Carbofuran Exposure Alters Autophagy Flux in Human Cornea In Vitro and Ex Vivo

Abstract

Purpose:

Carbofuran (CF) exposure causes ocular injury, but the underlying mechanism remains elusive. This study examined CF’s cytotoxicity and autophagy modulation in human cornea using in vitro and ex vivo organ culture models.

Methods:

Healthy human cadaver corneas were used for generating primary corneal stromal fibroblasts (hCSFs) and ex vivo organ culture. Dose-dependent CF (0–200 µM) cytotoxicity was evaluated using phase-contrast microscopy, live/dead, MTT, PrestoBlue, and Cyto-Tox-Glo assays. The half-maximal inhibitory concentration (IC₅₀) was determined via regression analysis. The messenger RNA (mRNA) and protein expression of autophagy genes, LC3, BECN1, and SQSTM1, were analyzed by quantitative real-time PCR and immunofluorescence. Transmission electron microscopy evaluated ultrastructural features.

Results:

Carbofuran reduced hCSFs’ viability in a dose-dependent manner. Cytotoxicity was not detected at ≤10 µM concentrations, whereas a significant progressive cytotoxicity was observed at concentrations ≥10 µM (P < 0.01, P < 0.001). Morphological changes corroborated with cytotoxicity assays. The IC₅₀ of CF was 58.6 µM to 24 h. Significant alterations in mRNA (P < 0.01, P < 0.001, or P < 0.0001) and protein levels of LC3, BECN1, and SQSTM1 were noted in hCSFs at two tested concentrations. The semiquantitative immunofluorescence analysis of LC3, BECN1, and SQSTM1 proteins was consistent with the changes in the transcript levels. Topical CF (IC₅₀) to human corneal tissues resulted in no remarkable loss of corneal clarity but significantly upregulated LC3, BECN1, and SQSTM1 expression (P < 0.01) in human cornea ex vivo. CF-exposed human cornea in organ culture revealed higher autophagosomes and electron-dense vesicles in stromal keratocytes than naive corneas in transmission electron microscopy.

Conclusion:

Carbofuran cytotoxicity to human cornea encompasses autophagy modifications in vitro and ex vivo.