Chlorpyrifos Induced Testicular Cell Apoptosis through Generation of

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Agricultural and Environmental Chemistry

Chlorpyrifos Induced Testicular Cell Apoptosis through Generation of Reactive Oxygen Species and Phosphorylation of AMPK Rui Chen, Yang Cui, Xuelian Zhang, Yanghai Zhang, Mingyue Chen, Tong Zhou, Xianyong Lan, Wuzi Dong, and Chuanying Pan J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b03407 • Publication Date (Web): 31 Oct 2018 Downloaded from http://pubs.acs.org on November 1, 2018

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Journal of Agricultural and Food Chemistry

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Chlorpyrifos Induced Testicular Cell Apoptosis through

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Generation of Reactive Oxygen Species and Phosphorylation

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of AMPK

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Rui Chen †, Yang Cui†, Xuelian Zhang†, Yanghai Zhang†, Mingyue Chen†, Tong Zhou†,

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Xianyong Lan†, Wuzi Dong†, Chuanying Pan†*

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†

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Shaanxi, 712100, China.

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*The corresponding author.

College of Animal Science and Technology, Northwest A&F University, Yangling,

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The first author:

Rui Chen

E-mail: [email protected]

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The second author:

Yang Cui


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The third author:

Xuelian Zhang


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The fourth author:

Yanghai Zhang


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The fifth author:

Mingyue Chen


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The sixth author:

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The seventh author:

Xianyong Lan

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The eighth author:

Wuzi Dong


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The ninth author:

Chuanying Pan


Tong Zhou

E-mail: [email protected] E-mail: [email protected]

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The corresponding author Chuanying Pan at College of Animal Science and Technology,

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Northwest A&F University, No. 22 Xinong Road, Yangling, Shaanxi 712100, P.R.

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China.

ACS Paragon Plus Environment


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ABSTRACT

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Chlorpyrifos (CPF) is the most frequently applied insecticide. Aside from effects on the

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neuronal cholinergic system, previous studies suggested a potential relationship

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between CPF exposure and male infertility; however, the molecular mechanism

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remains elusive. The aim of this study was to investigate the toxic effect of CPF on

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testicular cells and the potential mechanism via in vitro and in vivo experiments. The

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cytotoxic effects of CPF on mouse-derived spermatogonial cell lines (GC-1), Sertoli

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cell lines (TM4) and Leydig cell lines (TM3) were assessed by CCK-8 assay, flow

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cytometry, TUNEL assay, quantitative RT-PCR, and western blot. The exposure to CPF

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(10 to 50 μM) for 12 or 24 h resulted in significant death in all the three testicular cell

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lines. The number of TUNEL-positive apoptotic cells were dose-dependent and

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increased with raised CPF concentration. Further investigation indicated that CPF

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induced cell cycle arrest and then promoted cell apoptosis. Additionally, CPF increased

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reactive oxygen species (ROS) and lipid peroxidation (MDA) production, and reduced

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mitochondrial membrane potential. The mechanism involved in cell apoptosis induced

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by CPF was an increment of phosphorylated AMP-activated protein kinase (p-AMPK)

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levels in the tested cells. In vivo, the expression of steroid hormone bio-synthesis related

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genes in testis, spleen, and lung in F0 and F1 mice were downregulated when there was

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an intraperitoneal injection or dietary supplementation of CPF. This study provides a

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potential molecular mechanism of CPF-induced toxicity in testicular cells and a

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theoretical basis for future treatment of male infertility.


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KEYWORDS: chlorpyrifos, male reproduction toxicity, testicular cell lines,

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oxidative stress, AMPK, F0 and F1 mice



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INTRODUCTION

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According to Phillips McDougall of Agribusiness informa, the global pesticide market

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has increased from $25.1 billion to $56.6 billion, with an overall growth rate of 125%

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over the past 14 years (https://phillipsmcdougall.agribusinessintelligence.informa.com).

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Although the wide use of pesticides brings in more economic value, pesticide residue

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can cause irreparable damage to the environment, and long-term health problems for

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humans and animals.1 Currently, much effort has been given to study the effects of the

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environmental factors on health, which promotes a healthy environment for the

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reproduction of healthy offspring. Regrettably, Levine et al (2017) found that the sperm

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count of western men dropped by 50% over the last half century.2 A major cause for the

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observed defects in male reproductive function is exposure to industrial and agricultural

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toxins, and the by-products of other technological advancements.3 Investigators have

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hypothesized that the wide use of chemicals, pesticides, heavy metals, and

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hyperthermia, are key factors that result in male infertility.4

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Chlorpyrifos [O,O-diethyl-O-(3,5,6-trichloro-2-pyridyl)-phos-phorothioate] (CPF)

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is a broad spectrum organophosphorus pesticide (OP) that is commonly used in

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agricultural, industrial, and domestic applications.5,6 In 2016, the global sales of CPF

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were $686 million, and it was predicted that the compound annual growth rate (CAGR)

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of CPF will reach 6.1% between 2014 and 2020 (http://cn.agropages.com/). Initially,

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CPF was used for pest control, because the peripheral cholinergic nervous systems were

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the target of CPF in insects.7,8 CPF is able to inhibit acetylcholinesterase (AChE)

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activity, which results in an accumulation of acetylcholine and subsequent hyperactivity

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in the cholinergic system.6 Additionally, it has been reported to cause hepatotoxicity,9

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developmental toxicity,10,11 genotoxicity,11 immunological abnormalities12 and cell

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signaling transduction.13

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In multicellular organisms, the development of gonads and germ cells is essential

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for the transmission of genetic information to the next generation and ultimately for the

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survival of species.14 As the most important reproductive organ in male individuals,

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testis attract a great deal of attention. CPF was administered orally to male mice at


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different doses for 4 weeks, the number of live fetuses were decreased at high dose

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group, compared with that of control group, which also accompanied by an increased

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number of dead fetuses in CPF treated mice. Additionally, the sperm counts and sperm

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motility were markedly reduced, the sperm malformation rate in exposed males was

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also went up significantly.15 This phenomenon was further confirmed by Sai et al (2014)

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using rats as a model.16 Moreover, Sai et al (2014) also demonstrated that testosterone

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(T) levels decreased, and there was a statistical difference between the treatment group

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and the control group.16 The association between metabolite of CPF and serum

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reproductive hormone levels were also explored in adult men, which showed an inverse

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association between CPF metabolite and T concentration.17 In addition, the effect of

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CPF on testicular oxidative damage was also studied. The expression levels of

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glutathione (GSH) and antioxidant enzymes presented a downward trend in testis of

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CPF-treated rats.18 Recently, research indicated that extended exposure of CPF given

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rise to damage in the process of spermatogenesis, which probably through interference

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with sex hormones and AchE enzyme levels, thus, resulting in reduction of fertility.19

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Adedara et al (2017) also proved that CPF mediated toxicity along the hypothalamic-

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pituitary-testicular axis in rats via activating of lipid peroxidation, decreasing the

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antioxidant enzymes activities and leading to changes in testicular histology.20

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Although the effects of CPF on male reproduction toxicity have been detected, the

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mechanisms are unclear. Additionally, only the effects of CPF on the brain between the

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F0 and F1 generation of mice have been studied,6,21 other organs have not been reported.

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This study was designed to explore the potential mechanisms of CPF-induced

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toxic effects in the three most important cell types in mouse testis, which are the mouse-

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derived spermatogonial (GC-1), Sertoli (TM4) and Leydig (TM3) cell lines.

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Additionally, for a deeper understanding of CPF toxicity in vivo, intraperitoneal

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injections and the dietary intake of CPF were performed on mice to detect alterations

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in mice testis. These results can further extend the knowledge of CPF induced toxicity

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and provide a theoretical basis for the treatment of male infertility.

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MATERIALS AND METHODS



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Cell Culture

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The GC-1, TM4 and TM3 cells (ATCC, VA, USA) were cultured in high-glucose

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medium (Hyclone, MA, USA) supplemented with 10% Fetal Bovine Serum (FBS;

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Gibco, CA, USA), and 1% Penicillin/Streptomycin (Hyclone, MA, USA), under

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controlled conditions (37 oC, 5% CO2). At a 90% confluence, the cells were sub-

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cultured with 0.25% trypsin (Gibco, CA, USA) for further experiments. The density of

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the cells in the 96-well plate and the 6-well plate were 0.8×104 and 20×104, respectively.

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Cells were allowed to attach overnight.

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Drug Treatment

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The CPF was purchased from Shanghai Aladdin Bio-Chem Technology Co., LTD

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(Shanghai, China). As a lipophilic molecule, the combination of serum proteins can

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neutralize CPF activity, therefore, the cells were transferred to a serum-free medium

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when CPF treatment.22 The CPF was dissolved in dimethyl sulfoxide (DMSO) and the

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final concentration of DMSO did not exceed 0.1%. The incubation time when CPF (0-

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100 µM) treatment ranged from 0 to 24 hours and is indicated in the figures. The DMSO

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0.1% (vehicle) was added to the control group.

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Cell Proliferation Assay

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The GC-1, TM4 and TM3 cells were seeded into 6-well plate overnight. Cells were then

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treated with CPF (10, 25 or 50 µM) or the vehicle for 12 or 24 h. Subsequently, the cells

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were harvested with 0.25% trypsin (Gibco, CA, USA) for total cell count. The number

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of cells (N) were counted using a hemocytometer. The cell numbers were normalized

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and graphed as a ratio of Nt/N0, the DMSO was defined as N0, and the treatment group

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defined as Nt.23 The analyses were performed in triplicate.

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Cell Viability Assay

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Cell viability of the three cell lines were detected by Cell counting kit-8 (CCK-8, C0037,

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Beyotime Institute of Biotechnology, Shanghai, China) after CPF treated for 12 or 24

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h. Viability of DMSO group was measured as control. Cells in each well were cultured

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in serum-free culture medium containing 10 µL of CCK-8 reaction solution for 2 h at

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37 oC, then the absorbance at 450 nm was measured by a microplate reader. Each

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measurement was repeated three times. The data were calculated according to the


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following equation: Cell viability (%) = [(ODtreatment-ODblank)/(ODcontrol-

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ODblank)] × 100%.24

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Annexin-V-FLUOS and Propidium Iodide (PI) Double Staining Assay

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According to manufacturers’ instructions, cells were harvested, washed with PBS and

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then re-suspended in a 500 µL 1×binding buffer (Annexin-V-FLUOS 5 µL and PI 5 µL;

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Biobox, Nanjing, China). Subsequently, cells were incubated for 30 minutes at room

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temperature in the dark, and analyzed by flow cytometry (BD FACSAria™ III, BD

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Biosciences, USA). The data were analyzed using FCS express 5.0 Software (De Novo

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Software, Glendale, CA, USA).

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TUNEL Staining

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According to prospectus, the apoptosis cells were measured using In Situ Cell Death

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Detection Kit (Vazyme, Jiangsu, China). The 4’6’-diamidino-2-phenylindole (DAPI;

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CWBIO, Beijing, China) was used to visualize nucleus. Digital images were captured

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using a Nikon Eclipse 80i fluorescence microscope camera (Tokyo, Japan).25

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Quantitative RT-PCR (qRT-PCR)

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The TRIzol (TaKaRa, Dalian, China) was used to collect the RNA samples of cell after

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CPF treatment. The qRT-PCR was carried out as previously described using the primers

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presented in Table 1.26

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Cell Cycle Assay

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Cultures were analyzed for cell cycle after 12 or 24 h. Cells were trypsinized, washed

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with precooled PBS and treated with a cell cycle staining kit. Finally, cells analyzed by

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flow cytometry (Becton Dickinson, FACSCalibur). Data were analyzed using ModFit

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Software (Verity Software House).

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Measurement of Reactive Oxygen Species (ROS) Production

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The intracellular ROS level was measured by ROS Assay Kit (S0033, Beyotime

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Institute of Biotechnology, Shanghai, China) following the manufacturer's protocol.

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When 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) was oxidized by ROS to

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2′,7′-dichlorofluorescein (DCF), the higher fluorescence intensity could be observed at

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530 nm. The cells were suspended with high-glucose medium containing 1 µL of

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DCFH-DA (final concentration was 10 μM/L), and were incubated for 30 min at 37 oC



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in the dark. Finally, the multi-detection microplate reader (Synergy HT, BioTek,

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Vermont, USA) was used to quantify the relative levels of fluorescence (485 nm

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excitation and 535 nm emission). 27

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Measurement of Malonaldehyde (MDA) Level

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Malonaldehyde was determined by a Lipid Peroxidation MDA Assay Kit (S0131,

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Beyotime Institute of Biotechnology, Shanghai, China). The cells were prepared as

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described in kit instructions. The MDA concentration of each sample was evaluated by

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a multi-detection microplate reader (SpectramMax M5) at 532 nm, using 490 nm as the

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control. 28

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Determination of Mitochondrial Membrane Potential (MMP)

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The MMP (ΔΨm) was detected using fluorescent probe: JC-1 (5,5’,6,6’-Tetrachloro-

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1,1’,3,3’-tetraethyl benzimidazolyl carbocyanine iodide) (C2006, Beyotime Institute of

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Biotechnology, Shanghai, China). The increase green fluorescence intensity (FL1) was

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always accompanied by mitochondrial depolarization, which can be detected by multi-

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detection microplate reader. 29

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Western Blot

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Antibodies against BAX (2772), BCL2 (3498) and p-AMPK (50081) were purchased

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from Cell Signaling Technology (Beverly, MA, USA), and p-LKB1 (sc-271924; Santa

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Cruz Biotechnology, USA) and GAPDH (Cell Signaling Technology, USA) were also

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employed in the experiments.

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The cells were washed with PBS, and then lysed with RIPA (P0013B, Beyotime

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Institute of Biotechnology, Shanghai, China), which contained 1 mmol/L of PMSF

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(ST506, Beyotime Institute of Biotechnology, Shanghai, China). The 10% SDS-PAGE

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was used to separate the lysates of cells, which followed by transferred to PVDF

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membranes (Millipore, USA).25 The membranes stained with the reagents in Western

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Bright ECL Kit were then visualized using Bio-Rad Chemidoc (Bio-Rad, USA).

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Animal Feeding

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The Wild-type C57BL/6 mice (6 weeks) were maintained under controlled

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environment (22±2 oC, 55±10% humidity, 12 h reversed light-dark cycle), and fed

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with ad libitum food and water at a pathogen-free facility. The experimental animals


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and procedures used in this study were approved by the Faculty Animal Policy and

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Welfare Committee of Northwest A&F University. The care and use of experimental

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animals fully complied with local animal welfare laws, guidelines, and policies.

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Procedures and Experimental Groups for Intraperitoneal Injection of CPF in

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Mice

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Previous findings have shown that CPF at a dose of 17.5 mg/kg given orally to male

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rats for 30 days could induce severe testicular damage.30 In addition, Sai et al.

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demonstrated that CPF administered orally to male rats at different dose for 90 days

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had adverse effects on the reproductive system.31 The daily exposure doses of CPF in

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developing countries like Sri Lanka is 94,000 ng/kg ∙ day-1,32 indicating that exposure

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levels are lower than 17.5 mg/kg. However, farmers in Sri Lanka used higher levels of

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CPF than what is recommended for crop protection.19 Therefore, in this study, 3-, 6-,

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and 12-mg/kg doses were selected. The CPF could be absorbed by body via different

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approaches, including injection, ingestion, inhalation, and dermal absorption.33

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Injection and oral ingestion were selected as the exposure mode in this study.

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For the CPF intraperitoneal injection experiments, animals were randomly divided

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into four groups that contained 6 mice each (a control group and three exposure groups).

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In the exposure groups, different doses of CPF (3-, 6-, and 12-mg/kg) were injected

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intraperitoneally into eight-week-old mice once a day, and consecutive for 35 days

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(lasted for one spermatogenic cycle). The control mice were administered an equal

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volume of redistilled water and DMSO.

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Procedures and Experimental Groups for Dietary Supplementation of CPF in

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Mice

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For the CPF dietary supplementation experiments, the amount of CPF added to the diet

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corresponded to a dose of 3-, 6-, and 12 mg ∙ kg-1 / bw ∙ day-1 according to a food

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consumption of approximately 5 g/day for each mouse.34 The CPF diet was

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administered to eight-week-old male and female mice of the F0 generation for 80

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consecutive days. The offspring (F1 generation) of F0 mice were sacrificed at the time

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of weaning.

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At the end of the treatment period, mice were subjected to a 3 h fast before being



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deeply anesthetized with carbon dioxide prior to being euthanized. The tissue samples

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of mice were fixed in Bouin’s solution or transferred into TRIzol (TaKaRa, Dalian,

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China) for molecular analysis.

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Hematoxylin and Eosin (H&E) Staining

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Testis samples collected from mice were fixed in Bouin’s solution overnight before

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being dehydrated and embedded in paraffin. The 5 μm cross-sections were adhered to

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precoated glass slides. The H&E staining of the paraffinembedded sections were

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conducted to observe histology.35

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For the CPF intraperitoneal injection experiments, the samples of control group,

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3- and 6-mg/kg CPF injection group were collected at 2 weeks and 35 days, respectively,

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and three samples were taken for each group. While for 12-mg/kg CPF injection group,

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except for sampling at 2 weeks, the mice were not in good condition at 18 days and

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were not reared for 35 days, thus, the samples at 18 days were taken (n=3).

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For the CPF dietary supplementation experiments, the testis samples of control

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group, 3-, 6-, and 12 mg ∙ kg-1 / bw ∙ day-1 CPF diet feeding F0 mice were collected

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after 80 days of feeding. Three samples in each group were taken. The offspring (F1

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generation) of F0 mice were sacrificed at the time of weaning, and three samples were

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collected in each group.

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Statistical Analysis

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Statistical treatment of the data was performed with SPSS 19.0 software (SPSS,

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Chicago, IL, USA) using one-way ANOVA and Student t-test. The Student t-test was

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used for the two-group comparisons, while the ANOVA with a Tukey HSD post-hoc

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test was applied to multi-group comparisons.36 All data were expressed as the mean ±

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standard error (SE) of the three independent experiments and were considered

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statistically significant when the P value was less than 0.05 (*) and 0.01 (**).

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RESULTS

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CPF Induced Morphological Changes and Cytotoxicity

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A dose responsive effect, with a CPF concentration that ranged from 0-100 µM, was

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tested on GC-1, TM4, and TM3 cells at different treatment times to evaluate the toxicity


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of CPF. As CPF is a lipophilic molecule, the combination of CPF and serum proteins

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may compromise CPF activity, therefore, the cells were transferred to a serum-free

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medium when CPF treatment.22 In order to avoid the negative effects of long-term

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serum starvation during CPF treatment, the GC-1 cells were exposed for 24 h, while

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TM4 and TM3 cells were exposed for 12 h. Cell viability decreased with raised

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concentrations and prolonged treatment of CPF. Since 100 µM CPF resulted in a

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decrease in cell viability greater than 50% for GC-1 and TM4 cells (data not shown),

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50 µM and lower concentrations were chosen for further assayed in the subsequent

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experiments.

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As shown in Figure 1, a low CPF concentration (10 µM) had no obvious effect on

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cell morphology. Nevertheless, the shape and density of the cells changed with raised

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CPF concentration: lower cell density, cell shrinkage, round cells and a shedding

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morphology.

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Cell viability was also assessed in this study using a CCK-8 assay. The results

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from the CCK-8 assay showed that the 10 and 25 µM concentration of CPF significantly

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decreased the viability of the GC-1 and TM4 cells, and this response was still present

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at higher concentrations (Figure 2A,B). The viability of TM3 cells were slightly

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affected by CPF (Figure 2C).

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To further assess the CPF-induced cytotoxicity in the three cell types, the total cell

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number was counted. Consistent with the cell viability results, the CPF (0-50 µM)

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concentration dependently decreased the total cell number of the GC-1 and TM4 cells

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(Figure 2A´,B´). Unlike the moderate decrease in cell viability, the total number of TM3

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cells was statistically significant decreased with the raised concentration of CPF (Figure

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2C´).

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CPF Promoted Cell Apoptosis

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First of all, the TUNEL staining was used to assess whether cell apoptosis caused

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morphological changes of CPF treated cells. The principle of Terminal

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deoxynucleotidyl transferase (TdT) dUTP nick end labeling (TUNEL) is to attach dUTP

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to the 3’ ends of double- and single-stranded DNA breaks using TdT enzyme in cells.37

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The 25 µM CPF concentration dramatically increased the number of TUNEL positive



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cells in GC-1 cell line, and this response was still present at higher concentrations (50

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µM) (Figure 3A). For TM4 cells, the number of TUNEL positive cells were increased

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obviously when exposed to 50 µM CPF, compared with control group, 10 and 25 µM

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CPF group (Figure 3B). Nevertheless, no significant changes were observed in TM3

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cells when exposed to different concentrations of CPF (Figure 3C). The above

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experimental results hinted that the sensitivity of these three cell lines to CPF was

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different. GC-1 cells were the most sensitive cell line, followed by TM4 cells, while

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TM3 cells were the least sensitive.

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Subsequently, cell apoptosis was further monitored with an Annexin V/PI stain

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followed by flow cytometry analysis in the tested cells. The translocation of

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phospholipid phosphatidylserine to the outer plasma membrane was regarded as the

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early feature of apoptosis.25 In GC-1 cells, the percentage of early apoptosis cells

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(Annexin V+/PI−) that was induced by the different concentrations (0, 10, 25, and 50

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µM) of CPF were 2.635%, 2.770%, 2.255%, and 2.880%, respectively (Figure 4A,B;

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Table 2). There was an upward trend in the percentage of late apoptosis (Annexin

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V+/PI+) when cells were exposed to CPF (0 µM: 10.200%, 10 µM: 13.450%, 25 µM:

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22.400% and 50 µM: 32.400%) (Figure 4A,B; Table 2). Unlike GC-1 cells, no obvious

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changes in the percentage of late apoptosis (Annexin V+/PI+) were observed in TM4

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cells, while a very significant increase in the percentage of early apoptosis (Annexin

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V+/PI−) was detected when TM4 cells exposed to CPF (0 µM: 18.867%, 10 µM:

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18.800%, 25 µM: 29.200% and 50 µM: 29.750%) (Figure 4C,D; Table 2). For the TM3

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cells, the percentage of early apoptosis (Annexin V+/PI−) that was induced by the

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different concentrations (0, 10, 25, and 50 µM) of CPF were 3.715%, 3.410%, 7.005%,

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and 10.900%, respectively (Figure 4E,F; Table 2). A very significant increasing trend

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in the percentage of late apoptosis (Annexin V+/PI+) was detected when TM3 cells

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exposed to CPF (0 µM: 1.625%, 10 µM: 1.075%, 25 µM: 2.950% and 50 µM: 8.620%)

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(Figure 4E,F; Table 2).

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Next, the expressions of apoptosis-related genes were also detected in GC-1 and

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TM4 cells. The results demonstrated that the expression levels of the cell apoptosis

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genes (p53, Puma, Caspase 3 and Caspase 9) had at least a 2‑fold increase in both cell


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lines when exposed to high concentrations of CPF, compared to that of control groups

314

(Figure 5A,6A).

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As shown in Figure 5 and Figure 6, verification at the protein level further proved

316

that cell apoptosis was significantly induced by CPF in GC-1 and TM4 cells. The 10

317

and 25 µM concentration of CPF led to a decrease in BCL2 expression, while there was

318

noticeable change in BAX in GC-1 cells (Figure 5C). Importantly, the ratio of

319

BAX/BCL2 was up-regulated, and the expression of Caspase 9 increased when GC-1

320

cells were exposed to CPF (Figure 5B,C). Similarly, the ratio of BAX/BCL2 increased

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in TM4 cells. In contrast to GC-1 cells, the expression of BCL2 was not influenced, but

322

there was up-regulation of BAX in the TM4 cells (Figure 6B).

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CPF Induced Cell Cycle Arrest

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An additional study was carried out to explore the effects of CPF on cell cycle in the

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three cell lines. To evaluate this action, cell cycle was assessed by PI staining and

326

measured by flow cytometry. For GC-1 cells, the percentage of cells in the G1 phase

327

was dose-dependent and increased with an increase in the CPF treatment concentration

328

(Figure 7A,B). The proportion of GC-1 cells in the G1 phase ranged from 43.447% to

329

71.053% in the 10 µM to 50 µM CPF concentration treatments, respectively, and the

330

control group was 33.247% (Table 3). Additionally, the increased percentage of cells in

331

the G1 phase was accompanied by a decreased percentage of cells in both S and G2

332

phases (Figure 7A,B). For TM4 cells, CPF induced S cell cycle arrest with a

333

concentration-dependency (Figure 7C,D). Exposure to CPF dramatically shuffled the

334

cells in the cell cycle compartments as evidenced by a considerable accumulation of

335

cells in S phase (0 µM: 19.807%, 10 µM: 19.233%, 25 µM: 44.880% and 50 µM:

336

52.237%), and a remarkable reduction of cells in G1 phase (0 µM: 54.013%, 10 µM:

337

56.723%, 25 µM: 44.343% and 50 µM: 40.217%) and G2 phase (0 µM: 26.180%, 10

338

µM: 24.047%, 25 µM: 10.773% and 50 µM: 7.550%) (Table 3). For TM3 cells, consist

339

with the results of TM4 cells, a significant increment of cells in S phase was observed

340

at the 50 µM CPF treatment (35.595%) (Table 3), compared with control group

341

(11.460%). In addition, CPF resulted in a decreasing proportion of G1 phase cells,

342

though no statistically significant differences were detected at G2 phase (Figure 7E,F).



343

In light of the above observations, the expression of the cell cycle regulators was

344

examined in the GC-1, TM4 and TM3 cells with or without the CPF treatment. Cyclin

345

is a family of protein that controls the progression of cells through cell cycle.38 In the

346

three cells, expression levels of p53 and cyclin-dependent kinase inhibitor p21CIP, a

347

product of a p53-activated gene, increased when cells were exposed to CPF (Figure 8).

348

39,40

349

and CCNE1 (encoding Cyclin E1) was observed (Figure 8A). Nevertheless, the

350

expression of CCNA2 (encoding Cyclin A2) and CCNB1 (encoding Cyclin B1) was

351

decreased in CPF treated TM4 and TM3 cells (Figure 8B,C). These data suggest that

352

CPF suppressed cell proliferation via cell cycle arrest.

353

CPF Up-regulated ROS Level and Promoted AMPK Phosphorylation

354

To elucidate the underlying mechanism of cell apoptosis induced by CPF, the

355

phosphorylation of AMP-activated protein kinase (AMPK) was analyzed. The presence

356

of p-AMPK was evaluated by a western blot analysis of specific antibodies in GC-1

357

and TM4 cells exposed to CPF at different concentrations for 12 or 24 h. As shown in

358

Figure 9A, the 25 and 50 µM concentrations of CPF induced a significant increment of

359

AMPK phosphorylation in GC-1 cells compared to that of the control group, whereas

360

there was no change in the 10 µM CPF concentration group.

For GC-1 cells, a decrease in expression levels of CCND1 (encoding Cyclin D1)

361

Many environmental compounds could modify the oxidative balance, which led

362

to the inhibition of cell proliferation and induce apoptosis,41 therefore, we also detected

363

whether CPF could affect the redox balance in GC-1 cells. The ROS generation during

364

the GC-1 cells were treated with different concentrations of CPF were studied, and a

365

significant increase in cells treat with CPF was also noticed, compared to the control

366

group (Figure 9D,E,F). As the reduction of MMP is often associated with apoptosis,42

367

MMP during the CPF treatment was evaluated. The results revealed that the addition of

368

CPF could reduce MMP in GC-1 cells (Figure 9B). Moreover, elevated MDA level was

369

associated with an increase in the concentration of CPF (Figure 9C). These data

370

suggested that ROS production was involved in the regulation of CPF-induced cell

371

apoptosis.

372

To prove whether AMPK and ROS regulated the survival of TM4 cells after the


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373

CPF treatment, p-AMPK and ROS levels in TM4 cells were detected. Consistent with

374

the results of GC-1 cells, the addition of 25 µM CPF concentration resulted in up-

375

regulation of p-AMPK (Figure 10A,B), and ROS levels also increased in TM4 cells

376

compared to that of the control (Figure 10C).

377

Intraperitoneal Injection of CPF reduced the Cell Number of Testicular

378

Seminiferous Tubules

379

There were no significant differences in the organ coefficients, and morphologic

380

changes were observed in the testis for the 3 and 6 mg/kg CPF concentration injection

381

group and control group at 2 weeks and 35 days (Figure S1; Table S1,S2). Interestingly,

382

one mouse died after the intraperitoneal injection at 18 days in the 12 mg/kg group, the

383

neural reflex of the other mice in that group was slow, and the number of cells in the

384

testicular seminiferous were also decreased compared to that of the control, the

385

percentage of damaged seminiferous tubules was 28.54% (Figure 11A,B). The qRT-

386

PCR results certified that the expression of the germ, Sertoli, Leydig and apoptosis

387

related genes were altered in the 12 mg/kg CPF concentration injection group (Figure

388

11C). These data suggested that acute toxicity accumulation of CPF may damage male

389

reproduction in mice.

390

Dietary Supplementation of CPF Resulted in Expression Changes in Steroid

391

Hormone Bio-synthesis related genes in F0 and F1 Mice

392

In 2006, Meeker et al. analyzed the association of male reproductive hormones with

393

CPF and its metabolic product.17 In this study, the expression of steroid hormone bio-

394

synthesis related genes, such as StAR, HSD3B1 and HSD17B3, was detected. The

395

results showed that the expression of these three genes was markedly reduced in the F0

396

generation after fed CPF for 80 days (Figure 12A). Additionally, the expression of genes

397

that is associated with steroid hormone synthesis, the development of germ cells, Sertoli

398

cells and Leydig cells, cell proliferation and apoptosis, showed a downward trend in

399

the 12 mg/kg CPF concentration treatment group compared to that of the control group

400

(Figure 12B).

401

Figure 12C provides an overview of the effects of dietary CPF on the other organs

402

in mice. Overall, the expression of StAR, HSD3B1 and HSD17B3 decreased in the testis,



403

spleen, and lung in the F0 generation after the 12 mg/kg CPF concentration group when

404

fed for 80 days compared to that of the control group. In the kidney, the expression of

405

HSD3B1 and HSD17B3 showed a downward trend in the CPF treatment group

406

compared to that of the control group, but, interestingly, StAR expression levels were

407

significantly elevated in the CPF-treated kidney samples compared to that of the control

408

group.

409

F1 generation samples were collected at the time of weaning (3 and 4 w). There

410

were no significant differences in the body and tissue weights following the 1 and 6

411

mg/kg CPF exposure for any of the mice (Table S3). Nevertheless, in three-week-old

412

F1 mice, with an increase in CPF concentration, the expression of HSD3B1 and

413

HSD17B3 showed a downward trend (Figure 13A). However, no significant differences

414

were observed in four-week-old F1 mice (Figure 13B).

415

Additionally, no significant differences in morphologic changes were observed

416

using HE staining in neither F0 mice nor F1 mice testis tissue (data not shown).

417

DISCUSSION

418

Chlorpyrifos (CPF) is extensively used for various purposes. The widespread use of

419

CPF has stimulated research on the possible existence of effects related to reproductive

420

toxic activity.4,43,44 However, male reproductive toxicity mechanisms induced by CPF

421

has not been thoroughly studied. In this study, the toxic effects of CPF in vitro and in

422

vivo were detected.

423

Various stressors, including pesticides, are existed in the environment and are able

424

to cause DNA damage.45 Researchers demonstrated that the percentages of sperms with

425

DNA damage in CPF-exposed animals were significantly higher than control group.1

426

This phenomenon was also proved by using CPF and cypermethrin combined rat as

427

model. Significant increments in sperm DNA fragmentation index were manifested in

428

exposure group.46 In addition, CPF-induced DNA damage and apoptosis were observed

429

in larval Drosophila midgut tissues, which was proved by a markedly increase in the

430

Comet parameters, viz, tail length (mm), TM (arbitrary units) and tail DNA (%) of the

431

exposed individuals.43 In 2015, Li et al. indicated that the addition of CPF in the culture


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432

medium induced a dramatically concentration- and time-dependent augment in HeLa

433

and HEK293 cell apoptosis, which was also accompanied by single-strand DNA breaks

434

in CPF treated cells compared to that of the control.47 Normally, p53 plays a pivotal

435

role in cell cycle regulation and the induction of apoptosis when mammalian cells are

436

subjected to stress conditions, such as hypoxia, radiation, chemotherapeutic drugs, or

437

DNA damage.48,49 In the process of the cell cycle, the Cyclin-dependent kinase inhibitor

438

p21CIP (a downstream gene of p53) could be combined with a series of Cyclin-cdk

439

complexes to inhibit the activity of protein kinase, thereby arresting the cell cycle.39,40

440

Additionally, the p53 tumor suppressor could also mediate apoptosis through Bax

441

transactivation, the release of mitochondrial cytochrome c, and caspase-9 activation,

442

which is usually followed by the activation of caspase-3, -6, and -7.50

443

In this study, the addition of CPF to the culture medium led to a decrease in cell

444

number and viability in GC-1, TM4 and TM3 cells. The expression of p53 and p21CIP

445

increased when all testicular cells were exposed to CPF. Moreover, the expression

446

levels of cell apoptosis genes (p53, Puma, Caspase 3 and Caspase 9) increased when

447

GC-1 and TM4 cells exposed to high concentrations of CPF, compared to that of with

448

the control groups. Afterwards, the effect of the CPF on cell cycle was studied. The

449

results showed that CPF could induce G1 phase, S phase and S phase arrest in GC-1,

450

TM4 and TM3 cells, respectively. In addition, consistent with the results of flow

451

cytometry, a decrease in the expression levels of CCND1 (encoding Cyclin D1) and

452

CCNE1 (encoding Cyclin E1) in GC-1 cells, as well as a decrease in the expression

453

levels of CCNA2 (encoding Cyclin A2) and CCNB1 (encoding Cyclin B1) in both TM4

454

and TM3 cells were also observed. The differences of the results were related to the

455

different cell types. These data indicated that CPF may induce DNA damage first by

456

arresting the cell cycle, suppressing cell proliferation, and promoting cell apoptosis,

457

until, finally, there is a reduction in the number of GC-1, TM4, and TM3 cells. Future

458

studies should investigate the DNA damage induced by CPF in these cells.

459

Although no information about CPF induced oxidative stress on testicular cells

460

were reported, the in vivo experiments were extensive. The activation of lipid

461

peroxidation and the decrease of antioxidant enzymes activities were observed in CPF



462

treated rats.20 Association between reproductive toxicity and antioxidant enzymes

463

catalase activities, superoxide dismutase (SOD), glutathione peroxidase (GPx), and

464

glutathione S-transferase (GST) as well as glutathione (GSH) level exposed to CPF

465

compounds has been demonstrated in rats.51 Consistent with this, an obvious positive

466

correlation between ROS generation and CPF addition were detected in this study.52,53

467

To further examine the causes of CPF-induced oxidative stress, MMP was measured.

468

The data indicated that CPF mediated cell apoptosis was associated with a reduction in

469

MMP. Lipid peroxidation is a free radical-driven reaction that leads to membrane

470

damage by a reaction of oxygen with polyunsaturated fatty acids.54 Previous studies

471

had also indicated that the increment of MDA content was correlated to cellular

472

membrane damage in exposed cells, which could then derivate cells to death.55 An

473

augment of MDA was detected when GC-1 cells were exposed to CPF at a dose capable

474

of increasing the ROS generation.

475

The aforementioned results have shown that the addition of CPF could promote

476

cell apoptosis. The mechanism involved in CPF-induced cell apoptosis was also

477

examined in this study. Numerous studies have revealed that activation of MAPK and

478

EGFR/ERK1/2 are involved in CPF-induced apoptosis. CPF could induce human

479

neuroblastoma SH-SY5Y cell apoptosis through the MAPK pathway.55 A recent study

480

proved that CPF inhibited breast cancer cells MCF-7 and MDA-MB-231 proliferation

481

via an incremental phosphorylation of p-ERK1/2 levels that were mediated by oxidative

482

stress.5 In contrast, the function of AMP-activated protein kinase (AMPK) in CPF

483

induced toxic reactions has not been reported. AMPK is a crucial enzyme protein that

484

links energy induction with metabolic reactions, and it is also involved in regulating the

485

processes of glycometabolism, fat metabolism, protein metabolism, and maintaining

486

the homeostasis of energy in cells.56 There are increasing indications that some types of

487

cellular stress activate AMPK by ROS, such as H2O2.56 In cultured cells, AMPK is

488

activated by reactive oxygen species (ROS) such as H2O2. In 2008, research indicated

489

that Britannin (Bri) could induce apoptosis in liver cancer cell lines via AMPK

490

activation that was regulated by ROS.57 Moreover, Chen et al. proved that a hypoxia

491

treatment triggered AMPK activation in H9c2 cells in a time dependent manner and led


Page 18 of 47

Page 19 of 47


492

to cardiomyocyte injury.58 Consistent with this, AMPK was activated when testicular

493

cells were exposed to CPF in this study. Although this study confirmed the role of

494

AMPK in CPF-induced reproductive toxicity, the upstream and downstream signaling

495

pathways of AMPK are unknown and needs to be studied further.

496

CPF inhibits AchE activity and reduces monoamine levels that are needed for

497

adequate hypothalamic-pituitary-gonadal axis (HPGA) activity, which in turn directs

498

the toxicity of male hormones, induces sperm DNA damage, or causes sperm epigenetic

499

changes.4,17 To this point, the expression of steroid hormone bio-synthesis related genes

500

was detected in this study between the CPF treatment group and the control group. The

501

data from the in vivo experiments initially demonstrated that the expression of steroid

502

hormone bio-synthesis related genes in the testis, spleen, and lung of F0 mice was

503

downregulated when there was intraperitoneal injection or dietary supplementation of

504

CPF. It is worth noting that the intraperitoneal injection of CPF in mice led to a

505

significantly increasing trends in the expression of StAR in the CPF treatment group,

506

compared to that of control group. Nevertheless, the exact mechanism needs to be

507

studied further.

508

For F1 generations, the expression of HSD3B1 and HSD17B3 showed a downward

509

trend with an increase in CPF concentration of three-week-old mice, while no

510

significant differences were observed in four-week-old F1 mice. Considering the

511

expression of these two genes in testis was related to the development of Leydig cells,

512

which showed an increase tendency during the transition from progenitor Leydig cells

513

(PLCs) (around postnatal day 14) to immature Leydig cell (ILCs) (around postnatal day

514

28).59,60 It is well known that the cells with lower differentiation degree were more

515

sensitive to drugs. Back to our study, compared to ILCs, PLCs were more sensitive to

516

CPF. Thus, the number of PLCs decreased in three-week-old mice, resulted in the

517

decrease expression of these two genes.

518

In conclusion, we propose a model for the regulation of CPF for the survival of

519

testicular cells (Figure 14). The addition of CPF in the culture medium resulted in a

520

reduction in cell number and cell viability. CPF decreased the cellular membrane

521

potential of mitochondria, up-regulated the levels of ROS and MDA, activated the



522

phosphorylation of AMPK, and thereby, arrested the cell cycle, suppressed cell

523

proliferation, promoted cell apoptosis, and regulated the survival of testicular cells. In

524

vivo, the expression of steroid hormone bio-synthesis related genes in testis, spleen and

525

lungs of the F0 and F1 mice was downregulated when there was an intraperitoneal

526

injection or dietary supplementation of CPF. This study replenishes the research on the

527

regulation of CPF in male reproduction and provides a theoretical basis for the

528

treatment of male infertility in the future.


Page 20 of 47

Page 21 of 47


529

AUTHOR INFORMATION

530

Corresponding Authors

531

*E-mail: [email protected].

532

Funding

533

This work was supported by the National Natural Science Fund (regional project)

534

(No.31760650), The Key Research and Development Program of Shaanxi Province

535

(agricultural field) (No.2017NY-064).

536

Notes

537

The authors declare no competing financial interest.

Tel: 86-13772098751



538

ABBREVIATIONS USED

539

CPF, chlorpyrifos; OP, organophosphorous pesticide; AChE, acetylcholinesterase; GC-

540

1, mouse-derived spermatogonial cell lines; TM4, mouse Sertoli cell lines; TM3, mouse

541

Leydig cell lines; DMSO, dimethyl sulfoxide; qRT-PCR, quantitative RT-PCR; ROS,

542

reactive oxygen species; MDA, malonaldehyde; SOD, superoxide dismutase; GPx,

543

glutathione peroxidase; p-AMPK, phosphorylated AMP-activated protein kinase;

544

DCFH-DA, dichlorodihydrofluorescein diacetate; DCF, dichlorofluorescein; T,

545

testosterone; FSH, follicle-stimulating hormone; LH, luteinizing hormone; GSH,

546

glutathione; SHBG, sex hormone-binding globulin; ILCs, immature Leydig cell; PLCs,

547

progenitor Leydig cells; HPGA, hypothalamic-pituitary-gonadal axis; TUNEL,

548

terminal deoxynucleotidyl transferase (TdT) dUTP nick end labeling.


Page 22 of 47

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549

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reactive oxygen species, protein carbonylation and DNA damage on digestive gland

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Y. L.; Chen, P. H.; Teng, Y. N. Reactive oxygen species mediate Terbufos-induced

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apoptosis in mouse testicular cell lines via the modulation of cell cycle and pro-

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apoptotic proteins. Environ. Toxicol. 2016, 31, 1888-98.

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(43) Gupta, S. C.; Mishra, M.; Sharma, A.; Deepak Balaji, T. G.; Kumar, R.; Mishra, R.

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K.; Chowdhuri, D. K. Chlorpyrifos induces apoptosis and DNA damage in Drosophila

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through generation of reactive oxygen species. Ecotoxicol. Environ. Saf. 2010, 73,

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(44) Eaton, D. L.; Daroff, R. B.; Autrup, H.; Bridges, J.; Buffler, P.; Costa, L. G.; Coyle,

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Spencer, P. S. Review of the toxicology of chlorpyrifos with an emphasis on human

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exposure and neurodevelopment. Crit. Rev. Toxicol. 2008, 38, Suppl 2:1-125.

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(45) Wilson, D. M. 3rd.; Sofinowski, T. M.; McNeill, D. R. Repair mechanisms for oxidative DNA damage. Front. Biosci. 2003, 8, d963-81.

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(46) Alaa-Eldin, E. A.; El-Shafei, D. A.; Abouhashem, N. S. Individual and combined

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effect of chlorpyrifos and cypermethrin on reproductive system of adult male albino

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rats. Environ. Sci. Pollut. Res. Int. 2017, 24, 1532-1543.

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DNA damage and cell apoptosis. Chemosphere 2015, 135, 387-93.

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730



731

Table 1. Primers Used in qRT-PCR Gene names

Forward primers (from 5’ to 3’)

Reverse primers (from 5’ to 3’)

Caspase 9

CCACTGCCTCATCATCAAC

TGTGCCATCTCCATCAAA

Caspase 3

AGTTCCCGGGTGCTGTCTAT

GCCATGGTCTTTCTGCTCAC

p53

ATGCGGTTCGGGTCCAAAAT

CTAAATGGCAGTCGTTCTCTCC

Puma

AGCAGCACTTAGAGTCGCC

CCTGGGTAAGGGGAGGAGT

CCND1

TGCTGCAAATGGAACTGCTT

CCACAAAGGTCTGTGCATGCT

CCNE1

GTGGCTCCGACCTTTCAGTC

CACAGTCTTGTCAATCTTGGCA

P21CIP

CCTGGTGATGTCCGACCTG

CCATGAGCGCATCGCAATC

CCNB1

AAGGTGCCTGTGTGTGAACC

GTCAGCCCCATCATCTGCG

CCNA2

GCCTTCACCATTCATGTGGAT

TTGCTGCGGGTAAAGAGACAG

Akt

ATGAACGACGTAGCCATTGTG

TTGTAGCCAATAAAGGTGCCAT

PLZF

CTGCGGAAAACGGTTCCTG

GTGCCAGTATGGGTCTGTCT

GDNF

TCCAACTGGGGGTCTACGG

GCCACGACATCCCATAACTTCAT

Stra8

ACAACCTAAGGAAGGCAGTTTAC

GACCTCCTCTAAGCTGTTGGG

HSD3B1

TGGACAAAGTATTCCGACCAGA

GGCACACTTGCTTGAACACAG

HSD17B3

AGGTTCTCGCAGCACCTTTTT

CATCGCCTGCTCCGGTAATC

StAR

GGTTCTCAGCTGGAAGACACT

ACCTCGTCCCCATTCTCCTG

LHR

GCCTCAGCCGACTATCACTC

GGAGGTTGTCAAAGGCATTAGC

ꞵ-Actin

TTGCTGACAGGATGCAGAAG

ACTCCTGCTTGCTGATCCACAT

732


Page 30 of 47

Page 31 of 47


733

Table 2. Percent of Cell Apoptosis at Different Periods When GC-1, TM4 and TM3 Cells

734

Exposure to Different Concentrations of CPF Group

Early apoptosis (%)

Late apoptosis (%)

Total apoptosis (%)

DMSO

2.635±0.085b

10.200±0.100d

12.835±0.185c

CPF 10 µM

2.770±0.050b

13.450±0.450c

16.220±0.400c

CPF 25 µM

2.255±0.155ab

22.400±0.600b

24.655±0.755b

CPF 50 µM

2.880±1.010a

32.400±1.400a

35.280±2.410a

DMSO

18.867±1.425b

2.480±0.384b

21.947±1.808b

CPF 10 µM

18.800±0.900b

2.185±0.145b

20.985±1.045b

CPF 25 µM

29.200±1.500a

1.610±0.210b

30.610±1.490a

CPF 50 µM

29.750±0.350a

4.995±0.495a

34.745±0.845a

DMSO

3.715±0.745c

1.625±0.885b

5.340±1.630c

CPF 10 µM

3.410±0.580c

1.075±0.085b

4.485±0.495c

CPF 25 µM

7.005±0.395b

2.950±0.680b

9.955±1.075b

CPF 50 µM

10.900±0.400a

8.620±0.200a

19.520±0.600a

GC-1 cells

TM4 cells

TM3 cells

735

Note: The values with different letters (a, b, c and d) within the same column differ significantly at

736

P < 0.05 or P < 0.01.



737

Page 32 of 47

Table 3. Cell Cycle Distributions of GC-1, TM4 and TM3 Cells Using CPF Group

G1 (%)

S (%)

G2 (%)

DMSO

33.247±0.964d

46.587±2.222a

20.167±1.380a

CPF 10 µM

43.447±1.191c

41.730±1.168a

14.827±0.815b

CPF 25 µM

55.543±0.987b

35.987±1.698b

8.473±1.042c

CPF 50 µM

71.053±0.919a

23.707±1.669c

5.240±0.780c

DMSO

54.013±0.238a

19.807±0.855c

26.180±0.635a

CPF 10 µM

56.723±0.644a

19.233±0.456c

24.047±0.259a

CPF 25 µM

44.343±0.367b

44.880±0.921b

10.773±0.703b

CPF 50 µM

40.217±0.519c

52.237±0.524a

7.550±0.448c

DMSO

63.500±0.970a

11.460±0.550c

25.040±1.520

CPF 10 µM

64.047±0.158a

12.043±0.358c

23.910±0.219

CPF 25 µM

52.610±0.771b

24.140±0.573b

23.253±0.327

CPF 50 µM

41.265±0.005c

35.595±0.145a

23.140±0.140

GC-1 cells

TM4 cells

TM3 cells

738

Note: The values with different letters (a, b, c and d) within the same column differ significantly

739

at P < 0.05 or P < 0.01.


Page 33 of 47


740

741 742

Figure 1. The phenotype of GC-1, TM4 and TM3 cells after CPF exposed for 12 or 24 h. (A) GC-

743

1 cells treated with CPF of different concentrations for 24 h. (B) TM4 and TM3 cells treated with

744

CPF of different concentrations for 12 h. Bar=100 μm.



745 746

Figure 2. The cell viability and total cell number of GC-1, TM4 and TM3 cells after CPF treated

747

for 12 or 24 h. A and A´, B and B´, C and C´ represent cell viability and total cell number of GC-1,

748

TM4 and TM3 cells, respectively. Note: the values with different letters (a, b, c and d) differ

749

significantly at P < 0.05 or P < 0.01 level. NS means no significant differences.


Page 34 of 47

Page 35 of 47


750 751

Figure 3. TUNEL staining of GC-1, TM4 and TM3 cell lines treated with different concentrations

752

of CPF. A, B and C were the results of GC-1, TM4 and TM3 cells, respectively. Red: TUNEL

753

positive cells; Blue: nuclear (counterstaining of DNA).



754 755

Figure 4. Flow cytometry was used to detect apoptosis of GC-1, TM4 and TM3 cells after CPF

756

treatment. A and B, C and D, E and F were the results of GC-1, TM4 and TM3 cells, respectively.

757

The data were presented as the percentage of apoptotic cells. Early apoptotic cells were in green

758

square and late apoptotic cells were in red square.


Page 36 of 47

Page 37 of 47


759 760

Figure 5. The qPCR and western blot were used to detect the expression of apoptosis-related genes

761

in CPF treated GC-1 cells. (A) The expression of p53, puma, Caspase 3 and Caspase 9 of GC-1

762

after CPF treatment. (B) Expression of cleaved-Caspase 9 protein were detected in GC-1 cells.

763

Intensity analysis of cleaved-Caspase 9 ratio was calculated by Image J. ** P < 0.01 (C) Expression

764

of BAX and BCL2 protein were detected in GC-1 cells after CPF treated for 24 h. Intensity analysis

765

of BAX/BCL2 ratio was calculated by Image J. Note: the values with different letters (a, b, c and d)

766

differ significantly at P < 0.05 or P < 0.01 level.



767 768

Figure 6. The qPCR and western blot were used to detect the expression of apoptosis-related genes

769

in CPF treated TM4 cells. (A) The expression of p53, puma, Caspase 3 and Caspase 9 of TM4 after

770

CPF treatment. (B) Expression of BAX and BCL2 protein were detected in TM4 cells after CPF

771

treated for 12 h. Note: the values with different letters (a, b, c and d) differ significantly at P < 0.05

772

or P < 0.01 level.


Page 38 of 47

Page 39 of 47


773 774

Figure 7. CPF modifications on cell cycle distribution of GC-1, TM4 and TM3 cells. Cells were

775

exposed to CPF (10, 25 and 50 µM) or vehicle for 12 or 24 h. A and B, C and D, E and F were the

776

results of GC-1, TM4 and TM3 cells, respectively. Cells were stained with propidium iodide (PI),

777

and analyzed for DNA content by flow cytometry. Note: the values with different letters (a, b, c and

778

d) differ significantly at P < 0.05 or P < 0.01 level. NS means no significant differences.



779 780

Figure 8. The qRT-PCR was used to detect the expression of cell cycle regulators in CPF treated

781

GC-1, TM4 and TM3 cells. The expression of p21CIP, CCND1, CCNE1, CCNA2 and CCNB1 were

782

detected. A, B and C were the results of GC-1, TM4 and TM3 cells, respectively. Note: the values

783

with different letters (a, b, c and d) differ significantly at P < 0.05 or P < 0.01 level.


Page 40 of 47

Page 41 of 47


784 785 786

Figure 9. The expression of p-AMPK, mitochondrial membrane potentials, ROS level and MDA

787

level were detected in GC-1 cells after CPF treatment. (A) presented the expression of p-AMPK

788

protein in CPF treated GC-1 cells. (B) Detection of mitochondrial membrane potentials. (C)

789

Detection of MDA level. (D) Flow cytometry was applied to assess oxidative stress after CPF treated.

790

(E) and (F) indicated ROS levels in CPF treated cells compared to control group.



791 792

Figure 10. The expression of p-AMPK and ROS level were detected in TM4 cells after CPF

793

treatment. (A) Western blot analysis of p-AMPK expression in CPF treated TM4 cells. (B) p-AMPK

794

intensity analysis was calculated by Image J.**P

Chlorpyrifos Induced Testicular Cell Apoptosis through Generation of

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