Histone acetylation plays an important role in MC-LR-induced apoptosis and cycle disorder in SD rat testicular cells

Journal Pre-proof Histone acetylation plays an important role in MC-LR-induced apoptosis and cycle disorder in SD rat testicular cells Yueqin Wang, Haohao Liu, Xiaohui Liu, Xiaofeng Zhang, Jinxia Wu, Le Yuan, Xingde Du, Rui Wang, Ya Ma, Xinghai Chen, Xuemin Cheng, Donggang Zhuang, Huizhen Zhang PII:

S0045-6535(19)32312-4

DOI:

https://doi.org/10.1016/j.chemosphere.2019.125073

Reference:

CHEM 125073

To appear in:

ECSN

Received Date: 1 June 2019 Revised Date:

4 October 2019

Accepted Date: 7 October 2019

Please cite this article as: Wang, Y., Liu, H., Liu, X., Zhang, X., Wu, J., Yuan, L., Du, X., Wang, R., Ma, Y., Chen, X., Cheng, X., Zhuang, D., Zhang, H., Histone acetylation plays an important role in MCLR-induced apoptosis and cycle disorder in SD rat testicular cells, Chemosphere (2019), doi: https:// doi.org/10.1016/j.chemosphere.2019.125073. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

Credit Author Statement: Yueqin Wang: Conceptualization, Methodology, Investigation, Writing-Original Draft Preparation, Visualization. Haohao Liu: Methodology, Writing-Review & Editing, Visualization. Xiaohui Liu: Validation. Xiaofeng Zhang: Software. Jinxia Wu: Data Curation. Le Yuan: Software. Xingde Du: Data Curation. Rui Wang: Software. Ya Ma: Software. Xinghai Chen: Writing-Revise. Xuemin Cheng: Data Curation. Donggang Zhuang: Data Curation. Huizhen Zhang: Conceptualization, Supervision, Funding Acquisition.

1

Histone acetylation plays an important role in MC-LR-induced

2

apoptosis and cycle disorder in SD rat testicular cells

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Yueqin Wang1, Haohao Liu1, Xiaohui Liu2, Xiaofeng Zhang1, Jinxia Wu1, Le Yuan1,

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Xingde Du1, Rui Wang1, Ya Ma1, Xinghai Chen3, Xuemin Cheng1, Donggang

5

Zhuang1, Huizhen Zhang1*

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1

College of Public Health, Zhengzhou University, Zhengzhou, 450001, China

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2

School of Basic Medical Sciences, Henan University of Chinese Medicine,

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Zhengzhou, Henan, 450046, China

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3

Department of Chemistry and Biochemistry, St Mary's University, San Antonio, TX,

10

USA

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Corresponding author:

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Email: [email protected]

13 14 15 16 17 18 19 20 21 22 23 24 25 1

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Abstract

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Microcystin-leucine arginine (MC-LR) is a variant of microcystins (MCs),

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which poses a serious threat to the reproductive system. Histone acetylation

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modification can regulate the expression of apoptosis-related genes. However the

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mechanisms of histone acetylation involving MC-LR-induced apoptosis were not

31

understood. This study investigated the change of histone acetylation and its role in

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apoptosis and cell cycle arrest induced by MC-LR. MC-LR enhanced the activity of

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histone deacetylase (HDAC), decreased the activity of histone acetylase (HAT),

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up-regulated the expression of HDAC1, and down-regulated the expressions of Ac-H3

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and Ac-H4 in vitro and vivo. Meanwhile, MC-LR induced testicular tissue injury and

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increased the expressions of apoptosis-related genes, such as Bax, Caspase3 and

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Caspase8, ultimately causing cells apoptosis in testicular tissues. Furthermore,

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MC-LR also induced cell cycle arrest in S phase, increased the expression of

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P21Wif1/Cip1, and inhibited the expression of cyclinD1, cyclinE1, CDK2 and E2F1.

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Importantly, HDAC inhibitor Trichostatin A (TSA) could ameliorate MC-LR-induced

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apoptosis and cell cycle arrest by reverse-regulating the expression of these proteins.

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These results indicated that MC-LR could activate the mitochondrial apoptotic

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pathway and disorder the cell cycle pathway to induce the cell apoptosis by enhancing

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HDAC activity and reducing histone acetylation of normal testicular cells in SD rats.

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Hence, histone acetylation has a vital function in MC-LR-induced apoptosis in SD rat

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testicular cells, which provides a new insight on the reproductive toxicity of male

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induced by MC-LR. 2

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Keywords: Microcystin-leucine arginine (MC-LR); Trichostatin A (TSA); Histone

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deacetylase (HDAC); Histone acetylation; Apoptosis; Cell cycle arrest

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1. Introduction

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Microcystins (MCs) are a class of natural cyclic heptapeptide toxins secreted by

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freshwater cyanobacteria (Apeldoorn et al., 2007). Microcystin-leucine arginine

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(MC-LR) is the most toxic isomer among more than 100 MC isomers (Hoeger et al.,

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2005; Puddick et al., 2014). The World Health Organization (WHO) stipulated that the

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limit of MC-LR in drinking water is 1 µg/L (Dietrich and Hoeger, 2005). However,

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the concentration of MCs in natural water is often much higher (Duong et al., 2013;

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Zhang et al., 2015). Water eutrophication induced the increase of cyanobacterial

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blooms, which may be associated with a risk of different types of cancers in humans

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(Merel et al., 2013). In addition, the chemical properties of MCs are relatively stable

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and their natural degradation process is very slow, representing potential health

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hazards for humans (Buratti et al., 2017). MC-LR could accumulate and produce toxic

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effects in tissues such as liver, gonads, kidney, muscles, etc. (Funari and Testai, 2008).

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After liver, testis is the second most important target organ of MC-LR toxicity.

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Epigenetic modification is a common mechanism for regulating transcription

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levels, including DNA methylation, histone modification and non-coding RNA

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(Stillman, 2018), and has crucial effects on cell cycle regulation, prolonging cell life

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and inhibiting programmed cell death (Probst et al., 2009; Gao et al., 2016). Histone

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modifications include acetylation, phosphorylation, ubiquitination, methylation, and

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ribosylation (Turner, 2000; Nathan et al., 2003; Zhang, 2003). Histone acetylation has 3

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become a target site for many signaling pathways by affecting the interaction of DNA

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with histones and other DNA-binding proteins (Bannister and Kouzarides, 2011).

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Epigenetic changes regulate the expression of apoptosis-related genes, which may

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play a vital role in MC-LR-induced testicular cell apoptosis. However, the changes of

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histone acetylation and epigenetic mechanisms in MC-LR-induced apoptosis are not

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well elucidated.

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The acetylation of histones is regulated by histone acetylase (HAT) and histone

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deacetylase (HDAC) (Lei et al., 2010). HDAC, a key regulatory enzyme, induces

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histone deacetylation, which plays an important role in epigenetic regulation and

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regulation of transcription factors. Additionally, reducing the level of histone

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acetylation could inhibit gene transcription and exacerbate the uptake of toxicants by

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dopamine neurons, leading to neuronal cell death (Troke et al., 2006). Inactivation of

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HDAC2 could induce human lung cancer cell apoptosis (Jung et al., 2012) and

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cellular senescence (Harms and Chen, 2007) through activation of p53 and Bax.

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MC-LR induced apoptosis in rat spermatogonia (Zhou et al., 2012) and HDAC1 was

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also closely related to apoptosis (Kim, 2007). Our previous study showed that MC-LR

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could up-regulate the acetylation of p53 and Ku70, which induced apoptosis in

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co-cultured Sertoli–germ cells (Liu et al., 2018a). However, the role of class I HDACs

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in controlling apoptosis of germ cells induced by MC-LR is not well understood.

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MC-LR also increased the expressions of apoptosis-related mRNAs and proteins to

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induce apoptosis in Sertoli cells (Zhang et al., 2011). Previous studies showed that

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MC-LR could activate the mitochondrial apoptotic pathway through oxidative stress, 4

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inducing reproductive toxicity (Chen et al., 2013; Lone et al., 2015; Liu et al., 2018b).

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However, whether MC-LR-caused disorder of histone acetylation participated in

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MC-LR-induced cells apoptosis through the mitochondrial apoptotic pathway remains

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unclear.

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HDAC, as an important component of nucleosomes, could directly participate

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in the regulation of cell cycle (Lee et al., 2012). Silencing HDAC2 by siRNA

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increased the expression of p21 WAF1/Cip1 and apoptosis in cervical cancer cells (Huang

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et al., 2005). HDACs also affected the expression of cell cycle-associated proteins

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Cyclins and Cyclin-dependent kinases (CDKs), activated the Caspase cascade and

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up-regulated the expression of the pro-apoptotic protein family to induce apoptosis in

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multiple myeloma cells (Tandon et al., 2016). Additionally, HDACs could interact

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with a variety of transcriptional regulatory factors, such as p21WAF/Cip1, Stat3, E2F,

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retinoblastoma (Rb) etc., which participate in the cell cycle regulation resulting in

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apoptosis (Lee et al., 2012). A previous study demonstrated that MC-LR induced cell

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cycle arrest of Chinese hamster ovary cells in G2/M phase (Li et al., 2014). However,

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the effect of MC-LR on the cell cycle of testicular cells in SD rats and its molecular

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mechanism are still confusing.

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Trichostatin A (TSA) effectively inhibits the total activity of HDACs and

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increases the level of protein acetylation. The increase of histone acetylation caused

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by HDAC inhibitors promoted the activation of potential cell survival factors and the

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anti-apoptotic protein Bcl-2 family (Ying et al., 2010). Histone acetylation is closely

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related to apoptosis and cell cycle, but no studies have been published on possible 5

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histone acetylation involvement in MC-LR-induced apoptosis by activating the

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mitochondrial apoptotic pathway and disrupting cell cycle pathway. In this study, the

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effects of MC-LR on HAT activity and histone acetylation level in testicular cells of

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SD rats will be examined. Moreover, cell apoptosis and cell cycle-related factors in

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SD rat testes or in co-cultured SD rat Sertoli-germ cells will be evaluated after

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treatment of MC-LR with or without TSA. This study will reveal the mechanism of

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histone acetylation modification in SD rat testicular cell toxicity induced by MC-LR.

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2. Materials and methods

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2.1. Reagents

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MC-LR (purity >96%) was purchased from Beijing Express Technology Co.

124

(Beijing, China), which was dissolved in PBS to 1 mM as the solution and stored at

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-20 ℃. TSA was obtained from InvivoGen (San Diego, USA), which was dissolved in

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dimethyl sulfoxide (DMSO) (Sigma-Aldrich, USA) and then diluted in saline to the

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desired concentration (the final DMSO concentration was <0.1%). NE-PER Nuclear

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and Cytoplasmic Extraction Reagents were obtained from Thermo scientific

129

(Massachusetts, USA). EpiQuik™ HAT/HDAC Activity/Inhibition Assay Kits were

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purchased from GepiGentek (New York, USA). Cell Cycle detection kit was

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purchased from Keygen Biotech (Nanjing, China). Apoptosis kit was bought from

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Beyotime (Shanghai, China). Anti-acetyl-Histone H4 (Cat. # 06-598) and

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Anti-acetyl-Histone H3 (Cat. # 06-599) were obtained from Millipore (Massachusetts,

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USA). Anti-HAT (SAB4503405) was obtained from Sigma (Alexandria, USA).

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Anti-CDK2 Rabbit pAb (GB13237), Anti-CyclinD1 Rabbit pAb (GB11079) and 6

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Anti-CyclinE1 Rabbit pAb (GB13305) were obtained from Servicebio (Wuhan,

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China). P21wif1/Cip1 Antibody (AF6290), CDK4 Antibody (DF6102), E2F1 Antibody

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(DF6797) and Rb Antibody (AF7698) were obtained from Affinity (OH, USA). Bax

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Antibody (ab32503),

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Anti-Caspase-3 (ab2302), Anti-HDAC1 (ab19845), Anti-HDAC2 (ab16032),

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Anti-HDAC3 (ab32369) and β-actin Antibody (ab6276) were obtained from Abcam

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Company (Cambridge, UK).

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2.2. Isolation and treatment of co-cultured Sertoli-germ cells

Bcl-2

Antibody (ab7973),

Anti-Caspase-8

(ab25901),

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Co-cultured Sertoli-germ cells were performed as previously reported (Liu et al.,

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2018a). Briefly, both sides of the testis were taken out from the specific pathogen free

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(SPF) male Sprague-Dawley (SD) rats (18-20 days), washed with PBS, cut into pieces,

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and then digested with 0.25% trypsin in an incubator at 37 °C for 30 min. Next, the

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testicular fragments were further digested with collagenase (0.1%) at 37 °C for

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another 30 min. A stainless steel filter was used to filter the homogenate. Then, cells

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were collected and washed twice by PBS. After centrifugation for 5 min at 1000 r/min,

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DMEM/F-12 medium supplemented with 10% fetal bovine serum (FBS) were used to

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resuspend cells, which were cultured in an incubator at 37 °C. The 50% inhibitive

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concentration (IC50) of MC-LR for Sertoli–germ cells was 36 µg/mL (36 µg/mL≈36

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µM) according to our previous study (Liu et al., 2018a). The cells were separately

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treated with different MC-LR concentrations with or without TSA (0, 1/4IC50, 1/2IC50,

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IC50, IC50+TSA, TSA). Cells were pretreated with TSA 2 h before MC-LR in the

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IC50+TSA group. 7

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2.3. Cell viability assay

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Cell Counting Kit-8 (CCK8) (Dojindo Laboratories, Kumamoto, Japan) was

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used to detect the viability of co-cultured Sertoli-germ cells treated with TSA. When

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the density of cells reached 80-90% in the 96-well plate, the fresh medium containing

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TSA (0-1.060 µM) was used to culture cells for 24 h. Then, the CCK-8 solution was

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added to each well to analyze the viability of cells according to manufacturer’s

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instructions.

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2.4. Cell cycle analysis

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The co-culture of Sertoli-germ cells were plated in the 6-well plates and were

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divided into four groups. When the cells density in each well reached up to 80-90%,

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the cells were incubated in MC-LR solution (36 µM) with or without TSA (0.132 µM)

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for 24 h. The cells were digested with trypsin free of EDTA, washed by PBS and

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fixed at 4 °C with 70% ethanol overnight. Then, cells were kept away from light at

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37 °C for 30 min after propidium iodide (PI) staining solution and RNaseA stock

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solution were added. In the end, the cell cycle analysis was performed with BD

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AccuriTM C6 flow cytometer (BD Biosciences, San Jose, USA).

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2.5. The extraction of nuclear proteins from tissues and cells

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In vitro, each group had three independent experiments for repetition.

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Trypsin-EDTA was used to harvest the adherent cells about 2×106 cells per well, and

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the supernatant was discarded after centrifugation at 500 g for 3 min. The cell

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precipitation was added to 200 µL of cold CERI and shaken by a high-speed vortex

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for 15 s to fully suspend the cells. Then, the samples were placed on ice for 10 min 8

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and 11 µL cold CERII was added in each tube. Next, the tube was shaken by a

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high-speed vortex for 5 s and incubated on ice for 1 min. The tube was shaken by a

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high-speed vortex for 5 s and then centrifuged at 13500 g for 10 min, the supernatant

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was removed and 50 µL cold NER was added to each tube. After shaking by a

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high-speed vortex for 5 s, the tubes were centrifuged at 13500 g for 10 min. The

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samples were incubated on ice for 10 min after shaking by a high-speed vortex for 15

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s, which was repeated three times. After the last centrifugation, the supernatant

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(nuclear extract) was transferred to a new pre-chilled EP tube and stored at -80 ℃ for

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later use.

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In vivo, 3 parallel testicular tissue samples were from three rats for repetition in

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each group, 100 mg of tissue from each sample was placed in a tube with 1mL CER I,

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and then the tissue was homogenized with a homogenizer (Servicebio, Wuhan, China).

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The extraction of tissue nucleoprotein was performed as described above for the

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extraction method of cell nucleoprotein. The volume ratio of CER I: CER II: NER

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reagents at 1000:55:500 µL.

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2.6. Animal treatment

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Male SD rats were purchased from the Experimental Animal Center of Henan

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Province (Zhengzhou, China), and they were provided with water ad libitum and kept

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on a 12-h light/dark cycle at the barrier environment animal laboratory in college of

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public health in Zhengzhou University (license number: SCXK (YU) 2018-0005).

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The rats were randomly divided into four groups (each group had 6 rats) and injected

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intraperitoneally (i.p.) daily with MC-LR or TSA for 14 d: control group (normal 9

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saline), TSA (1 mg kg-1) group, TSA (1 mg kg-1) + MC-LR (40 µg kg-1) group and

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MC-LR (40 µg kg-1) group.

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The dose of MC-LR (40 µg kg-1) was chose according our previous study (Liu et

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al., 2018a). TSA (1 mg kg-1) was selected as treatment dosage according to previously

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published reports (Hyeon Ju Kim et al., 2007). Rats were injected with TSA 2 h

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before MC-LR in TSA+MC-LR group. When the last injection was completed, the

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testes of rats were excised and stored at -80 °C for later experiments.

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2.7. TUNEL assay

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Cells apoptosis of testes were tested by the terminal deoxynucleotidyl transferase

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dUTP nick end labeling (TUNEL) (Roche, Switzerland) (Liu et al., 2018a). Briefly, in

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each group, three testes from three different rats were immersed in 4%

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paraformaldehyde to be fixed for 24 h, blocked with 3% H2O2 dissolved in methanol

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for 10 minutes and immersed in 0.1% Triton X-100 to be permeabilized for 2 minutes

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at room temperature. Then, each section was added a terminal deoxynucleotidyl

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transferase (TdT)-labeled nucleotide mixture and incubated at 37 °C for 1 hour to

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carry out a labeling reaction. Three slices taken from each testis were observed using

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the fluorescent microscope (Olympus, Tokyo, Japan). The total cells and the positive

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cells would be distinguished by using Image-Pro Plus 6.0 (Media Cybernetics, Inc.,

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Rockville, MD, USA). The apoptosis rate (%) was expressed as the percentage of

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positive cells (number of positive cells/total number of cells of 100).

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2.8. Hematoxylin and Eosin staining

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The fresh testes were washed with cold PBS, fixed in 4% paraformaldehyde 10

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overnight, equilibrated by 30% phosphate-buffered sucrose solution and embedded in

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paraffin. And then the testes were cut into 6-µm sections. The xylene and 100%

226

alcohol were used to dehydrate the sections. Finally, histopathological observation

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was performed by hematoxylin and eosin (H&E) staining, and the morphological

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changes of the testes were observed with microscopy (Nikon Eclipse E100, Tokyo,

229

Japan).

230

2.9. HAT/HDAC activity assay

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The EpiQuik™ HAT/HDAC activity/inhibition assay kits (EpiGentek, NY, USA)

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were used to detect the activities of total HAT or HDAC. Nucleoprotein was extracted

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from co-cultured SD rat Sertoli-germ cells or the testicular tissue of SD rats. Nucleo

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proteins (10 µg) were added to 96-well ELISA plates. The OD values were read using

235

a plate reader (BioTek, Winooski, VT, USA), and the activity of HAT or HDAC was

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expressed as optical density OD/min/mg protein.

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2.10. Western blotting

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Total protein samples or nucleoprotein samples were isolated from the testes or

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co-cultured Sertoli-germ cells. BCA Protein Assay Kit (Beyotime, Shanghai, China)

240

was used to detect the concentration of protein. Protein (30 µg per sample) was

241

electrophoresed and transferred to polyvinylidene fluoride (PVDF) membranes. TBST

242

containing 5% BSA was used to block the membranes for 2 h at room temperature,

243

and primary antibody was used for immunoblotting. Finally, the membranes were

244

treated with HRP-coupled secondary antibodies for 90 min. The enhanced

245

chemiluminescence detection kit (Beijing ComWin Biotech, Beijing, China) was used 11

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to detect the protein bands. The Bio-Rad Quantity One software (Bio-Rad, Hercules,

247

CA, United States) was used to analyze the intensity of the bands. All values were

248

quantified with Quantity One, and all proteins were normalized to that of β-actin.

249

Then, the data from treatment groups were normalized to the mean of control group.

250

Data were represented as mean ± standard deviation (SD, n=3) for each group.

251

2.11. Real time-quantitative PCR

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RNA was extracted from testicular tissue or co-cultured Sertoli-germ cells of

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SD rats using TriZol reagent (TaKaRa, Dalian, China). RevertAid first Strand cDNA

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Synthesis kit (TaKaRa, Dalian, China) was used to synthesize of cDNA. SYBR

255

premix Ex Taq II (TaKaRa, Dalian, China) was used to perform Real

256

Time-quantitative PCR (RT-qPCR) on a QuantStudio 7 Flex real time PCR system

257

(Life Technologies, California, USA). Three samples from each group were assayed.

258

The primer sequences of PCR are presented in Table 1. The volume of RT-qPCR

259

reaction was 10 µL and β-actin was used as the endogenous reference gene. The fold

260

change was determined using the 2−∆∆Ct method.

261

262

263

264

265

12

Table1. The sequences of primers used in Real Time-quantitative PCR

266

Genes

267

Forward primers(5′-3′)

Reverse primers (5′-3′)

β-actin

GCCATGTACGTAGCCATCCA

GAACCGCTCATTGCCGATAG

HDAC1

ATGAAGCCTCACCGAATCCGAATG

CTTGGTCATCTCCTCAGCGTTGG

HDAC2

AGCGACATTCCTACGACCTCCTTC

GGATGACCCTGGCCGTAATAATAA

HDAC3

CTGAACCATGCACCCAGTGTC

CGGCATCCATGCTGCTCTTA

Bax

CCAGGACGCATCCACCAAGAAG

GCTGCCACACGGAAGAAGACC

Bcl-2

ACGGTGGTGGAGGAACTCTTCAG

GGTGTGCAGATGCCGGTTCAG

Caspase3 GTACAGAGCTGGACTGCGGTATTG

AGTCGGCCTCCACTGGTATCTTC

Caspase8 CGACGATTACGAACGATCAAGCAC

TCTCTTGCTCTCCTGGCGAGTC

PP21wif1/cip1

TCCTGGTGATGTCCGACCTGTTC

GCGGCTCAACTGCTCACTGTC

Rb

GCCTCCTACCTTGTCACCAATACC

ATGTTACCTCCAGGAATCCGCAAG

CyclinE1 CCGACCTCTCAGTCCGATCCAG

TCCACGCACGCTGAATCATCATC

CyclinD1 CCTGACTGCCGAGAAGTTGTGC

CTGCTTGTTCTCATCCGCCTCTG

CDK2

TCCGGCTCGACACTGAGACTG

ATCCAGCAGCTTGACGATGTTAGG

CDK4

GCCTGTGGTTGTTACGCTCTGG

CTGCGAAGATACAGCCAACACTCC

E2F1

CTCGCAGATCGTCATCATCTCCAC

CGAAGAGCAGCACGTCAGGATC

2.12. Statistical Analysis

268

The SPSS 21.0 statistical software package (Armonk, NY, USA, 2012) was used

269

for statistical analysis. Mean and standard deviation (SD) were used to evaluate the

270

data. First, the normality of data was tested by Kolmogorov-Smirnow test and 13

271

Homogeneity of variance test was carried by Levene test. When the variances showed

272

homoscedasticity, One-way ANOVA was applied to analyze the statistical differences

273

among groups, and then the Student-NewmanKeuls (SNK) test was performed to test

274

the statistical significance. When the variances were not uniform, Games-Howell test

275

was used to test the statistical significance. When the dependent variables were

276

non-normal distribution, Kruskal-Wallis was used for data analysis. P<0.05 indicated

277

statistically significant.

278

3. Results

279

3.1. The effect of histone acetylation transition in MC-LR-induced cell cycle arrest in

280

SD rat co-cultured Sertoli-germ cells.

281

To explore the effect of MC-LR on histone acetylation regulatory enzymes, the

282

co-cultured SD rats Sertoli-germ cells were exposed to MC-LR for 24 h, and

283

nucleoprotein was extracted to detect the effect of MC-LR on the level of histone

284

deacetylation. As shown in Fig 1A and 1B, the activity of HDAC was increased after

285

cells were exposed to 18 µM and 36 µM MC-LR respectively and the activity of HAT

286

was significantly decreased in the MC-LR treatment group (9, 18, 36 µM). The results

287

of the RT-qPCR (Fig 1C) showed that the mRNA expression of HDAC1, HDAC2, and

288

HDAC3 increased with the increase of MC-LR concentration. Western blotting results

289

(Fig 1D and 1E) indicated that the protein expression of HDAC1 was significantly

290

increased in MC-LR treatment group (9, 18, 36 µM). HDAC2 and HDAC3 were only

291

significantly decreased in MC-LR (36 µM) treatment group. Taken together, these

14

292

results indicated that MC-LR enhanced the activity of HDACs and decreased the

293

activity of HAT. Consequently, the HDAC inhibitor TSA was used to explore the role

294

of epigenetic mechanisms in MC-LR-induced SD rat testicular cells apoptosis.

295

Cell Counting Kit-8 (CCK8) was used to detect the cell viability of TSA on

296

co-cultured Sertoli-germ cells. As shown in Fig 1F, TSA (1-0.066 µM) slightly

297

increased the viability of cells (P>0.05). When the concentration reached 0.132 µM,

298

cell viability decreased slightly, but the changes were not statistically significant

299

(P>0.05). The activity of cells declined significantly when the concentration of TSA

300

ranged from 0.265 µM to 1.060 µM (P<0.05). Hence, 0.132 µM of TSA was selected

301

for subsequent experiments. Then, activities of HAT and HDAC, relative proteins and

302

mRNAs levels were detected after cells were treated to MC-LR with or without TSA.

303

As shown in Fig 1G and 1H, MC-LR increased the activity of HDAC and decreased

304

the activity of HAT. Furthermore, TSA pretreatment decreased the MC-LR-induced

305

the increase of HDAC activity and indirectly increased the activity of HAT, which

306

indicated that TSA could act as an effective inhibitor to decrease the induction effect

307

of MC-LR on the HDAC activity. The RT-qPCR results showed that MC-LR

308

enhanced the mRNA expression of HDAC1, HDAC2 and HDAC3. When cells were

309

pretreated with TSA, the induction effects of MC-LR on the expression HDAC1,

310

HDAC2 and HDAC3 were dramatically alleviated (Fig 1I). The results of western

311

blotting showed that MC-LR increased the expression of HDAC1 and reduced the

312

histone acetylation of H3 and H4 level (Fig 1J and 1K). Furthermore, compared to 36

313

µM MC-LR group, TSA could rescue MC-LR-induced decrease of histone acetylation. 15

314

The expressions of HDAC2 and HDAC3 were decreased in the cell groups pretreated

315

with MC-LR and TSA. Fig 1L and 1M showed that MC-LR could inhibit the cell

316

cycle of SD rats in S phase, and TSA pretreatment could decrease the

317

MC-LR-induced arrest.

318

16

319

320 321

Fig1. The effect of histone acetylation transition in MC-LR-induced cell cycle 17

322

arrest in SD rat co-cultured Sertoli-germ cells. (A-E) The effect of MC-LR on

323

histone acetylation transition in co-cultured Sertoli-germ cells. (A) The activity of

324

HDAC, (B) the activity of HAT, (C) the expression of mRNA (HDAC1, HDAC2 and

325

HDAC3) and (D) the expression of proteins (HDAC1, HDAC2, HDAC3), (E) the

326

quantitative analysis of the proteins (HDAC1, HDAC2, HDAC3) were detected in

327

co-cultured Sertoli-germ cells treated by MC-LR. (F) The effect of TSA on the

328

activity of co-cultured Sertoli-germ cells. (G) The activity of HDAC, (H) the activity

329

of HAT, (I) the expression of mRNA (HDAC1, HDAC2, HDAC3), (J) the protein

330

expression of histone acetylation and its regulatory enzyme, (K) quantitative analysis

331

of the protein and enzyme experiments, (L) cell cycle analysis, (M) quantitative

332

analysis of cell cycle experiments were performed on the co-cultured Sertoli-germ

333

cells treated by MC-LR with or without TSA. All genes and proteins were normalized

334

to that of β-actin. All values were quantified with Quantity One, and data were

335

represented as mean ± standard deviation (SD, n = 3) for each group. (*P<0.05 vs. the

336

control group; # P<0.05 vs. the 36 µM MC-LR group)

337

3.2 Histone acetylation participated in MC-LR-induced testicular tissue injury and

338

apoptosis in SD rats

339

SD rats were treated daily with MC-LR with or without TSA by i.p.

340

administration for 14 d. As shown in Fig 2A and 2B, MC-LR could significantly

341

increase the activity of HDAC and decrease the activity of HAT. Furthermore, TSA

342

pretreatment decreased the activity of HDAC and indirectly increased the activity of

343

HAT, when compared to the MC-LR (40 µg kg-1) group. However, the expressions of 18

344

HDAC1, HDAC2 and HDAC3 were not changed in rats exposed to MC-LR with or

345

without TSA (Fig 2C). Next, the proteins expressions of HDAC1, HDAC2, HDAC3,

346

HAT, Ac-H3 and Ac-H4 were examined via western blotting (Fig 2D and 2E), the

347

results showed that MC-LR increased the expression of HDAC1, decreased the

348

expression of HAT, Ac-H3 and Ac-H4, when compared to the control group.

349

Furthermore, TSA pretreatment decreased MC-LR-increased expression of HDAC1

350

and increased MC-LR-decreased expression of HAT, Ac-H3 and Ac-H4 (P<0.05).

351

However, the expressions of HDAC2 and HDAC3 were not different among groups.

352

The testicular histomorphology was performed using hematoxylin and eosin

353

(H&E) staining. As shown in Fig 2G, in MC-LR (40 µg kg-1) group, the

354

spermatogenic cells arranged in a disordered manner, and sperm cells were

355

agglomerated into the lumen of the seminiferous tubules (black arrow). Thin

356

spermatogenic epithelium and decreased spermatogenic cells were observed (red

357

arrow). TSA was pretreated before MC-LR in SD rats, Sertoli–germ cells were

358

regularly arranged, interstitial cells did not proliferate or atrophy, and were closely

359

connected with seminiferous tubules, compared to those of MC-LR-treated rats. It

360

was suggested that TSA could improve the pathological damage of testicular tissue

361

caused by MC-LR. TUNEL assay was used to detect the effect of MC-LR and TSA on

362

the apoptosis of testicular tissue of rats. As shown in Fig 2H and 2F, MC-LR could

363

significantly increase the number of cells apoptosis. However, in the TSA+MC-LR

364

group, the apoptotic cells were dramatically alleviated in the testes, when compared to

365

the MC-LR group. The results showed that histone acetylation was indeed involved in 19

366

MC-LR-induced testicular tissue injury and apoptosis in SD rats.

367 368

20

369

370 371

Fig2. Histone acetylation was involved in MC-LR-induced testicular tissue injury 21

372

and cell apoptosis in SD rats. (A) the activity of HDAC, (B) the activity of HAT, (C)

373

expressions of genes (HDAC1, HDAC2 and HDAC3), (D) protein expressions of

374

histone acetylation and its regulatory enzyme were detected after SD rats were treated

375

by MC-LR with or without TSA. (E) Quantitative analysis of proteins expression

376

levels and (F) quantitative analysis of cell apoptosis rates. (G) Histopathological

377

damage was observed by H&E staining. (H) TUNEL assay was used to detect cell

378

apoptosis in testicular tissue (x200, blue is the nucleus and green is the apoptotic

379

cells). All genes and proteins were normalized to that of β-actin. All values were

380

quantified with Quantity One, and data were represented as mean ± standard deviation

381

(SD, n = 3) for each group. (*P<0.05 vs. the control group; #P<0.05 vs. the 40 µg kg-1

382

MC-LR group)

383

3.3 Histone acetylation mediated MC-LR-induced cells apoptosis via the

384

mitochondrial apoptotic pathway

385

Expressions of apoptosis-related genes and proteins in mitochondrial apoptotic

386

pathway were detected after the co-cultured SD rat Sertoli-germ cells or SD rats were

387

exposed to MC-LR with or without TSA. The results of experiments in vitro (Fig 3A)

388

and in vivo (Fig 3B) showed that MC-LR increased the mRNA expressions of Bax,

389

Caspase3 and Caspase8, and decreased the mRNA expression of anti-apoptotic gene

390

Bcl-2. MC-LR also increased the proteins expression of Bax, Caspase3, Caspase8 in

391

vitro (Fig 3C) and in vivo (Fig 3D), decreased the expression of anti-apoptotic protein

392

Bcl-2. Interestingly, HDAC inhibitor TSA pretreatment partly alleviated apoptosis via

393

regulating MC-LR-induced change of apoptosis-related genes and proteins in vivo and 22

394

vitro.

395 396

Fig3. The effects of TSA and MC-LR on the genes and proteins levels of Caspase

397

pathway in co-cultured Sertoli-germ cells and SD rats testicular tissues. (A)

398

Expressions of cell apoptosis-related genes in vitro and (B) expressions of cell

399

apoptosis-related genes in vivo were detected by RT-qPCR. (C) Expressions of cell

400

apoptosis-related proteins in vitro and (D) expressions of cell apoptosis-related

401

proteins in vivo were detected by western blotting. (E) Quantitative analysis of

402

proteins expression levels in vitro. (F) Quantitative analysis of proteins expression

403

levels in vivo. (*P<0.05 vs. the control group; #P<0.05 vs. the 36 µM MC-LR group or

404

the 40 µg kg-1 MC-LR group) 23

405

3.4 Histone acetylation participates in MC-LR-induced cell cycle arrest

406

In order to investigate the molecular mechanism of TSA on MC-LR-induced

407

cells cycle arrest, the cell cycle-related genes were detected after SD rats and

408

Sertoli-germ cells were exposed to MC-LR with or without TSA. As shown in Fig 4A,

409

MC-LR significantly increased the expressions of Rb, E2F1 and CDK2, and

410

decreased the expression of CyclinD1 corresponding to increased concentrations of

411

MC-LR in vitro. The expressions of CyclinE1, CDK4 and P21waf1/cip1 were

412

significantly increased in the 36 µM MC-LR group. TSA pretreatment negatively

413

feedback regulated MC-LR-induced abnormal expressions of genes, when compared

414

the 36 µM MC-LR group. In vivo (Fig 4B), MC-LR decreased the expressions of

415

CyclinD1, E2F1 and CDK2, and increased the expression of P21waf1/cip1 in mRNA

416

level. TSA pretreatment increased the expressions of CyclinD1, E2F1 and CDK2, and

417

decreased the expression of P21waf1/cip1 in mRNA level, when compared with the

418

MC-LR group. However, there was no significant difference in the expressions of Rb

419

and CDK4 in SD rats treated with MC-LR.

420

The protein expressions were detected by western blotting. In vitro (Fig 4C) and

421

in vivo (Fig 4D) results showed that MC-LR significantly increased the expression of

422

P21waf1/cip1 and decreased the protein expression levels of E2F1, CDK2, CyclinD1 and

423

CyclinE1. Furthermore, TSA pretreatment suppressed the expression of P21waf1/cip1

424

and increased the expressions of E2F1, CDK2, CyclinD1 and CyclinE1. But there was

425

no significant difference in the expression of Rb in SD rats treated with MC-LR. The

426

CDK4 expression decreased in testicular cells after MC-LR exposure, but it was not 24

427

changed in co-cultured Sertoli-germ cells after MC-LR exposure in vitro. Additionally,

428

TSA pretreatment increased the expression of CDK4, when compared the MC-LR

429

group. These results indicated that the P21waf1/ cip1-CDKs-E2F1 pathway is significant

430

for the protection role of TSA in MC-LR-induced cell cycle disorders. It also revealed

431

that histone acetylation participated in MC-LR-induced cell cycle disorders and

432

apoptosis of testicular cells in SD rats.

433 434

Fig4. The effect of TSA and MC-LR on cell cycle-related genes and proteins

435

levels in co-cultured Sertoli-germ cells and SD rat testicular tissues. (A)

436

Expressions of cell cycle-related genes in vitro and (B) expressions of cell

437

cycle-related genes in vivo were detected by RT-qPCR. (C) Expressions of cell 25

438

cycle-related proteins in vitro and (D) expressions of cell cycle-related proteins in

439

vivo were detected by western blotting. (E) Quantitative

440

expression levels in vitro. (F) Quantitative analysis of proteins expression levels in

441

vivo. (*P<0.05 vs. the control group; #P<0.05 vs. the 36 µM MC-LR group or the 40

442

µg kg-1 MC-LR group)

443

4. Discussion

analysis

of

proteins

444

Histone acetylation can be regulated by both HAT and HDAC. The disruption

445

of the dynamic balance between HAT and HDAC may disorder the regulation of gene

446

expression and the normality of cell cycle gene transcription (Chen et al., 2015 ).

447

Studies showed that histone acetylation is involved in inducing apoptosis of primary

448

haploid sperm in mice (Xia et al., 2012) and impairing sperm formation during sperm

449

development (FENIC et al., 2004; Fenic et al., 2008). In this study, MC-LR increased

450

the total activity of HDAC, indirectly decreased the total activity of HAT and

451

decreased acetylation of histone H3 and H4 to induce apoptosis and cycle disorder in

452

SD rat testicular cells. However, HDAC inhibitor TSA could decrease the activity of

453

HDAC, indirectly increased the activity of HAT and the acetylation of histone H3 and

454

H4 to inhibit MC-LR-induced SD rat testicular cell apoptosis and cell cycle arrest.

455

Apoptosis is a common mechanism in many physiological and pathological

456

processes. The mitochondrial apoptotic pathway is a classical apoptotic pathway and

457

histone acetylation played an important role in the activation of apoptotic cells

458

(Glozak and Seto, 2007), which were consistent with the results of this study. HDAC1

459

and HDAC2 have different biological functions, they could promote apoptosis of 26

460

different cell types (Huang et al., 2005; Senese et al., 2007) and even have opposite

461

effects of embryonic differentiation (Huang et al., 2005; Humphrey et al., 2008). The

462

results in this study showed that MC-LR significantly up-regulated the expression of

463

HDAC1, and the activation of HDAC activity could increase the apoptosis.

464

Meanwhile, MC-LR decreased the acetylation levels of histone H3 and H4, promoted

465

the expressions of apoptosis-related genes (Bax, Caspase3 and Caspase8) and

466

inhibited the expression of anti-apoptotic genes (Bcl-2) in mitochondrial apoptotic

467

pathway. Interestingly, HDAC inhibitor TSA pretreatment partly alleviated apoptosis

468

via regulating MC-LR-induced change of apoptosis-related genes. Therefore, histone

469

acetylation was involved in the apoptosis of testicular cells in SD rats by activating

470

the mitochondrial Caspase signaling pathway.

471

Cell cycle includes G1, S, G2 and M phases. CyclinD1, CyclinE and

472

cyclin-dependent kinases (CDK2 and CDK4) are key proteins in the regulation of cell

473

cycle from G1 to S phases and from G2 to mitosis (Nurse, 1994). The results of

474

experiments showed that MC-LR up-regulated the expression of p21WAF/Cip1 and

475

disordered the expression of key regulatory proteins of cell cycle to induce the

476

testicular cell cycle arrest at S phase. Furthermore, cell cycle disorder was corrected

477

and the expression of p21WAF/Cip1 was down-regulated after inhibiting the activity of

478

HDAC. S phase is a critical period for the synthesis of DNA and histone proteins, in

479

which HDAC affects the binding of DNA and histones to induce cell cycle arrest

480

(Lucchini and Sogo, 1995 ).The regulation of non-coding RNA (Zhou et al., 2014;

481

Stillman, 2018) or other histone modifications (Zhang, 2003) may result in 27

482

inconsistent changes in transcript level and protein level caused by MC-LR. The

483

genes expressions of CyclinD1, E2F1, CDK2 and CDK4 in testicular cells may be

484

more important in elucidation of the molecular mechanism of MC-LR. MC-LR is

485

known to be a phosphatase inhibitor, and phosphorylation of Rb could release free of

486

E2F1 to promote cell cycle (Zhang and Dean, 2001). Therefore, it is possible that

487

MC-LR might affect the phosphorylation of Rb to induce cell cycle arrest. In

488

summary, MC-LR up-regulated the activity of HDAC, disordered the expression of

489

cell cycle-related genes and arrested the cell cycle of testicular cells in SD rats.

490

Previously, we reported that MC-LR induced Sertoli-germ cells apoptosis via

491

increasing the protein acetylation levels of p53 and Ku70 (Liu et al., 2018a). However,

492

the total activity of HDAC was increased and the acetylation levels of histone H3 and

493

H4 were decreased in this study. Based on these data, we propose that MC-LR may

494

induce apoptosis by increasing the acetylation level of p53 and Ku70 and decrease the

495

acetylation levels of histone H3 and H4. MC-LR is a highly specific inhibitor of

496

eukaryotic serine/threonine phosphatase 1 and 2A (PP1 and PP2A) (MacKintosh et al.,

497

1990). The intracellularly biochemical and molecular mechanisms of MCs- induced

498

apoptosis mainly include mitochondrial, p53, reactive oxygen species (ROS) and

499

endoplasmic reticulum Stress (ERs) pathways (Ding and Nam Ong, 2003; Chen et al.,

500

2016b; Chen et al., 2016c; Chen and Xie, 2016; Wu et al., 2019). P53 is an important

501

regulator in apoptosis. Cytosolic p53 can activate Bax to induce mouse fibroblasts

502

death via transcription-independent pathway (Speidel et al., 2006). Moreover, MC-LR

503

may induce Sertoli-germ cells apoptosis through p53-dependent transcription and 28

504

p53-independent transcription. Gcn5 (One well-conserved HAT) and PP2A formed

505

enzyme complex, and their coordinated functions are critical for cell viability (Chrun

506

et al., 2017). HDAC inhibitors potently reduced ROS production (Chen et al., 2016a).

507

Therefore, we speculated that histone acetylation may mediate MC-LR-induced SD

508

rat testicular cell apoptosis through p53-dependent or p53-independent transcription

509

signaling pathway, or through oxidative stress induced by the combination of MC-LR

510

and pp1/pp2A, the mechanisms need to be confirmed by more studies.

511 512

Fig5. The signaling pathway of histone acetylation acts on MC-LR-induced 29

513

apoptosis and cycle disorder in SD rat testicular cells. As we all know, MC-LR

514

could inhibit the activity of PP1 and 2A. The results showed that MC-LR enhanced

515

HDAC activity and reduced the acetylation levels of histone H3 and H4 in SD rat

516

testicular cells, subsequently increasing the expression of Bax/Bcl-2, Caspase-3 and

517

Caspase-8, and inducing cell cycle arrest. TSA pretreatment inhibited the activity of

518

HDAC, and prevented MC-LR-induced apoptosis by reducing expression of

519

Bax/BCL-2, Caspase3, Caspase8 and cell cycle arrest.

520

5. Conclusion

521

The present study demonstrated that histone acetylation may play an important

522

role in MC-LR-induced apoptosis and cell cycle disorder in SD rats. MC-LR

523

enhanced HDAC activity and reduced the level of histone acetylation and its

524

regulatory gene expression. This activated the mitochondrial apoptotic pathway and

525

increased the expression of apoptosis-related factors to induce apoptosis. Additionally,

526

MC-LR also caused cell cycle arrest in S phase to induce testicular cell apoptosis in

527

SD rats. This study provided some insights and theoretical basis about the epigenetic

528

mechanisms of MC-LR-induced apoptosis in testicular cells.

529

Conflict of interest

530 531

The authors declare that there are no conflicts of interest. Acknowledgements

532

We would like to thank that this work was supported by the National Nature

533

Science Foundation of China (Grant nos. 81472948 and 81773384), and the Henan

534

natural science foundation (Grant No. 162300410267). 30

535

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33

Highlights: MC-LR enhanced the activity of HDAC and reduced the acetylation levels of histone H3, H4.
MC-LR induced SD rat testicular cell apoptosis.
MC-LR induced SD rat testicular cell cycle arrest in S phase.
Trichostatin A inhibited MC-LR-induced SD rat testicular cell apoptosis and cell cycle arrest.


Declaration of interests
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: