The effects of hyperbaric oxygen on the crystalline of cultured rabbit lenses: a possible catalytic role for copper

Oxidative effects on lens proteins have been linked with the formation of h uman age-related cataract, particularly nuclear cataract. This study invest igated the effects of hyperbaric oxygen (HBO)-induced oxidative stress on n uclear and cortical alpha-, beta- and gamma-crystallins of cultured rabbit lenses, using high performance liquid chromatography (HPLC). The lenses wer e treated with 50 atm of either 100 % N-2 (control) or 100% O-2 (experiment al) for 3, 6, 16 and 48 hr. The levers of reduced glutathione (GSH) and wat er-soluble (WS) protein decreased more rapidly in the nucleus of the O-2-tr eated lens than in the cortex. The first significant loss of WS protein in each of the two regions occurred when levels of GSH had decreased by at lea st 90 % in either the nucleus (at 6 hr) or the cortex (at 16 hr). HPLC anal ysis of the nuclear WS proteins indicated that beta-crystallins were the fi rst proteins affected by the oxidative stress. Soon after HBO-treatment was initiated (at 6 hr) and prior to insolubilization of protein, nuclear beta - and gamma-crystallins moved to the higher molecular weight alpha-crystall in fraction; 2-D gel electrophoresis and Western blotting indicated the pre sence of disulfide-crosslinked and non-crosslinked beta- and gamma-crystall ins in this fraction. Significantly different HBO-induced effects were obse rved on lens cortical crystallins compared to those for the nucleus. For ex ample, gamma-crystallins in the cortex shifted very soon after HBO-treatmen t (at 3 hr) to slightly higher molecular weights, possibly the result of pr otein/glutathione mixed disulfide formation; however, this phenomenon was n ot observed in the nucleus. Cortical beta- and gamma-crystallins remained i n solution longer than nuclear proteins following HBO-treatment of the lens es, presumably the result of protection from the fear-fold higher level of GSH (22 vs 6 mM) present in the lens periphery. Surprisingly, there was no movement of beta- and gamma-crystallins to alpha(H)- and alpha-crystallin f ractions in the cortex of the O-2-treated lens, in contrast to that observe d for the nucleus. Cortical crystallins appeared to go directly from being soluble to being insoluble with no high molecular weight intermediate stage . The data suggested a possible chaperone-like function for alpha-crystalli n in the nucleus of the stressed lenses, but not in the cortex. HBO-induced effects on lens nuclear supernatants, which mimicked those observed for in tact lenses, could be nearly completely prevented by the copper-chelator ba thocuproine, but not by the iron-chelator deferoxamine. Overall, the result s provide additional evidence demonstrating an increased susceptibility of the lens nucleus to oxidative stress; the greater protective ability of the cortex may be linked to a higher capacity for beta- and gamma-crystallin/g lutathione mixed disulfide formation, inhibiting disulfide-crosslinked inso lubilization. The data also implicate copper as a catalyst for the autoxida tion of-SH groups in the lens, and suggest that alpha-crystallin chaperone- like activity may play a greater role in the lens nucleus than in the corte x in preventing oxidative insolubilization of crystallins. (C) 2000 Academi c Press .