Identification of apoptotic pathways in zearalenone-treated mouse sertoli cells

Abstract

Zearalenone (ZEN), one of the most prevalent non-steroidal oestrogenic mycotoxins, is primarily produced by Fusarium fungi. Due to its toxicity as an oestrogenic compound and wide distribution in feed and foods, the reproductive toxicology of ZEN exposure is of public concern. The aim of the present study was to investigate the effect of ZEN on Sertoli cells to identify apoptotic pathways induced by this compound. We found that ZEN reduced the viability and caused apoptosis in Sertoli cells in vitro. Notably, we observed that such effects were associated with a significant increase in reactive oxygen species (ROS) and the number of cells that showed positive staining for γH2AX and RAD51, enzymes essential for repairing DNA damage. There was a parallel decrease in the expression of occludin and connexin 43, proteins that are present in the testis–blood barrier and gap junctions of Sertoli cells, respectively. Overall, the present study confirms that ZEN exposure can have serious deleterious effects on mammalian Sertoli cells and offers novel insight about its molecular targets in these cells.

INTRODUCTION

Zearalenone (ZEN) is a mycotoxin produced by several species of Fusarium fungi, such as Fusarium culmorum that grow on oats, corn and other edible agricultural feed and food. Several studies have shown that chronic or acute exposure to ZEN or other non-steroidal oestrogenic compounds before and after birth and up to adulthood can impair spermatogenesis and, consequently, fertility in animals, including humans (Kowalska et al., 2016; Men et al., 2019; Zhou et al., 2020). In animals exposed to ZEN in early pregnancy, ZEN could affect the development of placenta and embryo, and even lead to embryo deformity or death (Zhang et al., 2014). In addition, increased incidence of testicular cancer in humans has been attributed to elevated levels of such compounds in the environment (Giannandrea et al., 2013).

In particular, early studies reported that in utero exposure to diethylstilbestrol (DES) or ZEN resulted in abnormal testicular differentiation and disturbed fertility in mice (Pérez-Martínez et al., 1996). Moreover, several studies have demonstrated that ZEN and its metabolites alter hormone production, including testosterone, thus acting as endocrine disruptors (Frizzell et al., 2011; Yang et al., 2007a, 2007b). Interestingly, Koraïchi et al. (2013) reported that neonatal exposure of rats to ZEN induces long-term modulation of ATP-binding cassette (ABC) transporter expression in the testis. Moreover, Zatecka et al. (2014) observed that besides reproductive parameters, ZEN also affects the expression of selected testicular genes in adult mice. More recent studies have confirmed that several alterations in testicular morphological parameters and reproductive disorders in farm animals and humans are attributable to ZEN (Koraïchi et al., 2013; Filipiak et al., 2009; Minervini and Dell’Aquila, 2008; Zinedine et al., 2007).

It is likely that the main effect of ZEN results from its oestrogenic activity. ZEN and its derivatives compete with 17-β-oestradiol (E2) to bind oestrogen receptors (ERs). The testicular targets of ZEN and oestrogenic compounds can be germ cells at all stages of development and differentiation as well as the somatic cells of the seminiferous tubules (Sertoli cells [SCs]) and interstitial Leydig cells. In fact, although SCs and Leydig cells express ERs and can be considered the major targets of oestrogen and oestrogenic compounds, ERs are found in immature germ cells, including spermatogonia, spermatocytes, and spermatids, as well as ejaculated spermatozoa in humans and many other animal species (Carreau et al., 2011a, 2011b). Moreover, ERs are present in primordial germ cells and gonocytes of foetal rodent and human testes (La Sala et al., 2010a, 2010b). Few studies have investigated the effects of ZEN on SCs. Some papers have reported that rodents exposed to this compound show a reduction in the SC number due to apoptosis (Koraïchi et al., 2013; Jee et al., 2010; Kim et al., 2003). In vitro, ZEN reduced the proliferation of prepuberal rat SCs in a dose-dependent manner, causing both apoptosis and necrosis associated with an increase in the Bax/Bcl-2 ratio, as well as the expression of Fas ligand (FasL) and caspase-3, caspase-8, and caspase-9 activity (Xu et al., 2016). Also, kidney cell exposure to ZEN led to increased caspase-3 activity, MDA, and IL-10, IL-6, TNF-alpha and Bax mRNA levels, but decreased TAC content and down-regulated expression of GSH-Px and CAT and Bcl-2 mRNA (Ben Salah-Abbès et al., 2020). ZEN treatment significantly increased the mRNA expression of EDN1, IER3, TGFβ and BDNF genes and significantly reduced the mRNA expression of IGF-1 and SFRP2 genes in granulosa cells (Zhang et al., 2017b). Moreover, ZEN impaired specific secretory functions of adult rat SCs by disrupting the cytoskeleton structure (Zheng et al., 2016).

Most in vivo studies have reported that developmental period exposure to ZEN in pregnant mothers may affect not only maternal reproductive function but also that of the male and female offspring. However, the effects of ZEN on the spermatogonia of male mice are not clear. The objective of this study was to explore the toxic effects of ZEN on adult mouse SCs.

MATERIALS AND METHODS

Cell culture and exposure of cells to ZEN

SCs (TM4 cell line, obtained from Korean Cell Line Bank (Seoul, South Korea)) were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Hyclone, UT, USA) supplemented with 10% foetal bovine serum (FBS) and antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin) at 37°C in a 5% CO₂ atmosphere. The cells were seeded at 1 × 10⁴ cells per well and incubated for 24 hr prior to the experiments. The cells were then washed with phosphate-buffered saline (PBS; pH 7.4) and incubated in fresh DMEM plus FBS containing different concentrations of ZEN (Sigma, Z2125, USA) for the indicated time.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays

The MTT and LDH assays were used to investigate cell viability, as described by Zhang et al. (2015). In brief, for both assays, 1 × 10⁴ cells were seeded in each well of a 96-well plate. After ZEN exposure (24 or 48 hr), for the MTT assay (mitochondrial activity), the medium was removed and replaced with 100 μL of fresh medium containing MTT; the plate was then incubated for 3 hr at 37°C. The absorbance was measured at 450 nm using a Multiskan FC multiplate reader (PerkinElmer, Waltham, MA, USA). The LDH assay (to evaluate the loss of membrane integrity) was carried out using the LDH cytotoxicity assay kit (Abcam, LDH Assay kit, Cambridge, MA, USA). LDH activity was determined in the spent medium by measuring the absorbance at 490 nm.

Measurement of reactive oxygen species (ROS) levels

Intracellular ROS levels were measured using a detection kit purchased from Beyotime (Jiangsu, China; number S0033). Briefly, 1 × 10⁶ cells were seeded in each well of a 6-well plate for 24 hr and then incubated with fresh medium containing 10 μM dichloro-dihydro-fluorescein diacetate (DCFH-DA) at 37°C in the dark for 20 min. Subsequently, cells were treated with different concentrations of ZEN for 12 or 24 hr. At the end of incubation, the cells were washed with PBS and the fluorescence intensity measured using a Nikon Eclipse E400 fluorescence microscope equipped with an image analysis software (Image J, National Institutes of Health, Bethesda, MD, USA).

JC-1 assay

The JC-1 assay (to measure the mitochondrial membrane potential [ΔΨm]) was used to evaluate activation of intrinsic apoptosis and carried out according to the manufacturer’s instructions (JC-1 Mitochondrial Membrane Potential Assay Kit, Abnova, Taipei, Taiwan). Cells were cultured with different concentrations of ZEN for 24 hr under the conditions described above. They were then transferred onto a coverslip housed in a 4-well plate and incubated in DMEM containing 10 µM JC-1 at 37°C for 15 min. The coverslips were washed with PBS and rapidly mounted with Vectashield medium for observation under a Nikon Eclipse E400 fluorescence microscope.

Terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assay

The TUNEL assay was employed to detect apoptotic cells, using an in situ detection kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Cells were treated with different concentrations of ZEN for 24 hr before being subjected to the assay. Samples were analysed under a Nikon Eclipse E400 fluorescence microscope.

Immunohistochemistry for γH2AX and RAD51

Cells treated with ZEN for 24 hr were fixed in 4% paraformaldehyde, blocked with 1% bovine serum albumin (BSA) for 30 min, and then incubated with primary antibodies against γH2AX (Abcam ab26350, London, UK) or RAD51 (Abcam ab88572) at 4°C overnight. After washing with PBS, cells were incubated with Cy3-labelled goat anti-rabbit IgG (Beyotime, A0516) diluted 1:50 at 4°C for 1.5 hr. Finally, samples were counterstained with DAPI and visualised under a confocal microscope (Carl Zeiss LSM780, Instrument Development Center, NCKU).

RNA sequencing (RNA-seq) analysis

RNA-seq was performed with the Hiseq 4000 platform by Novogene (Beijing, China). The expression of each gene was determined using featureCounts software; the DESeq package was used to identify differentially expressed mRNA (DEmRNA) between the two groups (control and 40 µM ZEN, treated for 24 hr) (Chongtham et al., 2020; Li et al., 2020). Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) were analysed with Cluster-Profiler, using a paired t-test with p < 0.05 between the groups was considered significant) (Zhang et al., 2019).

Western blot analysis

For western blot analysis, the treated cell (control and 40 µM ZEN, treated for 24 hr) were harvested and were incubated in radioimmunoprecipitation assay (RIPA) lysis buffer in the presence of a protease inhibitor. Protein concentrations were measured using the BCA Protein Assay Reagent (Pierce, Rockford, IL, USA). The cell lysates were then analysed using sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The following primary antibodies were used: anti-γH2AX (Abcam ab26350), anti-occludin (Abcam ab31721), anti-connexin 43 (Cx43; Abcam ab219493), anti-Bax (Cell Signaling Technology, 2772, Boston, MA, USA), anti-Bcl2 (ImmunoWay YT0470, Suzhou Jiangsu, China), and anti-β-actin (Abcam ab8227).

Statistics

Independent experiments were repeated at least three times (with triplicates within each experiment), and the data are represented as the mean ± standard deviation (SD). Data were analysed by t-test or one-way analysis of variance (ANOVA) and followed by the Tukey test for multiple comparisons to determine the differences between groups (denoted by an asterisk) using GraphPad Prism software (San Diego, CA, USA).

RESULTS AND DISCUSSION

Cytotoxic effects of ZEN on SCs

The MTT assay is used to detect cell survival and growth. It has been widely used as a measure of cytotoxicity and cell viability after exposing living cells to toxic substances (Rai et al., 2018; Jo et al., 2015). The detection principle is that succinate dehydrogenase in mitochondria of living cells can reduce exogenous MTT to water-insoluble bluish purple crystal formazan and deposit it in cells, but dead cells have no such function. For a certain cell number range, the amount of MTT crystal formation is proportional to the number of cells. According to the measured absorbance (optical density [OD]), one can judge the number of living cells. The larger the OD value is, the stronger the cell activity is. Similarly, LDH detection can also be used to detect cell activity. LDH is a stable cytoplasmic enzyme that is present in all cells. When the cell membrane is damaged, LDH is rapidly released into the cell culture medium. The degree of cell damage can be judged by detecting the activity of LDH in cell culture supernatant (Specian et al., 2016).

Studies have demonstrated that ZEN can perturb the differentiation of cells, inhibit cell viability, reduce the generation of reproductive cells, and induce cell death via apoptosis and necrosis (Zheng et al., 2018). In a previous study, treatment with ZEN (3–300 μM) significantly decreased cell viability, with a half-maximal inhibitory concentration (IC50) of 80 μM (Sang et al., 2016). Fig. 1A and 1B shows the viability of SCs incubated in various concentrations of ZEN evaluated by the MTT and LDH assays, respectively. The assays indicated a loss of viability parameters, respectively mitochondrial activity and membrane leakage, in SCs incubated with increasing ZEN concentrations for 24 or 48 hr.

Fig. 1

Effects of zearalenone (ZEN) on cell viability and lactate dehydrogenase (LDH) activity in Sertoli cells (SCs). A. SCs were incubated with different ZEN concentrations for 24 or 48 hr, and then cell viability was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. B. SCs were incubated with different ZEN concentrations for 12 or 24 hr, and the LDH activity was determined. *p < 0.05.

ZEN Induced apoptosis and increased ROS production in SCs

To evaluate whether the loss of cell viability was due to ZEN-induced activation of apoptotic pathways in SCs, we used the JC-1 and TUNEL assays. The JC-1 assay revealed a marked loss of ΔΨm in SCs after exposure for 24 hr to ZEN (Fig. 2A-a). These changes indicate activation of intrinsic apoptosis. Moreover, after the same incubation time, there were more TUNEL-positive SCs, which showed extensive DNA breakdown typical of apoptosis (Fig. 2B-b). Bax is the key executor of mitochondrial regulation of cell death through its activity of penetrating mitochondrial outer membrane. Bax gene is one of the most important genes promoting apoptosis and belongs to bcl-2 gene family. The encoded Bax protein can form heterodimer with Bcl-2 and inhibit Bcl-2. It is found that the proportional relationship between Bax/Bcl-2 protein is the key factor to determine the inhibitory effect on apoptosis (Matsuyama et al., 2016; Vogel 2002). Ongoing apoptosis in SC exposed to ZEN (40 µM) for 24 hr was confirmed by the increasing Bax/Bcl2 ratio (Fig. 6). ROS can lead to different oxidative alkali damage and 2-deoxyribose modification, and large amounts of ROS are significantly related to the development of diseases (Hafstad et al., 2013). ROS can attack DNA bases and destroy DNA skeleton, resulting in single strand breaks in animal cells (Broedbaek et al., 2009). Finally, the analysis of the ROS level in SCs indicated that 12 or 24 hr exposure to ZEN significantly increased free radical production (Fig. 3), suggesting that in such cells apoptosis induced by the drug can be triggered by enhanced ROS production.

Fig. 2

Evaluation of the mitochondrial membrane potential (∆ψm) and terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assay. A. The ∆ψm was evaluated using JC-1 in treated cells. Red fluorescence indicates JC-1 aggregates within mitochondria in healthy cells, whereas green fluorescence indicates JC-1 monomers in the cytoplasm and the loss of ∆ψm. The scale bars are 100 µm. a. The ratio of JC-1 monomers to JC-1 aggregates. B. Apoptosis was assessed using a TUNEL assay; nuclei were counterstained with Hoechst and apoptotic Sertoli cells (SCs) are labelled in red, while cell nuclei are labelled in blue. The scale bar was 100 µm. b. Average intensity of TUNEL fluorescence in SCs cells. *p < 0.05, **p < 0.01.

Fig. 3

Evaluation of the reactive oxygen species (ROS) levels in Sertoli cells (SCs) after zearalenone (ZEN) treatment. A. Intracellular ROS levels were measured with fluorescence imaging using the dichloro-dihydro-fluorescein diacetate (DCFH-DA) probe in cells cultured in the presence of ZEN for 12 or 24 hr. The scale bars are 100 µm. B. Average intensity of fluorescence in SCs. The results are expressed as the mean ± standard deviation of three independent experiments. There was a significant difference in the ROS generation of treated compared with untreated cells, assessed using Student’s t-test (**p < 0.01).

In previous studies, ZEN-induced apoptosis in SCs reduced the transcription and expression of the anti-apoptotic protein Bcl-2, and increased the transcription and expression of the pro-apoptotic proteins Bax, tBID, Fas, FasL, Fas-associated protein with death domain (FADD), and caspase-8. ZEN also increased the activation of caspase-8 and caspase-9 and promoted the release of cytochrome c from the mitochondria to the cytoplasm (Cai et al., 2019; Yi et al., 2020). Zearalenone induced apoptosis and necrosis of porcine ovarian granulosa cells in a dose-dependent manner, thereby reducing cell proliferation (Zhu et al., 2012).

Moreover, ZEN induced autophagy via activation of the nuclear factor kappa B (NF-κB) signalling pathway (Wang et al., 2019). In the prostatic endothelial PNT1A cell line, ZEN-induced oxidative stress was associated with a decrease in the expression of oxidative stress defence enzymes, cell cycle arrest in the G2/M phase, and reduced cell migration (Kowalska et al., 2019). ROS-mediated endoplasmic reticulum (ER) stress induced by ZEN activated AMP-activated protein kinase (AMPK) via Ca²⁺/CaM-dependent protein kinase kinase β (CaMKKβ) leading to autophagy in TM4 cells (Feng et al., 2020) ROS regulated ZEN-induced cell cycle arrest and apoptosis through ER stress and the ATP/AMPK signal ways (Zheng et al., 2018). ZEN exposure can reduce antioxidant enzyme activities, accumulation of ROS, and decreasing mitochondrial membrane potential to induce mitochondrial damage (Fan et al., 2017).

ZEN induced extensive DNA breakage in SCs

When incubated with ZEN for 24 hr, more SCs were positive for γH2AX (Fig. 4A-a) and RAD51 (Fig. 4B-b) compared with the control cells. Western blotting analysis confirmed higher expression of γH2AX in ZEN-treated compared with control SCs (Fig. 6). This suggests that ZEN at first causes DNA double-strand breaks (γH2AX foci) in SCs that they attempt to repair (RAD51 foci) or that in such cells, phosphorylation of H2AX and RAD51 recruitment are part of the apoptotic pathway, as has been reported in other cell types (Rogakou et al., 2000). DNA stores an organism’s genetic information, which is necessary for survival and reproduction, and organisms will protect their DNA at any cost. γH2AX and RAD51 are DNA repair proteins. γH2AX induction is one of the earliest events after DNA double-strand breaks and plays a central role in sensing and repairing DNA damage (Kopp et al., 2019; Godin et al., 2016). Organisms are constantly exposed to a large number of DNA damaging agents with endogenous and exogenous factors that can affect health and lead to cancer, certain diseases and aging. However, cells have a complex system of DNA repair, damage tolerance, cell cycle checkpoints and cell death pathways to protect DNA and reduce the harmful consequences of DNA damage (Chatterjee and Walker, 2017). As biomarkers, γH2AX and RAD51 can clearly reflect the degree of DNA damage and repair. ZEN exposure can lead to chromosomal deformity, mutations, and DNA double-strand breaks. A study revealed that ZEN increased the number of γH2AX-positive cells within mouse ovaries (Zhang et al., 2017a). Moreover, the expression of γ-H2AX and RAD51 was increased in ZEN exposed granulosa cells (Liu et al., 2018).

Fig. 4

Nuclear DNA damage in Sertoli cells (SCs) after zearalenone (ZEN) treatment using immunofluorescence for γ-H2AX and RAD51. A. Nuclear γ-H2AX foci in cells exposed to ZEN for 24 hr. The scale bars are 100 µm. a. Average intensity of γ-H2AX fluorescence in SCs. B. Nuclear RAD51 foci in cells exposed to ZEN for 24 hr. The scale bars are 100 µm. b. Average intensity of RAD51 fluorescence in SCs. **p < 0.01.

ZEN exposure disturbed mRNA expression in SCs

To understand the mechanism by which ZEN causes cytotoxicity in SCs, RNA expression was analysed with RNA-seq. The cluster dendrogram showed that the three replicates from the control and ZEN treatment groups were closely linked (Fig. 5A). A MA plot was used to show the DEmRNA distribution: 3,533 were upregulated and 3,469 were downregulated in the cells treated with 40 µM ZEN compared with the control group (Fig. 5B and 5C). ZEN significantly induced the expression of γH2AX (Fig. 5D), a finding that is consistent with the increased protein expression noted with western blotting (Fig. 6). The enriched GO terms included ribonucleoprotein complex biogenesis, regulation of mitotic cell cycle, regulation of DNA metabolic process, DNA repair, negative regulation of cell cycle, autophagy, and others (Fig. 5E). KEGG analysis was used to examine pathway enrichment (Fig. 5F). Included in the top 20 enriched KEGG pathways related to cell activity are focal adhesion, cell cycle, Wnt signalling pathway, the tumour necrosis factor (TNF) signalling pathway, and p53 signalling pathway (Fig. 5F, red boxes) (Akrami et al., 2019; Liu et al., 2018; Khedri et al., 2019). Wnt signalling is involved in a multitude of developmental processes through regulating cell proliferation, differentiation and apoptosis (Kahn, 2014). And also Tnf signalling pathway and p53 signalling pathway are involved in the process of apoptosis (Kiraz et al., 2016). Based on these changes, ZEN exposure affected mRNA expression in SCs and induced apoptosis (Fig. 5F). We also performed gene set enrichment analysis (GSEA). Genes related to the cell cycle were downregulated (Fig. 5G). Analysis of the genes related to the cell cycle can be acquired by STRING: the DEmRNAs Cdc7, Cdc20, Cdc14a, and Cdk1 are key genes in the cell cycle arrest regulatory network (Fig. 5H). Hence, ZEN exposure influenced the expression of genes involved in the cell cycle.

Fig. 5

Zearalenone (ZEN) exposure altered messenger RNA (mRNA) expression in Sertoli cells (SCs). A. Correlation analysis between the groups (CON = control; ZEN = 40 µM ZEN). B. The MA plot displays the distribution of differentially expressed mRNA (DEmRNA) between the ZEN treatment and the control groups. C. The bar graph indicates the number of statistically significant DEmRNAs. D. The relative expression level of γH2AX in the two groups. E. The histogram demonstrates the Gene Ontology (GO) enrichment analysis for the DEmRNAs. F. The histogram demonstrates the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis for DEmRNAs. G. The results of gene set enrichment analysis (GSEA) between the groups. H. The protein–protein interaction (PPI) network of the cell cycle-related DEmRNAs visualised by Cytoscape. **p < 0.01.

Fig. 6

Western blotting in Sertoli cells (SCs) treated with zearalenone (ZEN). A. Western blotting of Bax, Bcl2, γH2AX, occludin, and connexin 43 (Cx43) expression in SCs. Cells were treated with 40 µM ZEN for 24 hr. B. Protein expression is relative to β-actin, which served as the loading control. **p < 0.01.

ZEN decreased the level of occludin and Cx43 in SCs

The blood-testis barrier (BTB) is one of the tightest blood-tissue barriers in the mammalian body. BTB separates the blood vessels and seminiferous tubules in the human testis. It is composed of SCs, the vascular endothelial basement membrane, connective tissue, and the seminiferous tubule basement membrane. Environmental pollutants are hypothesised to induce testicular injury via their initial actions at the BTB to elicit subsequent damage to germ cell adhesion, thereby leading to germ cell loss, reduced sperm count, and male infertility or subfertility (Long et al., 2016). Perfluorooctanesulfonic acid (PFOS), a persistent organic pollutant, dose dependently increased BTB permeability and decreased expression of the occludin, a tight junction protein, and Cx43, the main SC gap junction protein (Qiu et al., 2016). Heavy metals can perturb the BTB. Cadmium, a major industrial pollutant and environmental toxicant, can cause significant BTB disruption concomitant with obvious sperm abnormity and dynamic impairment, and epigenetic dysregulation of Mdr1b in the BTB was a potential cause of dyszoospermia upon Cd exposure (Fang et al., 2020). And also BTB has been recognized as an intensive target for the toxicity of Endocrine-disrupting chemicals (EDCs) (Li et al., 2016). ZEN repressed the expression of vimentin and tight junction protein 11, which lead to a destruction of the BTB (Long et al., 2016). As shown in Fig. 6, ZEN decreased the expression of Occludin and Cx43. Which was consistent with the data of Wu (2019), BPAF (Bisphenol AF) exposure decreased the expression of Cx43 and Zo-1. These findings suggest that ZEN alters the BTB established by SC–SC and SC–germ cell interactions. Whether this is the cause or the consequence of SC apoptosis induced by ZEN remains to be investigated.

In conclusion, the present study confirms that ZEN exposure induces apoptosis in SCs and offers novel insight about its molecular targets in such cells (ROS production, DNA damage, and the BTB) that can explain the adverse effects of the ZEN on spermatogenesis and fertility reported in previous works.

ACKNOWLEDGMENTS

This work was supported by High level talents research fund project of Qingdao Agricultural University in China (1120043), Open research work of Institute of Animal Husbandry and Veterinary Research of Hubei Academy of Agricultural Sciences (KLAEMB-2018-05) and Science & Technology Fund Planning Projects of Qingdao City (21-1-4-ny-7-nsh).

Conflict of interest

The authors declare that there is no conflict of interest.

REFERENCES

•  Akrami,  H.,  Mehdizadeh,  K.,  Moradi,  B.,  Borzabadi Farahani,  D.,  Mansouri,  K.,  Ghalib Ibraheem Alnajar,  S. and  Alnajar,  S. (2019): PlGF knockdown induced apoptosis through Wnt signaling pathway in gastric cancer stem cells. J. Cell. Biochem., 120, 3268-3276.
•  Ben Salah-Abbès,  J.,  Belgacem,  H.,  Ezzdini,  K.,  Abdel-Wahhab,  M.A. and  Abbès,  S. (2020): Zearalenone nephrotoxicity: DNA fragmentation, apoptotic gene expression and oxidative stress protected by Lactobacillus plantarum MON03. Toxicon, 175, 28-35.
•  Broedbaek,  K.,  Poulsen,  H.E.,  Weimann,  A.,  Kom,  G.D.,  Schwedhelm,  E.,  Nielsen,  P. and  Böger,  R.H. (2009): Urinary excretion of biomarkers of oxidatively damaged DNA and RNA in hereditary hemochromatosis. Free Radic. Biol. Med., 47, 1230-1233.
•  Cai,  G.,  Si,  M.,  Li,  X.,  Zou,  H.,  Gu,  J.,  Yuan,  Y.,  Liu,  X.,  Liu,  Z. and  Bian,  J. (2019): Zearalenone induces apoptosis of rat Sertoli cells through Fas-Fas ligand and mitochondrial pathway. Environ. Toxicol., 34, 424-433.
•  Carreau,  S.,  Bois,  C.,  Zanatta,  L.,  Silva,  F.R.,  Bouraima-Lelong,  H. and  Delalande,  C. (2011a): Estrogen signaling in testicular cells. Life Sci., 89, 584-587.
•  Carreau,  S.,  Bouraima-Lelong,  H. and  Delalande,  C. (2011b): Estrogens: new players in spermatogenesis. Reprod. Biol., 11, 174-193.
•  Chatterjee,  N. and  Walker,  G.C. (2017): Mechanisms of DNA damage, repair, and mutagenesis. Environ. Mol. Mutagen., 58, 235-263.
• Chongtham, M., Wang, H., Thaller, C., Hsiao, N., Vachkov, I., Pavlov, S., Williamson, L., Yamashima, T., Stoykova, A., Yan, J., Eichele, G. and Tonchev, A. (2020): Transcriptome response and spatial pattern of gene expression in the primate subventricular zone neurogenic niche after cerebral ischemia. Front. Cell. Dev. Biol., 8, 01.
•  Fan,  W.,  Shen,  T.,  Ding,  Q.,  Lv,  Y.,  Li,  L.,  Huang,  K.,  Yan,  L. and  Song,  S. (2017): Zearalenone induces ROS-mediated mitochondrial damage in porcine IPEC-J2 cells. J. Biochem. Mol. Toxicol., 31.
•  Fang,  Y.,  Xiang,  Y.,  Lu,  X.,  Dong,  X.,  Zhang,  J. and  Zhong,  S. (2020): Epigenetic dysregulation of Mdr1b in the blood-testis barrier contributes to dyszoospermia in mice exposed to cadmium. Ecotoxicol. Environ. Saf., 190, 110142.
•  Feng,  N.,  Wang,  B.,  Cai,  P.,  Zheng,  W.,  Zou,  H.,  Gu,  J.,  Yuan,  Y.,  Liu,  X.,  Liu,  Z. and  Bian,  J. (2020): ZEA-induced autophagy in TM4 cells was mediated by the release of Ca²⁺ activates CaMKKβ-AMPK signaling pathway in the endoplasmic reticulum. Toxicol. Lett., 323, 1-9.
•  Filipiak,  E.,  Walczak-Jedrzejowska,  R.,  Oszukowska,  E.,  Guminska,  A.,  Marchlewska,  K.,  Kula,  K. and  Slowikowska-Hilczer,  J. (2009): Xenoestrogens diethylstilbestrol and zearalenone negatively influence pubertal rat’s testis. Folia Histochem. Cytobiol., 47, S113-S120.
•  Frizzell,  C.,  Ndossi,  D.,  Verhaegen,  S.,  Dahl,  E.,  Eriksen,  G.,  Sørlie,  M.,  Ropstad,  E.,  Muller,  M.,  Elliott,  C.T. and  Connolly,  L. (2011): Endocrine disrupting effects of ZENralenone, alpha- and beta-Zearalenol at the level of nuclear receptor binding and steroidogenesis. Toxicol. Lett., 206, 210-217.
•  Giannandrea,  F.,  Paoli,  D.,  Figà-Talamanca,  I.,  Lombardo,  F.,  Lenzi,  A. and  Gandini,  L. (2013): Effect of endogenous and exogenous hormones on testicular cancer: the epidemiological evidence. Int. J. Dev. Biol., 57, 255-263.
•  Godin,  S.K.,  Sullivan,  M.R. and  Bernstein,  K.A. (2016): Novel insights into RAD51 activity and regulation during homologous recombination and DNA replication. Biochem. Cell Biol., 94, 407-418.
•  Hafstad,  A.D.,  Nabeebaccus,  A.A. and  Shah,  A.M. (2013): Novel aspects of ROS signalling in heart failure. Basic Res. Cardiol., 108, 359.
•  Jee,  Y.,  Noh,  E.M.,  Cho,  E.S. and  Son,  H.Y. (2010): Involvement of the Fas and Fas ligand in testicular germ cell apoptosis by zearalenone in rat. J. Vet. Sci., 11, 115-119.
•  Jo,  H.Y.,  Kim,  Y.,  Park,  H.W.,  Moon,  H.E.,  Bae,  S.,  Kim,  J.,  Kim,  D.G. and  Paek,  S.H. (2015): The unreliability of MTT assay in the cytotoxic test of primary cultured glioblastoma cells. Exp. Neurobiol., 24, 235-245.
•  Kahn,  M. (2014): Can we safely target the WNT pathway? Nat. Rev. Drug Discov., 13, 513-532.
•  Khedri,  A.,  Khaghani,  S.,  Kheirollah,  A.,  Babaahmadi-Rezaei,  H.,  Shadboorestan,  A.,  Zangooei,  M.,  Afra,  H.S.,  Meshkani,  R. and  Ghahremani,  M.H. (2019): Signaling Crosstalk of FHIT, p53, and p38 in etoposide-induced apoptosis in MCF-7 cells. J. Cell. Biochem., 120, 9125-9137.
•  Kim,  I.H.,  Son,  H.Y.,  Cho,  S.W.,  Ha,  C.S. and  Kang,  B.H. (2003): Zearalenone induces male germ cell apoptosis in rats. Toxicol. Lett., 138, 185-192.
•  Kiraz,  Y.,  Adan,  A.,  Kartal Yandim,  M. and  Baran,  Y. (2016): Major apoptotic mechanisms and genes involved in apoptosis. Tumour Biol., 37, 8471-8486.
•  Kopp,  B.,  Khoury,  L. and  Audebert,  M. (2019): Validation of the γH2AX biomarker for genotoxicity assessment: a review. Arch. Toxicol., 93, 2103-2114.
•  Koraïchi,  F.,  Inoubli,  L.,  Lakhdari,  N.,  Meunier,  L.,  Vega,  A.,  Mauduit,  C.,  Benahmed,  M.,  Prouillac,  C. and  Lecoeur,  S. (2013): Neonatal exposure to zearalenone induces long term modulation of ABC transporter expression in testis. Toxicology, 310, 29-38.
•  Kowalska,  K.,  Habrowska-Górczyńska,  D.E. and  Piastowska-Ciesielska,  A.W. (2016): Zearalenone as an endocrine disruptor in humans. Environ. Toxicol. Pharmacol., 48, 141-149.
•  Kowalska,  K.,  Habrowska-Górczyńska,  D.E.,  Urbanek,  K.A.,  Domińska,  K.,  Sakowicz,  A. and  Piastowska-Ciesielska,  A.W. (2019): Estrogen receptor β plays a protective role in zearalenone-induced oxidative stress in normal prostate epithelial cells. Ecotoxicol. Environ. Saf., 172, 504-513.
•  Li,  J.,  Sheng,  N.,  Cui,  R.,  Feng,  Y.,  Shao,  B.,  Guo,  X.,  Zhang,  H. and  Dai,  J. (2016): Gestational and lactational exposure to bisphenol AF in maternal rats increases testosterone levels in 23-day-old male offspring. Chemosphere, 163, 552-561.
•  Li,  N.,  Liu,  X.L.,  Zhang,  F.L.,  Tian,  Y.,  Zhu,  M.,  Meng,  L.Y.,  Dyce,  P.W.,  Shen,  W. and  Li,  L. (2020): Whole-transcriptome analysis of the toxic effects of zearalenone exposure on ceRNA networks in porcine granulosa cells. Environ. Pollut., 261, 114007.
•  Liu,  S. and  Guan,  W. (2018): STING signaling promotes apoptosis, necrosis, and cell death: an overview and update. Mediators Inflamm., 2018, 1202797.
•  Liu,  X.L.,  Wu,  R.Y.,  Sun,  X.F.,  Cheng,  S.F.,  Zhang,  R.Q.,  Zhang,  T.Y.,  Zhang,  X.F.,  Zhao,  Y.,  Shen,  W. and  Li,  L. (2018): Mycotoxin zearalenone exposure impairs genomic stability of swine follicular granulosa cells in vitro. Int. J. Biol. Sci., 14, 294-305.
•  Long,  M.,  Yang,  S.,  Wang,  Y.,  Li,  P.,  Zhang,  Y.,  Dong,  S.,  Chen,  X.,  Guo,  J.,  He,  J.,  Gao,  Z. and  Wang,  J. (2016): The protective effect of selenium on chronic zearalenone-induced reproductive system damage in male mice. Molecules, 21, 1687-1703.
•  Matsuyama,  S.,  Palmer,  J.,  Bates,  A.,  Poventud-Fuentes,  I.,  Wong,  K.,  Ngo,  J. and  Matsuyama,  M. (2016): Bax-induced apoptosis shortens the life span of DNA repair defect Ku70-knockout mice by inducing emphysema. Exp. Biol. Med. (Maywood), 241, 1265-1271.
•  Men,  Y.,  Zhao,  Y.,  Zhang,  P.,  Zhang,  H.,  Gao,  Y.,  Liu,  J.,  Feng,  Y.,  Li,  L.,  Shen,  W.,  Sun,  Z. and  Min,  L. (2019): Gestational exposure to low-dose zearalenone disrupting offspring spermatogenesis might be through epigenetic modifications. Basic Clin. Pharmacol. Toxicol., 125, 382-393.
•  Minervini,  F. and  Dell’Aquila,  M.E. (2008): Zearalenone and reproductive function in farm animals. Int. J. Mol. Sci., 9, 2570-2584.
•  Pérez-Martínez,  C.,  García-Iglesias,  M.J.,  Ferreras-Estrada,  M.C.,  Bravo-Moral,  A.M.,  Espinosa-Alvarez,  J. and  Escudero-Díez,  A. (1996): Effects of in-utero exposure to zeranol or diethylstilboestrol on morphological development of the fetal testis in mice. J. Comp. Pathol., 114, 407-418.
•  Qiu,  L.,  Qian,  Y.,  Liu,  Z.,  Wang,  C.,  Qu,  J.,  Wang,  X. and  Wang,  S. (2016): Perfluorooctane sulfonate (PFOS) disrupts blood-testis barrier by down-regulating junction proteins via p38 MAPK/ATF2/MMP9 signaling pathway. Toxicology, 373, 1-12.
•  Rai,  Y.,  Pathak,  R.,  Kumari,  N.,  Sah,  D.K.,  Pandey,  S.,  Kalra,  N.,  Soni,  R.,  Dwarakanath,  B.S. and  Bhatt,  A.N. (2018): Mitochondrial biogenesis and metabolic hyperactivation limits the application of MTT assay in the estimation of radiation induced growth inhibition. Sci. Rep., 8, 1531.
•  Rogakou,  E.P.,  Nieves-Neira,  W.,  Boon,  C.,  Pommier,  Y. and  Bonner,  W.M. (2000): Initiation of DNA fragmentation during apoptosis induces phosphorylation of H2AX histone at serine 139. J. Biol. Chem., 275, 9390-9395.
•  La Sala,  G.,  Farini,  D. and  De Felici,  M. (2010a): Rapid estrogen signalling in mouse primordial germ cells. Exp. Cell Res., 316, 1716-1727.
•  La Sala,  G.,  Farini,  D. and  De Felici,  M. (2010b): Estrogenic in vitro assay on mouse embryonic Leydig cells. Int. J. Dev. Biol., 54, 717-722.
•  Sang,  Y.,  Li,  W. and  Zhang,  G. (2016): The protective effect of resveratrol against cytotoxicity induced by mycotoxin, zearalenone. Food Funct., 7, 3703-3715.
•  Specian,  A.F.,  Serpeloni,  J.M.,  Tuttis,  K.,  Ribeiro,  D.L.,  Cilião,  H.L.,  Varanda,  E.A.,  Sannomiya,  M.,  Martinez-Lopez,  W.,  Vilegas,  W. and  Cólus,  I.M. (2016): LDH, proliferation curves and cell cycle analysis are the most suitable assays to identify and characterize new phytotherapeutic compounds. Cytotechnology, 68, 2729-2744.
•  Vogel,  M.W. (2002): Cell death, Bcl-2, Bax, and the cerebellum. Cerebellum, 1, 277-287.
•  Wang,  X.,  Jiang,  L.,  Shi,  L.,  Yao,  K.,  Sun,  X.,  Yang,  G.,  Jiang,  L.,  Zhang,  C.,  Wang,  N.,  Zhang,  H.,  Wang,  Y. and  Liu,  X. (2019): Zearalenone induces NLRP3-dependent pyroptosis via activation of NF-κB modulated by autophagy in INS-1 cells. Toxicology, 428, 152304.
•  Wu,  D.,  Huang,  C.J.,  Jiao,  X.F.,  Ding,  Z.M.,  Zhang,  S.X.,  Miao,  Y.L. and  Huo,  L.J. (2019): Bisphenol AF compromises blood-testis barrier integrity and sperm quality in mice. Chemosphere, 237, 124410.
•  Xu,  M.L.,  Hu,  J.,  Guo,  B.P.,  Niu,  Y.R.,  Xiao,  C. and  Xu,  Y.X. (2016): Exploration of intrinsic and extrinsic apoptotic pathways in zearalenone-treated rat sertoli cells. Environ. Toxicol., 31, 1731-1739.
•  Yang,  J.Y.,  Wang,  G.X.,  Liu,  J.L.,  Fan,  J.J. and  Cui,  S. (2007a): Toxic effects of zearalenone and its derivatives alpha-zearalenol on male reproductive system in mice. Reprod. Toxicol., 24, 381-387.
•  Yang,  J.,  Zhang,  Y.,  Wang,  Y. and  Cui,  S. (2007b): Toxic effects of zearalenone and alpha-zearalenol on the regulation of steroidogenesis and testosterone production in mouse Leydig cells. Toxicol. In Vitro, 21, 558-565.
•  Yi,  Y.,  Wan,  S.,  Hou,  Y.,  Cheng,  J.,  Guo,  J.,  Wang,  S.,  Khan,  A.,  Sun,  N. and  Li,  H. (2020): Chlorogenic acid rescues zearalenone induced injury to mouse ovarian granulosa cells. Ecotoxicol. Environ. Saf., 194, 110401.
•  Zatecka,  E.,  Ded,  L.,  Elzeinova,  F.,  Kubatova,  A.,  Dorosh,  A.,  Margaryan,  H.,  Dostalova,  P.,  Korenkova,  V., Hoskova and Peknicova, J. (2014): Effect of Zearalenone on reproductive parameters and expression of selected testicular genes inmice. Reprod. Toxicol., 45, 20-30.
•  Zhang,  F.L.,  Li,  N.,  Wang,  H.,  Ma,  J.M.,  Shen,  W. and  Li,  L. (2019): Zearalenone exposure induces the apoptosis of porcine granulosa cells and changes long noncoding RNA expression to promote antiapoptosis by activating the JAK2-STAT3 pathway. J. Agric. Food Chem., 67, 12117-12128.
•  Zhang,  G.L.,  Sun,  X.F.,  Feng,  Y.Z.,  Li,  B.,  Li,  Y.P.,  Yang,  F.,  Nyachoti,  C.M.,  Shen,  W.,  Sun,  S.D. and  Li,  L. (2017a): Zearalenone exposure impairs ovarian primordial follicle formation via down-regulation of Lhx8 expression in vitro. Toxicol. Appl. Pharmacol., 317, 33-40.
•  Zhang,  G.L.,  Zhang,  R.Q.,  Sun,  X.F.,  Cheng,  S.F.,  Wang,  Y.F.,  Ji,  C.L.,  Feng,  Y.Z.,  Yu,  J.,  Ge,  W.,  Zhao,  Y.,  Sun,  S.D.,  Shen,  W. and  Li,  L. (2017b): RNA-seq based gene expression analysis of ovarian granulosa cells exposed to zearalenone in vitro: significance to steroidogenesis. Oncotarget, 8, 64001-64014.
•  Zhang,  X.F.,  Choi,  Y.J.,  Han,  J.W.,  Kim,  E.,  Park,  J.H.,  Gurunathan,  S. and  Kim,  J.H. (2015): Differential nanoreprotoxicity of silver nanoparticles in male somatic cells and spermatogonial stem cells. Int. J. Nanomedicine, 10, 1335-1357.
•  Zhang,  Y.,  Jia,  Z.,  Yin,  S.,  Shan,  A.,  Gao,  R.,  Qu,  Z.,  Liu,  M. and  Nie,  S. (2014): Toxic effects of maternal zearalenone exposure on uterine capacity and fetal development in gestation rats. Reprod. Sci., 21, 743-753.
•  Zheng,  W.,  Pan,  S.,  Wang,  G.,  Wang,  Y.J.,  Liu,  Q.,  Gu,  J.,  Yuan,  Y.,  Liu,  X.Z.,  Liu,  Z.P. and  Bian,  J.C. (2016): Zearalenone impairs the male reproductive system functions via inducing structural and functional alterations of sertoli cells. Environ. Toxicol. Pharmacol., 42, 146-155.
•  Zheng,  W.L.,  Wang,  B.J.,  Wang,  L.,  Shan,  Y.P.,  Zou,  H.,  Song,  R.L.,  Wang,  T.,  Gu,  J.H.,  Yuan,  Y.,  Liu,  X.Z.,  Zhu,  G.Q.,  Bai,  J.F.,  Liu,  Z.P. and  Bian,  J.C. (2018): ROS-Mediated Cell Cycle Arrest and Apoptosis Induced by Zearalenone in Mouse Sertoli Cells via ER Stress and the ATP/AMPK Pathway. Toxins (Basel), 10, 24.
•  Zhou,  Y.,  Zhang,  D.,  Sun,  D. and  Cui,  S. (2020): Zearalenone affects reproductive functions of male offspring via transgenerational cytotoxicity on spermatogonia in mouse. Comp. Biochem. Physiol. C Toxicol. Pharmacol., 234, 108766.
•  Zhu,  L.,  Yuan,  H.,  Guo,  C.,  Lu,  Y.,  Deng,  S.,  Yang,  Y.,  Wei,  Q.,  Wen,  L. and  He,  Z. (2012): Zearalenone induces apoptosis and necrosis in porcine granulosa cells via a caspase-3- and caspase-9-dependent mitochondrial signaling pathway. J. Cell. Physiol., 227, 1814-1820.
•  Zinedine,  A.,  Soriano,  J.M.,  Moltó,  J.C. and  Mañes,  J. (2007): Review on the toxicity, occurrence, metabolism, detoxification, regulations and intake of zearalenone: an oestrogenic mycotoxin. Food Chem. Toxicol., 45, 1-18.