Snake Venom Extract as Possible Alternative to Mitomycin-C in Glaucoma Filtering Surgery: In vitro Study

Abstract

Purpose:: To compare the inhibiting fibroblast properties between crude Bothrops moojeni venom extract and Mitomycin-C (MMC) through cell viability assay.

Methods:: Human Foreskin Fibroblast cells were cultured in 96-well plates (1 x10⁵ cells/well/200µL) in RPMI medium supplemented with antibiotics plus 10% fetal calf serum and incubated in a humidified 5% CO₂ incubator at 37ºC for 18 h, when non-adherent cells were removed during the change of medium. These cells were treated with serial two-fold dilutions of crude venom extract (10 to 0.019 µg/mL) or MMC (500 to 0.98 µg/mL) for 24 h. Cells treated with the medium only served as control. Cell viability was then evaluated by MTT (Methyl Thiazol Tetrazolium) assay. After another change of medium, cells were incubated with 100 µL of 0.5 mg/mL MTT for 4 h at 37ºC and 5% CO₂. Floating substances were removed and 100 µL of 10% SDS (sodium dodecyl sulfate) in a dimethylformamide:water (1:1) solution were added to dissolve insoluble blue-violet Formazan cristals in viable cells. All readings were taken in a spectrophotometer at 570 nm and the optical density measurements analyzed as cell viability percentage compared to the control.

Results:: Cell viability was inversely proportional to the concentration of both crude venom extract and MMC. Human fibroblast viability was less than 50% when treated with concentrations above 1.0 µg/ml of the crude venom extract and above 500 µg/ml of MMC. When compared to untreated cells (control), there was a statistically significant reduction in fibroblast viability for both crude venom extract (P=0.0102) and MMC (P=0.0420). However, there were no statistically significant differences between fibroblast viability using concentrations less than 0.078 µg/ml (P=0.0550) for the crude venom extract and less than 0.019 mg/ml (P=0.1695) for the MMC.

Conclusions:: Both of the drugs tested in vitro act inhibiting fibroblastic proliferation, but the crude venom extract showed higher ability to reduce the fibroblast viability since lower concentrations were required to achieve the same effect over fibroblasts as compared to the MMC. Further studies should be conducted to evaluate if crude Bothrops moojeni venom extract may be useful in the surgical treatment of glaucoma as a substitute of MMC.

Keywords: wound healing • cell survival • drug toxicity/drug effects