Bisphenol A (BPA) is an endocrine disruptor that negatively affects spermatogenesis, a process where Sertoli cells play a central role. Thus, in the present study we sought to ascertain whether BPA could modulate the endocannabinoid (eCB) system in exposed mouse primary Sertoli cells. Under our experimental conditions, BPA turned out to be cytotoxic to Sertoli cells with an half-maximal inhibitory concentration (IC₅₀) of ~6.0 µM. Exposure to a non-cytotoxic dose of BPA (i.e., 0.5 μM for 48 h) increased the expression levels of specific components of the eCB system, namely: type-1 cannabinoid (CB₁) receptor and diacylglycerol lipase-α (DAGL-α), at mRNA level, type-2 cannabinoid (CB₂) receptor, transient receptor potential vanilloid 1 (TRPV1) receptors, and DAGL-β, at protein level. Interestingly, BPA also increased the production of inhibin B, but not that of transferrin, and blockade of either CB₂ receptor or TRPV1 receptor further enhanced the BPA effect. Altogether, our study provides unprecedented evidence that BPA deranges the eCB system of Sertoli cells towards CB₂- and TRPV1-dependent signal transduction, both receptors being engaged in modulating BPA effects on inhibin B production. These findings add CB₂ and TRPV1 receptors, and hence the eCB signaling, to the other molecular targets of BPA already known in mammalian 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.

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.

Background: Neoadjuvant chemotherapy plus dual HER2 blockade is standard for HER2-positive early breast cancer (EBC), but the impact of hormone receptor (HR) status and PIK3CA mutations with anthracycline-free regimens remains unclear. Methods: We retrospectively analyzed 56 patients with stage II–III HER2-positive EBC treated with neoadjuvant pertuzumab–trastuzumab–taxane (THP) at a single institution. Pathological complete response (pCR, ypT0/is ypN0) was the primary endpoint; secondary endpoints were safety and early disease-free/overall survival (DFS/OS), while associations of HR status and PIK3CA mutations with pCR were explored. Results: The overall pCR rate was 60.7%, in line with major dual-HER2 neoadjuvant trials. HR-negative patients achieved higher pCR rates than HR-positive patients (85.7% vs. 45.7%; p = 0.007; odds ratio 7.125), identifying HR status as the main clinical factor associated with response. Among 36 patients with PIK3CA testing, pCR rates appeared similar in mutated and wild-type tumors (62.5% vs. 60.7%), but the small number of mutated cases precludes firm conclusions. At a median follow-up of 42 months, only five DFS and one OS event had occurred, so survival analyses are exploratory and should be interpreted cautiously. THP demonstrated an excellent safety profile, with minimal grade 3–4 toxicity, and no clinically relevant hematological, cardiac, or pulmonary events. Conclusions: Anthracycline-free THP is a highly active, well-tolerated neoadjuvant option for HER2-positive EBC, with particularly high pCR rates in HR-negative disease. HR status emerged as a key determinant of pCR, whereas the role of PIK3CA mutations remains inconclusive and requires confirmation in larger prospective studies.