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

Abstract

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.

Keywords: COX-2; GSH; SOD; Sertoli TM4 cells; TNF-α; apoptosis; econazole; oxidative stress; synergistic effects; tebuconazole.

Figure 1

Exposure to azole fungicides induces early and sustained ROS production over time in TM4 cells. The graph represents the over-production of ROS in SCs not exposed (CTR) or exposed to azole compounds ECO and TEB, alone and in mixture (MIX), for an extended time-course from 30 min up to 24 h; intracellular ROS levels were measured with a fluorimeter detecting DCFH-DA dye fluorescence intensity (arbitrary units, a.u.). To ensure a positive control, cells were incubated for 1.5 h with 500 µM tert-butyl hydroperoxide (t-BHP). Values are considered as the media ± SE, from three independent experiments; OneWay ANOVA, followed by Dunnett’s test; * p < 0.05 vs. CTR.

Figure 2

Imbalance of enzymatic antioxidant activities in azole-exposed TM4 cells. Azole-treatments significantly affected SOD activity levels, causing an altered SOD/(GPx + CAT) ratio. The specific activities of the tested enzymes were analyzed in samples exposed or not exposed (CTR) to azoles after 3 h of incubation. Values expressed as specific activities or ratios, as indicated, represent the media ± SE from three independent experiments; OneWay ANOVA followed by Dunnett’s test or Holm–Sidak methods; ANOVA on ranks (Kruskal–Wallis test followed by Tukey Test) (CAT activity); * p < 0.05 vs. CTR.

Figure 3

Azoles’ exposure results in significant alterations of GSH homeostasis. Levels of (A) reduced (GSH), (B) oxidized glutathione (GSSG), (C) GSH/GSSG, and (D) GR/(GPx + GST) ratios in CTR and azoles-treated groups after 3 h of incubation. Values from three independent experiments are expressed as the media ± SE; OneWay ANOVA, followed by Dunnett’s test; ANOVA on ranks (Kruskal–Wallis test followed by Tukey Test) (GR/(GPx + GST) ratio); * p < 0.05 vs. CTR.

Figure 4

Differential up-regulation of TNF-α and COX-2 expression by azoles in TM4 cells. Protein expression levels of TNF-α (A) and COX-2 (B(i)) in TM4 treated cells at 6 h of incubation, compared with the CTR. (B(ii)) Representative images of COX-2 blot band in CTR and treated samples with azoles at 6 h of incubation. Values from three independent experiments are expressed as the media ± SE; OneWay ANOVA followed by Dunnett’s test or Holm–Sidak methods; * p < 0.05 vs. CTR.

Figure 5

Anti-oxidative and cytoprotective effects of NAC in TM4 cells exposed to azoles. NAC treatment partially rescues azole-induced cytotoxicity in TM4 cells by decreasing ROS production (3 h; (A)), improving cell viability (48 h; (B,C(i))), and attenuating inflammatory mediators’ expression (6 h; (D(i),E)). (C(ii)) Representative images of FACS profiles of samples not exposed (CTR) or exposed to azoles for 48 h and stained with annexin V and 7-AAD, in the presence (+) or in the absence (−) of NAC. (D(ii)) Representative images of COX-2 blot band in CTR and treated samples with the tested antifungal compounds at 6 h of exposure, in the presence (+) or in the absence (−) of NAC. Values from three independent experiments are determined by ratios between exposed (X_NAC) and not exposed to NAC (X) groups ([X_NAC − X/X]) in azole and CTR samples, and expressed as the media ± SE; OneWay ANOVA followed by Dunnett’s test; * p < 0.05 vs. CTR.