Titanium dioxide nanoparticles induce apoptosis through ROS-Ca2+-p38/AKT/mTOR pathway in TM4 cells.

Titanium dioxide nanoparticles (TiO2 NPs) can cause apoptosis in TM4 cells; however, the underlying mechanism has not been entirely elucidated. The purpose of this study was to investigate the effects of TiO2 NPs on ROS, Ca2+ level, p38/AKT/mTOR pathway, and apoptosis in TM4 cells and to evaluate the role of Ca2+ in p38/AKT/mTOR pathway and apoptosis. After exposure to different concentrations (0, 50, 100, 150, and 200 µg/mL) of TiO2 NPs for 24 h, cell viability, ROS, Ca2+ level, Ca2+-ATPase activity, p38/AKT/mTOR pathway-related proteins, apoptosis rate, and apoptosis-related proteins (Bax, Bcl-2, Caspase 3, Caspase 9, and p53) were detected. The ROS scavenger NAC was used to determine the effect of ROS on Ca2+ level. The Ca2+ chelator BAPTA-AM was used to evaluate the role of Ca2+ in p38/AKT/mTOR pathway and apoptosis. TiO2 NPs significantly inhibited cell viability, increased ROS level, and elevated Ca2+ level while suppressing Ca2+-ATPase activity. TiO2 NPs regulated the p38/AKT/mTOR pathway via increasing p-p38 level and decreasing p-AKT and p-mTOR levels. TiO2 NPs significantly enhanced the apoptosis. NAC attenuated Ca2+ overload and reduction in Ca2+-ATPase activity caused by TiO2 NPs. BAPTA-AM alleviated TiO2 NPs-induced abnormal expression of p38/AKT/mTOR pathway-related proteins. BAPTA-AM assuaged the apoptosis caused by TiO2 NPs. Altogether, this study revealed that TiO2 NPs elevated intracellular Ca2+ level through ROS accumulation. Subsequently, the heightened intracellular Ca2+ level was observed to exert regulation over the p38/AKT/mTOR pathway, ultimately culminating in apoptosis. These results provides a complementary understanding to the mechanism of TiO2 NPs-induced apoptosis in TM4 cells.

Cadmium exposure induces pyroptosis of TM4 cells through oxidative stress damage and inflammasome activation.

Cadmium (Cd) is a harmful metal that seriously affects the male reproductive system, but the mechanism of how Cd exposure damages Sertoli cells is not fully understood. This study used TM4 cells to explore the mechanism of Cd damage to Sertoli cells. We found that Cd was concentration- and time-dependent on TM4 cell viability. Cd exposure increased intracellular reactive oxygen species (ROS) levels, lactate dehydrogenase (LDH), and Interleukin-1ß (IL-1ß) release in TM4 cells, decreased mitochondrial function, and increased pyroptosis. N-acetylcysteine (NAC), MCC950 and BAY 11-7082 (BAY) alleviate the release of IL-1ß and LDH induced by Cd. NAC reduced Cd induced increases in ROS, NLRP3, Caspase-1, Heme oxygenase-1(HO-1), superoxide dismutase (SOD2), and increased mitochondrial function. The activation of GSDMD is the main causes of pyroptosis, and NAC significantly inhibit its activation and formation. Our results suggest that Cd exposure induces a toxic mechanism of GSDMD-mediated pyroptosis in TM4 cells by increasing ROS levels and activating the inflammasome.

Compound probiotics regulate the NRF2 antioxidant pathway to inhibit aflatoxin B1-induced autophagy in mouse Sertoli TM4 cells.

This study investigated the effects of compound probiotics (CP) on AFB1-induced cytotoxicity in Sertoli TM4 cells. The L9 (3 × 3) orthogonal test was conducted to determine the optimal CP required for high AFB1 degradation in the artificial gastrointestinal fluid in vitro. The maximal AFB1 degradation rate was 40.55 % (P < 0.05) when the final viable count was 1.0 × 105 CFU/mL for Bacillus subtilis, Lactobacillus casein, and Saccharomyces cerevisiae. The effects of CP and the CP supernatant (CPS) on TM4 cell viability were evaluated to achieve the optimal protective conditions. When CPS4 (corresponding to CP viable counts of 1.0 × 104 CFU/mL) was added to the TM4 cells for 24 h, the cell viability reached 108.86 % (P < 0.05). AFB1 reduced TM4 cell viability in a concentration- and time-dependent manner at an AFB1 concentration ranging from 0 to 1.5 µM after 48-h AFB1 exposure. The optimal AFB1 concentration/times for low- and high damage models were 0.5 and 1.25 µM both for 24 h, which decreased viability to 76.04 % and 65.35 %, respectively. however, CPS4 added to low- and high-damage models increased the cell viability to 97.43 % and 75.12 %, respectively (P < 0.05). Transcriptome sequencing was performed based on the following designed groups: the control, 0.5 µM AFB1, 1.25 µM AFB1, CPS4, and CPS4+0.5 µM AFB1. The Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis was further performed to identify significantly enriched signaling pathways, which were subsequently verified. It was shown that AFB1 induced apoptosis by blocking the PI3K-AKT-mTOR pathway and upregulating autophagy proteins such as LC3B, Beclin1, and ATG5 while inhibiting autophagic flux. CPS4 promoted AFB1 degradation, activated the p62-NRF2 antioxidant, and inhibited ROS/TRPML1 pathways, thereby reducing ROS production and inflammation and ultimately alleviating AFB1-induced autophagy and apoptosis. These findings supports the potential of probiotics to protect the male reproductive system from toxin damage.

Assessment of the toxicity of different antiretroviral drugs and their combinations on Sertoli and Leydig cells.

The human immunodeficiency virus continues to pose a significant global public health challenge, affecting millions of individuals. The current treatment strategy has incorporated the utilization of combinations of antiretroviral drugs. The administration of these drugs is associated with many deleterious consequences on several physiological systems, notably the reproductive system. This study aimed to assess the toxic effects of abacavir sulfate, ritonavir, nevirapine, and zidovudine, as well as their combinations, on TM3 Leydig and TM4 Sertoli cells. The cell viability was gauged using 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) and neutral red uptake (NRU) assays. Reactive oxygen species (ROS) production was assessed via the 2',7'-dichlorofluorescein diacetate (DCFDA) test, and DNA damage was determined using the comet assay. Results indicated cytotoxic effects at low drug concentrations, both individually and combined. The administration of drugs, individually and in combination, resulted in the production of ROS and caused damage to the DNA at the tested concentrations. In conclusion, the results of this study suggest that the administration of antiretroviral drugs can lead to testicular toxicity by promoting the generation of ROS and DNA damage. Furthermore, it should be noted that the toxicity of antiretroviral drug combinations was shown to be higher compared to that of individual drugs.

Long-term exposure to polystyrene microplastics triggers premature testicular aging.

BACKGROUND: Plastic pollution is greatly serious in the ocean and soil. Microplastics (MPs) degraded from plastic has threatened animals and humans health. The accumulation of MPs in the tissues and blood in animals and humans has been found. There is therefore a need to assess the toxicological effects of MPs on the reproductive system. RESULTS: In this study, we explored the effect of polystyrene microplastics (PS-MPs) on premature testicular aging in vitro and in vivo. In vitro, we found that testicular sertoli cells (TM4 cells) was prematurely senescent following PS-MPs treatment by the evaluation of a range of aging marker molecules (such as Sa-ß-gal, p16 and 21). TM4 cells were then employed for in vitro model to study the potential molecular mechanism by which PS-MPs induce the premature senescence of TM4 cells. NF-κB is identified as a key molecule for PS-MPs-induced TM4 cellular senescence. Furthermore, through eliminating reactive oxygen species (ROS), the activation of nuclear factor kappa B (NF-κB) was blocked in PS-MPs-induced senescent TM4 cells, indicating that ROS triggers NF-κB activation. Next, we analyzed the causes of mitochondrial ROS (mtROS) accumulation induced by PS-MPs, and results showed that Ca2+ overload induced the accumulation of mtROS. Further, PS-MPs exposure inhibits mitophagy, leading to the continuous accumulation of senescent cells. In vivo, 8-week-old C57 mice were used as models to assess the effect of PS-MPs on premature testicular aging. The results illustrated that PS-MPs exposure causes premature aging of testicular tissue by testing aging markers. Additionally, PS-MPs led to oxidative stress and inflammatory response in the testicular tissue. CONCLUSION: In short, our experimental results revealed that PS-MPs-caused testicular premature aging is dependent on Ca2+/ROS/NF-κB signaling axis. The current study lays the foundation for further exploration of the effects of microplastics on testicular toxicology.

[KEAP1/PGAM5/AIFM1 mediated oxeiptosis pathway in TDCIPP-induced reduction of TM4 cell viability].

OBJECTIVE: To investigate the toxic effects and potential mechanisms of tri(1, 3-dichloro-2-propyl) phosphate(TDCIPP) exposure on the mouse testicular supporting cell line(TM4 cells). METHODS: TM4 cells were treated with different concentrations of TDCIPP(0, 12.5, 25 and 50 µmol/L), or 50 µmol/L TDCIPP combined with antioxidant N-acetylcysteine(NAC) for 24 h. Cell viability was assessed using the CCK8 assay, intracellular ROS levels were detected using the DCFH-DA probe, and the protein levels of oxeiptosis-related proteins, such as KEAP1, PGAM5, AIFM1 and phosphorylated AIFM1(p-AIFM1), were detected using Western blot. RESULTS: TDCIPP dose-dependently reduced TM4 cell viability(P<0.05). ROS levels in TM4 cells treated with 12.5, 25 and 50 µmol/L TDCIPP were 9.44±1.42, 17.25±1.81 and 18.38±2.66, respectively, significantly higher than the control group's 5.08±0.90(P<0.05). ROS levels in the 5 mmol/L NAC+50 µmol/L TDCIPP group were 14.70±0.50, significantly lower than the corresponding TDCIPP group's 26.44±0.73(P<0.05). The activity of TM4 cells in KEAP1siRNA+TDCIPP group and PGAM5siRNA+TDCIPP group were 77.00±1.73 and 76.67±1.53, respectively, significantly higher than TDCIPP group 68.67±1.53(P<0.05). The relative expression of KEAP1 protein in TM4 cells treated with 25 and 50 µmol/L TDCIPP were 0.77±0.04 and 0.82±0.02, respectively, significantly higher than the control group's 0.57±0.01(P<0.05). The relative expression of PGAM5 protein in TDCIPP-treated TM4 cells were 1.17±0.04, 1.38±0.03 and 1.41±0.03, respectively, significantly higher than the control group's 0.81±0.02(P<0.05). The relative expression of AIFM1 protein were 0.42±0.01, 0.63±0.01 and 0.68±0.02, respectively, significantly higher than the control group's 0.34±0.02(P<0.05). The relative expression of p-AIFM1 protein were 1.73±0.02, 1.52±0.02 and 0.73±0.01, respectively, significantly lower than the control group's 2.25±0.02(P<0.05). In the 5 mmol/L NAC+50 µmol/L TDCIPP group, the relative expression of KEAP1, PGAM5 and AIFM1 proteins in TM4 cells were 0.61±0.01, 0.58±0.01 and 0.48±0.03, respectively, significantly lower than the TDCIPP group's 0.86±0.12(P<0.05), 0.74±0.02(P<0.05) and 0.92±0.01(P<0.05). The relative expression of p-AIFM1 protein in the 5 mmol/L NAC+50 µmol/L TDCIPP group was 0.45±0.11, significantly higher than the TDCIPP group's 0.23±0.01(P<0.05). CONCLUSION: The reduction of TM4 cell viability induced by TDCIPP may be related to ROS-mediated regulation of the KEAP1/PGAM5/AIFM1 pathway, leading to oxeiptosis.

Mechanism of 2,4-Dichlorophenoxyacetic acid-induced damage to rat testis via Fas/FasL pathway and the protective effect of Lycium barbarum polysaccharides.

The herbicide 2,4-Dichlorophenoxyacetic acid (2,4-D) is widely used to control broadleaved weeds and has been associated with male infertility. We studied the molecular mechanisms of 2,4-D induced male reproductive system damage and the protective effects of Lycium barbarum polysaccharides (LBP) using Sprague Dawley rats and TM4 cells. Treatment with 2,4-D caused architectural and functional changes in the testis, including collapsed and atrophied seminiferous tubules with reduced number of spermatozoa, scarce sperm in the epididymal duct, low levels of serum testosterone, decreased superoxide dismutase and glutathione peroxidase activity, high malondialdehyde content, and increased apoptosis in the testis and epididymis. The expression of Fas, FasL, FADD, Pro-caspase-8, Cleaved-Caspase-8, Pro-Caspase-3, and Cleaved-Caspase-3 were significantly increased in the testicular tissue of 2,4-D-treated rats. The proliferative activity of TM4 cells decreased with an increase in dose and time of 2,4-D exposure, along with enhanced Fas/Fas ligand expression and a decreased concentration of inhibin B in TM4 cell culture medium. Depletion of Fas by specific shRNA transfection reversed the effects of 2,4-D in TM4 cells, further confirming the involvement of death receptor pathway in 2,4-D-mediated apoptosis of sertoli cells. Treatment with LBP also reversed the effects of 2,4-D in testicular cells, resulting in improved cell architecture along with enhanced proliferative capacity. Moreover, in response to LBP treatment of Sertoli cells, the content of inhibin B increased, the level of reactive oxygen species and malondialdehyde decreased, the activities of superoxide dismutase and glutathione peroxidase increased, and the rate of apoptosis as well as the expression of Fas/Fas ligand signaling pathway proteins decreased.

Synergistic Activity of Ketoconazole and Miconazole with Prochloraz in Inducing Oxidative Stress, GSH Depletion, Mitochondrial Dysfunction, and Apoptosis in Mouse Sertoli TM4 Cells.

Triazole and imidazole fungicides represent an emerging class of pollutants with endocrine-disrupting properties. Concerning mammalian reproduction, a possible causative role of antifungal compounds in inducing toxicity has been reported, although currently, there is little evidence about potential cooperative toxic effects. Toxicant-induced oxidative stress (OS) may be an important mechanism potentially involved in male reproductive dysfunction. Thus, to clarify the molecular mechanism underlying the effects of azoles on male reproduction, the individual and combined potential of fluconazole (FCZ), prochloraz (PCZ), miconazole (MCZ), and ketoconazole (KCZ) in triggering in vitro toxicity, redox status alterations, and OS in mouse TM4 Sertoli cells (SCs) was investigated. In the present study, we demonstrate that KCZ and MCZ, alone or in synergistic combination with PCZ, strongly impair SC functions, and this event is, at least in part, ascribed to OS. In particular, azoles-induced cytotoxicity is associated with growth inhibitory effects, G0/G1 cell cycle arrest, mitochondrial dysfunction, reactive oxygen species (ROS) generation, imbalance of the superoxide dismutase (SOD) specific activity, glutathione (GSH) depletion, and apoptosis. N-acetylcysteine (NAC) inhibits ROS accumulation and rescues SCs from azole-induced apoptosis. PCZ alone exhibits only cytostatic and pro-oxidant properties, while FCZ, either individually or in combination, shows no cytotoxic effects up to 320 µM.

Vitamin C mitigates heat damage by reducing oxidative stress, inducing HSP expression in TM4 Sertoli cells.

Heat stress is a major stressor that can lead to male reproductive dysfunction. Sertoli cells play a crucial role in spermatogenesis by providing germ cells with structural and nutritional support, and contributing to blood-testis barrier formation. Vitamin C (Vc) is an antioxidant capable of neutralizing reactive oxygen species and preventing lipid peroxidation widely used because it is inexpensive and highly accessible. In the present study, we investigated the protective effect of Vc on TM4 cells following heat stress. Pretreatment with Vc could effectively inhibit apoptosis (p < 0.01), lipid peroxidation, and lactate dehydrogenase (LDH) activity. However, a significant increase in the malondialdehyde (MDA) level and LDH activity (p < 0.01) was observed in TM4 cells without Vc-pretreatment, in conjunction with vacuole degeneration and karyopyknosis. In addition, both the messenger RNA and protein levels of CryAB, Hsp27, Hsp70, and Hsp110 substantially increased in the 3 and 12 hr recovery groups (p < 0.01). Vc also prevented microtubule aggregation following heat stress. These results suggest that pretreatment with Vc-protected TM4 cells against heat stress by reducing the level of oxidative stress and inducing heat shock protein expression.

Downregulation of steroid hormone receptor expression and activation of cell signal transduction pathways induced by a chiral nonylphenol isomer in mouse sertoli TM4 cells.

Nonylphenols (NPs) are considered as important environmental toxicants and potential endocrine disrupting compounds which can disrupt male reproductive system. 4-[1-Ethyl-1-methylhexy] phenol (4-NP65 ) is one of the main isomers of technical nonylphenol mixtures. In the present study, effect of NPs was evaluated from an isomer-specific viewpoint using 4-NP65 . Decreased mRNA expression levels of estrogen receptor (ER)-α, ER-ß, androgen receptor (AR) and progesterone receptor (PR) were observed in the cells exposed to 4-NP65 for 24 h. Furthermore, 4-NP65 treatment evoked significant decrease in protein expression levels of ER-α and ER-ß. Levels of mullerian inhibiting substance and transferrin were found to change significantly in 4-NP65 challenged cells. Additionally, JNK1/2-MAPK pathway was activated due to 4-NP65 exposure, but not ERK1/2 and p38-MAPK pathways. Meanwhile, 4-NP65 increased the p-Akt level and showed no effects on the Akt level which indicated that Akt pathway was activated by 4-NP65 . In conclusion, these findings have shown that 4-NP65 exposure affected expression of cell receptors and cell signaling pathways in Sertoli TM4 cells. We proposed that molecular mechanism of reproductive damage in Sertoli cells induced by NPs may be mediated by cell receptors and/or cell signal transduction pathways, and that the effects were dependent on the side chain of NP isomers. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 469-476, 2017.

Nonylphenol regulates cyclooxygenase-2 expression via Ros-activated NF-κB pathway in sertoli TM4 cells.

The aim of this study was to investigate the signaling pathways involved in the cyclooxygenase (COX)-2 regulation induced by nonylphenol (NP) in mouse testis Sertoli TM4 cells. Our results showed that treatment of TM4 cells with NP increased COX-2 protein expression and interleukin-6 (IL)-6 and prostaglandin E2 (PGE2) secretion in a dose-dependent manner. Pretreatment with reactive oxygen species (ROS) scavenger, N-acetylcysteine (NAC), attenuated NP-induced ROS production, COX-2 expression, and IL-6 and PGE2 release in TM4 cells. Exposure to NP stimulated activation of NF-κB, whereas the NF-κB inhibitor, pyrrolidine dithiocarbamate, attenuated NP-enhanced COX-2 expression and IL-6 and PGE2 release in TM4 cells in a dose-dependent manner. Furthermore, NAC blocked NP-induced activation of NF-κB. In addition, inhibition of COX-2 mitigated NP-induced IL-6 release. In conclusion, NP induced ROS generation, activation of NF-κB pathway, COX-2 upregulation, and IL-6 and PGE2 secretion in TM4 cells. NP may regulate COX-2 expression via ROS-activated NF-κB pathway in Sertoli TM4 cells.

Nickel induces blood-testis barrier damage through ROS-mediated p38 MAPK pathways in mice.

Nickel (Ni) is an essential common environmental contaminant, it is hazardous to male reproduction, but the precise mechanisms are still unknown. Blood-testis barrier (BTB), an important testicular structure consisting of connections between sertoli cells, is the target of reproductive toxicity caused by many environmental toxins. In this study, ultrastructure observation and BTB integrity assay results indicated that NiCl2 induced BTB damage. Meanwhile, BTB-related proteins including the tight junction (TJ), adhesion junction (AJ) and the gap junction (GJ) protein expression in mouse testes as well as in sertoli cells (TM4) were significantly decreased after NiCl2 treatment. Next, the antioxidant N-acetylcysteine (NAC) was co-treated with NiCl2 to study the function of oxidative stress in NiCl2-mediated BTB deterioration. The results showed that NAC attenuated testicular histopathological damage, and the expression of BTB-related proteins were markedly reversed by NAC co-treatment in vitro and vivo. Otherwise, NiCl2 activated the p38 MAPK signaling pathway. And, NAC co-treatment could significantly inhibit p38 activation induced by NiCl2 in TM4 cells. Furthermore, in order to confirm the role of the p38 MAPK signaling pathway in NiCl2-induced BTB impairment, a p38 inhibitor (SB203580) was co-treated with NiCl2 in TM4 cells, and p38 MAPK signaling inhibition significantly restored BTB damage induced by NiCl2 in TM4 cells. These results suggest that NiCl2 treatment destroys the BTB, in which the oxidative stress-mediated p38 MAPK signaling pathway plays a vital role.

Deoxynivalenol Induces Blood-Testis Barrier Dysfunction through Disrupting p38 Signaling Pathway-Mediated Tight Junction Protein Expression and Distribution in Mice.

Deoxynivalenol (DON) is widely present in cereals and processed grains. It can disrupt the blood-testicular barrier (BTB), leading to sterility in males; however, the mechanism is unknown. In this study, 30 Kunming mice and TM4 cells were exposed to 0 or 4.8 mg/kg (28 d) and 0-2.4 µM (24 h) of DON, respectively. Histopathological findings showed that DON increased BTB permeability in mice, leading to tight junction (TJ) structural damage. Immunofluorescence results indicated that DON disrupted the localization of zonula occludens (ZO)-1. The results of protein and mRNA expression showed that the expression of ZO-1, occludin, and claudin-11 was reduced, and that the p38/GSK-3ß/snail and p38/ATF-2/MLCK signaling pathways were activated in mouse testes and TM4 cells. Pretreatment with the p38 inhibitor SB203580 maintained TJ integrity in TM4 cells after exposure to DON. Thus, DON induced BTB dysfunction in mice by disrupting p38 pathway-mediated TJ expression and distribution.

Oxidative Stress, Cytotoxic and Inflammatory Effects of Azoles Combinatorial Mixtures in Sertoli TM4 Cells.

Triazole and imidazole fungicides are an emerging class of contaminants with an increasing and ubiquitous presence in the environment. In mammals, their reproductive toxicity has been reported. Concerning male reproduction, a combinatorial activity of tebuconazole (TEB; triazole fungicide) and econazole (ECO; imidazole compound) in inducing mitochondrial impairment, energy depletion, cell cycle arrest, and the sequential activation of autophagy and apoptosis in Sertoli TM4 cells (SCs) has recently been demonstrated. Given the strict relationship between mitochondrial activity and reactive oxygen species (ROS), and the causative role of oxidative stress (OS) in male reproductive dysfunction, the individual and combined potential of TEB and ECO in inducing redox status alterations and OS was investigated. Furthermore, considering the impact of cyclooxygenase (COX)-2 and tumor necrosis factor-alpha (TNF-α) in modulating male fertility, protein expression levels were assessed. In the present study, we demonstrate that azoles-induced cytotoxicity is associated with a significant increase in ROS production, a drastic reduction in superoxide dismutase (SOD) and GSH-S-transferase activity levels, and a marked increase in the levels of oxidized (GSSG) glutathione. Exposure to azoles also induced COX-2 expression and increased TNF-α production. Furthermore, pre-treatment with N-acetylcysteine (NAC) mitigates ROS accumulation, attenuates COX-2 expression and TNF-α production, and rescues SCs from azole-induced apoptosis, suggesting a ROS-dependent molecular mechanism underlying the azole-induced cytotoxicity.

Nanosized Titanium Dioxide Induced Apoptosis and Abnormal Expression of Blood-Testis Barrier Junction Proteins Through JNK Signaling Pathway in TM4 Cells.

Nanosized titanium dioxide (nano-TiO2) has been widely used in consumer products. It can cross the blood-testis barrier (BTB), and it has adverse effects on the male reproductive system. However, the specific mechanism has not been fully elucidated. The purpose of this study was to understand the role of the JNK signaling pathway in the apoptosis and abnormal expression of BTB junction proteins induced by nano-TiO2 in TM4 cells. After different concentration of nano-TiO2 treatments, the cell viability, apoptosis, mitochondrial membrane potential (Δψm), BTB junction proteins (Claudin-11, ZO-1, ß-catenin), apoptosis-related proteins (Bax, Bcl-2, cleaved caspase-9, cleaved caspase-3), and phosphorylated (p)-JNK protein were examined. The results showed that cell viability, apoptosis rates, Δψm, and apoptosis-related protein levels changed in a concentration-dependent manner. Cell viability decreased significantly from 100 µg/mL nano-TiO2 group. Apoptosis rates increased significantly from 150 µg/mL nano-TiO2 group, and Δψm decreased significantly from 150 µg/mL nano-TiO2 group. The protein levels of Bax, cleaved caspase-9, and cleaved caspase-3 increased significantly from 150 µg/mL nano-TiO2 group, and the protein level of Bcl-2 decreased significantly from 100 µg/mL nano-TiO2 group. The protein level of p-JNK increased significantly from 100 µg/mL nano-TiO2 group. Abnormal expression of ZO-1 and ß-catenin started from 150 µg/mL nano-TiO2 group, and abnormal expression of Claudin-11 started from 100 µg/mL nano-TiO2 group. Cells were treated with JNK inhibitor SP100625 to determine whether the changes of the above indicators in the concentration of 150 µg/mL nano-TiO2 group can be reversed. We found that SP100625 at 20 µM significantly reversed these effects. These results highlighted that nano-TiO2 could activate the JNK signaling pathway to induce mitochondria-mediated apoptosis and abnormal expression of BTB junction proteins in TM4 cells.

Exposure to zinc oxide nanoparticles affects testicular structure, reproductive development and spermatogenesis in parental and offspring male rats.

Background: This study aimed to comprehensively evaluate the toxicity exerted by zinc oxide nanoparticles (ZnO NPs) on rat testis and its effects on fertility and progeny development. Methods: Different concentrations of ZnO NPs were administered by gavage to Sprague Dawley (SD) rats to examine the adverse effects resulting from pre- and post-natal exposure. Systemic distribution of ZnO NPs, developmental performance, sperm parameters, reproductive performance, histopathological examination, and sex hormone levels were determined scheduled in the experimental rats and their male offspring. The comparative in vitro cytotoxicity of the ZnO NPs was determined among C18-4, TM3, and TM4 cells. The toxicity exerted by ZnO NPs on germ cells in vitro and the effects on the expression of cytoskeleton and blood-testis barrier (BTB)-related proteins were also determined. Results: After oral gavage, ZnO NPs mainly accumulated in the liver and testes of rats; 350 mg/kg ZnO NPs adversely affected the epididymal weight, sperm motility, and hormone levels but did not affect the fertility of rats. In addition, 350 mg/kg ZnO NPs significantly reduced the reproductive and developmental performance of offspring male rats. Testicular histopathological and electron microscopic ultrastructure examinations showed more significant abnormal structural changes than those observed in parental rats. The results of in vitro cell experiments further showed that ZnO NPs exerted cytotoxic effects on germ cells, and led to DNA damage, nucleoskeleton and cytoskeleton alterations, and could regulate actin changes through changes in LC3B. Conclusions: It is possible that ZnO NPs act directly on TM4 cells by penetrating the BTB, causing damage to the cytoskeleton and disrupting the dynamic balance of the BTB, thereby destroying the microenvironment necessary for spermatogenesis, which may lead to poor reproduction in rats.

Role of PI3K/Akt-Mediated Nrf2/HO-1 Signaling Pathway in Resveratrol Alleviation of Zearalenone-Induced Oxidative Stress and Apoptosis in TM4 Cells.

Zearalenone (ZEA) is a common mycotoxin that induces oxidative stress (OS) and affects the male reproductive system in animals. Resveratrol (RSV) has good antioxidant activity and can activate nuclear factor erythroid 2-related factor (Nrf2) to protect cells through the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway. The objective of this study was to investigate the protective effect and the mechanism of RSV on OS and apoptosis in TM4 cells induced by ZEA. Prior to being exposed to ZEA, TM4 cells were pretreated with RSV or the PI3K/Akt inhibitor LY294002. Cell viability was measured by Cell Counting Kit-8 (CCK-8) assays. Flow cytometry was used to determine the level of apoptosis and intracellular reactive oxygen species (ROS). The expression of poly ADP-ribose polymerase (PARP), caspase-3, BCL2-associated X (Bax)/B-cell lymphoma-2 (Bcl-2), and PI3K/Akt-mediated Nrf2/heme oxygenase 1 (HO-1) signaling pathway-related proteins was evaluated by Western blotting. Nrf2 siRNA transfection and LY294002 treatment were used to investigate the role of the Nrf2/HO-1 and PI3K/Akt signaling pathways in RSV alleviation of ZEA-induced OS. The results showed that pretreatment with RSV significantly reduced the expression of apoptosis-related proteins and increased cell viability. Catalase (CAT) activity and glutathione (GSH) levels were also increased, whereas malondialdehyde (MDA) and ROS levels decreased (p < 0.05). RSV also upregulated Akt phosphorylation, Nrf2 nuclear translocation, and HO-1 expression under conditions of OS (p < 0.05). Transfection with Nrf2 siRNA abolished the protective effects of RSV against ZEA-induced cytotoxicity (p < 0.05), ROS accumulation (p < 0.05), and apoptosis (p < 0.05). LY294002 completely blocked the RSV-mediated increase in Nrf2 nuclear translocation (p < 0.05), HO-1 expression (p < 0.05), and cytoprotective activity (p < 0.05). Collectively, the above findings indicate that RSV can protect against ZEA-induced OS and apoptosis in TM4 cells by PI3K/Akt-mediated activation of the Nrf2/HO-1 signaling pathway.

4-[1-Ethyl-1-methylhexy]-phenol induces apoptosis and interrupts Ca2+ homeostasis via ROS pathway in Sertoli TM4 cells.

Biological effect of an individual nonylphenol (NP) isomer extremely relies upon the side chain structure. This research was designed to evaluate the impact of NP isomer, 4-[1-ethyl-1-methylhexy]-phenol (NP65), on Sertoli cells in vitro. Sertoli TM4 cells were exposed to various concentration (0, 0.1, 1, 10, or 20 µM) of NP65 for 24 h, and the outcomes indicated that treatment of NP65 induced reactive oxygen species (ROS) generation, oxidative stress, and apoptosis for Sertoli TM4 cells. In addition, it was found that NP65 exposure affected homeostasis of Ca2+ in Sertoli TM4 cells by increasing cytoplasm [Ca2+]i, inhibiting Ca2+-ATPase activity and decreasing cyclic adenosine monophosphate (cAMP) concentration. Pretreatment with ROS scavenger, N-acetylcysteine (NAC), attenuated NP65-induced oxidative stress as well as apoptosis for TM4 cells. Furthermore, NAC blocked NP65-induced disorders of Ca2+ homeostasis by attenuating the growth of intracellular [Ca2+]i and the inhibition of Ca2+-ATPase and cAMP activities. Thus, we have demonstrated that NP65 induced apoptosis as well as acted as a potent inhibitor of Ca2+-ATPase activity and resulted in disorder of Ca2+ homeostasis in Sertoli TM4 cells; ROS participated in the process. Our results supported the view that oxidative stress acted an essential role within the development of apoptosis and Ca2+ overload in TM4 cells as a consequence of NP65 stimulation.

Apigenin induces oxidative stress in mouse Sertoli TM4 cells.

BACKGROUND AND AIM: Apigenin (API) is an estrogenic compound found in many plants. Sertoli cells reside in the testis and are a key target of environmental toxicants. This study aimed to examine the cytotoxicity, especially oxidative stress of API in mouse Sertoli TM4 cells. MATERIALS AND METHODS: Mouse Sertoli TM4 cells were treated with 50 and 100 µM API for 48 h. Cell viability, lactate dehydrogenase (LDH) activities, glutathione reductase (GR) activities, production of reactive oxygen species (ROS), and malondialdehyde (MDA) levels were evaluated using various assays. RESULTS: Treatment with API at both 50 and 100 µM decreased viability and GR activity but increased LDH activity, ROS production, and MDA levels in mouse Sertoli TM4 cells. CONCLUSION: Exposure to API induced oxidative stress in mouse Sertoli TM4 cells.

Zearalenone Exposure Disrupts Blood-Testis Barrier Integrity through Excessive Ca2+-Mediated Autophagy.

Zearalenone (ZEA), a common mycotoxin in grains and animal feeds, has been associated with male reproductive disorders. However, the potential toxicity mechanism of ZEA is not fully understood. In this study, in vivo and in vitro models were used to explore the effects of ZEA on the blood-testis barrier (BTB) and related molecular mechanisms. First, male BALB/C mice were administered ZEA orally (40 mg/kg·bw) for 5-7 d. Sperm motility, testicular morphology, and expressions of BTB junction proteins and autophagy-related proteins were evaluated. In addition, TM4 cells (mouse Sertoli cells line) were used to delineate the molecular mechanisms that mediate the effects of ZEA on BTB. Our results demonstrated that ZEA exposure induced severe testicular damage in histomorphology and an ultrastructural, time-dependent decrease in the expression of blood-testis barrier junction-related proteins, accompanied by an increase in the expression of autophagy-related proteins. Additionally, similar to the in vitro results, the dose-dependent treatment of ZEA increased the level of cytoplasmic Ca2+ and the levels of the autophagy markers LC3-II and p62, in conjunction with a decrease in the BTB junction proteins occludin, claudin-11, and Cx43, with the dislocation of the gap junction protein Cx43. Meanwhile, inhibition of autophagy by CQ and 3-MA or inhibition of cytoplasmic Ca2+ by BAPTA-AM was sufficient to reduce the effects of ZEA on the TM4 cell BTB. To summarize, this study emphasizes the role of Ca2+-mediated autophagy in ZEA-induced BTB destruction, which deepens our understanding of the molecular mechanism of ZEA-induced male reproductive disorders.