cell (cells/mL)

0 272.2 17.83 1.17E+07 7.58E+09

0.2 294.8 18.67 1.20E+07 8.62E+09

2 305.5 16.67 2.32E+07 5.53E+09

20 268.8 19.67 3.01E+07# 1.83E+09#

0+GTDE 268.1 19.69 1.06E+07 6.54E+09

0.25+GTDE 259.4 22.67 8.13E+06 5.23E+09

2.5+GTDE 274.2 20.33 7.13E+06 7.20E+09

25+GTDE 292.4 19.5 9.85E+06** 5.80E+09*

Table. In vivo physiological conditions in samples treated 2 weeks GTDE and PM2.5

Control

GTDE (One week)

GTDE (Two weeks)

Control

GTDE (One week)

GTDE (Two weeks)


Tseng et al, submitted 2017

記憶測試：靈芝增加子代老鼠的長期記憶能力

0

10

20

30

40

50

60

0 2.5 25

Late

ncy

(s)


GMI 0 μg/kg

GMI 0.33 μg/kg

GMI (µg/kg)

0 0.33 3.3

0

2.5

25

PM

2.5

(m

g/m

3)

Max

Min

* *#

#

rno-miR-9a-3p↓ rno-miR-9a-3p↓

rno-miR-489-3p↓

rno-miR-30d-5p↓

rno-miR-346↓

rno-miR-339-5p↓

rno-miR-191a-5p↓

rno-miR-433-3p↓

rno-miR-151-3p↓

rno-miR-107-3p↓

rno-miR-181a-5p↓

rno-miR-139-5p↓

Yu et al, in manuscript

Cytotoxicity

Antioxidants

GSH

Summary

PM2.5

p53/mdm2 ApoptosisAutophagy

PM2.5

GTDE



Aim:

Chronic Exposure to PM2.5 or Its Major Component Cause Genotoxicity, Mutagenesis and Carcinogenicity in Mammalian Cells

?

Normal cell

PM2.5

Mutant cellMalignant

tumor cell

Benign

tumor cell

CarcinogenesisMutagenesis

?

?

The impact of Chinese PM2.5 in mutagenesis and carcinogenesis

In Vitro model: gpt-transgenic CHO AS52 cell line with 6-TG selection

Tai

l m

om

ent

(pix

el)

0

100

200

300

0

20

40

60

80

100

Tai

l %

C

0

10

20

30

40

0 10 20 30

Mutant 1

mutant 2

Mutant 3

Control

AA8 UV5 EM9 AS52

Time 0 h 24 h 48 h 72 h 0 h 24 h 48 h 72 h 0 h 24 h 48 h 72 h 0 h 24 h 48 h 72 h

Chemical Concentration Survival(%)

DEHP

0.1 mM 102.47 101.66 97.43 100.88 89.52 97.61 100.21 99.43 102.98 101.04 99.67 103.11 92.31 90.76 97.75 98.96

1 mM 101.41 102.65 102.55 100.97 87.09 97.19 98.04 103.91 103.95 101.49 101.56 104.03 87.29 86.83 96.28 97.62

10 mM 100.49 100.59 97.49 103.63 76.1 98.33 97.14 105.89 81.22 103.64 99.01 100.32 75.45 84.77 91.43 99.61

50 mM 100.97 105.52 98.08 104.29 73.86 99.94 103.06 101.97 75.61 100.63 101.41 97.82 59.60 83.17 85.85 92.93

MEHP

0.1 mM 97.37 96.16 94.38 90.69 95.51 96.28 78.95 99.85 93.54 97.15 96.2 97.82 96.74 96.90 90.87 90.87

1 mM 95.9 84.78 79.45 93.18 74.46 92.01 82.69 85.06 91.09 91.98 86.96 92.03 85.56 95.07 84.95 84.95

10 mM 57.2 35.87 5.18 0.37 59.07 22.04 6.64 0.481 51.66 5.81 3.093 5.44 24.69 4.54 0.69 0.69

50 mM 45.05 x x x 49.93 x x x 25.45 x x x 6.66 x x x

MEHP+

NAC

0.1 mM 92.76 94.27 93.2 104.65 96.91 98.04 86.87 98.27 91.7 93.71 100.08 95.99 98.28 97.01 96.70 99.82

1 mM 89.93 87.28 88.01 100.86 89.59 94.12 86.41 105.82 94.09 93.7 98.05 93.58 92.96 95.92 87.30 97.45

10 mM 73.85 85.42 21.77 31.35 79.7 96.13 21.84 29.18 80.53 92.78 31.78 25.94 45.26 29.57 16.13 27.18

50 mM 31.71 40.05 12.85 18.49 33.06 35.85 14.88 20.69 33.37 33.46 13.59 23.92 30.09 25.08 12.43 22.96

DEHP, Di-(2-ethylhexyl) phthalate, the most common phthalate plasticizer used in the production of polyvinyl chloride (PVC). Several in

vivo studies have shown that DEHP absorbed in the body is metabolized to produce MEHP, mono-ethylhexyl phthalate and other metabolite.

The metabolite can be absorbed through the villi and travel in the circulation system then causes series damages in endocrine and

reproductive system. In addition, investigations also demonstrated an association with elevated induction of rat hepatic cancer, testicular

cancer, and developmental toxicity in reproductive system under MEHP exposure. However, the mechanism regarding to carcinogenicity

induced by DEHP or its metabolite MEHP remains unclear up to date. We therefore are investigating the cell viability and mutagenicity of

DEHP and MEHP in Chinese hamster ovary (CHO) cells. Cytotoxicity and mutagenicity induced by these chemicals in AS52 cell which

inserted gpt gene. Results show that the parental chemical, DEHP, as well as MEHP derivatives caused dose-dependent decreases in cell

survival, but with very different potencies: MEHP has the high potency; and DEHP lower. Exposure to these compounds induced dose-

dependent production of ROS, as determined by Cm-H2DCF-DA fluorescence. It was noted however that simultaneous treatment of ROS

scavenger N-acetylcysteine (NAC) shows the protection against DEHP and MEHP induced DNA damages. In 6-TG selection assay, data

indicate that exposure MEHP cause dose-dependent mutant frequency as compare to the control. In summary, DEHP and MEHP are

cytotoxic to the cells and require induction of ROS to exert their genotoxicity effects.

DEHP and Its Metabolite MEHP Cause Genotoxicity and

Mutagenesis in Mammalian Chinese Hamster Ovary Cells

1. Bioscience Technology, Chung Yuan Christian University, Zhongli district, Taoyuan city, Taiwan 320232. Biomedical Engineering, Chung Yuan Christian University, Zhongli district, Taoyuan city, Taiwan 32023

*Corresponding author

張祐容1(Yu-Jung Chang)、林佩穎1(Pei-Ying Lin)1 、曾嘉儀2 (Chia-Yi Tseng)、招名威1* (Ming-Wei Chao)

ABSTRACT

Figure 1. Metabolism of DEHP hydrolytic/oxidative pathway

leading to MEHP and secondary metabolite formation. The image is

modified from Matthew et al., 2010.

Metabolism of DEHP

Cell viability

MEHP causes mutagenicity in the gpt in AS52 cells

Figure 6. Mutagenicity of

AS52 cells treated with MEHP

and MEHP+NAC. A) AS52

cells carry a single copy of the

bacterial gpt gene functionally

expression B) MEHP increased

mutant frequencies in a dose

dependent and NAC protection

against mutagenicity. C) All

mutated gpt sequences were

obtained from 22 independent

clones.

Figure 3. Cell viability of DEHP or MEHP-treated CHO cell lines

(AA8, UV5, EM9, AS52). DEHP is not cytotoxic but MEHP has

higher potency. We found that MEHP cause none of nucleotide

excision damage and base excision damage. Addition of NAC

provides protection against the MEHP-induced toxicity. This data

suggests that MEHP-caused cytotoxicity is according to the ROS

generated by MEHP. The cell viability was determined by the MTS

assay and the data was presented percentage of the control.

A

C

Mutagens

When gpt is active

When gpt is inactivated

(mutated)

CHO cells (dish)

were treated with

MEHP

No Colony

Use 6-TG as

selection reagent

for the gpt gene

6-TG mutant

colonies

gpt Assay (6-Thioguanine selection) for mutagenicity

CHO cells

No. of mutations (% of total)Control MEHP Average

Type of

mutationA B 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 C M

Single base

pair

substitutions

1

(33)

7

(100)

56

(28)

55

(33)

57

(28)

63

(34)

60

(34)

52

(26)

59

(32)

65

(37)

65

(33)

56

(30)

58

(31)

74

(52)

46

(42)

2

(67)

1

(50)

4

(80)

4

(67)

1

(50)

3

(60)

6

(50)4±3.0 39.4±6.2

Transversio

n

G:C → T:A 0 1(14) 10(5) 7(4) 11(5) 5(3) 10(6) 11(6) 9(5) 18(10)15(8) 13(7) 12(6) 12(9) 7(6) 0 0 0 0 0 0 0 0.5±0.5 7±1.3

G:C →

C:G0 0 9(5) 12(7) 12(6) 16(9) 13(7) 10(5) 11(6) 10(6) 15(8) 11(6) 12(6) 13(9)13(12)1(33) 0 0 0 0 0 1(8) 0 8±1.3

A:T → T:A 0 0 11(6) 12(7) 7(3) 9(5) 8(4) 6(3) 7(4) 7(4) 11(6) 7(4) 7(4) 12(9) 8(7) 1(33) 1(50) 1(20) 0 0 1(20) 0 0 5.8±1.0

A:T → C:G 0 0 12(6) 11(7) 10(5) 14(7)12(57)10(5) 11(6) 8(4) 4(2) 9(5) 10(5) 12(9) 5(5) 0 0 1(20) 1(17) 1(50) 0 0 0 6.6±1.1

Transition

G:C → A:T 0 5(71) 11(6) 9(5) 10(5) 12(6) 16(9) 10(5) 12(6)14(48)11(6) 9(5) 9(5) 13(9) 9(8) 0 0 0 1 0 0 0 2.5±2.5 7.3±1.3

A:T → G:C 0 1(14) 3(2) 4(2) 7(3) 7(4) 1(1) 5(3) 9(5) 8(4) 9(5) 7(4) 8(4) 12(9) 4(4) 0 0 2(40) 2(33) 0 0 0 0.5±0.5 4.4±0.8

Insertion

(total base)

1

(33)0

124

(63)

92

(55)

127

(63)

107

(57)

100

(56)

127

(64)

105

(57)

86

(48)

107

(54)

113

(61)

106

(57)

46

(33)

41

(37)0 0 0 0 0 40 42 0.5±0.5 64.4±11.7

Deletion

1 base pair 0 0 3(2) 2(1) 3(1) 1(1) 2(1) 3(2) 5(3) 7(4) 4(2) 4(2) 3(2) 3(2) 5(5) 0 1(50) 0 0 0 0 0 0 2.3±0.5

2 base pair 0 0 2(1) 3(2) 2(1) 3(2) 3(2) 1(1) 2(1) 4(2) 8(4) 1(1) 3(2) 2(1) 4(4) 0 0 1(20) 0 0 0 0 0 2±0.4

3 base pair 0 0 2(1) 3(2) 2(1) 1(1) 1(1) 4(2) 1(1) 3(2) 2(1) 2(1) 2(1) 3(2) 2(2) 1(33) 0 0 1(17) 0 0 0 0 1.5±0.3

Multiple

changes1(33) 0 11(6) 13(8) 11(5) 12(6) 13(7) 11(6) 13(7) 13(7) 12(6) 9(5) 13(7) 13(9)12(11) 0 0 0 1(17) 1(50) 0 1 0.5±0.5 8±1.3

Total 3(100) 7 198 168 202 187 179 198 185 178 198 185 185 141 110 3 2 5 6 2 5 12 5±2.5 117.5±19.5

The research was supported from Ministry of Science and Technology (NSC102-2320-B-033-MY3)

Chung Yuan Christian University

0

20

40

60

80

100

120

140

0.1 1 10 50

MEHP

MEHP+5mM NAC

MEHP+10mM NAC

0

5000

10000

15000

20000

25000

30000

0 0.1 1 10 50

MEHP

MEHP+5mM NAC

MEHP+10mM NAC

0

0.01

0.02

0.03

0.04

0.05

0 0.1 1 10 50

MEHP

MEHP+5mM NAC

MEHP+10mM NAC

0

2000

4000

6000

8000

10000

12000

14000

0 50 100 150

H2O2MEHP 10mMMEHP 100mMDEHP 10mMDEHP 100mMH2O2+NAC

Concentration of MEHP (mM)

Intr

acel

lula

r R

OS

gen

erat

ion

Su

per

ox

ide

anio

n

(fo

ld o

f co

ntr

ol)

A B

Lip

id p

ero

xid

atio

n

Co

nce

ntr

atio

n o

f M

DA

(n

M)

Ex

trac

ellu

lar

RO

S g

ener

atio

n

(in

ten

sity

)

ETime (min) Concentration of MEHP (mM)

Concentration of MEHP (mM)

D

Figure 2. (A) Extracellular ROS production was measured by using Cm-H2DCFDA. DEHP

and MEHP do not produce extracellular ROS generation. (B) MEHP induces intracellular ROS

production in AS52 cells in a dose dependent manner and addition of NAC decreases the ROS

production. (C) Fluorescence microscopy also confirm the result from Figure 2B. Cells treated

with 10 mM H2O2 was used as positive controls. Nuclei were stained with 1-μg/ml Hoechst

33258. (D) Superoxide anion production were measured by MitoSOX assay. (E) MEHP-

induced ROS increase lipid peroxidation in AS52 cells. MEHP increases MDA in a dose

dependent. Lipid peroxidation was detected by using MDA assay kit.

MEHP causes ROS generation in vitro

10 mM

10 μm

Co

ntr

ol

10

mM

50

mM

Con

trol

(+)

ROS Production Nuclei Stain

10 μm

400X

10 μm

ROS Production Nuclei Stain Merge

C

A B

0

20

40

60

0 24 0 24 0 24

AA8 UV5

EM9 AS52

ME

HP

IC5

0(m

M)

Cont

siRNA

XPD

siRNA

PARP

siRNA

Time (h)

0

20

40

60

0 24 0 24

AA8 UV5

EM9 AS52

MEHP

NAC

+-

+

ME

HP

IC5

0(m

M)

IC50 for XPD－/＋ and PARP1－/＋ CHO cell

Figure 5. IC50 of MEHP-treated XPD－/＋ and PARP1－/＋ CHO cell

lines. IC50 was determined by each cell viability. (A) CHO Cells

were transfected with XPD and PARP siRNA by using

Lipofectamine 3000 reagent (Invitrogen) according to the

manufacturer’s instructions. The data indicates that IC50 of XPD－/＋

and PARP1－/＋ CHO cell was decreased after MEHP treatment. (B)

is non-siRNA treated cells. Apparently, MEHP treatment was

decreased IC50 of XPD－/＋ and PARP1－/＋ CHO cells compare with

non-siRNA treated cells. Addition of NAC protects CHO cells and

increases IC50 for MEHP treatment.

Day

Wei

gh

t (g

)

0

1

2

3

0 10 20 30Tu

mo

r V

olu

me

(cm

3)

Figure 8. Tumorigenicity of mutant AS52

cells in vivo. Mutant cells were injected

subcutaneously into immunodeficient nude

mice (A) Upper panel: representative

subcutaneous flank tumor in mice. However,

the tumor size is various. Scale bar = 1 cm. (B)

The first tumor is palpable from the 15th day

after injection. Plots of tumor volumes (cm3)

determined by measurements with a caliper,

the range is from 0.14-2.4 cm3. Body weight

by plotted and it shows no difference between

the samples. (C) The 8 micrometer tumor

sections were examined by

immunohistochemical(c-Myc) and H&E

staining. The tumor sections show that the

cancerous cells are characterized by large

nuclei, having irregular size and shape.

Mutant AS52 cells induce tumorigenesis in vivoA B

C

Mutant 1 Mutant 2 Mutant 3 Control

A549 Mutant 1 Mutant 3Mutant 2

H&

Est

ain

DA

PI

c-M

yc

Mer

ge

400X

Figure 7. Analysis of AS52

mutant samples: ①AS52 ②Mutant control named as

STAS52-C ③Mutant AS52

named as STAS52. (A) The data

shows that normal AS52

compare with mutant control and

mutant. The miRNA expressions

are significantly alteration

between mutant and normal

AS52. Furthermore, the miRNA

expressions between mutant

control and normal AS52 are not

significantly alteration. (B) The

table show the fold change in

different miRNAs. There are 96

miRNAs was down-regulated

compare with normal AS52.

0

20

40

60

80

100

MEHP induces DNA single strand break

Figure 4. MEHP cause DNA damages measured with using

Comet assay. Formation of single DNA strand breaks but no

observed any double DNA strand breaks by MEHP. Cells

treated 10, 25 mM of MEHP only 50–150 comets were

collected and analyzed from each experiment. (A) Fluorescence

microscopy for single DNA damage. Magnification= 400x.

Quantification of Figure 4A and 4B: (C) and (E) The comet tail

percent of DNA; (D) and (F) The comet moment is a function

of the distance and intensity of DNA from the center of the

comet head.

Co

ntr

ol

25

mM

10

mM

-NAC +NAC

A Single strand DNA damage Double strand DNA damage

-NAC +NAC

B

Co

ntr

ol

(+)

Tai

l %

E

F

D

Tai

l m

om

ent

(pix

el)

0

10

20

30

40

50

+

1.MEHP causes the generation of intracellular ROS

such as superoxide anion and do not generate

extracellular ROS.

2.MEHP has the high potency; and DEHP lower. But it

does not have nucleotide excision and base excision

toxicity.

3.MEHP induces DNA double strand break.

4.MEHP causes PARP1＋/－ and XPD＋/－ CHO cell

lines lower IC50 compare with non-treated CHO cells.

5.MEHP increases gpt-AS52 mutant frequency in a

dose dependent manner and NAC against

mutagenicity.

6. AS52 mutant clones have an ability of tumorigenicity

in vivo.

Summary

mmu-miRNA-130a-3p

SMAD4Smad2/3

ATG2B

miR-130a-3p (and other miRNAs w/seed AGUGCAA)

ZEB1 PPARA

Microarray analysisNormalized Intensity log2 (Ratio)

ID Name AS52 STAS52

STAS52 /

AS52

PH_mr_0000996 mmu-miR-130a-3p 1698 521 -1.7044812

PH_mr_0002298 mmu-miR-27a-3p 1701 548 -1.6341353

PH_mr_0002920 mmu-miR-25-3p 803.75 260 -1.6282352

PH_mr_0001256 mmu-miR-92a-3p 2207.32 727 -1.6022685

PH_mr_0000042 mmu-miR-125b-5p 18658.47 6283 -1.5703052

PH_mr_0000150 mmu-let-7i-5p 1802.62 628 -1.5212588

PH_mr_0000206 mmu-let-7f-5p 4698.36 1660 -1.500974

PH_mr_0000446 mmu-miR-22-3p 1957.46 694 -1.4959753

PH_mr_0000581 mmu-miR-181a-5p 837.6 299 -1.486116

PH_mr_0000367 mmu-miR-34b-5p 820.75 304 -1.4328715

PH_mr_0001696 mmu-miR-20a-5p 2123.19 790 -1.4263089

PH_mr_0000169 mmu-let-7c-5p 4210.54 1597 -1.398641

PH_mr_0000163 mmu-let-7b-5p 2421.32 920 -1.396088

PH_mr_0002380 mmu-miR-23a-3p 4369.67 1662 -1.3946039

PH_mr_0000203 mmu-let-7a-5p 4703.7 1852 -1.344712

PH_mr_0000379 mmu-miR-574-5p 4455.21 1796 -1.3107061

PH_mr_0001695 mmu-miR-93-5p 1276.77 527 -1.2766238

PH_mr_0000017 mmu-miR-100-5p 865.38 359 -1.2693499

PH_mr_0000207 mmu-let-7d-5p 3319.33 1396 -1.2495931

PH_mr_0001589 mmu-miR-16-5p 1667.08 704 -1.243676

PH_mr_0002893 mmu-miR-125a-5p 4953.41 2095 -1.2414718

PH_mr_0001702 mmu-miR-106a-5p 1646.32 717 -1.1991998

PH_mr_0002170 mmu-miR-34c-5p 671.44 305 -1.1384492

PH_mr_0002203 mmu-miR-27b-3p 550.83 253 -1.1224698

PH_mr_0002528 mmu-miR-23b-3p 2102.51 989 -1.0880702

PH_mr_0002259 mmu-miR-320-3p 893 425 -1.0711973

PH_mr_0002947 mmu-miR-19b-3p 1996.14 958 -1.0591153

PH_mr_0009550 mmu-miR-7650-5p 777.13 373 -1.0589803

PH_mr_0000425 mmu-miR-146b-5p 303.6 146 -1.0562034

PH_mr_0001874 mmu-miR-24-3p 1864.68 913 -1.0302413

PH_mr_0002734 "mmu-miR-199a-3p,

mmu-miR-199b-3p"360.5 179 -1.0100397

PH_mr_0000495 mmu-miR-181d-5p 247 123 -1.0058527

STAS5

2

STAS52-

C

AS52

A B

Figure 7. Analysis of AS52 mutant samples: ①AS52

②Mutant control named as STAS52-C ③Mutant

AS52 named as STAS52. (A) The data shows that

normal AS52 compare with mutant control and mutant.

The miRNA expressions are significantly alteration

between mutant and normal AS52. Furthermore, the

miRNA expressions between mutant control and

normal AS52 are not significantly alteration. (B) The

table show the fold change in different miRNAs. There

are 96 miRNAs was down-regulated compare with

normal AS52.

048

121620

0 1 10 25Muta

nt

Fra

ctio

n (

10

-5) MEHP

MEHP+5 mM NAC

Concentration of compound (mM)

B

Mutagens: AFB1, TCDD,

PAH, BPA, PM2.5, Aniline,

Aminophenol, DEHP…

0.5 Kb PCR fragment

0.8 Kb PCR fragment

The gpt gene and linked gpt rearrangement in AS52 cells

Kelecsenyi, Z. et al. Mutagenesis (2000) 15:25-31

Chao et al., Tox Sci, 2012

Skipper et al, Chem Res Toxicol. 2006

Cui et al, Chem Res Toxicol. 2007

3,5-DMA N-OH-3,5-DMA

3,5-DMAP

Chao et al, ToxSci, 2012

Mutation type

Number of mutations (% of total)

Control 3,5-DMA+S9 N-OH-3,5-

DMA

3,5

-DMAP

- NAC - NAC - NAC

Single base pair substitution 12 (52) 19 (54) 21 (40) 25 (54)

Transversions G:C to T:A 6 (26) 4 (11) 8 (15) 14 (30)

G:C to C:G 3 (13) 1 (3) 0 (0) 2 (4)

A:T to T:A 2 (9) 2 (6) 0 (0) 1 (2)

A:T to C:G 0 (0) 1 (3) 0 (0) 2 (4)

Transitions G:C to A:T 1 (4) 4 (11) 7 (13) 2 (4)

A:T to G:C 0 (0) 7 (20) 6 (11) 4 (8)

Insertion 4 (17.5) 6 (17) 5 (10) 4 (8)

Deletion 5 (21.5) 9 (26) 20 (38) 20 (43)

1 bp 4 (17.5) 4 (11) 10 (19) 10 (22)

2 bp 0 (0) 5 (15) 0 (0) 0 (0)

3 bp 2 (9) 0 (0) 10 (19) 6 (13)

Multiple sequence change 1 (4) 1 (3) 6 (11) 1 (2)

Total 23 (100) 35 (100) 52 (100) 46 (100)

PM2.5 contains Dimethylaniline

Palmiotto et al, Chemosphere, 2001

The idea for Long-term ROS generation in the cells

Chao et al , ToxSci, 2014

Monocyclic aromatic amine-induced ROS can be detected within 5 days

0.

100.

200.

300.

400.

0.

40.

80.

120.

160.

0 1 3 5 7

RO

S p

rod

uction

(%

of

Co

ntr

ol)

Su

rviv

al (%

of C

ontr

ol) Cytotoxicity

ROS production

3,5-DMAP

Control

+NAC

DMAP

DMAP

Chao et al , ToxSci, 2014

Histone+DMAP+Ascorbic acid

Histone+DMAP

5.4216544

x103

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

Counts vs. Acquisition Time (min)4 5 6 7 8 9

+ESI EIC(267.1660-267.1730) SNR (5.536min) = 190.3

5.536

21024

Ye et al, Chemical Research Toxicology, 2012

Acknowledgements

Chia-Yi Tseng 王志軒 Gerald WoganMarion Gordon

林欽鴻莊妤甄

廖家巍

余佳語

童思瑜

Wen-Hsung Chan

林佩穎

吳均豪 Pinpin LinJolie WangBonnie Firestein

PM -Induced Imbalance of Intracellular Reactive Oxygen ...Nov 26, 2018 · D B C Time (h) 0. 0.25 0.5 0.75 1. 1.25 0 2 4 8 12 24 DEP ent DEP+NAC 0. 17.5 35. 52.5 70. 87.5 0 2 4 8

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