The p53–APAF-1–BAX Axis Mediated Apoptosis in HEK293 Kidney Cells Exposed to Methylisothiazolinone

Abstract

Objectives: Methylisothiazolinone (MIT) is a widely used synthetic biocide in industrial, household, and cosmetic products. There is insufficient information about its nephrotoxic potential, although it causes skin sensitivities or ecological problems. We investigated MIT-induced nephrotoxicity and molecular mechanisms in human embryonic kidney (HEK293) cells. Materials and Methods: Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptotic/necrotic cells were defined by Annexin V/ Propidium iodide (PI) assay, inflammatory responses were quantified by LEGENDPlex™, and the protein expression levels of apoptotic-, cell survival-, and angiogenic-related biomarkers were determined by enzyme-linked immunosorbent assay (ELISA). Results: The IC50 was 301.67 µM. MIT triggers concentration-dependent apoptosis about 34.46% at the highest exposed concentration, with late apoptosis being the primary mechanism of cell death. Interleukin-6 (IL-6) and IL-8 levels were significantly elevated at 75 µM as 3.43-fold and 2.34-fold, respectively, while monocyte chemoattractant protein-1 (MCP-1) levels were dramatically suppressed to 97%. The intrinsic apoptotic pathway was activated by significant upregulation of p53 (14.8-fold), Β-cell lymphoma 2 (BCL-2) associated X protein (BAX) (10.0-fold), and apoptotic protease activating factor-1 (APAF-1) (6.1-fold), along with a disruption in the BAX/BCL-2 balance. The elevation in cyclooxygenase (COX), COX-2, Src Kinase (SRC), and vascular endothelial growth factor receptor 2 (VEGFR2) levels showed stress-induced pro-survival signaling. Conclusion: The results imply a possible link between systemic absorption and renal-related safety considerations, supporting that existing safety thresholds may benefit from further evaluation for products with potential mucosal or ingestive exposure.

Keywords

• Methylisothiazolinone
• nephrotoxicity
• apoptosis
• p53 signalling
• HEK293

References

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