(226) Investigation of Proliferative and Cytotoxic Effects of Low and High Doses of Boric Acid Exposure on Mouse Gc-1spg Spermatogonial Cells

Introduction Boron is an essential trace element for plants and plays important physiological roles in animals and humans. It contributes to membrane stability, cell proliferation, bone formation, immune response, and liver function. However, high-dose exposure has been linked to reproductive toxicity, particularly impairing testicular function. Experimental studies have shown that excessive boric acid intake disrupts spermatogenesis, yet the molecular mechanisms remain poorly defined. This study investigated how boric acid exposure influences cell viability, redox homeostasis, and inflammatory signaling in spermatogonial stem cells under in vitro conditions. Objective To evaluate the effects of low (1 mM) and high (50 mM) concentrations of boric acid on cell proliferation, cell cycle regulation, membrane integrity, oxidative stress parameters, and inflammatory markers in the mouse GC-1spg spermatogonial stem cell line. Methods GC-1spg cells were exposed to 1 mM and 50 mM boric acid for 24 and 48 hours. Cell viability was assessed using the MTT Cell Viability Assay, while cell cycle distribution was analyzed by Flow Cytometric Cell Cycle Analysis. Membrane integrity was evaluated through Propidium Iodide (PI) Staining Assay. Oxidative and antioxidant status were quantified using the Total Antioxidant Status (TAS) and Total Oxidant Status (TOS) assays. The expression of Nuclear factor erythroid 2–related factor 2 (Nrf2) and Nuclear factor kappa B (NF-κB) was measured by ELISA to determine antioxidant and inflammatory pathway activity. Results Boric acid treatment caused a significant, dose- and time-dependent decrease in cell viability (p<0.05). At 1 mM, viability remained close to 100%, whereas at 50 mM it decreased to 60.4% at 24 hours and 50.4% at 48 hours. Flow cytometry showed G₂ phase accumulation with low-dose exposure (22.5%) and G₁ phase arrest at high dose (63.7%). PI staining indicated increased membrane permeability in the high-dose group, suggesting impaired membrane integrity. Redox assays revealed that 1 mM boric acid increased TAS level compared to the controls (1.24 ± 0.13 vs 0.74 ± 0.04, p=0.001), while 50 mM boric acid did not cause a significant difference (0.73 ± 0.04 vs 0.74 ± 0.04, p=0.97). Furthermore, 50 mM boric acid significantly elevated TOS levels compared to the controls (0.26 ± 0.013 vs 0.18 ± 0.006, p=0.002), whereas 1 mM boric acid did not cause a significant difference (0.17 ± 0.002 vs 0.18 ± 0.006, p=0.92). ELISA assays revealed that 50 mM boric acid decreased Nrf2 expression significantly (1.27 ± 0.06 vs 1.90 ± 0.10, p=0.02), reflecting suppressed antioxidant activation, whereas 1 mM boric acid did not cause a significant difference (2.04± 0.08 vs 1.90 ± 0.10, p=0.92). NF-κB expression levels did not change significantly across boric acid concentrations (p>0.05). Conclusions Boric acid exerts dose and time-dependent biochemical effects on spermatogonial stem cells. Low concentrations of boric acid support antioxidant activation by increasing TAS levels, whereas high concentrations support oxidant activation by increasing TOS levels, decreasing Nrf2 expression, and causing membrane damage and cell cycle arrest. The findings suggest that boric acid–induced redox imbalance and inflammation could contribute to male fertility impairment, underscoring the importance of defining safe exposure thresholds for boron compounds. Disclosure No