Regulation of Cataractogenesis in Cultured Bovine Lenses by ATB 337

Abstract

Purpose : Previous reports have shown that substrates for the endogenous production of hydrogen sulfide (H₂S) such as L-cysteine (Zhang et al., Mol Vis, 14:862, 2008) and NSAIDs such as ibuprofen (Harding JJ et al., Acta Ophthalmol, 67:518, 1989) can mitigate cataractogenesis. However, the role of the hybrid compound consisting of the NSAID, diclofenac and a H₂S-donating moiety, ATB 337 on cataractogenesis has not been elucidated. In this study, we investigated the effect of the ATB 343 on cataractogenesis in cultured bovine lenses.

Methods : Freshly isolated bovine lenses were cultured in a DMEM buffer solution as follows: Group I: Control (DMEM); Group II-III: hydrogen peroxide (H₂O₂); Group IV-VI: ATB 337 (10^-8M to 10^-6M; Group VII: ascorbic acid (AA; 10 mM) in presence and absence of H₂O₂ (10 mM or 50 mM). Lenses were incubated in a CO² chamber and assessed at 3, 6, 24, 48 and 72 hour-time points. Qualitative assessments were conducted by visual inspection of lenses and photographic images captured against a black grid, while quantitative assessment was conducted by measurement of transmittance using a plate reader (Synergy H1 hybrid reader; Bio Tek Instruments, Inc) at every time point.

Results : DMEM-cultured lenses exhibited a time-dependent decrease in transmittance (420nm) and a corresponding loss of lens optical clarity up to 72 hours. Unlike the endogenous antioxidant, AA (10 mM) which attenuated time-dependent lens degradation up to 24 hours, ATB 337 (10^-6M to 10^-8M) significantly (p<0.001; n=12) decreased time-dependent lens degradation. H₂O₂ (10 mM and 50 mM) significantly (p<0.001; n=12) decreased light transmittance in a time-dependent manner, achieving an inhibition of 41.98% and 46.37% respectively after 72 hours. Interestingly, ATB 337 (10^-8 M to 10^-6 M) partially reversed H₂O₂ (10 mM and 50 mM)-induced degradation up to 72 hours. After 48 hours, the rank orders of protection from H₂O₂ (10 mM)-induced cataractognesis was: ATB (10^-7M)>ATB (10^-8M)>AA (10 mM)>ATB (10^-6M) while that from H₂O₂ (50 mM)-induced cataractogenesis was ATB (10^-6M)>ATB (10^-7 M)>ATB (10^-8M >AA (10 mM).

Conclusions : ATB 337 partially protected cultured bovine lenses from peroxide-induced cataractogenesis. Studies are in progress to determine the mechanisms by which ATB 337 protects cultured lenses from degradation.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.