Abstract
Zearalenone (ZEA) mainly injures the reproductive system of mammals. In the present study, we aimed to explore the mechanism by which zinc inhibits ZEA-induced reproductive damage in KK-1 cells for the first time. The results shown that both zinc sulfate and zinc gluconate addition increased the intracellular zinc concentration and influenced the expression of zinc transporters (Slc30a1 and Slc39a1) in a time-dependent manner. Co-incubation of zinc with ZEA significantly reduced the ZEA-induced reactive oxygen species and malondialdehyde elevation by promoting the transcription of Mtf1 and Mt2. Meanwhile, two different zincs inhibited the ZEA-induced loss of mitochondrial membrane potential and elevation of late-stage apoptosis via activating the mitochondrial apoptotic pathway by recovering the mRNA and protein expression of pro-apoptotic genes (Bax, Casp3, Casp9). Zinc also recovered cells from S-phase cell cycle arrest. In addition, both of them promoted the ZEA-induced estrogen production but regulated the expression of steroidogenic enzymes (Star, Cyp11a1, Hsd3b1, Cyp17a1) in different way. All these results indicated that zinc could inhibit the reproductive toxicity of ZEA.
Similar content being viewed by others
Introduction
ZEA, a mycotoxin produced by several species of Fusarium, is commonly found as a contaminant in cereals. Animals and humans are exposed to ZEA by consuming cereals and their by-products. Reproductive system is the main target of this mycotoxin, many previous studies have evidenced that both male and female fertility are injured by ZEA and its derivatives. For example, Benzoni et al. used direct measures of sperm integrity to show the potential adverse effect of ZEA exposure on boar fertility and discovered that alpha-Zearalenol and ZEA, at picomolar levels, negatively influenced chromatin structure stability and viability, respectively, whereas beta-Zearalenol negatively influenced the sperm motility at micromolar levels1. Minervini et al. demonstrate a negative effect of ZEA and its derivatives on meiotic progression of bovine oocytes by induced dose-dependent oocyte maturation delay and chromatin abnormalities at levels ranging from 0.3 to 30 μg/ml2. ZEA even delayed the in-utero development of rats, Collins et al. found that the number of viable fetuses, the fetal body weight and the maturity of skeleton were all significantly decreased after gavaging ZEA at a dose of 4 or 8 mg/kg to pregnant female Sprague Dawley (SD) rats3.
Due to its widespread threat to health, the detoxification of ZEA has been of major interest to researchers. Physical, chemical and biological methods have been developed to reduce and/or eliminate the toxic effects of contaminated products, improve food safety and minimize economic losses4. However, the process of detoxification is often accompanied by a loss of palatability and nutritional value of the food crop. The addition of nutrients to contaminated foods is one approach that reduces the toxicity of mycotoxins. Antioxidants, such as N-acetyl cysteine, vitamins C and E, have been reported to protect animals and cultured cells against the toxic effects of the T-2 toxin and ZEA5,6,7.
Zinc, as an essential element, binds to 10% of proteins in the mammalian proteome and is a cofactor for over 300 enzymes and more than 2,000 transcription factors. Zinc deficiency causes growth retardation, immune dysfunction, cognitive impairment, metabolic disorders and infertility8. Zinc plays vital roles in the synthesis and metabolism of many types of hormones in vivo. Kralik et al. proved that zinc deficiency decreased the concentrations of triiodothyronine (T3) and free thyroxine (fT4) in serum by approximately 30% compared with zinc-adequate controls in SD rats, which indicated that zinc influences the levels of thyroid hormones9. Other studies have also shown that growth hormones and thymic hormones were affected by zinc supplementation or zinc deficiency10,11.
It has also been shown that the addition of zinc in feed was beneficial for animal reproduction and development. supplementation zinc in an organic form at the recommended level (300 mg of Zn/kg) for cows increased the milk yield and immunity12. The addition of zinc (800 ppm ZnCl2) inhibited the reduction of testis weight, the decrease of the epididymal sperm number and the remarkable degenerative lesions on the seminiferous tubules induced by low-dose mercury in male CD-1 mice13. Maternal zinc deficiency during pregnancy produced effects ranging from infertility, to embryonic/fetal death, to intrauterine growth retardation and teratogenesis14. Moreover, Batra et al. observed that zinc has protective effects against heavy metal-induced testis damage in male Portan rats13 due to its potential antioxidant properties. However, the potential for zinc to inhibit the reproductive toxicity of ZEA and the potential mechanisms are still unclear.
Due to the involvement of zinc in improving reproduction and the protective effects of zinc against several toxins, such as H2O2, heavy metals and ethanol15,16,17, we examined the effects of zinc on ZEA-induced reproductive toxicity. Because the ovary is a major target of ZEA toxicity, we employed immortalized murine ovarian granular KK-1 cells as an in vitro model to investigate the influence of zinc supplementation on the ZEA-induced reproductive damage at the molecular level. The present study is the first to explore both the protective effects of two sources of zinc and their mechanism of action in KK-1 cells from following aspects, including the cellular zinc response and homeostasis, oxidative stress, cell apoptosis and steroidogenesis.
Results
Zinc supplementation and zinc depletion both reduced the KK-1 cell viability
Because zinc and zinc chelator, N,N,N′N′-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN), can both be toxic to cells, we determined the optimal concentrations of these substances for subsequent experiments by performing a CCK8 assay. Growing cultures of KK-1 cells were exposed to increasing concentrations of zinc sulfate (ZnSO4) (from 0 to 150 μM), zinc gluconate (GZn) (from 0 to 150 μM) and TPEN, a specific zinc chelator, (from 0 to 20 μM) for 24 h. All of the treatments caused a decrease in the cell viability in a dose-dependent manner (Fig. 1A–C), which indicated that both zinc overload and zinc depletion induce the death of KK-1 cells. Therefore, we choose the maximal non-toxic concentration of 25 μM ZnSO4, 25 μM GZn and near the IC50 concentration (2.5 μM) of TPEN for further studies. As shown in Fig. 1D, the results of co-incubation indicated that 25 μM of the two sources zinc addition could significantly prevent the decrease in cell viability induced by ZEA. We chose to use 20 μM ZEA, which was a concentration slightly lower than the IC50, based on the previous results of CCK8 studies (data not shown) and the report by Li et al.18.
Both sources of zinc increased the intracellular zinc level in a time-dependent manner
To assess the relationship between zinc homeostasis and organic or inorganic zinc exposure, we used Zinpyr-1, a new membrane-permeant fluorescent sensor for Zn2+, to effectively label Zn2+in living cultured KK-1 cells. The uptake of Zinpyr-1 by cells is proportional to the zinc ion concentration. Figure 1E shows the findings in cells loaded with 25 μM ZnSO4, 25 μM GZn or 2.5 μM TPEN for different periods of time (1, 6, 12 and 24 h). The zinc fluorescence only decreased after 24 h of culture in the control, indicating that the fluorescence was stable over time. Both sources of zinc increased the intracellular zinc fluorescence in a time-dependent manner and the changes showed similar tendencies. A 1-h treatment made no significant difference compared with the control, but after a 6-h treatment, the zinc fluorescence increased markedly and it reached the peak value, which were 2.97 and 2.35-fold that observed in control cells, after another 6 h in culture. Between 12 h and 24 h, the zinc fluorescence decreased slightly, but remained high. Compared to the zinc supplementation groups, TPEN treatment decreased the zinc fluorescence from the start, with cultures beginning to show a significant reduction after 1-h of incubation.
We next evaluated the effects of the two types of zinc supplements on the transcription levels of the major zinc transport proteins, Slc30a1 and Slc39a1, by RT-PCR. We treated KK-1 cells with 25 μM ZnSO4 and 25 μM GZn for different lengths of time (1, 6, 12 and 24 h) (Fig. 1F,G) and found that the transcriptional level of Slc30a1 had increased dramatically by 6 h after the addition of 25 μM zinc (Fig. 1F). The Slc30a1 transcription level reached the peak values of 6.29- and 5.71-fold higher than the control (p < 0.05) after 12 h of treatment with ZnSO4 and GZn, respectively and it decreased and returned to the basal level after 24 h. This was similar to the previously reported zinc response of HeLa cells19. These results confirmed that zinc treatment induced Slc30a1 transcription. Of note, after 6 h of incubation, the transcription level of Slc39a1 had increased to peak values of 1.39- and 1.68-fold higher compared to the control (p < 0.05), but the level suddenly decreased below the basal level after 12 h and returned to the basal level after 24 h. This was likely due to the zinc ions aggregating at approximately 12 h. Based on these findings, we speculated that KK-1 cells reached a new zinc homeostasis as the intracellular zinc concentration increased, without any apparent effects on the cell viability after 24 h zinc supplementation.
Both sources of zinc protected KK-1 cells from oxidative damage
ROS generation was investigated using the 2′, 7′-dichlorodihydro fluorescein diacetate (DCFH-DA) fluorescent probe, which detects peroxide radicals and various other active oxygen radicals. Upon interaction with ROS, the DCFH-DA is converted to fluorescent DCF. Therefore, the level of intracellular ROS can be evaluated by analyzing the fluorescence of DCF. As shown in Fig. 2A, the level of intracellular ROS notably increased with the addition of ZEA and zinc supplementation significantly reduced the ZEA-induced ROS production. Zheng et al. also showed that zinc ions could protect cells from ROS damage20. There were no significant differences between the cells treated with inorganic zinc and organic zinc. Lipid peroxidation was measured by determining the MDA production (Fig. 2B). Similar to the ROS generation, zinc supplementation significantly reduced the ZEA-induced MDA production, also with no significant differences noted between inorganic and organic zinc.
The transcription of Mtf1, Mt2 and Sod1 was examined to clarify the effects of zinc and ZEA treatment (Fig. 2). The KK-1 cells were first treated with 25 μM ZnSO4 and 25 μM GZn for different time period (1, 6, 12 and 24 h). The Mt2 mRNA level increased dramatically during the period from 1 h to 6 h after zinc treatment, reaching levels of 19.50- and 21.43-fold higher for ZnSO4 and GZn, respectively, than the corresponding levels in the control. On the contrary, the transcription level of Sod1 decreased by nearly 30% compared to the control in response to zinc treatment and the time course had no effect on the Sod1 mRNA level. Second, it was observed that the exposure of KK-1 cells to 20 μM ZEA decreased the mRNA expression of Mt2 to half and the level of Mtf1 to 63% of the control, it also increased the mRNA expression of Sod1, although the change was not significant. The addition of both sources of zinc apparently inhibited the decrease in the mRNA expression of Mt2 and Mtf1 and slightly reduced the expression of Sod1 induced by ZEA. There were no differences between the two sources of zinc with regard to the inhibition of ZEA-induced oxidative stress.
The two sources of zinc protect KK-1 cells from apoptosis
As shown in Fig. 3A, both types of zinc significantly reversed the ZEA-induced loss of mitochondrial membrane potential (Δψm). In detail, a marked decrease in the Δψm by approximately 50% occurred when cells were treated with 20 μM ZEA. However, co-incubation with 25 μM ZnSO4 or GZn for 24 h rescued the ZEA-induced Δψm loss by 19.88% and 21.20%, respectively, compared to the ZEA group. There was no difference between the effects of the two types of zinc. A decrease in the Δψm is an early indicator of cell apoptosis19, so we also tested the effects of zinc supplementation on the KK-1 cells by comparing the apoptosis rates. As shown in Fig. 3B, most of the cells were in the late stage of apoptosis. The rate was 2.84% in the control group and increased to 8.78% in the ZEA group, which was reduced to 5.43% and 4.09% in the Z + Zn and Z + GZn groups, respectively (p < 0.05). The total cell death, including early apoptotic and late apoptotic cells, showed the same tendency.
All of the above results indicated that the addition of zinc efficiently protected KK-1 cells from apoptosis. To confirm this finding, we accumulated more evidence by performing a RT-PCR analysis of the expression of three pro-apoptotic genes: Bax, Casp9 and Casp3. Two sources of zinc inhibited the increased transcription levels of Bax (Fig. 3C), Casp3 (Fig. 3D) and Casp9 (Fig. 3E). Western blotting shown that both inorganic and organic zinc could down-regulate the expression of BAX and inhibit the activation of CASP9 (Fig. 4A,B). On the other hand, although adding zinc alone could reduce the level of Casp3 mRNA, co-incubation with zinc could not significantly prevent the ZEA-induced increase in expression (Fig. 3D). In summary, zinc exerted cytoprotective effects against ZEA-induced apoptosis and GZn was better than ZnSO4 in inhibiting the Casp9 expression and late-stage apoptosis.
Zinc supplementation prevents the ZEA-induced S-phase arrest
The cell cycle distribution was examined by flow cytometry. As shown in Fig. 4C, the treatment with ZEA increased the number of cells in the S phase from 25.70% to 47.26% and decreased the number of cells in the G0/G1 and G2/M phases from 66.10% and 8.2% to 48.59% and 4.15%, respectively. In addition, co-incubation of ZnSO4 and GZn with ZEA reduced the percentage of cells in the S phase to 35.95% and 35.24%, whereas the number of cells in the G0/G1 phases increased to 60.12% and 62.24% and those in the G2/M phases increased to 6.18% and 7.60%, respectively. Zinc supplementation could significantly prevent the ZEA-induced S-phase arrest, with no significant differences noted between organic zinc and inorganic zinc.
Both sources of zinc affect the steroidogenesis in KK-1 cells
As a type of reproductive cell, KK-1 cells are unique because they have the ability to synthesize steroid hormones. We therefore assessed the effects of the two sources of zinc on the estrogen production by a radioimmunosorbent assay (RIA) (Fig. 5E). Both zinc and ZEA could promote estrogen production, with levels of 5.51, 5.87 and 8.71 pg/mg protein respectively, in the ZnSO4-, GZn- and ZEA-treated groups in comparison to the control level of 3.72 pg/mg protein. Moreover, co-incubation with these zinc sources and ZEA led to synergistic increases to 15.53 and 19.72 pg/mg protein, respectively.
In addition to evaluating the hormone synthesis, we also examined the mRNA levels of four important genes encoding steroidogenic enzymes required for progesterone or estrogen production in KK-1 cells (Star, Cyp11a1, Cyp17a1 and Hsd3b1)in the different groups (Fig. 5A–D). We validated the expression of Star and Cyp11a1 at the protein level by Western blotting (Fig. 5F,G). Interestingly, 25 μM ZnSO4 and 25 μM GZn affected the expression levels of all four enzymes, but in different ways (Table 1). Co-incubation with ZnSO4 mainly increased the mRNA and protein levels of Star while co-incubation with GZn elevated the mRNA levels of Cyp11a1,Cyp17a1and Hsd3b1. The mRNA and protein expression of Star and Cyp11a1 displayed different trends. This may have resulted from the delayed induction of enzyme synthesis21. In the early 1990s, Hanukoglu et al. found that brief (30 min) stimulation of adrenocortical cells with adrenocorticotropin (ACTH) led to both a rapid increase in steroid secretion and a delayed increase in enzyme synthesis that peaked 36 h later22. The above results indicated that both of the sources of zinc promoted estrogen production, but they influenced the steroidogenic enzyme levels in different ways.
Discussion
The effects of ZEA-induced reproductive toxicity have been widely investigated in vivo, especially in sensitive animal species, such as swine23. Likely because of the multiple interactions that occur in whole organisms during ZEA exposure, the results of in vitro studies may only be partial agreement with those of in vivo studies, but cell-specific responses can only be understood from in vitro investigations. Moreover, in vitro experiments may contribute to understanding the direct biological effects of ZEA and defining its mechanism of action, which would help understand the effects of ZEA on the testes and ovaries24,25. Thus in the present study, cultured KK-1 cells were employed to explore the protection mechanism of two distinct sources of zinc on ZEA-induced reproductive toxicity in vitro.
Oxidative stress, inducing an imbalance between the level of ROS and the cellular defense system26,27, has been proposed as a possible mechanism of reproductive damage in both human and animals28. Municipal landfill leachate induced the reproductive toxicity to male rats by increased sperm abnormalities and disrupted the antioxidant systems of rats sperm with concomitant elevation in hydrogen peroxide and malondialdehyde levels29. The cyclophosphamide -treated group showed enhancement of lipid peroxidation leading to testicular reproductive toxicity while total flavonoids of epimedium restored these oxidative damages by up-regulating the expression of antioxidant enzymes, especially SOD3 and GPX130. Yang et al. illustrated that carbon disulfide directly induced DNA damage in endometrial cells and enhanced the action through oxidative stress, both of which were responsible for carbon disulfide-induced embryo loss in pregnant female mice31.
In this study, co-incubation of zinc with ZEA significantly reduced the ZEA-induced ROS generation and lipid peroxidation, indicating that zinc increased the antioxidant processes in KK-1 cells. Then, we explored the potential mechanism of action by detecting the mRNA levels of Mt2, Mtf1 and Sod1. Mt2, ubiquitous and stress-inducible in mice, encodes one type of metallothioneins which in favor of scavenging ROS32. As shown in Fig. 2, treatment with either of the two sources of zinc strongly increased the transcription level of Mt2 and zinc addition significantly inhibited the ZEA-induced decrease in Mt2. Mtf1 has been proven to be an essential cell stress responder following oxidative stress33. When cells are treated with heavy metals, it is activated to bind the metal response element34, which induces the transcription of target genes, notably metallothioneins, which have MRE binding sites in the 5′ regulatory region15,35. In our study, the expression of Mtf1 changed in the same way as Mt2, which indicated that zinc regulated the transcription of Mt2 through the activation of the Mtf1. However, zinc supplementation inversely decreased the expression of Sod1 and there was no apparent change in the expression in ZEA-treated cells following zinc supplementation (Fig. 2E,F). Zheng et al. also found that Sod1 was not influenced by ZnSO4 in HepG2 cells20. However, there has been a lot of evidence proving that zinc increased the activity of Sod136. These results indicated that although zinc plays an important role in the function of Sod1, it has no obvious effects on the transcription of the gene.
In summary, zinc could enhance the resistance of KK-1 cells to the reproductive toxicity of ZEA by inhibiting the ZEA-induced oxidative stress via activation of the transcription expression of antioxidative genes: Mtf1 and Mt2.
Apoptosis is a highly regulated form of programmed cell death, which plays a key role in the development and homeostasis of reproductive systems in vitro and vivo. Ovarian granular cells, which grow around the mammalian oocytes in the ovary, can promote follicle growth and provide energy for the oocytes. In vivo, a high level of granular cell apoptosis will affect the normal maturation process of follicles, resulting in follicular atresia37. In vitro, many previous studies have demonstrated that xenobiotics could promote mouse Leydig or spermatogenic cells apoptosis which was one of the main mechanisms of their reproductive toxicity38,39. As an early event in apoptosis, mitochondrial dysfunction is always considered40. Chayma Bouaziz et al. reported that ZEA induced mitochondrial alterations in HepG2 cells, including a loss of the Δψm, PTPC opening and cytochrome c release41. Guo et al. demonstrated that zinc deficiency in osteoblastic MC3T3-E1 cells induced cell apoptosis via the mitochondria-mediated pathway, resulting in a reduction of the Δψm and increased levels of BAX in the mitochondrial fraction and increased levels of cytochrome c, apoptosis-inducing factor (AIF) and cleaved of CASP3 and CASP9 in the cytosolic fraction42. Similarly, our present study showed that zinc supplementation inhibited the increase in late-stage apoptosis and the loss of Δψm in KK-1 cells, which had been induced by ZEA. The mRNA expression of pro-apoptotic genes including Bax, Casp3 and Casp9, were determined by RT-PCR (Fig. 3C–E). In addition, the BAX level decreased markedly and the cleavage of CASP9 was inhibited after co-incubation. All of these results indicated that the two sources of zinc inhibited the apoptosis of KK-1 cells via the Caspase-dependent mitochondrial apoptotic pathway.
In a word, zinc could enhance the resistance of KK-1 cells to ZEA-induced reproductive toxicity by inhibiting the ZEA-induced apoptosis via regulating key genes in the Caspase-dependent mitochondrial apoptotic pathway. Moreover, the proliferation of a cell population is regulated by the balance among cell division, growth arrest, differentiation and programmed cell death43. Under our experimental conditions, both inorganic and organic zinc could inhibit the ZEA-induced S-phase cell cycle arrest. However, the exact mechanism leading to this effect will need to be elucidated in further studies.
In mice, the pathways of steroid hormone biosynthesis starts with cholesterol44. Trophic hormones (e.g., ACTH, luteinizing hormone (LH) and FSH) activate a chain of reactions that lead STAR to translocate cholesterol from the outer to the inner mitochondrial membrane, where it is converted to pregnenolone by CYP11A1, which is present in the inner mitochondrial membrane in all steroidogenic cells45. After this rate-limiting step, pregnenolone is converted to progesterone by HSD3B1 in the microsomal compartment. Progesterone is then partly converted to testosterone by CYP17A1 and HSD17B1. Finally, CYP19A1 converts testosterone to estradiol. However, in the ovary, CYP17A1 is expressed in the theca interna cells. Antibody and cDNA probes have shown very low expression of Cyp17a1 in the granulosa cells of humans and rats46,47,48. Thus, theca interna cells can synthesize androgens, but granulosa cells which produce estrogens are dependent on the androgen precursor supply from the theca interna. This process is called the two cell hypothesis of follicular estrogen production49. In our experiments, we added testosterone to activate the steroid hormone biosynthesis pathway and to serve as the substrate for estrogen synthesis.
In the present study, we investigated the effects of zinc on steroidogenic enzymes and estrogen production in vitro for the first time and the results are described in section 3.6 (Results). In brief, we found that both zinc and ZEA could promote estrogen production and co-incubation likely led to a synergistic effect. Previous studies have shown that adding exogenous 17β-estradiol may be protective against oxidative stress-induced damage and even apoptosis both in vivo and in vitro. For example, Wang et al. found that 17β-estradiol could ameliorate light-induced retinal damage in SD rats via its antioxidative effects and they showed that its underlying mechanism involves the regulation of the gene expression levels of antioxidant enzymes (SOD, CAT and GPX) and proteins (TRX and NRF2)50. Kanda et al. provided evidence that 17β-estradiol inhibits oxidative stress-induced apoptosis in keratinocytes by promoting Bcl2 expression51. Thus, we speculate that the slight increase in estrogen promoted by ZEA may have been a stress response to the ZEA-induced oxidative damage and both sources of zinc enhanced this response. However, further investigations will be needed to confirm whether this is the case.
Generally, there are considered to be three types of zinc sources; inorganic zinc (e.g., zinc sulfate, zinc oxide and zinc chloride) and organic zinc which includes zinc salts of organic acids (e.g., zinc gluconate, zinc citrate, zinc acetate) and zinc chelates of amino acids or peptides (e.g., zinc methionine, lysine zinc)52. Different zinc sources have been widely used in animal nutrition studies to alleviate zinc deficiency.
Lin et al. evaluated zinc methionine (ZnMet), zinc lysine (ZnLys), zinc glycine (ZnGly) and ZnSO4 as dietary zinc sources for Litopenaeus vannamei. Their results showed that when the shrimp were fed diets with 6.5 mg Zn/kg diet, organic zinc supplementation produced significantly higher growth, survival and immune parameters than ZnSO4 treatment. Shrimp supplemented with ZnMet had the highest weight gain and best immune parameters. However, there were no significant differences between the ZnLys and ZnGly groups. These results suggest that the zinc from ZnMet was a better source than the other zinc forms53.
The bioavailability of different zinc compounds (sulfate, gluconate and citrate) was also compared in the male rat prostate after adding three different doses (3.0, 15.0 and 50.0 mg Zn/kg b.w.) to their diet for 30 days. Only zinc gluconate and zinc citrate increased the zinc concentrations in the dorsolateral lobe of the prostate compared to controls54. In addition, Wegmüller et al. used the double-isotope tracer method with 67Zn and 70Zn and found that the median (IQR) fractional absorption of zinc from zinc oxide (49.9%) was significantly lower than that from zinc citrate (61.3%) and zinc gluconate (60.9%), but there was no marked difference between two organic zinc sources55. However, few studies have focused on the differences between sources of zinc in vitro. Yu et al. investigated the effects of different zinc sources and levels on the inhibition of the thymocyte apoptosis induced by glucocorticoid in vitro. Zinc sulfate and zinc methionine were supplemented into the medium at levels of 0, 50, 100, 500 and 1000 μM. The intracellular calcium concentrations of the cells cultured with zinc methionine were higher than those cultured with zinc sulfate at the same levels, but both of them could modulate the glucocorticoid-induced apoptosis of thymocytes. The mechanism might involve the exchange of intracellular calcium or the redox of cells and the two forms of zinc might have different effects on the regulation of these processes56.
Above all, we insured that zinc sulfate and zinc gluconate could inhibit the reproductive toxicity of ZEA on KK-1 cells.
Methods
Chemicals and reagents
Fetal bovine serum (FBS) was obtained from HyClone,USA; 100 U/ml penicillin, 100 μg/ml streptomycin, 250 ng/ml amphotericin B, high-glucose Dulbecco’s minimal essential medium (DMEM), 0.25% trypsin (w/v) and 0.52 mM EDTA were purchased from Macgene, PRC. ZnSO4·7H2O, GZn, follicle-stimulating hormone (FSH), testosterone (T) and the TPEN, were purchased from Sigma,USA.
Cell cultured and treatments
KK-1 cells were cultured in DMEM supplemented with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin and 250 ng/ml amphotericin B at 37 °C in a humidified 5% CO2 incubator (Sanyo, JP).
The cells were divided into different experimental groups according to the study requirements. The different agents used for the treatments were diluted with serum-free medium and cells were treated for 24 h. The cells were dispersed with 0.25% trypsin (w/v) and 0.52 mM EDTA.
Cell viability assay
Cell viability was determined using the Cell Counting Kit-8 (Beyotime, PRC) according to the manufacturer’s instructions. Briefly, 1 × 104 cells/well were seeded in a 96-well plate and grown at 37 °C for 24 h, then were washed once with phosphate-buffered saline (PBS) after being treated in 5 technical replicates. Subsequently, 10 μl of WST-8 and 100 μl of PBS were added to each well and the cells were incubated at 37 °C for 2 h. Finally, the dye absorbance was determined at 450 nm using a microplate reader (Thermo, USA).
Zn2+ concentration determination
The intracellular Zn2+ level in the KK-1 cells was measured using the Zn2+ indicator, Zinpyr-1 (Sigma, USA). Cells were cultured in 6-well plates at a density of 2 × 105 cells/well, grown at 37 °C for 24 h and treated in 3 technical replicates. Then, the cells were loaded with 12 mM Zinpyr-1 solution with serum-free medium and incubated at 37 °C for 20 min. Before detecting the fluorescence intensity using a FACSCalibur instrument (BD Biosciences, USA), the cells were rinsed twice with PBS to remove the fluorescent probe and were dispersed by trypsin. The emission was detected in the FL1. At least 1 × 104 cells in the selected gate were collected for further analyses.
ROS determination
ROS generation was measured using a fluorescent probe assay kit (Beyotime, PRC), which contained DCFH-DA. The protocol was as follows: Cells were cultured in 6-well plates at a density of 2 × 105 cells/well, grown at 37 °C for 24 h and treated in 3 technical replicates. The cells were then washed once with PBS and incubated for 30 min at 37 °C with 10 μM of the probe. Finally, before detecting the fluorescence intensity using the FACSCalibur instrument, the cells were rinsed twice with PBS and dispersed by trypsin. The fluorescence intensity in FL1 was proportional to the ROS production. At least 1 × 104 cells in the selected gate were collected for further analyses.
MDA assay
KK-1 cells were cultured at 6 × 105 cells/ml and seeded onto 60 mm dishes. The cells were treated for 24 h and then harvested by trypsinization, followed by washing twice with PBS. Cell lysates were homogenized and centrifuged at 15,000 × g for 15 min at 4 °C after being lysed in RIPA buffer (Beyotime, PRC)
The MDA assay was performed with a Lipid Peroxidation Kit (Nanjing Jiancheng, PRC) following the protocol provided by the manufacturer. Briefly, 100 μl of supernatant was mixed with 1 ml of malondialdehyde (MDA) working solution and was boiled at 95 °C for 45 min. Samples were cooled down to room temperature in a water bath and then centrifuged at 1,000 × g for 10 min. The absorbance at 532 nm was measured in a 96-well plate (250 μl/well) with a microplate reader (Thermo, USA). The results of the MDA assay were expressed as micromoles of MDA per milligram of protein, which was measured using a BCA Kit (Beyotime, PRC)57.
Δψm measurement
Δψm was measured using JC-1 dye. The cells were seeded on 6-well plates and exposed to different treatments. 24 h later, the cells were incubated with 5 μg/ml of JC-1 staining solution (Beyotime, PRC) for 20 min at 37 °C. After the cells were washed twice with the JC-1 staining buffer, the fluorescence densities of the JC-1 monomers (λex = 488 nm, λem = 529 nm) and JC-1 aggregates (λex = 524 nm, λem = 594 nm) were detected using a microplate reader. The Δψm of the KK-1 cells was expressed as the fluorescence intensity ratio of the JC-1 aggregates to that of the JC-1 monomers. Three technical replicates were carried out in each group.
Cell apoptosis
Cellular apoptosis was determined using the Annexin V-FITC Apoptosis Detection Kit (Nanjing Jiancheng, PRC). According to the protocol, KK-1 cells were cultured at 6 × 105 cells/ml and seeded onto 60 mm dishes. The cells were treated for 24 h and then harvested by trypsinization, followed by washing twice with cold PBS. Approximately 1 × 105 to 1 × 106 cells were resuspended in 200 μl of 1 × binding buffer and were transferred to a sterile flow cytometry glass tube. The cells were incubated with 5 μl Annexin V-FITC and 10 μl propidium iodide (PI) in the dark at room temperature. The apoptotic cells were identified and analyzed using a FACSCalibur instrument. The emission wavelengths of Annexin V-FITC and PI were detected in the FL1 and FL2 channels. The percentages of normal, early apoptotic, late apoptotic and necrotic cells were calculated using the CellQuest software program.
Cell cycle
A cell cycle analysis was conducted according to the method described by Cai et al.58 with slight modifications. Briefly, cells were cultured and synchronized before they were divided for six different experiments in three technical replicates, then were harvested in PBS-0.05% trypsin buffer. After centrifugation for 5 min at 1500 rpm at 4 °C, the supernatant was removed and the pellet was resuspended in ice-cold 70% ethanol. Cells were digested with 2 mg/ml RNase A at 37 °C for 20 min and were stained with 50 g/ml PI containing 0.1% Triton X-100 and EDTA 0.02 mg/ml. Samples were analyzed for their DNA content and cell cycle distribution using a FACSCalibur flow cytometer with the ModFit software program.
E2 determination by RIA
KK-1 cells were cultured at 1 × 105 cells/ml and seeded onto 12-well plates. The cells were treated for 24 h, followed by washing twice with PBS. Then, 1 ml/well of serum-free medium containing 75 ng/ml of FSH and 5 μg/ml of T was added for another 4 h at 37 °C. Finally, the serum-free medium solution was collected from every well and the level of E2 was measured following the manufacturer’s instructions for the E2 Kit (Beijing North Biological Institute, PRC) using a RIA method. The results were expressed as the micromoles of E2 per milligram of protein, which was measured using a BCA Kit.
RNA extraction and RT-PCR
Total RNA was isolated from the cells using the TRIzol reagent (CWBio, PRC) according to the manufacturer’s instructions. The integrity of the purified RNA was tested on a 1% agarose gel and was quantified spectrophotometrically at 260 nm and 280 nm. First-strand cDNA was synthesized from 2 μg of RNA using the Reverse Transcription System (Promega, USA) according to the manufacturer’s protocol using oligo (dT) as the primers. The RT-PCR reaction was performed using the Real Master Mix (SYBRGreen, Tiangen, PRC) with 200 nM of forward primer, 200 nM of reverse primer (Supplemental Table 1) and 100 ng cDNA as the template. The RT-PCR was performed on a 7500 Real-Time PCR System (Applied Biosystems, USA) with the following conditions: 95 °C for 5 min and then 40 cycles of 95 °C for 30 s, 60 °C/64 °C for 30 s and 72 °C for 30 s. The GAPDH gene was selected as the control gene. The relative expression of each target gene, which was calculated using the average value of the ΔCt between the target gene and the control gene (GAPDH), was expressed as 2−ΔΔCt.
Protein isolation and Western blotting
Following different treatments, the cells were lysed on ice with RIPA Lysis Buffer supplemented with 1 mM PMSF (Beyotime, PRC). Cells were then homogenized using a 1 ml syringe59,60. Cellular lysates were centrifuged at 13,000 × g for 10 min at 4 °C. The supernatant proteins were collected and quantified using a BCA Kit. Equal amounts (40 μg) of protein from each sample were loaded on 13% SDS-PAGE gels and blotted on nitrocellulose membranes for 1.5 h at 80 V. Nonspecific binding was blocked by incubating the samples in blocking buffer (1% BAS and Tris-buffered saline containing 0.1% Tween-20 (TBST)) for 1 h and the membranes were incubated for 1 h with one of the following primary antibodies: rabbit anti-GAPDH (Cell Signaling, 4970; 1:1000), rabbit anti-BAX (Cell Signaling, 2772; 1:1000), mouse anti-CASP9 (Cell Signaling, 9508; 1:1000), rabbit anti-STAR (Cell Signaling, 8449; 1:1000), or rabbit anti-CYP11A1 (Santa Cruz, 292456; 1:1000).
Statistics
The data were expressed as the means ± standard deviation. The experiments were repeated at least twice and each experiment included at least triplicate treatments. The data from different treatments were subjected to an analysis of variance (ANOVA) and the comparisons of the means were performed using Duncan’s multiple range test. All of the statistical analyses were performed using the SPSS 16.0 software program. In all figures, the characters above the error bar indicate that there are significant differences between the compared groups (p < 0.05).
Additional Information
How to cite this article: Li, Y. et al. Zinc inhibits the reproductive toxicity of Zearalenone in immortalized murine ovarian granular KK-1 cells. Sci. Rep. 5, 14277; doi: 10.1038/srep14277 (2015).
References
Benzoni, E. et al. Influence of in vitro exposure to mycotoxin zearalenone and its derivatives on swine sperm quality. Reprod Toxicol 25, 461–467 (2008).
Minervini, F., Dell’Aquila, M. E., Maritato, F., Minoia, P. & Visconti, A. Toxic effects of the mycotoxin zearalenone and its derivatives on in vitro maturation of bovine oocytes and 17 beta-estradiol levels in mural granulosa cell cultures. Toxicology in vitro : an international journal published in association with BIBRA 15, 489–495 (2001).
Collins, T. F. et al. Effects of zearalenone on in utero development in rats. Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association 44, 1455–1465 (2006).
Zinedine, A., Soriano, J. M., Molto, J. C. & Manes, J. Review on the toxicity, occurrence, metabolism, detoxification, regulations and intake of zearalenone: an oestrogenic mycotoxin. Food and chemical toxicology 45, 1–18 (2007).
Ghedira-Chekir, L. et al. Induction of a SOS repair system in lysogenic bacteria by zearalenone and its prevention by vitamin E. Chemico-biological interactions 113, 15–25 (1998).
Abid-Essefi, S. et al. DNA fragmentation, apoptosis and cell cycle arrest induced by zearalenone in cultured DOK, Vero and Caco-2 cells: prevention by Vitamin E. Toxicology 192, 237–248 (2003).
Venkataramana, M. et al. Zearalenone induced toxicity in SHSY-5Y cells: The role of oxidative stress evidenced by N-acetyl cysteine. Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association 65, 335–342 (2014).
Jeong, J. & Eide, D. J. The SLC39 family of zinc transporters. Molecular aspects of medicine 34, 612–619 (2013).
Kralik, A., Eder, K. & Kirchgessner, M. Influence of zinc and selenium deficiency on parameters relating to thyroid hormone metabolism. Hormone and metabolic research = Hormon- und Stoffwechselforschung = Hormones et metabolisme 28, 223–226 (1996).
Cabrera, Á. J. R. Zinc, aging and immunosenescence: an overview. Pathobiology of aging & age related diseases 5 (2015), 10.3402/pba.v5.25592.
Livingstone, C. Zinc Physiology, Deficiency and Parenteral Nutrition. Nutrition in Clinical Practice 30, 371–382 (2015), doi: 10.1177/0884533615570376.
Cope, C., Mackenzie, A., Wilde, D. & Sinclair, L. Effects of level and form of dietary zinc on dairy cow performance and health. Journal of dairy science 92, 2128–2135 (2009).
Batra, N., Nehru, B. & Bansal, M. The effect of zinc supplementation on the effects of lead on the rat testis. Reproductive Toxicology 12, 535–540 (1998).
Uriu-Adams, J. Y. & Keen, C. L. Zinc and reproduction: effects of zinc deficiency on prenatal and early postnatal development. Birth Defects Research Part B: Developmental and Reproductive Toxicology 89, 313–325 (2010).
Chung, M. J., Walker, P. A., Brown, R. W. & Hogstrand, C . Zinc-mediated gene expression offers protection against H2O2-induced cytotoxicity. Toxicology and applied pharmacology 205, 225–236 (2005).
Rudolf, E. & Červinka, M. The role of intracellular zinc in chromium (VI)-induced oxidative stress, DNA damage and apoptosis. Chemico-biological interactions 162, 212–227 (2006).
Szuster-Ciesielska, A., Plewka, K., Daniluk, J. & Kandefer-Szerszeń, M. Zinc inhibits ethanol-induced HepG2 cell apoptosis. Toxicology and applied pharmacology 229, 1–9 (2008).
Li, Y. et al. Analysis of individual and combined effects of ochratoxin A and zearalenone on HepG2 and KK-1 cells with mathematical models. Toxins 6, 1177–1192 (2014).
Devergnas, S. et al. Differential regulation of zinc efflux transporters ZnT-1, ZnT-5 and ZnT-7 gene expression by zinc levels: a real-time RT–PCR study. Biochemical pharmacology 68, 699–709 (2004).
Zheng, J. et al. Zinc protects HepG2 cells against the oxidative damage and DNA damage induced by ochratoxin A. Toxicology and applied pharmacology 268, 123–131 (2013).
Hanukoglu, I. Steroidogenic enzymes: structure, function and role in regulation of steroid hormone biosynthesis. The Journal of steroid biochemistry and molecular biology 43, 779–804 (1992).
Hanukoglu, I., Feuchtwanger, R. & Hanukoglu, A. Mechanism of corticotropin and cAMP induction of mitochondrial cytochrome P450 system enzymes in adrenal cortex cells. Journal of Biological Chemistry 265, 20602–20608 (1990).
Marin, D., Taranu, I., Pistol, G. & Stancu, M. Effects of zearalenone and its metabolites on the swine epithelial intestinal cell line: IPEC 1. Proceedings of the Nutrition Society 72, E40 (2013).
Penning, T. et al. Human 3α-hydroxysteroid dehydrogenase isoforms (AKR1C1–AKR1C4) of the aldo-keto reductase superfamily: functional plasticity and tissue distribution reveals roles in the inactivation and formation of male and female sex hormones. Biochem. J 351, 67–77 (2000).
Minervini, F. & Dell’Aquila, M. E. Zearalenone and reproductive function in farm animals. International journal of molecular sciences 9, 2570–2584 (2008).
Othmen, Z. O.-B., El Golli, E., Abid-Essefi, S. & Bacha, H. Cytotoxicity effects induced by Zearalenone metabolites, α Zearalenol and β Zearalenol, on cultured Vero cells. Toxicology 252, 72–77 (2008).
Yu, S.-W. et al. Outer mitochondrial membrane localization of apoptosis-inducing factor: mechanistic implications for release. ASN neuro 1, AN20090046 (2009).
Dorostghoal, M., Seyyednejad, S. M. & Jabari, A. Protective effects of Fumaria parviflora L. on lead-induced testicular toxicity in male rats. Andrologia 46, 437–446 (2014).
Adedara, I. A. et al. Sperm functional parameters and erythrocytes oxidant-antioxidant imbalance during municipal landfill leachate treatment withdrawal in rats. Environmental toxicology and pharmacology 37, 460–467 (2014).
Yuan, D. et al. Protective effects of total flavonoids from Epimedium on the male mouse reproductive system against cyclophosphamide-induced oxidative injury by up-regulating the expressions of SOD3 and GPX1. Phytotherapy research : PTR 28, 88–97 (2014).
Yang, L., Zhang, B., Yuan, Y., Li, C. & Wang, Z. Oxidative stress and DNA damage in utero and embryo implantation of mice exposed to carbon disulfide at peri-implantation. Human & experimental toxicology 33, 424–434 (2014).
Heuchel, R., Radtke, F. & Schaffner, W. in Inducible Gene Expression, Volume 1, 206–240 (Springer, 1995).
Dalton, T. P., Li, Q., Bittel, D., Liang, L. & Andrews, G. K. Oxidative Stress Activates Metal-responsive Transcription Factor-1 Binding Activity OCCUPANCY IN VIVO OF METAL RESPONSE ELEMENTS IN THE METALLOTHIONEIN-I GENE PROMOTER. Journal of Biological Chemistry 271, 26233–26241 (1996).
Sanden, M., Jorgensen, S., Hemre, G. I., Ornsrud, R. & Sissener, N. H. Zebrafish (Danio rerio) as a model for investigating dietary toxic effects of deoxynivalenol contamination in aquaculture feeds. Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association 50, 4441–4448 (2012).
Durnam, D. & Palmiter, R. Transcriptional regulation of the mouse metallothionein-I gene by heavy metals. Journal of Biological Chemistry 256, 5712–5716 (1981).
Saïd, L., Banni, M., Kerkeni, A., Saïd, K. & Messaoudi, I. Influence of combined treatment with zinc and selenium on cadmium induced testicular pathophysiology in rat. Food and Chemical Toxicology 48, 2759–2765 (2010).
Hidiroglou, M. & Knipfel, J. Z inc in mammalian sperm: a review. Journal of dairy science 67, 1147–1156 (1984).
Zhang, D. Y., Xu, X. L., Shen, X. Y., Ruan, Q. & Hu, W. L. Analysis of apoptosis induced by perfluorooctane sulfonates (PFOS) in mouse Leydig cells in vitro. Toxicology mechanisms and methods 25, 21–25 (2015).
Xu, Y. et al. The mechanism study of endosulfan-induced spermatogenic cell apoptosis of mouse in vitro. Toxicology and industrial health (2015), doi: 10.1177/0748233714567525.
Tomasello, F. et al. Outer membrane VDAC1 controls permeability transition of the inner mitochondrial membrane in cellulo during stress-induced apoptosis. Cell research 19, 1363 (2009).
Bouaziz, C. et al. Different apoptotic pathways induced by zearalenone, T-2 toxin and ochratoxin A in human hepatoma cells. Toxicology 254, 19–28 (2008).
Guo, B. et al. Cell apoptosis induced by zinc deficiency in osteoblastic MC3T3-E1 cells via a mitochondrial-mediated pathway. Molecular and cellular biochemistry 361, 209–216 (2012).
Spengler, D. et al. Regulation of apoptosis and cell cycle arrest by Zac1, a novel zinc finger protein expressed in the pituitary gland and the brain. The EMBO journal 16, 2814–2825 (1997).
Stocco, D. M. StAR protein and the regulation of steroid hormone biosynthesis. Annual Review of Physiology 63, 193–213 (2001).
Farkash, Y., Timberg, R. & Orly, J. Preparation of Antiserum to Rat Cytochrome P-450 Cholesterol Side Chain Cleavage and Its Use for Ultrastructural Localization of the Immunoreactive Enzyme by Protein A-Gold Technique*. Endocrinology 118, 1353–1365 (1986).
Voutilainen, R., Tapanainen, J., Chung, B.-C., Matteson, K. J. & Miller, W. L. Hormonal Regulation of P450scc (20, 22-desmolase) and P450cl7 (17α-hydroxylase/17, 20-lyase) in Cultured Human Granulosa Cells*. The Journal of Clinical Endocrinology & Metabolism 63, 202–207 (1986).
Hedin, L., Rodgers, R., Simpson, E. & Richards, J. Changes in content of cytochrome P450 (17) alpha, cytochrome P450scc and 3-hydroxy-3-methylglutaryl CoA reductase in developing rat ovarian follicles and corpora lutea: correlation with theca cell steroidogenesis. Biology of reproduction 37, 211–223 (1987).
Sasano, H. et al. Immunolocalization of aromatase, 17α-hydroxylase and side-chain-cleavage cytochromes P-450 in the human ovary. Journal of reproduction and fertility 85, 163–169 (1989).
Jerome, F. et al. CHAPTER 8—The Ovarian Life Cycle. In Yen & Jaffe’s Reproductive Endocrinology (Sixth Edition), 155–190 (2009), doi: 10.1016/B978-1-4160-4907-4.00008-5.
Wang, S. et al. 17β-Estradiol Ameliorates Light-Induced Retinal Damage in Sprague–Dawley Rats by Reducing Oxidative Stress. Journal of Molecular Neuroscience 55, 141–151 (2015).
Kanda, N. & Watanabe, S. 17β-estradiol inhibits oxidative stress-induced apoptosis in keratinocytes by promoting Bcl-2 expression. Journal of Investigative Dermatology 121, 1500–1509 (2003).
Lönnerdal, B. Dietary factors influencing zinc absorption. The Journal of nutrition 130, 1378S–1383S (2000).
Lin, S., Lin, X., Yang, Y., Li, F. & Luo, L. Comparison of chelated zinc and zinc sulfate as zinc sources for growth and immune response of shrimp (Litopenaeus vannamei). Aquaculture 406, 79–84 (2013).
Sapota, A. et al. The bioavailability of different zinc compounds used as human dietary supplements in rat prostate: a comparative study. BioMetals 27, 495–505 (2014).
Wegmüller, R., Tay, F., Zeder, C., Brnić, M. & Hurrell, R. F. Zinc absorption by young adults from supplemental zinc citrate is comparable with that from zinc gluconate and higher than from zinc oxide. The Journal of nutrition 144, 132–136 (2014).
Ze-peng, Y., Guo-wei, L., Hong-yu, H., Yun-yu, W. & Yong-hui, S. Effect of different zinc sources and levels on inhibition of the apoptosis induced by glucocorticoid of thymocytes in vitro. Biol Trace Elem Res 105, 215–227 (2005).
Wang, N., Wang, Y., Yu, G., Yuan, C. & Ma, J. Quinoprotein adducts accumulate in the substantia nigra of aged rats and correlate with dopamine-induced toxicity in SH-SY5Y cells. Neurochemical research 36, 2169–2175 (2011).
Cai, X. et al. Luteolin induced G2 phase cell cycle arrest and apoptosis on non-small cell lung cancer cells. Toxicology in Vitro 25, 1385–1391 (2011).
Lim, J. et al. Binding preference of p62 towards LC3-ll during dopaminergic neurotoxin-induced impairment of autophagic flux. Autophagy 7, 51–60 (2011).
Shen, X. L. et al. An iTRAQ-based mitoproteomics approach for profiling the nephrotoxicity mechanisms of ochratoxin A in HEK 293 cells. Journal of proteomics 78, 398–415 (2013).
Acknowledgements
This work was funded by the Program for New Century Excellent Talents in University (2014FG046). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors thank the Beijing North Biological Institutes for their kind support of the E2 detection.
Author information
Authors and Affiliations
Contributions
K.H., W.X., Y.L. and X.H. conceived and designed the experiments. YJ.L. performed the experiments. YJ.L. and X.Y. analyzed the data. L.Z. and YZ.L. contributed to the reagents/materials/analysis tools. YJ.L., X.Y. and W.X. wrote the paper.
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Electronic supplementary material
Rights and permissions
This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/
About this article
Cite this article
Li, Y., He, X., Yang, X. et al. Zinc inhibits the reproductive toxicity of Zearalenone in immortalized murine ovarian granular KK-1 cells. Sci Rep 5, 14277 (2015). https://doi.org/10.1038/srep14277
Received:
Accepted:
Published:
DOI: https://doi.org/10.1038/srep14277
This article is cited by
-
Mitigative effects of zinc on cadmium-induced reproductive toxicity in the male freshwater crab Sinopotamon henanense
Environmental Science and Pollution Research (2020)
-
Urinary mycoestrogens and age and height at menarche in New Jersey girls
Environmental Health (2019)
-
Zearalenone altered the cytoskeletal structure via ER stress- autophagy- oxidative stress pathway in mouse TM4 Sertoli cells
Scientific Reports (2018)
-
Zinc enhances the cellular energy supply to improve cell motility and restore impaired energetic metabolism in a toxic environment induced by OTA
Scientific Reports (2017)
-
Proanthocyanidin protects against acute zearalenone-induced testicular oxidative damage in male mice
Environmental Science and Pollution Research (2017)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.