Oxidative damage and inflammation are related to the pathogenesis of age-related macular degeneration (AMD). Epidemiologic studies suggest that insufficient dietary lutein and zeaxanthin intake or lower serum zeaxanthin levels are associated with increased risk for AMD. The objective of this work is to test the protective effects of lutein and zeaxanthin against photo-oxidative damage to retinal pigment epithelial cells (RPE) and oxidation-induced changes in expression of inflammation-related genes. To mimic lipofuscin-mediated photo-oxidation in vivo, we used ARPE-19 cells that accumulated A2E, a lipofuscin fluorophore and photosensitizer, as a model system to investigate the effects of lutein and zeaxanthin supplementation. The data show that supplementation with lutein or zeaxanthin in the medium resulted in accumulation of lutein or zeaxanthin in the RPE cells. The concentrations of lutein and zeaxanthin in the cells were 2–14-fold of that detected in the medium, indicating that ARPE-19 cells actively take up lutein or zeaxanthin. As compared with untreated cells, exposure of A2E-containing RPE to blue light resulted in a 40–60% decrease in proteasome activity, a 50–80% decrease in expression of CFH and MCP-1, and an ~ 20-fold increase in expression of IL-8. The photo-oxidation-induced changes in expression of MCP-1, IL-8 and CFH were similar to those caused by chemical inhibition of the proteasome, suggesting that inactivation of the proteasome is involved in the photo-oxidation-induced alteration in expression of these inflammation-related genes. Incubation of the A2E-containing RPE with lutein or zeaxanthin prior to blue light exposure significantly attenuated the photo-oxidation-induced inactivation of the proteasome and photo-oxidation induced changes in expression of MCP-1, IL-8, and CFH. Together, these data indicate that lutein or zeaxanthin modulates inflammatory responses in cultured RPE in response to photo-oxidation. Protecting the proteasome from oxidative inactivation appears to be one of the mechanisms by which lutein and zeaxanthin modulate the inflammatory response. Similar mechanisms may explain salutary effects of lutein and zeaxanthin in reducing the risk for AMD.

NF-κB is a family of important transcription factors involved in many cellular functions, such as cell survival, proliferation and stress responses. Many studies indicate that NF-κB is a stress sensitive transcription factor and its activation is regulated by reactive oxygen species. In previous studies, we and others demonstrated that this transcription factor can be activated by transient oxidative stress. However, the effects of sustained oxidative stress on NF-κB activation are not clear. The objective of this study is to determine the effects of sustained oxidative stress on NF-κB activation and to elucidate the signaling events affected by sustained oxidative stress. Human lens epithelial cells (HLEC) that were subjected to 4 h of continuous influx of hydrogen peroxide were used to investigate the effects of sustained oxidative stress on NF-κB activation. The data showed that, unlike transient oxidative stress, sustained exposure of HLEC to physiologically relevant levels of H 2 O 2 (50-100 μM for 4) did not induce the degradation of I-κB and activation of NF-κB, but attenuated TNFα-induced degradation of I-κB and activation of NF-κB. Sustained exposure of HLEC to these levels of H 2 O 2 also inactivated proteasome activity by 50-80%. Consistent with the role of the proteasome in degradation of I-κB and activation of NF-κB, treatment of HLEC with proteasome inhibitors also attenuated TNFα-induced I-κB degradation and NF-κB activation. The data also indicate that activation of NF-κB is essential for the cells to recover from oxidative stress. Inhibiting NF-κB activation during recovery from transient oxidative stress significantly reduced the cell viability. Together, these data indicate that sustained oxidative stress may inactivate the proteasome and subsequently inhibit NF-κB activation via impeding the degradation of I-κB. The oxidative inactivation of the proteasome and subsequent impairment of NF-κB activation may contribute to the death of lens epithelial cells, a common feature associated with cataract.

Oxidative stress and inflammation are implicated in the pathogenesis of many age-related diseases. Stress-induced overproduction of inflammatory cytokines, such as interleukin-8 (IL-8), is one of the early events of inflammation. The objective of this study was to elucidate mechanistic links between oxidative stress and overproduction of IL-8 in retinal pigment epithelial (RPE) cells. We found that exposure of RPE cells to H 2 O 2 , paraquat, or A2E-mediated photooxidation resulted in increased expression and secretion of IL-8. All of these oxidative stressors also inactivated the proteasome in RPE cells. In contrast, tert-butylhydroperoxide (TBH), a lipophilic oxidant that did not stimulate IL-8 production, also did not inactivate the proteasome. Moreover, prolonged treatment of RPE cells with proteasome-specific inhibitors recapitulated the stimulation of IL-8 production. These data suggest that oxidative inactivation of the proteasome is a potential mechanistic link between oxidative stress and up-regulation of the proinflammatory IL-8. The downstream signaling pathways that govern the production of IL-8 include NF-B and p38 MAPK. Proteasome inhibition both attenuated the activation and delayed the turnoff of NF-B, resulting in biphasic effects on the production of IL-8. Prolonged proteasome inhibition (>2 h) resulted in activation of p38 MAPK via activation of MKK3/6 and increased the production of IL-8. Chemically inhibiting the p38 MAPK blocked the proteasome inhibition-induced up-regulation of IL-8. Together, these data indicate that oxidative inactivation of the proteasome and the related activation of the p38 MAPK pathway provide a potential link between oxidative stress and overproduction of proinflammatory cytokines, such as IL-8.Oxidative stress, which refers to cellular damage caused by reactive oxygen species, has been implicated in the onset and progression of many age-related diseases, including age-related macular degeneration (AMD), 3 arthritis, atherosclerosis, and certain types of cancer (1-3). Inflammatory events are also known to participate in the pathogenesis of these age-related diseases. The activation of redox-sensitive transcription factors may be involved in triggering the expression of proinflammatory cytokines (2). However, the molecular links between oxidative stress and inflammation are not fully understood.Retina has the highest metabolic rate and oxygen consumption in the body. The high metabolic rate and oxygen consumption is usually accompanied by generation of reactive oxygen species. Chronic exposure to light could also increase the production of reactive oxygen species (1, 4). Therefore, retinal pigment epithelium (RPE) is a primary target of oxidative stress.Age-related accumulation of lipofuscin in RPE is another source of oxidative stress. Lipofuscin is a mixture of nondegradable protein-lipid aggregates derived from the ingestion of photoreceptor outer segments (5). A2E is the major fluorophore of lipofuscin and acts as a photosensitizer to generate reactive oxygen spe...