Avermectin Confers Its Cytotoxic Effects by Inducing DNA Damage

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Avermectin confers its cytotoxic effects by inducing DNA damage and mitochondria-associated apoptosis Yang Zhang, Mingming Luo, Wenping Xu, Mingjun Yang, Bo Wang, Ju-Fang Gao, Yaxiao Li, and Liming Tao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b02812 • Publication Date (Web): 23 Aug 2016 Downloaded from http://pubs.acs.org on August 25, 2016

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Journal of Agricultural and Food Chemistry

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Avermectin confers its cytotoxic effects by inducing DNA damage and

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mitochondria-associated apoptosis

3 †

†

†

†

†

‡

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Yang Zhang , Mingming Luo , Wenping Xu , Mingjun Yang , Bo Wang , Jufang Gao ,

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Yaxiao Li , Liming Tao

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†

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University of Science and Technology, Shanghai 200237, China

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‡

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200234, China

§

†, *

Shanghai Key Laboratory of Chemical Biology, School of Pharmacy, East China

College of Life and Environmental Sciences, Shanghai Normal University, Shanghai

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§ Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath, BA2

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7AY, UK

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* Corresponding authors.

ACS Paragon Plus Environment


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ABSTRACT ： Avermectin (AVM) has been widely used in agriculture and animal

14

husbandry based on its broad spectrum of effective anthelmintic activity and specificity

15

targets. However, AVM induction of cytotoxicity through DNA damage is still remains

16

elusive. Here we investigate the cytotoxic effects of AVM in human non-target cells in vitro.

17

We clarify that AVM inhibited the viability of Hela cells and enhanced apoptosis. We have

18

used alkaline comet assay and γH2AX foci formation to detect DNA damage of HeLa cells.

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As expected, we found AVM caused DNA double-strand breaks in Hela cells, as measured

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by significance of comet assay parameters (e.g., tail DNA) and increases of γH2AX foci in

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HeLa cells. Moreover, established assays of cytotoxicity were performed to characterize the

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mechanism of AVM toxicity on Hela cells. The results demonstrated the collapse of

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mitochondrial membrane potential and up-regulating the expression level of Bax/Bcl-2,

24

resulted in a release of cytochrome-c into cytosol as well as the activation of caspase-9/-3

25

and cleavage of PARP. In conclusion, we conclude that AVM has a potential risk to human

26

health that can induce human cell DNA damage and mitochondria-associated apoptosis.

27

Avermectin;

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KEYWORDS:

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mitochondrial pathway

Cytotoxic

effects;

DNA

30 31


damage;

Apoptosis;

The

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INTRODUCTION Avermectin (AVM) is a macrocyclic lactone compound was first discovered in the 1, 2

34

extracts of the fungus Streptomyces avermitilis,

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AVM, applied extensively as both anthelmintic aiming at internal as well as external

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parasites of livestock, and insecticide to reduce agricultural pests.

37

the nervous system of organisms which is well known that it potentiate the activity of

38

glutamate-gated chloride ion channels or gamma-aminobutyric acid (GABA) receptor.

39

Therefore, generally considered that the poisonousness of AVM is low for the health of

40

adults because neurons which are sensitive to GABA are confined to the mammalian central

41

nervous system.

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announced AVM is highly toxic to fish and bees and extremely toxic to mammals and

43

aquatic invertebrates.

44

more frequently in recent years,

45

have led to drug residues on our food.

46

and photo-oxidative mechanisms when exposed to light and air in the environment,

47

experiences almost no metabolism in the target organism, therefore the dose offered to the

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animal is discharged as parent compound, mainly in the excreta among which no more than

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2% is in urine, 13 which means a seriously potential risk to human health.

8

Avermectin B1, the main component of

3-5

.AVM acts mainly on

6, 7

The U. S. Environmental Protection Agency (EPA) Pesticide Fact Sheet

9

Meanwhile, the resistance to AVM has been reported more and 10

which demonstrates the excessive application of AVM 11

Although AVM is quickly degraded by oxidative 12

it

50 51

DNA damage is a significant parameter in genotoxicity assessment of environmental

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poison to human being. It could be induced by DNA double-strand breaks (DNA-DSBs) or

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DNA single-strand breaks (DNA-SSBs) resulting from the interaction of the pesticide or its



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metabolites and DNA.14 Under physiological circumstance or the DNA damage is limited,

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poly (ADP-ribose) polymerase (PARP) plays a protective role in the repair of the strand

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breaks in DNA either by its over-activation or by participating in an extracellular signal

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regulated cascade.15 Genotoxic compounds normally take effect on chromosomal DNA.

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Apoptosis is a major form of cell death induced by drugs(e.g. environmental poison)

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represented by the chromatin DNA cracking into internucleosomal fragmentation.16, 17 It is

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an important mechanism for maintaining balance of diverse biological processes and is

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critical for the cytotoxicity.18,

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dysregulation would result in pathological conditions including various diseases and

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cancer.20 We have known that two major pathways contained in apoptosis, intrinsic

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(mediated by mitochondrial) and extrinsic (mediated by death receptors). In the

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mitochondrial pathway, the release of death factors and the regulation of Bcl-2 family

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proteins would contribute to the triggering of apoptosis which induced by some intracellular

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signals, such as DNA damage.21, 22 For instance, the mainly death factor cytochrome-c once

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released into cytosol and Apaf-1, in the presence of dATP, are required for activation of

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caspase-9 and subsequently effector caspases-3. Finally, the activated effector caspases

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leading irreversibly to cell death.23, 24 The distribution of cytochrome-c between cytosol and

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mitochondria alters following Bax translocation, which transfers signals of death from

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cytosol to mitochondria.25

19

The apoptosis process is strictly regulated, and its

73 74

In this study, Human cervical carcinoma HeLa cells, an most commonly used model cell

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line for studying human cellular and molecular biology, were used to evaluate the


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toxicological effects of AVM in human non-target cells, and we found the AVM has

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potential genotoxicity and significant cytotoxicity to human cells in vitro. We characterized

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the mechanism of AVM toxicity on Hela cells with established assays of cytotoxicity, which

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was shown as it can induce DNA-DSBs and apoptosis. Moreover, the collapse of

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mitochondrial membrane potential and up-regulating the expression level of Bax/Bcl-2,

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resulted in a release of cytochrome-c as well as the activation of caspase-9/-3 and cleavage

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of PARP. Taken together, these results show that the Hela cells were undergoing

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mitochondrial apoptosis-associated DNA fragmentation.

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MATERIALS AND METHODS

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Chemicals and antibodies. Avermectin（AVM, 98% pure, containing 95% B1a）was obtained

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from

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3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium

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(Rh-123), 4’,6-Diamidino-2-phenylindole (DAPI), Phenylmethylsulfonyl fluoride (PMSF)

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and RIPA Lysis buffer were purchased from Sigma (St. Louis, MO, USA). All other

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chemicals and reagents reagents used were of analytical grade and obtained from Shanghai

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Titanchem Co. Ltd. (Shanghai, China). Antibodies were used as follows: caspase-9,

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cytochrome-c, PARP, Bcl-2, Bax, γH2AX and β-actin (Cell Signaling Technology, Beverly,

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MA, USA). Secondary anti-rabbit antibody was purchased from Sangon Biotech Co., Ltd

95

(Shanghai, China).

Shanghai

Pesticide

Research

Institution bromide

(Shanghai, (MTT),

China).

Rhodamine123

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Cell culture and Maintenance. Human cervical carcinoma cells (HeLa, ATCC, CCL-2)



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were maintained in DMEM medium (Hyclone, USA) supplemented with 10% fetal bovine

99

serum (Gibco, USA) and 1% antibiotics (streptomycin and penicillin) (Hyclone) at 37 °C in

100

an incubator with a humidified atmosphere of 5% CO2.

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Cell viability assay. The cytotoxic effects of AVM on the Hela cells' viability was

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performed by MTT assays as previously described.26 In brief, after Hela cells (1×105

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cells/mL) were treated with specified concentrations (2.5, 5, 10, 15, 20 and 40 µM) of AVM

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for 24 and 48 h, MTT reagent (20 µL /well, 5 mg/mL) was added to form formazan crystals

106

after 4 h incubation at 37°C. The culture media was aspirated before DMSO (150 µL/well)

107

was added to dissolve the formazan crystals, and the reading at 492 and 630 nm were taken

108

by a microplate reader (Synergy H1, Bio-Teck, Winooski, VT, USA).

109 110

Alkaline comet assay. The Alkaline single cell gel electrophoresis technique also known as

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comet assay is an experimental technology developed in recent years.27 Determination of

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DNA by migrating optical density, tail length or tail moment could quantify degree of DNA

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damage in individual cells. Briefly, after Hela cells were treated with specified

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concentrations (0, 5, 10, 20 and 40 µM) of AVM for 24 h, the cells were harvested, washed

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and suspended in PBS ( pH 7.4), subsequently mixed with 1% low melting agarose (1:6).

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Slides were preserved in refrigerators at 4°C for 20 min, after that performed the slides 2 h

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in lysis buffer (10% DMSO, 1% Triton X-100, 2.5 M NaCl, 10 mM Tris–HCl, 100 mM

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EDTA, pH 10) at 4 °C for in the dark. Use double distilled water (ddH2O) to wash it twice

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times, injection fresh alkaline electrophoresis solution (1 mM EDTA,300 mM NaOH, pH 13)


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into a horizontal electrophoresis tanks, the slides were immersed in 10 min then

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electrophoresed for 30 min at 20 V (1 V/cm) or 300 mA. After electrophoresis with

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neutralizing buffer and double distilled water washing three times, stained with 40 µL PI

123

solution (20 µg/mL), examined and photographed by fluorescence microscopy (Lecois,

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DM3000, GER). The degree of DNA damage was measured by an image analysis system

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(CASP, www.casp.of.pl).

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H2AX foci staining. The γH2AX foci formation as a typical sign of DNA-DSBs was

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measured by immunofluorescent assays as previously described.28 After Hela cells were

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treated with specified concentrations (0, 5, 10, 20 and 40 µM) of AVM for 6 h, cells were

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fixed in 4% paraformaldehyde for 15 min, washed twice with PBST (PBS buffer pH 7.4 and

131

0.1% Tween 20), and permeabilized in 1% Triton-X100 for 15 min. After blocked with 5%

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serum at room temperature for 1 h, the cells were incubated with a rabbit anti-γH2AX

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antibody (1:200) overnight in 4°C, and conjugated with Alexa Fluor 488-conjugated

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anti-rabbit secondary antibody (1:1000) at room temperature for 60 min. For the staining of

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the nuclei, DAPI (1 mg/mL) was added to the cells and incubated for 10 min at 37 °C in

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dark. The cells were then mounted in antifade media, and immunofluorescent graphic were

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taken using a confocal laser scanning microscope (Nikon Inc., Melville, N.Y.).

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Chromatin condensation detection. After Hela cells were treated with specified

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concentrations (0, 5, 10, 20 and 40 µM) of AVM for 24 h, cells were fixed with 4%

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paraformaldehyde for 10 min at 4°C. The fixed cells were washed three times with PBS



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(PH7.4) and then stained with 1µL of Hoechst 33258 (5 mg/ml) in 1 mL PBS (PH7.4) and

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incubated for 10 min at 37°C. The morphology of the treated cells was analyzed and

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photographed by fluorescence microscopy.

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Apoptosis assay. Apoptosis-associated changes were analyzed by flow cytometry using the

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Invitrogen™ Alexa Fluor 488 Annexin V/Dead cell apoptosis kit. Briefly, after cells were

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treated with specified concentrations (0, 5, 10, 20 and 40 µM) of AVM for 24 h, no less than

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1×106 cells were centrifuged at 100 × g and 4°C for 5 min and washed twice with PBS

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(PH7.4) for the apoptosis assay. Then, the cells were labeled with PI and Annexin V-FITC

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for 15 min at room temperature in the dark. Finally, apoptosis of cells was examined by flow

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cytometer (FACS Calibur, B.D). Data was assayed using the Flowjo software program.

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Mitochondrial membrane potential (△Ψm) analysis. The effects of AVM on

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mitochondrial membrane potential (△Ψm) were evaluated by fluorescence microscopy and

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flow cytometry combining with Rhodamine123 (Rh-123). After HeLa cells were treated

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with specified concentrations (0, 5, 10, 20 and 40 µM) of AVM for 6 h, cells were tinted

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with Rh-123 for 15 min at 37°C in dark. The fluorescent images of the treated cells was

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photographed by fluorescence microscopy. Meanwhile, the treated cells were collected by

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centrifugation after being washed and tinted with Rh-123 for flow cytometry assay. The

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fluorescent intensity was examined at an excitation wavelength of 488 nm and an emission

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wavelength of 530 nm. Data analysis was performed using the Flowjo software program.

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Western blotting. Total protein of AVM-stimulated cells were extracted in RIPA lysis

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buffer with 1mM PMSF, cytosolic proteins were isolated using the Mitochondria/Cytosol

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Fractionation Kit, and the protein concentrations were measured by the BCA protein assay.

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Proteins were mixed with 2× sample buffer, boiling for 5 min to make it inactive. Equivalent

168

quantities of protein were exposed to 8–15% SDS-PAGE and then transferred by

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electrophoresis onto polyvinylidene fluoride (PVDF) membranes (Millipore Corp, USA).

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The membranes were blocked with 5% non-fat dried milk in Tris-buffered saline-Tween

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(TBST; 150mM NaCl, 10mM Tris·HCl, 0.1% Tween-20, pH 7.5) for 1.5 h, incubated at 4°C

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with the following primary antibodies: caspase-9, cytochrome c, PARP, Bcl-2, Bax, and

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β-actin (diluted 1:1000; 1:300; 1:1000; 1:300; 1:300; 1:1000, respectively) overnight. After

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washing three times with TBST, HRP-conjugated secondary antibodies were incubated

175

respectively. Bound antibody was examined using an enhanced chemiluminescence (ECL)

176

solution. The densitometry analysis of bands’ integrated density values (IDVs) were

177

quantified by ImageJ software.

178 179

Caspase-3 activity assay. The enzymatic activity of caspases were examined by using the

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caspase activity kit. Concisely, after incubation with AVM for 6 h, the cells were extracted

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with extraction buffer at 4°C for 20 min, centrifuged at 12,000 g and 4°C for 20 min and

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harvested, then washed with PBS (PH 7.4). The proteins concentration were measured using

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the BCA protein assay. Activities of caspase-3 were gauged using substrate peptides

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Ac-DEVD-pNA for 4h at 37 °C and absorbance at 405 nm was measured by microplate

185

reader.



186 187

Statistical analysis. We conducted no less than three independent experiments for each

188

experiment and found a close relationship between foundings of each independent

189

experiment. The results are presented as the mean ± standard deviation (SD), with the whole

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statistical analyses process run under SPSS version 17.0, statistical program. To find the

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differences between groups, we used ANOVA followed by the Dunnet’s test (P ≤ 0.05 means

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statistically significant).

193 194

RESULTS

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Avermectin inhibited the viability of Hela cells. We used the MTT assay to evaluate the

196

cytotoxicity of AVM in Hela cells. Just as exhibited in Fig. 1, the AVM inhibited the

197

viability of HeLa cells both in a dose- and time-dependent way. The viability inhibition

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proportions of HeLa cells were 6.93 ± 2.18%, 11.47 ± 0.84%, 32.62 ± 1.81%, 53.56±5.48%,

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69.95±3.46% and 81.81±1.68% after 24 h of treatment with AVM at the concentrations of

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2.5, 5, 10, 15, 20 and 40µM, which finally reached to 14.99±4.05%, 25.28±4.64%,

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58.33±4.82%, 79.92±2.96%, 88.82±1.34% and 97.92±0.74% after 48 h treatments. IC50

202

values for AVM treatments of 24 and 48 h were 14.52 and 7.63µM respectively.

203 204

Avermectin induced DNA damage in Hela cells. The alkaline comet assay showed DNA

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fragments transferred and shaped into a comet-like form.17 In 0 µM AVM group, comet

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heads were focused with highly dense DNA accompanied by undamaged nuclei and the

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frequencies of comet cells were ~5%. In the AVM treatments, the impaired DNA transferred


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from the heads and constructed tails shaped like bloom in Fig.2A. The proportion of

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comet-positive cells remarkably rose after exposing to AVM for 24h by a dose-dependent

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means (Fig. 2C).The spread of AVM–treatment Hela cells, with respect to comet assay

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parameters including tail lengths and tail DNA are presented in Table 1.The study showed a

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dose-dependent raise in the dimension of comet tails with concomitant decrease in head

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dimension, which means that AVM inducing the generation of typical DNA fragmentation

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in Hela cells in a dose-dependent way.

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Avermectin induced DNA double-strand breaks in Hela cells. The immunofluorescent

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graphic of phosphorylation of histone H2AX in γH2AX-stained HeLa cells are demonstrated

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in Fig. 2B. It described that AVM induced a dose-dependent reaction of γH2AX foci. In 0

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µM AVM group, cells had little γH2AX foci within the nuclei. All the treatments with AVM

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led to the forming of foci formation, and added to the number of γH2AX foci in nuclei. Data

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in Fig. 2D demonstrated that AVM presented a significant dose-dependent influence on the

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forming of γH2AX foci formation, which means it induced DNA-DSBs in HeLa cells.

223 224

Avermectin induced apoptosis in HeLa cells. After treatment with 5, 10, 20 and 40µM of

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AVM for 24 h, a growing number of cells started to contract, flow from the bottom of the

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pallets (no pictures shown) and get fragmented. The influence that AVM exerted on Hela

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cells depended on flow cytometry assay. We went a step further to confirm that apoptosis

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was triggered by AVM by flow cytometry assay. The results exhibited that the percentage of

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apoptotic cells added from 3.22 ± 1.02% in untreated cells up to 5.91 ± 1.76%, 10.44 ±



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2.56%, 21.09 ± 2.08% and 47.46 ± 3.73% in 5, 10, 20 and 40µM AVM- processed cells,

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separately (Fig. 3A). These data suggested that AVM enhanced the apoptosis of HeLa cells

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in a dose-dependent way (Fig. 3B). Chromatin condensation assay was further performed to

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show the apoptosis of cell nuclei. HeLa cells were bare to AVM and tinted with Hoechst

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33258. Cells of AVM 0 µM group exhibited an evenly spread of the tinted and circular

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homogeneous nuclei. Apoptotic cells raised gradually in a dose-dependent method and

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exhibited representative alteration, with condensed and brightly stained or fragmented nuclei

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included (Fig. 3C). This result also showed that AVM caused chromatin DNA fragmentation

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of HeLa cells during apoptosis.

239 240

Avermectin induced mitochondrial dysfunction in Hela cells. We used flow cytometry

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and fluorescent microscopy to examine the quantification of mitochondrial membrane

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potential (△Ψm) in HeLa cells upon AVM treatment. Just as exhibited in Fig. 4,we

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discovered that exposure of Hela cells to AVM for 6 h led to both fluorescence intensity

244

declined and the ratio of △Ψm collapse increased in a dose-dependent way (Fig. 4C),

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suggesting that AVM caused the reduction of △Ψm in Hela cells.

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Influence of Avermectin on the degree of apoptosis-associated proteins in HeLa cells.

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Cytochrome-c is a key factor in mitochondrial-mediated apoptosis pathway anchored in the

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mitochondrial inner membrane.29 In order to examine the release of cytochrome-c from

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mitochondria into the cytosol, extracted cytosolic fractions of Hela cells treated with

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specified concentrations of AVM were analyzed by Western blot assay. Just as exhibited in


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Fig. 5A, cytochrome-c release was raised in a dose-dependent way in AVM-induced Hela

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cells.

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Bcl-2 family proteins play a vital role in apoptotic process to regulate the release of

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cytochrome-c. Among them, Bax/Bcl-2 is the key factors in regulating apoptosis which were

257

measured by Western blot assay. Just as exhibited in Fig. 5B, the expression level of protein

258

bax was elevated and the protein Bcl-2 was down-regulated in simultaneously.

259 260

Influence of Avermectin on caspase-3/-9 activation and cleavage of PARP in HeLa cells.

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Caspases play a vital role in modulating apoptosis. The activated caspase cleaves PARP, a

262

substrate protein of caspase-3, which is one of the hallmarks of apoptosis.30 The

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AVM-treated HeLa cells were examined for cleavage of PARP and the activation of

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caspase-9/-3 by using Western blot assay or colorimetric enzymatic assay. Just as exhibited

265

in Fig. 5(D and E), both cleavage of PARP and activation of caspase-9/-3 raised in a

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dose-dependent way after AVM treatment. This result demonstrated that the apoptosis

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AVM-induced in Hela cells was associated with the activation of caspase-9/-3 and cleavage

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of PARP.

269 270

DISCUSSION

271

Natural product pesticide usually make their targets not unique based on its complicated

272

structure, so it is hard to be available in practical application which means it has seriously

273

potential risk to non-target organism simultaneously. Recently, a large number of studies



274

showed AVM have adverse effects on non-target cells including Chinese hamster ovary cells

275

and male rat hepatocytes,31, 32 but it's has been barely emerged that reports revealed the

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cytotoxic effects to human of AVM and its toxicologically mechanism at molecular and

277

cellular lever. In our study, we investigated if the AVM has potential genotoxicity and

278

significant cytotoxicity to human non-target cells and the action mechanism of it whether

279

relies on mitochondrial apoptotic pathway.

280 281

Although AVM is negative for skin sensitization, it has previously been reported to

282

possess the potential acute toxicity (rat oral LD50 10.6 mg/kg males) 9 and inhibit viability

283

of various types of non-target mammalian cells across an extensive spread of concentrations

284

(1-100 µM).32, 33 In order to assess its biological safety on human cells, we primarily tested

285

the effect of AVM on the viability of HeLa cells. The outcome revealed that AVM has

286

ability to inhibit the viability of Hela cells both in a dose- and time-dependent way (Fig.1).

287

This illustrates that AVM has cytotoxicity to HeLa cells in vitro, which might be the

288

foundation of its potential risk to human. Moreover, based on this result, we screened out a

289

best group concentrations (0, 5, 10, 20 and 40 µM) to reflect the cytotoxic effects of AVM in

290

cellular level in the following experiments.

291 292

Genotoxicity of agrochemicals is supposed to be among the most severe side effects on

293

mankind. On condition that a chemical has reactivity with nuclear DNA, there could be

294

mutagenesis and carcinogens to the guardless living beings.34 The genotoxicity of AVM to

295

human cells has been studied seldomly and needs further explanation. The alkaline comet


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assay, as a sensitive means which can be applied to identify DNA fragments induced by the

297

interaction of pesticide and DNA,14 which would significantly increase our comprehension

298

of the genotoxicity effect of AVM. Observation of DNA strand breaks induced by AVM in

299

HeLa cells have been done using this method under alkaline conditions. As shown in Fig.

300

2(A and C), the decrease in head size with concomitant raise in the dimension of comet tails

301

in a dose-dependent manner. The pictures of typical comets distinctly illustrate the extent of

302

fragmentized DNA with increasing AVM concentrations. Besides, the γH2AX foci formation

303

has been recognized as a sensitive means to discover DNA-DSBs due to the development of

304

double-stranded DNA fragments causes the gathering of γH2AX within nuclei.35 Various

305

way that induce DNA damage can cause the phosphorylation of histone H2AX including

306

drugs (e.g., pesticide).36 Threshold of no less than four γH2AX foci per cell is best for

307

judging the DNA damage,37 which offers a precious metrics to recognize DNA damage

308

caused by AVM in cells (Fig.2B). Fig. 2(B and D) represented that AVM induced reaction of

309

γH2AX foci depending on doses given in HeLa cells, which means AVM resulted in

310

DNA-DSBs. The latest study indicates that γH2AX foci may lead to DNA-DSBs, which

311

could be verified by the comet assay. The outcome suggests that AVM may be a potently

312

genotoxic medium that leads to non-target human cellular DNA damage in vitro.

313 314

DNA fragments was also the primary representative biomorphic clue of apoptosis.38

315

Our further study verified whether AVM inhibits HeLa cells viability by causing cell

316

apoptosis. Results of flow cytometry analysis showed that the ratios of apoptotic cells

317

evidently raised according to various concentrations of AVM treatment (Fig.3A and B).



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Meanwhile, a sequence of morphologic alterations, with chromosomal DNA condensed and

319

fragmented nuclei included, can be detected (Fig.3C). Therefore, the initiatory discovery

320

insinuated that AVM induced the HeLa cell death potential in an apoptotic way.

321 322

As is known, apoptosis chiefly realizes through two mainly ways including the

323

intracellular

(mitochondrial-mediated)

pathway

and

the

extracellular

(death

324

receptor-mediated) pathway.39 The two principal features of the intracellular pathway are

325

△Ψm collapse and cytochrome-c release.40 Collapse of △Ψm leads to mitochondrial

326

structure alternation on account of conformational modifications of permeability transition

327

pore on mitochondrial membrane. When the permeability transition pore gets activated and

328

△Ψm collapse, cytochrome-c discharges into cytosol.41 Flow cytometry and fluorescent

329

microscopy analysis results demonstrated AVM can induce △Ψm collapse (Fig. 4A-C).

330

Besides, using mitochondria assay alone indicated that the discharge of mitochondrial

331

elements into cytosol is vital for invoking caspases, which is to say cytochrome-c discharge

332

is an indispensable part and may be adequate for activating caspase in HeLa cells apoptosis.

333

The data in our study indicated that the augment of cytochrome-c discharge into cytosol in

334

AVM-processed HeLa cells (Fig.5A). Meanwhile, the initiating agent caspase-9 and the

335

effector substance caspase-3 were both invoked and were cleavage of PARP (Fig.5D and E),

336

indicating that either of these caspases associated with apoptosis induced by AVM and the

337

repair of AVM-caused DNA damage was inhibited in Hela cells.

338 339

Bcl-2 family proteins are supposed to be concerned with the discharge of


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cytochrome-c.21 Previous researches indicated that the protein Bcl-2 mainly prevents △Ψm,

341

restrains the discharge of cytochrome-c and the protein Bax leads reallocation of

342

cytochrome-c into cytosol,25, 42 in which place it activates caspase proteases (e.g.,caspase-9

343

and caspase-3), and succeeding apoptosis.43 The data in our study demonstrated that AVM

344

significantly suppressed the performance of Bcl-2 but facilitated the performance of Bax

345

(Fig. 5B), which suggests AVM can raise the proportion of Bax/Bcl-2 in HeLa cells (Fig.

346

5C). Therefore, AVM greatly induced loss of △Ψm (Fig. 4B), discharges cytochrome-c into

347

cytosol (Fig. 5A) and activates caspase-9 and caspase-3 (Fig. 5D and E). These results

348

shows that mitochondrial dysfunction was incorporated in HeLa apoptosis caused by AVM.

349 350

In conclusion, we reveal that AVM has ability to induce DNA damage and apoptosis in

351

Hela cells. AVM as the most widely used bio-pesticide, the potential human health threat via

352

long-term accumulation was disregarded.44 Our study clarify that AVM causes the death of

353

Hela cells associated with the mitochondrial-mediated apoptotic pathways. These results

354

would not only supply indication for recognizing the safety of AVM to human beings, but

355

also provide a theoretical basis for understanding its mechanisms of toxicity. However, we

356

cannot figure out whether the apoptosis brought about has direct correlation with the

357

potential genotoxicity caused by AVM with the experimental data in hand, which will be the

358

focus of our future researches.

359 360

AUTHOR INFORMATION

361

Corresponding Author



362

* (L. Tao) E-mail addresses: [email protected]. Tel.: +86 2 164 253 756; fax: +86 2 159

363

883 730.

364

Funding

365

This work was financial supported by National Key Technology Research Development

366

Program of China (NO.2011BAE06B04).

367

Notes

368

The authors declare no competing financial interest.


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Fig. 1.

485

Cytotoxicity of AVM on Hela cells. Cell viability of Hela cells treated with various concentrations (2.5, 5,

486

10, 15, 20 and 40 µM) AVM for 24 and 48 h. Different small letters show significant differences between

487

any two groups (P≤0.05). The data are shown as the means ± SD of three independent experiments.

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Fig.2.

491

AVM induced DNA damage in Hela cells. DNA fragments were shown as comet images in alkaline gel

492

electrophoresis (200 ×) (A). Anti-γH2AX monoclonal antibody was used to detect DNA damage foci

493

immunofluorescence and DAPI was for nuclei staining (B). Percentage of comet-positive cells in the

494

treatment of AVM for 24 h analysis results were shown in the right panel (C). The relative density of

495

γH2AX in the treatment of AVM for 6h analysis results were shown in the right panel (D). Data were

496

represented as means ± SD from three independent experiments.*P ≤ 0.05 and **P < 0.01.

497



498

499 500

Fig. 3.

501

AVM induced apoptosis in Hela cells. The lower left panel shows the normal cells, the lower right panel

502

shows the early apoptotic cells and the upper right panel shows the late apoptotic cells or undergoing

503

necrotic cells (24 h) (A). The apoptosis cells ratio was shown in the right panel (B). Cell nuclei were

504

observed by fluorescence microscopy (200×). Typical apoptosis morphological changes were shown in

505

treated cells including chromatin condensation and DNA fragmentation (C). Data were represented as

506

means ± SD from three independent experiments.

**P ≤ 0.01.


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Fig. 4.

509

AVM induced △Ψm collapse on the HeLa cells. Cells were treated with AVM at various concentrations

510

(0, 5, 10, 20 and 40µM) for 6 h. Changes in △Ψm were evaluated by the flow cytometry method of

511

Rh-123(A). Data were considered significant at **P

Avermectin Confers Its Cytotoxic Effects by Inducing DNA Damage

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