title: Role of pirenoxine in the effects of catalin on in vitro ultraviolet-induced lens protein turbidity and selenite-induced cataractogenesis in vivo.
abstract: OBJECTIVE
  In this study, we investigated the biochemical pharmacology of pirenoxine (PRX) and catalin under in vitro selenite/calcium- and ultraviolet (UV)-induced lens protein turbidity challenges. The systemic effects of catalin were determined using a selenite-induced cataractogenesis rat model.
  
  
  METHODS
  In vitro cataractogenesis assay systems (including UVB/C photo-oxidation of lens crystallins, calpain-induced proteolysis, and selenite/calcium-induced turbidity of lens crystallin solutions) were used to screen the activity of PRX and catalin eye drop solutions. Turbidity was identified as the optical density measured using spectroscopy at 405 nm. We also determined the in vivo effects of catalin on cataract severity in a selenite-induced cataract rat model. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was applied to analyze the integrity of crystallin samples.
  
  
  RESULTS
  PRX at 1,000 μM significantly delayed UVC-induced turbidity formation compared to controls after 4 h of UVC exposure (p<0.05), but not in groups incubated with PRX concentrations of <1,000 μM. Results were further confirmed by SDS-PAGE. The absolute γ-crystallin turbidity induced by 4 h of UVC exposure was ameliorated in the presence of catalin equivalent to 1~100 μM PRX in a concentration-dependent manner. Samples with catalin-formulated vehicle only (CataV) and those containing PRX equivalent to 100 μM had a similar protective effect after 4 h of UVC exposure compared to the controls (p<0.05). PRX at 0.03, 0.1, and 0.3 μM significantly delayed 10 mM selenite- and calcium-induced turbidity formation compared to controls on days 0~4 (p<0.05). Catalin (equivalent to 32, 80, and 100 μM PRX) had an initial protective effect against selenite-induced lens protein turbidity on day 1 (p<0.05). Subcutaneous pretreatment with catalin (5 mg/kg) also statistically decreased the mean cataract scores in selenite-induced cataract rats on post-induction day 3 compared to the controls (1.3±0.2 versus 2.4±0.4; p<0.05). However, catalin (equivalent to up to 100 μM PRX) did not inhibit calpain-induced proteolysis activated by calcium, and neither did 100 μM PRX.
  
  
  CONCLUSIONS
  PRX at micromolar levels ameliorated selenite- and calcium-induced lens protein turbidity but required millimolar levels to protect against UVC irradiation. The observed inhibition of UVC-induced turbidity of lens crystallins by catalin at micromolar concentrations may have been a result of the catalin-formulated vehicle. Transient protection by catalin against selenite-induced turbidity of crystallin solutions in vitro was supported by the ameliorated cataract scores in the early stage of cataractogenesis in vivo by subcutaneously administered catalin. PRX could not inhibit calpain-induced proteolysis activated by calcium or catalin itself, and may be detrimental to crystallins under UVB exposure. Further studies on formulation modifications of catalin and recommended doses of PRX to optimize clinical efficacy by cataract type are warranted.
authors.affiliation: Department of Ophthalmology, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan.
authors.forename: Chao-Chien
authors.initials: CC
authors.lastname: Hu
authors.forename: Jiahn-Haur
authors.initials: JH
authors.lastname: Liao
authors.forename: Kuang-Yang
authors.initials: KY
authors.lastname: Hsu
authors.forename: I-Lin
authors.initials: IL
authors.lastname: Lin
authors.forename: Ming-Hsuan
authors.initials: MH
authors.lastname: Tsai
authors.forename: Wen-Hsin
authors.initials: WH
authors.lastname: Wu
authors.forename: Tzu-Tang
authors.initials: TT
authors.lastname: Wei
authors.forename: Yi-Shiang
authors.initials: YS
authors.lastname: Huang
authors.forename: Shih-Jiuan
authors.initials: SJ
authors.lastname: Chiu
authors.forename: Hsiang-Yin
authors.initials: HY
authors.lastname: Chen
authors.forename: Shih-Hsiung
authors.initials: SH
authors.lastname: Wu
authors.forename: Tzu-Hua
authors.initials: TH
authors.lastname: Wu
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002154:Calpain / Q000009:adverse effects / Q000494:pharmacology; D002386:Cataract / Q000139:chemically induced / Q000188:drug therapy* / Q000378:metabolism / Q000517:prevention & control; D004305:Dose-Response Relationship, Drug; D054796:Drug Dosage Calculations; D004591:Electrophoresis, Polyacrylamide Gel; D007908:Lens, Crystalline / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D009883:Ophthalmic Solutions / Q000008:administration & dosage / Q000627:therapeutic use*; D010078:Oxazines / Q000008:administration & dosage / Q000627:therapeutic use*; D059748:Proteolysis / Q000187:drug effects; D051381:Rats; D017207:Rats, Sprague-Dawley; D018038:Sodium Selenite / Q000008:administration & dosage / Q000009:adverse effects; D013057:Spectrum Analysis; D013552:Swine; D014466:Ultraviolet Rays; D038222:gamma-Crystallins / Q000737:chemistry / Q000378:metabolism*
chemical_list: D009883:Ophthalmic Solutions; D010078:Oxazines; D038222:gamma-Crystallins; C004580:pirenoxine; D002154:Calpain; D018038:Sodium Selenite; D002118:Calcium