Andrew J. O. Smith

Sulforaphane Can Protect Lens Cells Against Oxidative Stress: Implications for Cataract Prevention

PURPOSE Protecting the lens against oxidative stress is of great importance in delaying the onset of cataract. Isothiocyanates, such as sulforaphane (SFN), are proposed to provide cytoprotection against oxidative stress. We therefore tested the ability of SFN to perform this role in lens cells and establish its ability to delay the onset of cataract. METHODS The human lens epithelial cell line FHL124 and whole porcine lens culture systems were used. The ApoTox-Glo Triplex Assay was used to assess FHL124 cell survival, cytotoxicity, and apoptosis. The MTS assay was used to assess cell populations. To determine levels of DNA strand breaks, the alkaline comet assay was performed and quantified. Lactate dehydrogenase levels in the medium were evaluated to reflect cell damage/death. To assess level of gene expression, an Illumina whole-genome HT-12 v4 beadchip was used. Protein expression was determined by Western blot and immunocytochemistry. RESULTS Exposures of 30 μM H2O2 to FHL124 cells caused a reduction in cell viability and increased cytotoxicity/apoptosis; these effects were significantly inhibited by 24-hour pretreatment with 1 μM SFN. In addition, 1 μM SFN significantly reduced H2O2-induced DNA strand breaks. When applied to cultured porcine lenses, SFN protected against H2O2-induced opacification. Illumina whole-genome HT-12 v4 beadchip microarray data revealed eight genes upregulated following 24-hour exposure to 1- and 2-μM SFN, which included NQO1 and TXNRD1. This pattern was confirmed at the protein level. Nrf2 translocated to the nucleus in response to 0.5- to 2.0-μM SFN exposure CONCLUSIONS The dietary component SFN demonstrates an ability to protect human lens cells against oxidative stress and thus could potentially delay the onset of cataract.

Sigma 1 receptor stimulation protects against oxidative damage through suppression of the ER stress responses in the human lens.

Stimulation of sigma-1 receptors is reported to protect against oxidative stress. The present study uses cells and tissue from the human lens to elucidate the relationship between the sigma 1 receptor, ER stress and oxidative stress-induced damage. Exposure of the human lens cell line FHL124 to increasing concentrations of H(2)O(2) led to reduced cell viability and increased apoptosis. In response to 30 μM H(2)O(2), levels of the ER stress proteins BiP, ATF6 and pEIF2α were significantly increased within 4h of exposure. Expression of the sigma 1 receptor was markedly increased in response to H(2)O(2). Application of 10 and 30 μM (+)-pentazocine, a sigma 1 receptor agonist, significantly inhibited the H(2)O(2) induced cell death. (+)-Pentazocine also suppressed the oxidative stress induced reduction of pro-caspase 12 and suppressed the induction of the ER stress proteins BiP and EIF2α. When applied to cultured human lenses, (+)-pentazocine protected against apoptotic cell death, LDH release and against H(2)O(2) induced opacification. These data demonstrate that stimulation of the sigma 1 receptor provides significant protection from oxidative damage and is, therefore, a putative therapeutic approach to delay the onset of diseases that may be triggered by oxidative damage, including cataract formation.

NAD+-dependent DNA ligases of Mycobacterium tuberculosis and Streptomyces coelicolor.

Sequencing of the genomes of Mycobacterium tuberculosis H37Rv and Streptomyces coelicolor A3(2) identified putative genes for an NAD+‐dependent DNA ligase. We have cloned both open reading frames and overexpressed the protein products in Escherichia coli. In vitro biochemical assays confirm that each of these proteins encodes a functional DNA ligase that uses NAD+ as its cofactor. Expression of either protein is able to complement E. coli GR501, which carries a temperature‐sensitive mutation in ligA. Thus, in vitro and in vivo analyses confirm predictions that ligA genes from M. tuberculosis and S. coelicolor are NAD+‐dependent DNA ligases. Proteins 2003;51:321–326.

AGEs in human lens capsule promote the TGFβ2‐mediated EMT of lens epithelial cells: implications for age‐associated fibrosis

Proteins in basement membrane (BM) are long‐lived and accumulate chemical modifications during aging; advanced glycation endproduct (AGE) formation is one such modification. The human lens capsule is a BM secreted by lens epithelial cells. In this study, we have investigated the effect of aging and cataracts on the AGE levels in the human lens capsule and determined their role in the epithelial‐to‐mesenchymal transition (EMT) of lens epithelial cells. EMT occurs during posterior capsule opacification (PCO), also known as secondary cataract formation. We found age‐dependent increases in several AGEs and significantly higher levels in cataractous lens capsules than in normal lens capsules measured by LC‐MS/MS. The TGFβ2‐mediated upregulation of the mRNA levels (by qPCR) of EMT‐associated proteins was significantly enhanced in cells cultured on AGE‐modified BM and human lens capsule compared with those on unmodified proteins. Such responses were also observed for TGFβ1. In the human capsular bag model of PCO, the AGE content of the capsule proteins was correlated with the synthesis of TGFβ2‐mediated α‐smooth muscle actin (αSMA). Taken together, our data imply that AGEs in the lens capsule promote the TGFβ2‐mediated fibrosis of lens epithelial cells during PCO and suggest that AGEs in BMs could have a broader role in aging and diabetes‐associated fibrosis.

PARP-1 inhibition influences the oxidative stress response of the human lens

Poly(ADP-ribose) polymerase-1 (PARP-1) is best characterised for its involvement in DNA repair. PARP-1 activity is also linked to cell fate, confounding its roles in maintaining genome integrity. The current study assessed the functional roles of PARP-1 within human lens cells in response to oxidative stress. The human lens epithelial cell line FHL124 and whole human lens cultures were used as experimental systems. Hydrogen peroxide (H2O2) was employed to induce oxidative stress and cell death was assessed by LDH release. The functional influence of PARP-1 was assessed using targeted siRNA and chemical inhibition (by AG14361). Immunocytochemistry and western blotting were used to assess PARP-1 expression and the alkaline comet assay determined the levels of DNA strand breaks. PARP-1 was generally observed in the cell nucleus in both the FHL124 cell line and whole human lenses. PARP-1 inhibition rendered FHL124 cells more susceptible to H2O2-induced DNA strand breaks. Interestingly, reduction of PARP-1 activity significantly inhibited H2O2-induced cell death relative to control cells. Inhibition of PARP-1 in whole human lenses resulted in a reduced level of lens opacity and cell death following exposure to H2O2 relative to matched pair controls. Thus, we show that PARP-1 could play a role in the fate of human lens cells, and these first observations in human lenses suggest that it could impact on lens opacity. Further studies are required to elucidate the regulatory processes that give rise to these effects.

Ku80 Counters Oxidative Stress-Induced DNA Damage and Cataract Formation in the Human Lens.

PURPOSE Oxidative stress in the human lens leads to a wide range of damage including DNA strand breaks, which are likely to contribute to cataract formation. The protein Ku80 is a fundamental component of the nonhomologous end-joining pathway that repairs DNA double strand breaks. This study investigates the putative impact of Ku80 in cataract prevention in the human lens. METHODS The present study used the human lens epithelial cell line FHL124 and whole human lens organ culture. Targeted siRNA was used to deplete Ku80, with Western blot and immunocytochemistry employed to assess Ku80 expression levels. Oxidative stress was induced with hydrogen peroxide and DNA strand breaks measured by alkaline comet assay and γH2AX foci counts. Visual quality of whole human lenses was measured with image analysis software. RESULTS Expression of Ku80 was predominately found in the cell nucleus of both FHL124 cells and native human lens epithelium. Treatment of FHL124 cells and whole lens cultures with siRNA targeted against Ku80 resulted in a significant knockdown at the protein level. Application of oxidative stress (30 μM H2O2) created more DNA strand breaks when added to Ku80 knockdown cells than in scrambled siRNA control cells as determined by the alkaline comet assay and the number of γH2AX foci. In whole lens cultures, exposure to 1 mM H2O2 resulted in more lens opacity in Ku80 knockdown lenses than match-paired controls. CONCLUSIONS Depletion of Ku80 in the lens through acute change or a consequence of aging is likely to increase levels of DNA strand breaks, which could negatively influence physiological function and promote lens opacity. It is therefore feasible that Ku80 plays a role in retarding cataract formation.

Chemopreventive Activities of Sulforaphane and Its Metabolites in Human Hepatoma HepG2 Cells

Sulforaphane (SFN) exhibits chemopreventive effects through various mechanisms. However, few studies have focused on the bioactivities of its metabolites. Here, three metabolites derived from SFN were studied, known as sulforaphane glutathione, sulforaphane cysteine and sulforaphane-N-acetylcysteine. Their effects on cell viability, DNA damage, tumorigenicity, cell migration and adhesion were measured in human hepatoma HepG2 cells, and their anti-angiogenetic effects were determined in a 3D co-culture model of human umbilical vein endothelial cells (HUVECs) and pericytes. Results indicated that these metabolites at high doses decreased cancer cell viability, induced DNA damage and inhibited motility, and impaired endothelial cell migration and tube formation. Additionally, pre-treatment with low doses of SFN metabolites protected against H2O2 challenge. The activation of the nuclear factor E2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway and the induction of intracellular glutathione (GSH) played an important role in the cytoprotective effects of SFN metabolites. In conclusion, SFN metabolites exhibited similar cytotoxic and cytoprotective effects to SFN, which proves the necessity to study the mechanisms of action of not only SFN but also of its metabolites. Based on the different tissue distribution profiles of these metabolites, the most relevant chemical forms can be selected for targeted chemoprevention.