Durga K. Bhuyan

Regulation of hydrogen peroxide in eye humors. Effect of 3-amino-1H-1,2,4-triazole on catalase and glutathione peroxidase of rabbit eye.

Activities of catalase (H2O2: H2O2 oxidoreductase, EC 1.11.1.6) and GSH peroxidase (GSH: H202 oxidoreductase, EC 1.11.1.9) have been measured in iris, ciliary body, retina, corneal epithelium, corneal endothelium, lens capsule-epithelium and decapsulated lens. 3-Amino-1H-1,2,4-triazole is a specific inhibitor of catalase and a potent cataractogenic agent. We observed marked inhibition of catalase activity in these tissues 1--6 h after the administration of a single intravenous dose of 1 g 3-aminotriazole per kg body weight in rabbit. This was associated with a 2--3-fold increase in the H2O2 concentrations of aqueous humor and vitreous humor. The increased peroxide concentrations were restored to the physiological levels as the catalase activity of eye tissues gradually returned to normal with time after injection. Under the conditions, GSH peroxidase activity of the afore-mentioned eye tissues was unaltered, GSH and protein sulfhydryl of lens were not changed, and ascorbic acid of aqueous humor and vitreous humor was not significantly altered. Based on these findings our conclusion is that catalase of eye tissues regulates the endogenous H2O2 in eye humors to the physiological level. We speculate that H2O2 may be the triggering factor in cataract induced by 3-aminotriazole.

Crosslinking of aminophospholipids in cellular membranes of lens by oxidative stress in vitro

We have previously demonstrated by TLC an additional phospholipid spot between phosphatidylethanolamine (PE) and phosphatidylserine (PS) in human cataract. This was further identified as a fluorescent Schiff-base conjugate resulting from crosslinking of reactive carbonyl groups of malondialdehyde (MDA) with the primary amino groups of membrane phospholipids. Evidence presented here shows that such an adduct could be formed in rabbit lens subjected to oxidative stress in vitro. TLC analysis of a lipid extract of a crude membrane fraction obtained from the lens homogenate incubated with 1 mM H2O2, tert-butyl hydroperoxide (TBHP) or MDA for 1-6 h at 25 degrees C, showed that the oxidants and MDA produced time-dependent crosslinking of aminophospholipids. Under identical conditions of incubation with TBHP or MDA, development of the Schiff-base lipid fluorochrome in lens with peak emission at 470 nm when excited at 360 nm also showed a time-dependent increase. The PE.MDA.PS produced in cellular membranes of the lenses cultured for 3 h in Krebs-Ringer medium was 151 nmol/mumol PE, and addition of 1 mM H2O2, TBHP or MDA, increased it to 881, 610 and 375 nmol/mumol PE, respectively. Adduct was also formed when authentic samples of PE and PS were reacted with pure MDA. From the results it is clear that oxidants viz., H2O2 and TBHP, or MDA were effective in promoting crosslinking of lens membrane aminophospholipids by Schiff-base conjugation of primary amino groups with the carbonyl groups of the aldehyde, a breakdown product of lipid peroxides.

ASSESSMENT OF OXIDATIVE STRESS TO EYE IN ANIMAL MODEL FOR CATARACT

Publisher Summary There is increasing evidence for the involvement of oxygen free radicals in the mechanism of several diseases including some types of cataracts. This chapter describes methods for measurement of oxygen radicals in eye tissues to establish theft causal relationship to the human senile cataract. Such studies in humans have limitations. Diquat-induced cataract in the rabbit provides a suitable experimental model to demonstrate causal relationships of the reactive oxygen species to cataractogenesis. The most sensitive and direct physical technique—namely, electron spin resonance spectroscopy, is more elaborate, expensive, and not available in general laboratory facilities. The spectrophotometric techniques for estimation of O2-, for measurement of .OH, and for estimation of H2O2, are simple, sensitive, reproducible, and suitable for quantitation of cumulative productions of oxygen radicals and H2O2 in the eye tissues. This animal model for cataracts is also ideal for testing the therapeutic efficacy of potential anticataract drugs in vivo.

Growth factor receptor gene and protein expressions in the human lens

In this study the mRNAs encoding epidermal growth factor receptor (EGFR), basic fibroblast growth factor receptor (FGFR-2) and insulin-like growth factor receptor (IGFR-1) genes of the human normal lenses at ages varying from 0.5 to 72 years, were identified by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Regulation of EGFR gene expression in the lens did not change with aging, and of FGFR-2 and IGFR-1 genes also remained unaltered up to age 53 years. However, expressions of FGFR-2 and IGFR-1 genes were decreased at ages above 60 years. EGFR, FGFR-2 and IGFR-1 proteins were detected by immunoblot analysis in the epithelial cell membranes of lens at age varying from 40 to 72 years. There was no detectable amount of EGFR protein in fiber cell membranes of the lens, and the levels of FGFR-2 and IGFR-1 proteins were much lower than those in the epithelial cell membranes. The low levels of these receptor proteins in the fiber cell membranes of lens, suggest their possible role in keeping the differentiated function of these unique transparent cells. The findings of the increased protein levels with age of EGFR with the appearance of some degradation products at age 48 years and higher, and the increased FGFR-2 protein at age 60 years and higher in the epithelial cell membranes of lens, were of interest. It appears that this could be a compensatory protective response of the lens to aging process for lifelong continuation of normal growth by proliferation and differentiation of its epithelial cells into new fiber cells in the germinative zone at the equatorial region. Thus, these results could provide a basis for further studies on growth factor receptor gene and protein regulations in the mechanism of lens aging and progression of age-related human cataract.