Iron-Overload, Oxidative DNA Damage and Differential Transcript Expression of Iron Homeostasis Genes in Human Cortical Cataracts

Abstract

Purpose: The purpose of the study is to determine the level of iron, its role in oxidative damage and to profile and measure the level of transcript expression of genes responsible for iron homeostasis in lenses of human cataracts. Materials and methods: Human whole lenses (WLs) were obtained from donors’ eyes (n = 33) within 8 hours of death. A total of 167 (control, n = 35 and cataract, n = 132) anterior central capsules (ACC) harboring lens epithelial cells (LECs) and lens aspirate containing LECs, primary and secondary lens fiber cells (LFCs) were collected postoperatively. Control samples were obtained mainly from patients suffered from lenticular trauma or subjected for myopic corrections. Lenticular total iron was determined by Atomic Absorption Spectrophotometer (AAS). Comet assay using lens epithelial cells was performed to determine the oxidative DNA damages. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to profile and determine the level of transcript expression of genes of iron homeostasis in lenses. An in vitro culture model using fetal human lens epithelial cell line (FHL124) was used to validate the effect of iron on oxidative DNA damage and expression of iron homeostasis genes. Results: Cortical cataract (CC) had significantly higher levels of iron (58.89 ± 20.56 μg/g dry tissue weight, p = 0.00) than control lenses (17.82 ± 5.29 μg/g dry tissue weight) and revealed a significant increase in tail length (μ) (79.59 ± 21.31 vs. control, 66.56 ± 15.68; p = 0.005) and olive moment (29.69 ± 10.31 vs. control, 26.97 ± 8.12, p = 0.018) by Comet assay in LECs. Fetal human lens epithelial cell line (FHL124) treated with 500 μM (concentration equivalent to lenticular iron level in CC) of ferric chloride (FeCl3), revealed a significant increase in tail length (μ) (80.71 ± 8.63 vs. control, 62.55 ± 6.63; p = 0.01), tail DNA (%) (44.01 ± 18.02 vs. control 27.39 ± 5.14; p = 0.05), tail moment (36.87 ± 17.33 vs. control, 18.1 ± 3.41; p = 0.02) and olive moment (27.41 ± 6.72 vs. control, 18.88 ± 2.86; p = 0.02) suggestive of substantial DNA damage. QRT-PCR analysis displayed elevated level of iron responsive element (IRE) containing DMT1 (IRE-DMT1, 1.99 ± 0.34 fold change to control, p = 0.046) and diminished level of FTH (0.13 ± 0.02-fold change to control; p = 0.01) in lenses of CC. However, in vitro model, though displayed a conflicting data as compare to human samples, exhibited a conventional coordinated regulation of iron within the system. Conclusion: In conclusion, differential transcript expression of genes responsible for iron uptake (IRE-DMT1) and storage (FTH) might lead to an increase in iron over-load in human lenses. Iron-over load could potentially accelerate the cascade of mechanisms leading to oxidative damage, as evident by increased level of DNA damage in cortical cataract and also in FHL124 cells treated with FeCl3.