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Oxidative stress plays a central role in cataract formation suggesting that antioxidants might slow cataract progression. The anticataract activity of N-acetylcysteine amide (NACA) and (2 R, 2 Rʼ)-3,3ʼ-disulfanediyl bis(2-acetamidopropanamide) (diNACA) and/or N-acetylcysteine (NAC), were evaluated in porcine and rat lens models. Cataractogenesis via oxidation was induced with H 2 O 2 and/or glucose oxidase (GO). Porcine lenses were incubated in 0.1 mM, 1 mM, or 10 mM NAC, NACA or diNACA for 24 h. Lenses were then transferred to media containing 0.75 mM H 2 O 2 and 4.63U of GO in order to maintain a constant H 2 O 2 level for an additional 8 h. At the end of incubation, lenses were imaged under darkfield microscopy. Separately, rat lenses were extracted from 3-week-old Wistar rats and incubated with either 10 mM NACA or 10 mM diNACA for 24 h prior to treatment with 0.2U GO to generate a steady source of ∼0.6 mM H 2 O 2. Rat lenses were analyzed by LC-MS/MS to quantify changes in cysteine, cystine, glutathione (GSH) or oxidised glutathione (GSSG) levels in the lens epithelium, cortex or core. Pre-treatment with NACA or diNACA followed by oxidation with H 2 O 2 and/or GO to stimulate cataract formation afforded rapid assessment in ex vivo porcine (32 h) and rat (48 h) lens models. Pre-treatment of isolated porcine lenses with 0.1 mM, 1 mM or 10 mM of either NAC, NACA or diNACA followed by H 2 O 2 /GO treatment resulted in reduced lens opacity relative to the lenses exposed to H 2 O 2 /GO, with NACA and diNACA reducing opacities to a greater extent than NAC. Rat lenses incubated with 10 mM NACA or 10 mM diNACA without exposure to H 2 O 2 showed no signs of opacities. Pre-treatment of rat lenses with 10 mM NACA or 10 mM diNACA, followed by GO cataract induction resulted in reduced opacities compared to control (GO alone). LC-MS/MS analyses revealed that NACA, but not diNACA, increased cysteine, cystine and GSH levels in rat lens epithelium and cortex regions. Taken together, both NACA and diNACA inhibited cataract formation to a greater extent than NAC (all at 1–10 mM) in an ex vivo porcine lens model. Both NACA and diNACA (both at 10 mM) reduced cataract formation in rat lenses. Based on LC-MS/MS analyses, NACA-induced reduction in opacity observed in rat lenses was attributed to enhanced cysteine and GSH levels while the diNACA-induced reduction in opacity induced did not consistently increase cysteine, cystine and GSH levels and, therefore, appears to involve a different antioxidant mechanism. These screening studies warrant further testing of NACA and diNACA as anticataract agents. • NACA and diNACA were tested for their anti-cataract activity. • NACA and diNACA reduced opacities induced by H 2 O 2 in porcine and rat lenses. • NACA reduced opacities by enhancing cysteine and glutathione in the rat lens. • DiNACA reduced opacities by a pathway which may involve mixed disulphide formation. • Further work is warranted to further test NACA and diNACA as anticataract agents. [ABSTRACT FROM AUTHOR]