Atrazine Triggers the Extrinsic Apoptosis Pathway in Lymphocytes of

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Atrazine triggers the extrinsic apoptosis pathway in lymphocytes of the frogs Pelophylax nigromaculata in vivo Xiuying Jia, Dandan Wang, Nana Gao, Hui Cao, and Hanjun Zhang Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.5b00238 • Publication Date (Web): 18 Sep 2015 Downloaded from http://pubs.acs.org on September 23, 2015

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Chemical Research in Toxicology

1

Atrazine triggers the extrinsic apoptosis pathway in lymphocytes of

2

the frogs Pelophylax nigromaculata in vivo

3 4 5

Xiuying Jia†,‡, Dandan Wang†, Nana Gao†, Hui Cao†, Hangjun Zhang†,‡,*

6 7 8 9

†

College of Life and Environmental Sciences, Hangzhou Normal University,

Hangzhou, China, 310036 ‡

Key Laboratory of Hangzhou City for Ecosystem Protection and Restoration,

Hangzhou Normal University, Hangzhou, China, 310036

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*corresponding author.

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Email: [email protected]

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ACS Paragon Plus Environment


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ABSTRACT: ATR is extensively used worldwide as a herbicide, with a

2

global influence on the ecological environment. The widespread

3

distribution of herbicides may be one of the possible reasons for the

4

decline in the global amphibian population. The acute toxicity and

5

potential toxicological mechanisms of ATR on the immune system of

6

frogs are not well known. In this study, Pelophylax nigromaculata was

7

used as an experimental carrier and exposed to 0, 1, 10, 100, and 1000 µg

8

L-1 ATR solutions for 14 days. Lymphocyte viability was significantly

9

decreased, and the characteristics of apoptosis, such as DNA damage,

10

apoptosis percentages, DNA ladder and morphologic features were

11

measured in ATR-exposed groups. It is the result of the alterated

12

expression of some key proteins in the extrinsic apoptosis pathway. The

13

expressions of the key proteins Fas, Fas-L, c-FLIP, caspase-8, Bid, and

14

caspase-3 were significantly induced in a dose-dependent manner.

15

Moreover, c-FLIP was shown to modulate the Fas-dependent apoptosis of

16

lymphocytes. In summary, acute ATR exposure could damage

17

lymphocytes as a result of the apoptosis of lymphocytes via an extrinsic

18

signaling pathway. This study provided novel insights into the

19

immunological and toxicological responses of amphibians exposed to

20

triazine herbicides.

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Keywords: Atrazine; lymphocyte; Rana nigromaculata; apoptosis;

22

Fas/Fas-L 2


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1

INTRODUCTION

2

ATR is perhaps the most commonly used pesticide for controlling

3

broadleaf and grassy weeds in a variety of crops worldwide.1-3 The

4

chemical was detected in 90% of stream water in agricultural areas;

5

approximately 45% of the samples had >0.1 µg L-1 ATR.4 In the United

6

States, ATR is a commonly used pesticide, with up to 36,000 tons applied

7

annually since 1990.5 In Canada, Africa, and the Asia-Pacific region, the

8

quantity of ATR used is still large.6 The ATR concentration detected in

9

rainfall has ranged between 40 and 500 µg L-1 in surface water.7 ATR

10

can contaminate natural water sources through soil leaching, which

11

results in permanent water pollution.8

12

ATR is an immunosuppressant toxin that directly threatens the

13

survival of aquatic animals.9 ATR can increase the pathogen infection

14

ratio in frogs, such as that of chytrid fungal infection, in frogs.10-12

15

Furthermore, the infected individuals generally display morphological

16

abnormalities, high mortality rates, and low survival.13 Gonadal

17

development retardation (gonadal dysgenesis) and testicular oogenesis

18

(hermaphroditism) in leopard frogs (Rana pipiens) have been attributed to

19

ATR exposure (≥0.1 µg L-1).14 In addition, male frogs were completely

20

feminized and chemically castrated.9,15 These reports suggest that ATR

21

contributes to the decline in amphibian populations.9,16 Amphibians have

22

specific life history characteristics that increase their susceptibility to 3



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toxins. In particular, their permeable skin plays an important role in

2

absorbing moisture and gaseous interchange, but is always distinctly

3

exposed to the natural water environment. Chemical pollution is generally

4

suspected as one of the main contributing factors to amphibian population

5

decline.17 Pelophylax nigromaculatus is an important frog species that is

6

widely distributed in East Asia, especially in China. The species may be a

7

strong representative of frogs and even amphibians, in the region. P.

8

nigromaculatus is responsive and sensitive to chemicals.18,19 Therefore,

9

pesticides and herbicides, such as ATR, are easily concentrated in

10

amphibians and may negatively affect the physiological functions of

11

different tissues and organs.20

12

Exposure to ATR can stimulate the response of immune system of

13

amphibian.21 Lymphocytes are the primary functional cells in the specific

14

cell-mediated immune response.22 High ATR concentrations (4.7 g/mL)

15

can induce apoptosis in lymphocytes.23 Apoptosis can be induced through

16

the intrinsic or extrinsic pathway. An important regulatory factor of the

17

extrinsic apoptosis pathway in the immune system is the Fas

18

(APO-1/CD95) receptor–ligand system,24 which plays an important role

19

in maintaining the immune system stability through the clearance of

20

infected, aging, and potentially harmful lymphocytes.25,26 Fas is activated

21

by Fas-ligand (Fas-L), which recruits a number of the adapter protein

22

FADD. In turn, FADD binds a carboxy terminal death domain to Fas. 4


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FADD also recruits procaspase-8 via the interaction of DED, thereby

2

forming DISC and causing the autocatalytic activation of caspase-8. The

3

downstream targets of caspase-8 are directly and indirectly controlled by

4

the activation of the apoptosis executor caspase-3, which is mediated by

5

cleaved Bid.27 To tightly control apoptosis, intracellular inhibitory

6

molecules that regulate Fas-induced apoptosis; one such protein is c-FLIP,

7

which blocks the activation of caspase-8 in DISC, thereby inhibiting the

8

Fas-induced apoptosis.28

9

ATR exposure in vivo may disrupt the normal immune response via

10

direct injury to lymphocytes. For amphibians, the mechanism by which

11

ATR affects lymphocytes in the immune systems is generally unknown,

12

and related research is urgently needed. In the present study, the

13

ATR-induced lymphocyte apoptosis in the immune defense via the

14

Fas/Fas-L system is explored. This study will establish a toxicity model

15

for the ATR-induced immunotoxicity in frogs and may contribute to the

16

explain the population decline of amphibian species induced by triazine

17

herbicides.

18

MATERIALS AND METHODS

19

Chemicals.

20

2-chloro-4-ethylamino

21

formulation emulsifiable solution) was obtained from the Academy of

22

Agricultural Science (Hangzhou, Zhejiang, China). MTT was purchased

ATR

(CAS

Registry

Number:

-6-isopropylamino-s-triazine,

301-49A; commercial

5



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from Sigma Co. Sodium heparin was purchased from Nanjing Jiancheng

2

Bioengineering Inc. (Nanjing, Jiangsu, China). Lymphocyte separation

3

medium was purchased from Sangon Bioengineering Co. (Shanghai,

4

China). The AxyPrep genomic DNA extraction kit was purchased from

5

Sanhe Bioengineering Inc. (Hangzhou, Zhejiang, China).

6

Animals and Treatment. In summer, healthy adult P. nigromaculata

7

were collected from the Tianmu Mountain in the Hangzhou suburbs. The

8

frogs were kept in an aquarium (60 cm × 40 cm × 35 cm) in 2 cm to 3 cm

9

of dechlorinated tap water for 6 days to 7 days prior to the experiments,

10

for which only robust frogs were selected. Based on the 96 h LC50 ATR

11

acute test and the most reported ATR concentrations in the field water

12

samples, 150 frogs were randomly divided into 5 groups (n = 30 per

13

group) and exposed to 0, 1, 10, 100, and 1000 µg/L of ATR at a depth of

14

3 cm. The research was approved by the local government, and the

15

animals were handled in accordance with the guidelines set by the

16

Experimentation Ethics Review Committee. All the groups and

17

treatments were set up as shown in Table 1.

18

Table 1

19

The control group was exposed to dechlorinated tap water at 3 cm.

20

An indoor glass aquarium (60 cm × 40 cm × 35 cm) and containing

21

aerated water (experimental water temperature range from 20 °C to 25 °C,

22

pH 6.0 to 7.0; dissolved oxygen, 8 µg/mL) was used in the experiment for 6


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14 days. Static displacement was applied, such that the test solution was

2

replaced daily, and the animals were fed with earthworms twice daily. At

3

the end of the exposure period, the frogs were placed under short-term

4

ether anesthesia and sacrificed by pithing. Blood was collected from all

5

frogs by cardiac puncture into EDTA-2K anticoagulant syringes.

6

Serum Lymphocytes Separation. Briefly, 3 mL of the lymphocyte

7

separation medium (density, 1.060 g/mL) was added to a 15 mL

8

centrifuge tube, where 1 mL of whole blood was diluted with 3 mL of

9

phosphate buffer. The medium was slowly added to keep the layer of

10

diluted blood over the separation medium. At temperatures from 18 °C to

11

20 °C, the 15 mL tube was centrifuged for 30 min at 2500 rpm. The

12

middle of the white lymphocyte layer was drawn and transferred into a

13

1.5 mL tube and centrifuged at 3000 rpm for 5 min at 4 °C. The cells

14

were cultured in RPMI-1640 medium (Gibco) supplemented with 5%

15

fetal calf serum (Gibco), 100 U/mL penicillin and 100 µg/mL

16

streptomycin in 5% CO2 atmosphere at 27 °C for 5 h to remove the other

17

leukocyte populations. The non-adherent lymphocytes were carefully

18

collected and re-cultured in RPMI-1640 medium. The cells were stained

19

with Giemsa and observed under a microscope with a hemocytometer to

20

identify the purity of the lymphocytes. When an obviously overwhelming

21

majority of lymphocytes was observed, the final set of collected cells was

22

resuspended in 1 mL of PBS for subsequent tests. 7



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Cell Viability Analysis. The effect of ATR on lymphocytes was

2

determined by a modified MTT test, as previously suggested.29 Briefly,

3

the cell suspension was diluted to 106 /mL with PBS; 50 µL per well of

4

the diluted cell suspension was added to 50 µL of the culture medium in a

5

96-well culture plate. Subsequently, 10 µL of the MTT solution (5 mg/

6

mL) was added to each well before incubation for 4 h at 27 °C. After

7

removing the supernatant liquid, 100 µL of DMSO was added to dissolve

8

the formazan precipitate at 27 °C. Simultaneously, zero setting wells with

9

100 µL of culture medium, 10 µL of MTT, and 100 µL of DMSO were

10

prepared for testing. After shaking for 10 min, the absorbance was

11

measured at 570 nm with a microplate reader for enzyme-linked

12

immunosorbent assay. Cell viability formulation:

13

Cell viability(%) =

cells of treatment groups in each well cells of control groups in each well

× 100%

14

Apoptosis Detection and PI Staining for Flow Cytometry. The

15

apoptotic percentages of lymphocytes in different groups were analyzed

16

according to our previous method.30 Briefly, lymphocytes were washed

17

with ice-cold PBS, and fixed in 70% ethanol for 24 h at 4 °C. Cells were

18

washed twice with PBS and treated with 100 mg/mL of RNase (Sigma)

19

and 50 µg/mL of PI staining bfuffer for 30 min at room temperature. Cells

20

were then filtered with a B Falcon circular tube (No. 352235, Becton

21

Dickinson, New Jersey, USA) prior to analysis with a BD flow cytometer.

22

PI was excited at 488 nm and detected at 630 nm. 8


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1

Commet Assessment of DNA Damage. DNA damage was determined

2

by a single cell gel electrophoresis assay, as described by Singh et al.31

3

Fully frosted slides were covered with 1.5% normal melting point agarose.

4

After solidification, a second layer was placed on the first gel layer and

5

covered with a coverslip. The second layer contained 50 µL of the cell

6

PBS suspension (105/mL) mixed with 50 µL of 1% low melting point

7

agarose. After 10 min of solidification at 4 °C, the coverslips were

8

removed from the slides and immersed in ice-cold, freshly prepared lysis

9

solution with 1% Triton X-100 and 10% DMSO for 1.5 h at 4 °C to lyse

10

the cells and allow DNA unfolding. The slides were removed from the

11

lysis solution and washed thrice with ice-cold PBS before they were

12

placed on a horizontal gel electrophoresis tank filled with ice-cold fresh

13

electrophoresis buffer. Denaturation and electrophoresis were performed

14

on ice under dim light for 30 min at 4 °C. Electrophoresis was performed

15

at 25 V (300 mA) for 30 min. After electrophoresis, the slides were rinsed

16

gently thrice with the neutralization buffer to remove excess alkali and

17

detergents. Each slide was stained with ethidium bromide and covered

18

with a coverslip in dim light for 10 min. The slides were washed thrice

19

with distilled water to remove the excess ethidium bromide. A total of

20

100 randomly captured comets from each slide were examined with an

21

epifluorescence microscope (Zeiss, Oberkochen, Germany) connected to

22

an image analysis system (UVP BioImaging Systems). The following 9



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comet parameters were evaluated by the comet score analysis software:

2

tail length, tail intensity (% of DNA), and tail moment.

3

Agarose Gel Electrophoresis (DNA ladder). The AxyPrep genomic

4

DNA Kit was used to extract DNA from aliquots of 2×106 cells according

5

to the manufacturer’s protocol. A total of 3 mg of agarose and 30 mL of

6

0.5× Tris–acetic acid–ethylenediaminetetraacetic acid solution were

7

combined in a 200 mL Erlenmeyer flask. The solution was heated in a

8

microwave oven for 2 min to 4 min until the agarose dissolved.

9

Subsequently, 15 µL of 1 µg/mL ethidium bromide was added to the gels

10

until the solution cooled to 40 °C. The dissolved agarose was swirled for

11

mixing and poured onto a taped plate with casting combs in place. The

12

gel was allowed to solidify for 20 min to 30 min. The casting combs were

13

removed and the gel was placed in a horizontal electrophoresis apparatus.

14

A 1×TAE electrophoresis buffer was added to the reservoirs until the

15

buffer covered the agarose gel. A total of 2 µL of loading dye and 5 µL of

16

the DNA sample solution were mixed and loaded into the wells. Each gel

17

was electrophoresed for 30 min at 120 V and analyzed with a Gel Doc

18

1000 system. A 1 kb Ready-Load DNA ladder was used as the standard

19

size marker.

20

TEM Morphological Analysis. Lymphocytes were harvested and fixed

21

overnight in 2.5% glutaraldehyde at 4 °C. The fixed cell samples were

22

washed thrice with 0.1 M PBS (pH 7.4) for 15 min, post-fixed in 1.5% 10


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1

osmium tetroxide for 1 h, and washed again with 0.1 M PBS. After

2

dehydration in graded alcohol concentrations (20% to 100%) and

3

embedding in pure acetone, the samples were polymerized at 60 °C in an

4

incubator for 48 h. The ultrathin sections were prepared and stained with

5

3% uranyl acetate and 3% lead citrate. The sections were mounted on

6

copper grids for viewing under an H-7650 transmission electron

7

microscope (Hitachi, Japan).

8

Western Blot Analysis. A total protein extraction kit (containing the

9

Protease Inhibitor Cocktail) was used to extract proteins. The protein

10

concentration was determined with a BCA protein assays kit. Briefly,

11

60 µg of total protein per mini-gel well was loaded and resolved by 10%

12

SDS-PAGE for 3 h. The gel for SDS-PAGE was balanced in a

13

Tris–glycine buffer solution for 30 min, and the proteins were transferred

14

from the gels to polyvinylidene difluoride membranes, which were

15

treated with methyl alcohol for 20 s and balanced in Tris–glycine buffer

16

solution for 5 min under cold conditions at a constant voltage of 100 V

17

for 2 h. The blots were blocked in T-TBS containing 5% nonfat dry milk

18

for 1 h at room temperature and washed thrice with T-TBS for 5 min. The

19

membranes were incubated overnight in T-TBS containing 3% nonfat dry

20

milk and the respective primary antibodies (Anti-Fas-L antibody, Santa

21

Cruz sc-834, 1:1000; Anti-Fas antibody, Thermo Scientific MA1-34171,

22

1:300; Anti- caspase-8 antibody, Santa Cruz sc-56070, 1:500; Anti-c-FLIP 11



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Page 12 of 42

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antibody, Abcom #ab8421, 1:500; Anti-caspase-3 antibody, Santa Cruz

2

caspase-3, 1:800; Anti-Bid antibody, Santa Cruz sc-6291, 1:800) at 4 °C.

3

After incubation, the membranes were washed four times with T-TBS for

4

5 min. The membranes were incubated in T-TBS containing 2% nonfat

5

dry milk and secondary antibodies (1:5000) for 1 h at room temperature

6

and washed five times with T-TBS for 5 min. The internal control protein

7

β-actin was similarly treated. A SuperSignal® West Dura Extended

8

Duration Substrate was used according to the manufacture’s protocol, and

9

1 mL of the ECL working solution was prepared for membrane transfer

10

incubation. After 1 min, the excess ECL working solution was removed

11

and the membranes were sealed with cling film. The X-ray film was

12

placed in a darkroom and exposed for 5 min to 10 min before

13

development and fixation. The density of each band was quantified thrice

14

by densitometry with the Bandscan 5.0 software. β-Actin was used to

15

normalize the samples.

16

Statistical Analysis. Data were analyzed with Origin 8.0 by one-way

17

ANOVA followed by Dunnett’s t test to evaluate the differences between

18

groups. All data were expressed as mean ± S.D. Asterisks in the talbes

19

indicate statistically significant differences between the treatments and

20

the corresponding control group: *p < 0.05; **p < 0.01.

21

RESULTS

22

Viabilities of Lymphocytes. MTT assays were used to quantify the 12


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1

effects of ATR exposure on the lymphocyte viability of frogs. As shown

2

in Table 2, the lymphocyte viability decreased by 7.3％ to 17.5％ from

3

group II to group V, respectively, as compared with group I (the control

4

group). Significant differences in the lymphocyte viability were observed

5

between group I and all the treatment groups (II, III, IV, and V; p < 0.01,

6

r = 0.91). These results clearly indicate a dose–effect relationship in the in

7

vivo experiment.

8

Table 2

9

DNA Damage Assessment. Table 3 shows that the percentage of DNA

10

damage percentages in the treatment groups (II, III, IV, and V) is

11

significantly higher than in the control (p < 0.01). The comet tail length

12

and tail moment of the lymphocytes in frogs exposed to ATR was notably

13

increased with the increasing ATR concentration (p < 0.01). In group Ⅳ,

14

the tail length, percentage of DNA in the tail, and tail moment were

15

8.95 µm, 77.46%, and 0.44, respectively. Meanwhile, a dose–effect

16

relationship was observed between ATR exposure and DNA damage

17

level.

18

Table 3

19

DNA Fragmentation. DNA degradation by endonucleases is a

20

representative characteristic in apoptosis. In the present study, DNA

21

damage was evaluated by the electrophoresis of DNA isolated from frog

22

lymphocytes. ATR administration resulted in DNA damage, as shown in 13



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Page 14 of 42

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Fig. 1. Lanes 3 to 6 correspond to DNA from animals of groups II, III, IV,

2

and V, respectively. The genomic DNA of groups II, III, IV, and V (lanes

3

3 to 6) had 180 bp to 200 bp fragments. The ATR exposure evidently

4

resulted in the shearing of DNA as compared with group I (lane 2). The

5

data clearly showed that DNA damage is dose-dependent.

6

Fig. 1

7

Analysis of Apoptosis Percentage.

8

apoptotic lymphocytes in the group treated by exposure to different ATR

9

concentrations (0, 1, 10, 100, and 1000µg/L) are shown in Fig.2, which

10

were 3.27%, 3.66%, 5.21%, 12.43%, and 21.26%, respectively.

11

Compared with group I, the percentages of apoptotic lymphocytes of

12

group IV and V were significantly increased.

13

Fig.2

14

Analysis of Apoptotic Morphology. Changes in the micromorphological

15

structures of lymphocytes in groups III, IV, and V are apparent in Fig.3.

16

The cells of the control had normal morphology (Fig. 3(a)). The cells

17

displayed perinuclear chromatin margination and condensation in group

18

III (Fig. 3(b)). For group IV, serious nuclear chromatin condensation, as

19

well as dense granules and cytoplasmic vacuolation had developed (Fig.

20

3(c)). As expected, the cells in group V showed more serious

21

morphological changes, such as disappearance of the nuclear membrane,

22

chromatin condensation, and cellular organelle dissolution (Fig. 3(d)).

The average percentages of

14


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1

Fig.3

2

Analysis of Fas/Fas-L Protein Expression. The Fas antigen is a

3

cell-surface protein that mediates apoptosis, which can be activated by

4

interactions between Fas-L and Fas.32 The Western blot analysis of Fas

5

and Fas-L proteins in lymphocytes is shown in Fig. 4. The expressions of

6

Fas and Fas-L proteins increased with increasing ATR concentration, and

7

the β-actin control remained constant as expected (Fig. 4A).

8

Quantification of the Fas and Fas-L protein expression by densitometry of

9

lymphocytes in groups II, III, IV, and V verified the significant

10

dose-dependent increase in a significant level (p < 0.01; Fig. 4B).

11

Fig.4

12

Analysis of c-FLIP Protein Expression. c-FLIP has been identified as a

13

protease-dead, procaspase-8-like regulator of death ligand-induced

14

apoptosis.33 The Western blot of c-FLIP protein in lymphocytes of all

15

groups is shown in Fig. 4. Expression of the c-FLIP protein increased

16

with increasing ATR concentration, whereas that of the β-actin control

17

remained constant as expected (Fig. 5A). Quantification of the c-FLIP

18

protein expression by densitometry in lymphocytes of groups II, III, IV,

19

and V verified the significant dose-dependent increase (p < 0.01; Fig.

20

5B).

21

Fig.5

22

Analysis of Bid Protein Expression. Bid is a pro-apoptotic member of 15



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Page 16 of 42

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the Bcl-2 protein family.34 The Western blot of the Bid protein in

2

lymphocytes of all groups is shown in Fig. 6. Expression of the Bid

3

protein increased with increasing ATR concentration, whereas the β-actin

4

control remained constant as expected (Fig. 6A). Quantification of the

5

Bid protein expression by densitometry in lymphocytes of groups II, III,

6

IV, and V verified the significant dose-dependent increase in a significant

7

level (p < 0.01; Fig. 6B).

8

Fig.6

9

Analysis of Caspase-8 and Caspase-3 Protein Expression. Caspase-8

10

and caspase-3 are situated at pivotal junctions in apoptosis pathways.

11

Caspase-8

12

apoptosis-inducing ligands, whereas caspase-3 appears to amplify the

13

caspase-8

14

disassembly.35 The Western blots of caspase-8 and caspase-3 in the

15

lymphocytes of all groups are shown in Figs. 7 and 8, respectively. The

16

expression of caspase-8 and caspase-3 proteins both increased with

17

increasing ATR concentrations, whereas the β-actin control remained

18

constant as expected (Fig. 7A and Fig. 8A). The quantification of

19

caspase-8 protein expression by densitometry of lymphocytes in groups II,

20

III, IV, and V verified the significant dose-dependent increase in a

21

significant level respectively (p < 0.01; Fig. 7B). Similar to caspase-8, the

22

caspase-3 protein expression showed a significant dose-dependent

initiates

initiation

disassembly

signals

into

in

response

full-fledged

to

extracellular

commitment

to

16


Page 17 of 42

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1

increase in a significant level respectively (p < 0.01) after exposure to

2

ATR (Fig. 8).

3

Fig. 7

4

Fig. 8

5

DISCUSSION.

6

ATR has been identified as a disruptor of the immune system, one

7

which is hazardous to amphibians.36 Brodkin et al. found that ATR

8

decreased the phagocytic activity of immune cells.21 ATR also

9

significantly inhibits cell growth and alters cell cycle progression.37 In the

10

present study, ATR exposure negatively influenced the viability of

11

lymphocytes. A dose-dependent relationship was observed between cell

12

viability and

13

immunocyte production, which is a marker for immune system function.

14

The results illustrated that ATR decreases the viability of lymphocytes in

15

frogs and suppresses cell-mediated responses.

ATR concentration.

Lymphocyte

viability

reflects

16

Decreased cell viability can be explained by the ultrastructural

17

damage caused by ATR.38 In the present research, different levels of DNA

18

damage were observed. Rayburn et al. found similar results with flow

19

cytometry.39 The tail length, tail moment, and DNA damage ratio

20

increased with increasing ATR concentration compared with the controls,

21

thereby indicating a dose-response relationship. Similarly, exposure to

22

pesticides increased the incidence of chromosomal aberrations in 17



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Page 18 of 42

1

lymphocytes.40 Moreover, DNA fragments from 180 bp to 200 bp were

2

found in the lymphocytes of frogs exposed to ATR, but not in the control

3

group. The morphological characteristics of apoptosis such as shrinkage,

4

zeiosis, and nuclear collapse are observed in our present research.41

5

Lymphocytes develop obvious blebbing of the plasma membrane and

6

condensation into dense granules after ATR exposure. In the groups

7

exposed to a higher concentration of ATR, the plasma of apoptotic

8

lymphocytes began to dissolve. With BALB/C mice that were

9

administered oral doses of ATR (100, 200, and 400 mg/kg), Zhang et al.

10

observed chromatin margination and condensation into dense granules or

11

blocks, as well as mitochondrial vacuolization and the formation of

12

apoptotic bodies in splenocytes.42 Ultrastructural damage, including

13

abnormal morphology abnormal and DNA damage, has been identified as

14

characteristics of apoptosis. Therefore, the lymphocytes in immune

15

systems were damaged and apoptotic because of ATR exposure.41,43 In

16

Fig.2, flow cytometry analysis by PI staining further confirmed the

17

percentage of apoptotic lymphocytes in P. nigromaculatus. These

18

percentages increased with exposure to an increasing dosage of ATR in a

19

dose-dependent manner.

20

The present study investigated the Fas-associated apoptosis of

21

lymphocytes because Fas has an important role in the immune system.

22

Fas is a cell surface-bound receptor expressed on surfaces of thymocytes, 18


Page 19 of 42

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1

active T and B lymphocytes, NK cells, and monocytes.44 The

2

Fas-L-dependent activation of Fas can recruit FADD, which provides a

3

platform for the formation of DISC; DISC contains pro-caspase-8 and

4

c-FLIP.44 Western blot analysis showed that Fas and Fas-L protein

5

expression increased in the treatment groups; both proteins have a

6

dose-dependent relationship with ATR exposure. Other in vivo and in

7

vitro studies demonstrated that glucuronoxylomannan induces the

8

Fas/Fas-L protein expression in immunocytes.45 With the increase of Fas

9

and Fas-L protein expression, the expression of the caspase-8 protein is

10

activated, which then activates the expression of caspase-3 and Bid

11

proteins. Bid is cleaved into tBid by caspase-8. tBid induces the release of

12

cytochrome C from mitochondria, which interacts with Apaf-1 and dATP

13

thereby ultimately activating caspase-3.46 Meanwhile, caspase-3 can be

14

directly activated by caspase-8. The simultaneous expression of these

15

apoptosis-related proteins suggests that ATR initiates lymphocyte

16

apoptosis, and the apoptosis mechanism of lymphocytes in frogs occurs

17

via the signal transduction from Fas and Fas-L to FADD and down to

18

caspase-3 via a direct pathway and a Bid-dependent indirect pathway.

19

Caspase-3 activation, in turn, may promote the complete activation of

20

caspase-8 and Bid, thereby enhancing caspase-3 activity and apoptosis.47

21

Finally, caspase-3 induces the DNA fragmentation and morphological

22

changes associated with apoptosis,48 thereby indicating the degree of 19



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Page 20 of 42

1

lymphocyte damage in ATR-exposed frogs. Therefore, the apoptosis of

2

lymphocytes involves the immune systems of amphibians via the

3

Fas-dependent pathway to protect these cells from damage.

4

As a potent regulator of lymphocyte susceptibility to Fas-mediated

5

apoptosis, the caspase-8 inhibitor c-FLIP interferes with the caspase-8

6

link to FADD at the DISC level and blocks the downstream caspases

7

activation, which consequently prevents apoptosis.49 In the present study,

8

the relative protein expression of c-FLIP showed a dose-dependent

9

decrease, thereby indicating the effective work of the immune system; a

10

previous report showed that diseases were correlated with the sustained

11

high level expression of c-FLIP in cells.50,51 In addition to the modulation

12

of Fas-dependent apoptosis, c-FLIP also played a key role in lymphocyte

13

proliferation, which can also be induced by the Fas-dependent

14

pathway.52,53 These results suggests multiple biological functions of the

15

Fas/Fas-L signal. In fact, the signal conversion between cell death and the

16

growth associated with the death receptor Fas is regulated by c-FLIP.49

17

Currently, greater focus has been given to the role of C-FLIP in the

18

apoptosis of cancerous cells to explore novel disease therapy.51,54 c-FLIP

19

needs to be further investigated because this protein accelerates apoptosis

20

in affected cells via the Fas-dependent pathway, which can effectively

21

strengthen the viability of amphibians.

22

According

to

the

abovementioned

experimental

results,

a 20


Page 21 of 42

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1

hypothetical model is presented in Figure 9 for the extrinsic apoptosis

2

pathway induced by ATR in P. nigromaculata lymphocytes in vivo. The

3

down-regulation

4

Fas/Fas-L/Bid/casepase-3/caspase-8 are completely involved in this

5

pathway. ATR can trigger the expression of Fas and Fas-L to promote the

6

expression of casepase-8. Subsequently, the expression of the caspase-8

7

inhibitor c-FLIP is inhibited. However, Bid is stimulated to induce the

8

generation of cytochrome C. Consequently, caspase-3 activation may be

9

indirectly affected by the caspase-8/Bid/cytochrome C route or directly

10

induced by the Fas-L/Fas/caspase-8 pathway. Therefore, the apoptosis of

11

P. nigromaculata lymphocytes is induced by ATR via the activation by

12

caspase-3.

13

CONCLUSIONS

of

c-FLIP

and

the

up-regulation

of

14

This study demonstrated the immunotoxicity of ATR and the induced

15

immune defense of lymphocytes via the Fas/Fas-L system in frogs. The

16

key proteins, such as Fas, Fas-L, c-FLIP, caspase-8, Bid, and caspase-3,

17

were expressed in a dose-dependent manner; the role of c-FLIP in the

18

immune system of frogs was emphasized. Further studies are needed to

19

investigate the defense mechanisms of the amphibian immune system,

20

especially under natural conditions, to provide better protection for

21

amphibians.

22

FUNDING SOURCES 21



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Page 22 of 42

1

This work was funded by the Program for Excellent Young Teachers

2

in Hangzhou Normal University (No: JTAS 2011-01-012), the Excellent

3

Talent Program in Hangzhou Normal University, the Program for ‘131’

4

talents in Hangzhou City and the Grant of Chinese Scholarship Council

5

(No: 201208330300).

6

ACKNOWLEDGEMENT 7

We acknowledge the support from the following individuals: for 8

Western blot analysis, Dr. Jun Liu; and for flow cytometry alalysis, Wendi 9

Fang. 10 11

ABBREVIATIONS

12 13 14 15 16 17 18 19 20 21

Apaf-1, apoptotic protease activating factor-1; APO-1, apoptosis antigen-1; ATR, atrazine; BCA, bicinchoninic acid; Bcl-2, B-cell lymphoma 2; CAS, chemical abstracts service; CD95, cluster of differentiation 95; c-FLIP, cellular FLICE-like inhibitory protein; Ck, control check; DED, Death Effector Domain; DISC, death-inducing signaling complex; DMSO, dimethyl sulfoxide; DNA, deoxyribonucleic acid; ECL, electrochemiluminescence; EDTA, ethylenediaminetetraacetic acid; FADD, Fas-associated Death Domain; Fas-L, fas ligand; LC50, lethal concentration 50; MTT, 3-(4,5-Dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide; PBS, phosphate-buffered saline; S.D., standard deviation; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; TAE, tris acetate ethylenediaminetetraacetic acid; TEM, Transmission electron microscope;T-TBS, tris-buffered saline with tween-20

22 23

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Kataoka,T., Budd, R. C., Holler, N., Thome, M., Martinon, F.,

21

Irmler, M., Burns, K., Hahne, M., Kennedy, N., Kovacsovics, M., and 22

Tschopp, J. (2000) The caspase-8 inhibitor FLIP promotes activation 30


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1

of NF-κB and Erk signaling pathways. Curr. Biol. 10, 640-648. 2

(54)

Bagnoli, M., Canevari, S., and Mezzanzanica, D. (2010) Cellular

3

FLICE-inhibitory protein (c-FLIP) signalling: A key regulator of 4

receptor-mediated apoptosis in physiologic context and in cancer. Int.l 5

J. Biochem.Cell. B. 42, 210-213. 6 7 8

Table 1 Groups and treatment protocols of frogs exposed to different concentrations of atrazine Treatment Groups

Atrazine concentration (µg/L)

I 0

II 1

III 10

IV 100

V 1000

9 10

Table 2 Atrazine-induced reduction in lymphocyte viability of frogs Group I II III V V r

Animals 30 30 30 30 30

Cell viabilities (%) 87.50±2.11 80.17±0.96** 78.00±0.83** 73.12±0.75** 70.00±0.34** 0.91

11 12

Table 3 Frequency and degree of DNA damage to frog lymphocytes in all groups Group

Animals

I II III IV V r

30 30 30 30 30

DNA damage percentage (%)

Tail length(µm)

Tail moment

13.76±4.31 52.10±6.89** 59.34±6.19** 74.04±9.37** 77.46±8.66** 0.93

1.01±0.19 4.01±0.42** 5.58±0.52** 6.95±0.38** 8.95±0.60** 0.98

0.12±0.03 0.34±0.03** 0.37±0.02** 0.42±0.03** 0.44±0.03** 0.92

13 14

31



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

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Figure Legends:

2 3

Fig. 1. ATR-induced DNA fragmentation in lymphocytes, as determined by agarose gel

4

electrophoresis. Lane 1, 100 bp to 2000 bp DNA markers for molecular size; lane 2, DNA from

5

control lymphocytes; lanes 3 to 6, DNA ladder patterns in lymphocytes after 14 days of ATR

6

exposure at 1, 10, 100, and 1000 µg L-1, respectively.

7 8

Fig. 2. Apoptosis percentages of frog lymphocytes treated with ATR in vivo as determined by flow

9

cytometry. (GroupⅠ: 0 µg L-1; GroupⅡ:1µg L-1; Group Ⅲ: 10 µg L-1; Group Ⅳ: 100 µg L-1;

10

Group Ⅴ: 1000 µg L-1).

11 12

Fig. 3. Electron micrographs of lymphocytes treated with ATR. (a) Lymphocytes from control

13

group, showing normal morphology. (b) Lymphocytes treated with 10 µg L-1 ATR, showing

14

intense perinuclear chromatin margination (white arrows). (c) Lymphocytes treated with 100 µg

15

L-1 ATR, showing chromatin condensation into dense granules (double black arrows) and

16

vacuolizations (single black arrows). (d) Lymphocytes treated with 1000 µg L ATR, showing

17

chromatin condensation into dense granules (double black arrows) and vacuolizations (single

18

black arrows). Original magnification: 12000×.

-1

19 20

Fig. 4. Effects of ATR upon the Fas and Fas-L proptein expression in frog lymphocytes. Group I

21

denotes the control group; groups II to V denote ATR exposure at different concentrations

22

(Ⅱ,1µg L-1; Ⅲ, 10 µg L-1; Ⅳ, 100 µg L-1; Ⅴ, 1000 µg L-1). (A) Representative autoradiograph.

23

(B) Intensities of protein bands were quantified by densitometry.

24 25

Fig. 5. Effects of ATR on the c-FLIP protein expression in frog lymphocytes. Group I denotes the

26

control group; groups II to V denote ATR exposure at different concentrations (Ⅱ, 1µg L-1; Ⅲ, 10 32


Page 33 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

µg L-1; Ⅳ, 100 µg L-1; Ⅴ, 1000 µg L-1). (A) Representative autoradiograph. (B) Intensities of

2

protein bands were quantified by densitometry.

3 4

Fig. 6. Effects of ATR on the Bid protein expression in frog lymphocytes. Group I denotes the

5

control group; groups II to V denote ATR exposure at different concentrations (Ⅱ, 1µg L-1; Ⅲ, 10

6

µg L-1; Ⅳ, 100 µg L-1; Ⅴ, 1000 µg L-1). (A) Representative autoradiograph. (B) Intensities of

7


8 9

Fig. 7. Effects of ATR on the caspase-8 protein expression in frog lymphocytes. Group I denotes

10

the control group; groups II to V denote ATR exposure at different concentrations (Ⅱ, 1µg L-1; Ⅲ,

11

10 µg L-1; Ⅳ, 100 µg L-1; Ⅴ, 1000 µg L-1). (A) Representative autoradiograph. (B) Intensities of

12


13 14

Fig. 8. Effects of ATR on the caspase-3 protein expression in frog lymphocytes. Group I denotes

15

the control group; groups II to V denote ATR exposure at different concentrations (Ⅱ, 1µg L-1; Ⅲ,

16

10 µg L-1; Ⅳ, 100 µg L-1; Ⅴ, 1000 µg L-1). (A) Representative autoradiograph. (B) Intensities of

17 18


19 Fig. 9. Hypothetical model for ATR-induced apoptosis pathway in frog lymphocytes.

33



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Fig. 1. ATR-induced DNA fragmentation in lymphocytes, as determined by agarose gel electrophoresis. Lane 1, 100 bp to 2000 bp DNA markers for molecular size; lane 2, DNA from control lymphocytes; lanes 3 to 6, DNA ladder patterns in lymphocytes after 14 days of ATR exposure at 1, 10, 100, and 1000 µg L-1, respectively. 44x39mm (300 x 300 DPI)


Page 34 of 42

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Fig. 2. Apoptosis percentages of frog lymphocytes treated with ATR in vivo as determined by flow cytometry. (GroupⅠ: 0 µg L-1; GroupⅡ:1µg L-1; Group Ⅲ: 10 µg L-1; Group Ⅳ: 100 µg L-1; Group Ⅴ: 1000 µg L-1). 58x13mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Fig. 3. Electron micrographs of lymphocytes treated with ATR. (a) Lymphocytes from control group, showing normal morphology. (b) Lymphocytes treated with 10 µg L-1 ATR, showing intense perinuclear chromatin margination (white arrows). (c) Lymphocytes treated with 100 µg L-1 ATR, showing chromatin condensation into dense granules (double black arrows) and vacuolizations (single black arrows). (d) Lymphocytes treated with 1000 µg L-1 ATR, showing chromatin condensation into dense granules (double black arrows) and vacuolizations (single black arrows). Original magnification: 12000×. 49x49mm (300 x 300 DPI)


Page 36 of 42

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Fig. 4. Effects of ATR upon the Fas and Fas-L proptein expression in frog lymphocytes. Group I denotes the control group; groups II to V denote ATR exposure at different concentrations (Ⅱ,1µg L-1; Ⅲ, 10 µg L-1; Ⅳ , 100 µg L-1; Ⅴ, 1000 µg L-1). (A) Representative autoradiograph. (B) Intensities of protein bands were quantified by densitometry. 76x93mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Fig. 5. Effects of ATR on the c-FLIP protein expression in frog lymphocytes. Group I denotes the control group; groups II to V denote ATR exposure at different concentrations (Ⅱ, 1µg L-1; Ⅲ, 10 µg L-1; Ⅳ, 100 µg L-1; Ⅴ, 1000 µg L-1). (A) Representative autoradiograph. (B) Intensities of protein bands were quantified by densitometry. 116x165mm (300 x 300 DPI)


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Fig. 6. Effects of ATR on the Bid protein expression in frog lymphocytes. Group I denotes the control group; groups II to V denote ATR exposure at different concentrations (Ⅱ, 1µg L-1; Ⅲ, 10 µg L-1; Ⅳ, 100 µg L-1; Ⅴ, 1000 µg L-1). (A) Representative autoradiograph. (B) Intensities of protein bands were quantified by densitometry. 69x78mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Fig. 7. Effects of ATR on the caspase-8 protein expression in frog lymphocytes. Group I denotes the control group; groups II to V denote ATR exposure at different concentrations (Ⅱ, 1µg L-1; Ⅲ, 10 µg L-1; Ⅳ, 100 µg L-1; Ⅴ, 1000 µg L-1). (A) Representative autoradiograph. (B) Intensities of protein bands were quantified by densitometry. 69x82mm (300 x 300 DPI)


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Fig. 8. Effects of ATR on the caspase-3 protein expression in frog lymphocytes. Group I denotes the control group; groups II to V denote ATR exposure at different concentrations (Ⅱ, 1µg L-1; Ⅲ, 10 µg L-1; Ⅳ, 100 µg L-1; Ⅴ, 1000 µg L-1). (A) Representative autoradiograph. (B) Intensities of protein bands were quantified by densitometry. 71x87mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Fig. 9. Hypothetical model for ATR-induced apoptosis pathway in frog lymphocytes. 240x200mm (150 x 150 DPI)


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Atrazine Triggers the Extrinsic Apoptosis Pathway in Lymphocytes of

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