Toxic Effects and Possible Mechanisms of Deoxynivalenol Exposure

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Food Safety and Toxicology

Toxic effects and possible mechanisms of deoxynivalenol exposure on sperm and testicular damages in BALB/c mice Junhua Yang, Jianhua Wang, Wenbo Guo, A-Ru Ling, Ai-Qiong Luo, Dan Liu, Xianli Yang, and Zhihui Zhao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b04783 • Publication Date (Web): 01 Feb 2019 Downloaded from http://pubs.acs.org on February 3, 2019

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Journal of Agricultural and Food Chemistry

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Toxic effects and possible mechanisms of deoxynivalenol exposure on sperm and

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testicular damages in BALB/c mice

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Jun-hua Yang1, Jian-hua Wang1, Wen-bo Guo, A-ru Ling, Ai-qiong Luo, Dan Liu,

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Xian-li Yang*, Zhi-hui ZHAO*

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Institute for Agri-Food Standards and Testing Technology, Shanghai Academy of

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Agricultural Sciences, Shanghai, 201403, People’s Republic of China

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*Co-corresponding author

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E-mail addresses: [email protected] (J.-H. Yang), [email protected]

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(Z.-Z. Zhao)

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Co-first author.

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ABSTRACT

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Deoxynivalenol (DON, vomitoxin) is the most common mycotoxin in cereals

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and grains. DON contamination can cause a serious health threat to humans and farm

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animals. DON has been reported to exert significant toxicity effects on male

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reproductive system. However, the causes and mechanisms underlying efforts of

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DON on sperm and testicular damage remain largely unclear. In the present study, we

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deeply investigated into this issue. Eighty male BALB/c mice were randomly divided

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into control group (n=40) and DON treatment group (2.4 mg/kg bw, n=40). The ratio

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of testes and seminal vesicle to body, sperm survival and motility, and morphology of

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sperm and testis were observed in DON-treated and control mice. In addition, the

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concentrations of reactive oxygen species (ROS) and malondialdehyde (MDA), the

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activities of superoxide dismutase (SOD) and glutathione (GSH), as well as the

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expression levels of JNK/c-Jun signaling and apoptotic factors such as caspase-3,

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caspase-8, caspase-9, Bim and Bid were analyzed and compared between the two

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groups. The results demonstrated that a single topical application of DON

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significantly increased the percentage of abnormal sperm and decreased the motility

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of sperm, indicating the sperms are damaged by DON. Additionally, the reduced

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relative body weight of testis and severe destruction of testicular morphology were

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observed. Moreover, the increased levels of ROS and MDA levels and decreased

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activities of SOD and GSH were found in testicular tissues, suggesting that oxidative

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stress is induced by DON treatment. Furthermore, DON upregulated the expression of

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stress-induced JNK/c-Jun signaling pathway proteins as well as JNK/c-Jun

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phosphorylation proteins. Besides, DON could enhanced testicular apoptosis by

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increasing expression levels of apoptotic genes including Bim, cytochrome c, caspase

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3, caspase 8 and caspase 9. These results suggest that DON exposure can cause sperm

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damage, oxidative stress, testicular apoptosis and phosphorylation of JNK/c-Jun

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signaling pathway. The underlying mechanisms may be that DON induces sperm

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damage by exacerbating oxidative stress-mediated testicular apoptosis via JNK/c-Jun

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signaling pathway.

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KEYWORDS: Deoxynivalenol; Sperm damage; Oxidative stress; JNK/c-Jun

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pathway; Apoptosis; Testis

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INTRODUCTION

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Mycotoxin contamination can occur in different agricultural commodities during

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growth, storage and processing, if the environmental conditions are favorable for

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fungi growth. Deoxynivalenol (DON), also called vomitoxin, a secondary metabolite

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of several fusarium species, is one of the most important mycotoxins in cereal crops,

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and the most common type of contaminant in animal feedstuffs.1 DON exerts toxic

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effects on experimental animals, livestock and humans. Such negative effects can

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growth retardation, intestinal malabsorption, immunosuppression, neurological

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disorder and reproductive system problems.2 Annual economic loss of the livestock

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industries has been estimated as much as several hundred million dollars.3

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The toxic effects of DON on various animal species are mainly through inhibited

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protein synthesis, impaired membrane functions, altered intercellular communication,

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deregulated calcium homeostasis, and induced oxidative stress and enhanced cell

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apoptosis.4-6 The possible mechanism may rely on its binding with 60S ribosomal

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subunit to inhibit RNA transcription and protein synthesis and induce a rapid

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activation of mitogen-activated protein kinase (MAPKs) signaling pathways,

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including p38, c-Jun N-terminal kinase (JNK) and extracellular-signal regulated

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kinase (ERK).7,8 MARKs signaling pathways have been identified to contribute to

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wide variety of intracellular signaling, including proliferation, differentiation, cellular

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stress responses, and apoptosis.9 MAPKs signaling pathways also play a pivotal role

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in the mechanism of DON-induced toxicity. For instance, Bimczok et al.10 have

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reported that DON-induced apoptotic changes in blood-derived REH and Jurkat cells,



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by increasing the phosphorylation levels of JNK and p38 MAPKs, as well as c-Jun

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expression. Yang et al.11 found that the activation of JNK, p38 and ERK pathways by

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DON and other trichothecenes are associated with apoptosis induction.

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An increasing body of studies have suggested that oxidative stress is associated

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with apoptotic pathways.12-18 DON can disrupt the normal function of mitochondria

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and ROS releases, enhance lipid peroxidation, and reduce antioxidant enzyme

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activities in different cell lines.13-16 Gan et al.17 have found that oxidative stress can

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activate MAPKs signaling pathways. Wu et al.18 also reported that MAPKs signaling

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pathways are induced by oxidative stress and subsequently activated the

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caspase-mediated apoptotic pathways.18 Moreover, JNK and c-Jun are involved in the

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apoptotic process induced by T-2 toxin.18 However, no studies have been reported to

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date on DON-induced apoptosis via oxidative stress-mediated JNK/c-Jun signaling

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pathway.

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As a consequence of DON toxicity effects, the fertility rate is noticeably affected.

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Maternally toxic doses induce the embryotoxic effects and teratogenic effects

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(absence and fusion of ribs),19,20 and increase postnatal mortality in mice.19 Alm et

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al.21 and Malekinejad et al.22 have reported that the loss of porcine fertility is

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associated with DON-inhibited oocyte maturation in vitro. Germ cell degeneration,

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sperm retention and abnormal nuclear morphology have been observed in rats treated

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with 2.5 mg/kg bw DON.23 However, there is not much information available on the

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sperm damage in mice exposed to DON, and the mechanisms underlying

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DON-induced sperm damage have yet to be clarified. Therefore, this study aimed to


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investigate the effects of DON on toxicity and apoptosis of testicular tissues during

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spermatogenesis and sperm maturation, and to explore the roles of oxidative

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stress-mediated JNK/c-Jun signaling pathway in sperm damage and testicular cell

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apoptosis.

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MATERIALS AND METHODS

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Preparation of Chemicals.

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Deoxynivalenol was purchased from Sigma-Aldrich Co. LLC, China. Powdered

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of DON with 99.99% purity was dissolved in distilled water. All real-time PCR

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reagents were purchased from TaKaRa Biotechnology Co. Ltd., unless otherwise

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specified. Primary antibodies were obtained from Thermo Fisher Scientific (China)

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Co., Ltd., except where otherwise specified. All other chemicals, if not stated, were

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purchased from Sigma.

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Animals Treatment.

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Six-week-old male BALB/c mice were purchased from the department of

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laboratory animal science of Fudan University (Shanghai, China). Animals were

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maintained in polycarbonate cages containing aspen bedding, nestlets and wire-top

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lids, under standard conditions of temperature (21-24 °C), humidity (40-55%) and

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light cycle (12:12 h light-dark). All the mice were provided with ad libitum access to

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feed and water,

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A total of 80 adult male mice were randomly divided into DON and control

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groups (40 animals in each group). The mice in DON groups were intragastrically

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administered with 0.4 mL DON (2.4 mg/kg body weight) between 2 and 3 p.m. each



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day. Meanwhile, the animals in control group received an equal volume of distilled

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water once daily for 4 weeks. This dosage was selected based on our previous study,

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which was extrapolated from BALB/c mice model exposed to DON at 0.15, 0.6, 2.4

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mg/kg bw. In addition, a previous report has used 2.5 mg/kg bw DON in rat models.23

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All procedures on experimental animals were carried out in accordance with the

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Guidelines of Animal Ethics Committee in Shanghai Academy of Agricultural

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Science.

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Sampling.

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All mice were humanely sacrificed by cervical dislocation after 4 weeks of DON

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treatment. The body weight was measured, and total testicular weight was calculated

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by adding the weight of left testis and right testis after separating epididymides.

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Individual organ index values were calculated as follows:

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Organ index = organ weight / body weight × 100%.

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After weight measurement, testis samples were immediately frozen in liquid

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nitrogen and stored at -80 °C until further analysis. For histopathology, 4 fresh

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samples were independently selected from control and DON treatment groups. These

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samples were fixed in 10% buffered formalin (pH 7.4) and stored at 4 °C for 24-48 h.

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Meanwhile, other parts of fresh testis samples were immediately weighted and

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homogenized for enzyme activity assays.

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Subsequently, dissected cauda epididymides from the both sides of each mouse

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were separated into two groups, and immediately placed in a pre-warmed Petri dish

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(37 °C) containing 5 ml of calcium and magnesium-free Hank’s solution. Tissue


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samples were minced with scalpels for approximately 1 min, followed by incubation

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at 37 °C for 10 min.

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Epididymal Sperm Motility.

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After 10 min of diffusion, the motility and viability of sperm were assessed. One

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drop of sperm suspension was placed on a warmed microscope slide, with 22 mm ×

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22 mm cover slip. Each of at least 10 microscopic fields was observed at 400x

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magnification under a standard optical microscope (Olympus CX41). The parameters

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for sperm motility were the percentage of motile sperm and the percentage of

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progressive motile sperm. The assessment of sperm motility was carried out according

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to the WHO protocol.24-25

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Epididymal Sperm Morphology.

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Following previous observations, a small amount of sperm suspension was

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smeared onto a slide by using a pipette and then fixed with methanol. After drying for

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10 min, the smear was stained with 2% eosin for 1 h. All stained slides were

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examined for morphological abnormalities at 400x magnification under a standard

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optical microscope. Approximately 100-200 spermatozoa were counted for each

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animal, and the percentage of normal sperm cells was calculated. Abnormal sperm

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cells were classified as headless and double heads, amorphous shapes a tail, folded,

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short and double tails, and other aberrations.

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Epididymal Sperm Viability.

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For the assessment of sperm viability, 10 μL of sperm suspension was placed on

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a warmed microscope slide. Then, 10 μL of eosin-nigrosin staining solution was



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added and covered with a cover slip. All samples were observed under an optical

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microscope at 400x magnification. At least 200 sperm cells were counted for each

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animal, by discriminating live sperm (not stained) from death sperm (red stained).

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Histopathology of Testes.

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Testicular tissue samples were embedded in paraffin, sectioned at 5 µm thickness,

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deparaffinized with xylene and then stained by hematoxylin and eosin (HE) via

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standard histopathological procedures. The mounted slides were examined under a

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microscope, and the images were recorded by using Nikon Eclipse 5i (H5505, Nikon,

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Japan) microscope equipped with Digital Sight Camera System (DS-Fi1 Nikon,

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Japan).

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Antioxidant Status in Testicular Tissues.

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After thawing in 0.9% physiological saline (1:20, W/V), fresh testis samples

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were mechanically homogenized by a tissue grinder (Polytron, Polytron PT1200E;

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Brinkman Instruments, Littau, Switzerland). Subsequently, the homogenate was

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centrifuged at 12,000 g for 5 min at 4 °C to remove insoluble materials. After

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centrifugation, the supernatant was collected, and the protein concentration was

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determined using Easy II Protein Quantitative Kit (DQ111-01, Transgen Biotech,

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China). Bovine serum albumin was used as a protein standard.

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The concentrations of reactive oxygen species (ROS) and malondialdehyde

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(MDA), and the activities of superoxide dismutase (SOD) and glutathione peroxidase

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(GSH-Px) were measured using assay kits (Jiancheng, Nanjing, China). The levels of

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enzyme activities were calculated according to the manufacturer's instructions.


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RNA Extraction and Real-time Quantitative PCR.

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Total RNA were extracted from testis tissues by RNAiso Plus (Takara

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Biotechnology (Dalian, China) Co., Ltd.) according to the manufacturer’s instructions.

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cDNAs for each RNA sample were prepared using PrimeScriptTM RT Master Mix.

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The mRNA expression levels of caspase 3, caspase 8, caspase 9, Bim and Bid

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were quantified using ABI-prism 7500 Sequence Detection System (Applied

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Biosystems, Inc., Foster City, CA) and normalized to β-actin reference gene. As

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shown in Table 1, the primers sequences for caspase 3, caspase 8, caspase 9, Bim, Bid

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and β-actin were designed using Prime Premier 5.0, and then synthesized by

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Invitrogen Biotechnology Ltd. (Shanghai, China). Table 1. Primer sequences for real-time quantitative PCR

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Target

GenBank

PCR Products Primer Sequences

Genes

Accession

(bp) F: 5’-gttcctcctgtcgtctttattgctc-3’

caspase 3

NM_009810.2

131 R: 5’-aagggactggatgaaccacgac-3’ F: 5’-agatcctgtgaatggaacctggtat-3’

caspase 8

AF067834.1

136 R: 5’-gttcctcctgtcgtctttattgctc-3’ F: 5’-accgtgccctggactgcttt-3’

caspase 9

NM_015733.4

144 R: 5’-agccgctcccgttgaagata-3 F: 5’-gagatacggattgcacaggagc-3’

Bim

AF032459.1

155 R: 5’-ctccataccagacggaagataaagc-3’

Bid

NM_007544.3

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F: 5’-ccaaagcccttgatgaggtga-3’



R: 5’-gcaaagatggtgcgtgactgg-3’ F: 5’-gctacagcttcaccaccaca-3’ β-actin

M12481.1

208 R: 5’-aagggactggatgaaccacgac-3’

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Real-time quantitative PCR reaction was carried out in 20 μl reaction mixtures,

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containing 10 μL SYBR Premix Ex Taq (2×), 0.4 μL ROX Reference Dye (50×), 0.8

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μM of each forward and reverse primers, cDNA aliquots and nuclease-free water.

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Real-time PCR amplification was performed with an initial denaturation step at 95 °C

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for 30 s, followed by 40 cycles of 5 s at 95 °C and 31 s at 60-64 °C. The 2−ΔΔCt

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method was used to determine the relative expression of each gene compared to a

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reference gene.26 Mock RT and no template controls were included to monitor

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possible genomic DNA contamination. All samples were amplified in a single PCR

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run, and each amplications was repeated at least three times.

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Protein Extraction and Western Blotting.

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Quick-frozen testis samples (100 mg) were mechanically homogenized by a

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tissue grinder, after thawing in ice-cold ProteinExtTM Mammalian Total Protein

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Extraction Kit (DE 101-01, Transgen Biotech, China). After 30 min incubation on ice,

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the homogenate was centrifuged at 12,000 g for 5 min at 4°C to remove all insoluble

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materials. The supernatants were collected for protein determination using Easy II

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Protein Quantitative Kit. Bovine serum albumin was used as a protein standard.

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A total of 40 µg proteins were mixed with loading buffer and separated in 10%

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sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The


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separated proteins were transferred onto nitrocellulose membranes at 100 V for 50

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min by using a tank blotting system (Bio-Rad, Hercules, CA, USA). Membranes were

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blocked with 5% skim milk in TBS for 1 h at room temperature, and then probed

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overnight (4 °C) with either mouse anti-JNK1/JNK2 (AHO1362; Invitrogen; 1:100),

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mouse anti-c-Jun (702170; Invitrogen; 1:150), β-actin antibody (MA5-15739;

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Invitrogen; 1:2000), rabbit anti-phospho-JNK1/JNK2 (44-682G; Invitrogen; 1:250),

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rabbit anti-phospho-c-Jun (44-292G; Invitrogen; 1:500), mouse anti-cytochrome c

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(33-8500; Invitrogen; 1:600), rabbit anti-caspase 3 (ab49822; Abcam; 1:500), rabbit

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anti-caspase 8 (ab25901; Abcam; 1:400), rabbit anti-caspase 9 (ab25758; Abcam;

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1:400). After that, the blots were incubated with HRP-conjugated goat anti-rabbit IgG

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(GGHL-15P; Immunology Consultants Laboratory, Inc., USA; 1:5000) or

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HRP-conjugated goat anti-mouse IgG (BS50350; Bioworld Technology Co., Ltd.,

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Louis Park, MN, USA; 1:10 000) for 1 h at room temperature. Finally, positive bands

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were detected with enhanced chemiluminescence (ECL). The ECL signals recorded

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on X-ray film were scanned and analyzed with Kodak 1D Electrophoresis

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Documentation and Analysis System 120 (Kodak Photo Film Co. Ltd., USA) or

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ChemiDocTM Touch Imaging System with Image LabTM Touch Software (Bio-Rad

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Laboratories). The band density of each protein was normalized to β-actin

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(housekeeping protein).

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Statistical Analysis.

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All statistical analyses were performed using SPSS 16.0 for Windows (SPSS Inc., Chicago, IL, USA). The values for mRNA expression and protein levels were



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presented as fold-change relative to control group. All data were expressed as mean ±

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standard error of mean (SEM). Randomization test was used to compare the

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differences between two groups. P values of less than 0.05 were considered

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statistically significant.

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RESULTS

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DON Induced Sperm Malformation and Decreased Sperm Qualities.

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As shown in Figure 1, the sperm morphology of DON-treated mice revealed an

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increase of double-head, double-tail and fracture. The viability, motility and

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progressive motility of mouse sperm were significantly reduced (P

Toxic Effects and Possible Mechanisms of Deoxynivalenol Exposure

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