Nilesh M. Kalariya

Copper and zinc distribution in the human retina: Relationship to cadmium accumulation, age, and gender

The essential metals copper and zinc play vital roles in retinal cell survival and are crucial for the normal functioning of antioxidant enzymes. Retinal zinc deficiencies and decreased cellular antioxidative capacity have been linked to human retinal diseases including age-related macular degeneration (AMD). We recently reported that cadmium (a toxic metal with no known physiological function that interferes with copper and zinc metabolism) accumulates in human retinal tissues during aging. Moreover, cadmium content was higher in specific retinal tissues of aged women compared to men. Since cadmium, zinc and copper bind to similar proteins, we hypothesized that Cu and Zn content of human retinal tissues change as functions of cadmium accumulation during aging. Thus, we assessed the distribution of zinc and copper in the neural retina, retinal pigment epithelium (RPE) and choroid (Bruchs membrane-choroid; BMC) in male and female donors aged 1.5-87 years. Two independent methods, graphite furnace atomic absorption spectrometry and inductively-coupled plasma mass spectrometry, were used to measure Cd, Zn, and Cu in retinal tissues in human eyes from donors aged 1.5 to 87 years and the resulting values were normalized to protein concentration. Zn levels were approximately 5 times higher than Cu levels in the same tissues. The relative tissue distributions of these metals were: BMC>RPE>neural retina (Zn) and BMC>RPE=neural retina (Cu). In the choroid, mean Cu and Zn levels were higher in aged donors (>or=55 years old) than young donors (<55 years) and levels of these metals were strongly correlated with each other (r=0.90). In the neural retina, Cu and Zn both significantly decreased as a function of age. Several sex-related differences were found in the RPE. Specifically, copper levels were significantly higher in males than in females. In addition, both Zn and Cu levels in males were positively correlated with cadmium content, whereas this association did not occur in females. The results are consistent with co-regulation of zinc and copper stores in retinal tissues and suggest that the balance of these metals is associated with cadmium accumulation and gender. Thus, the roles of cadmium and gender differences in retinal metal balance warrant further investigation as factors in age-related retinal disease.

Cadmium-induced apoptotic death of human retinal pigment epithelial cells is mediated by MAPK pathway

Cadmium (Cd), released from cigarette smoke and metal industrial activities, is known to accumulate in human body organs including retina and is particularly higher in retinal tissues of age-related macular degeneration (AMD) eyes compared to non-AMD eyes. We have determined the cytotoxic effects of Cd on human retinal pigment epithelial (RPE) cells. Upon Cd treatment, there was a dose- and time-dependent decline in ARPE-19 cell viability as well as early apoptotic changes such as altered mitochondrial membrane potential (MMP) and Cytochrome C release in cytosol. Depletion of GSH by buthionine-[S,R]-sulfoximine (BSO) resulted in increased Cd toxicity in ARPE-19 cells. Cadmium also caused reactive oxygen species (ROS) generation and activation of mitogen-activated protein kinases (MAPKs) pathway including c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase 1/2 (Erk1/2), and p38 in ARPE-19 cells. Antioxidants such as N-acetylcysteine (NAC) significantly reduced Cd-induced toxicity. These results indicate that elevated ROS-induced activation of the MAPK signaling pathway could be associated with Cd-induced RPE cell apoptosis, one of the major contributing factors in AMD. The toxic effects of Cd on ARPE-19 cells indicate that environmental heavy metals such as Cd could be important potential factors in RPE cells death associated retinal diseases particularly related to smoking.

Cadmium-induced induction of cell death in human lens epithelial cells: implications to smoking associated cataractogenesis.

Cadmium is reported to accumulate in human eye tissues suggesting its implication in diverse ocular pathology. Using an in vitro cell culture model we investigated the effects of cadmium on human lens epithelial cells (HLECs) (HLE-B3). We observed cadmium-induced dose- as well as time-dependent decline in HLECs viability which was exacerbated significantly upon reduction of intracellular glutathione levels by buthionine sulfoximine (BSO). There was a dose-dependent significant increase in lactate dehydrogenase (LDH) release from HLECs suggesting cadmium-induced alteration of membrane integrity as well as necrotic cell death. The decline in cell viability was also due to apoptosis of the HLECs as determined by quantifying % apoptotic cells as well as PARP cleavage. Moreover, release of apoptosis inducing factor (AIF) into the cytosol was also detected. Cadmium was also observed to increase oxidative stress, lipid peroxidation and activation of MAPK pathway in HLECs. Antioxidants like N-acetylcysteine (NAC) and alpha-Tocopherol significantly prevented cadmium-induced toxicity in HLECs. Our findings suggest that cadmium-induced elevated oxidative stress as well as activation of MAPK signaling cascade eventually led to cell death of HLECs through apoptosis as well as necrosis. The loss of HLECs by cadmium could possibly explain its implication in cataract development particularly associated with smoking.

Antidiabetic Drug Metformin Suppresses Endotoxin-Induced Uveitis in Rats

PURPOSE To investigate the therapeutic effects of metformin, a commonly used antidiabetic drug, in preventing endotoxin-induced uveitis (EIU) in rats. METHODS EIU in Lewis rats was developed by subcutaneous injection of lipopolysaccharide (LPS; 150 μg). Metformin (300 mg/kg body weight, intraperitoneally) or its carrier was injected either 12 hours before or 2 hours after LPS induction. Three and 24 hours after EIU, eyes were enucleated and aqueous humor (AqH) was collected. The MILLIPLEX-MAG Rat cytokine-chemokine magnetic bead array was used to determine inflammatory cytokines. The expression of Cox-2, phosphorylation of AMPK, and NF-κB (p65) were determined immunohistochemically. Primary human nonpigmented ciliary epithelial cells (HNPECs) were used to determine the in vitro efficacy of metformin. RESULTS Compared with controls, the EIU rat AqH had significantly increased number of infiltrating cells and increased levels of various cytokines and chemokines (TNF-α, MCP-1, IL-1β, MIP-1α, IL-6, Leptin, and IL-18) and metformin significantly prevented the increase. Metformin also prevented the expression of Cox-2 and phosphorylation of p65, and increased the activation of AMPK in the ciliary bodies and retinal tissues. Moreover, metformin prevented the expression of Cox-2, iNOS, and activation of NF-kB in the HNPECs and decreased the levels of NO and PGE2 in cell culture media. CONCLUSIONS Our results for the first time demonstrate a novel role of the antidiabetic drug, metformin, in suppressing uveitis in rats and suggest that this drug could be developed to prevent uveitis complications.

Emerging Role of Antioxidants in the Protection of Uveitis Complications

Current understanding of the role of oxidative stress in ocular inflammatory diseases indicates that antioxidant therapy may be important to optimize the treatment. Recently investigated antioxidant therapies for ocular inflammatory diseases include various vitamins, plant products and reactive oxygen species scavengers. Oxidative stress plays a causative role in both non-infectious and infectious uveitis complications, and novel strategies to diminish tissue damage and dysfunction with antioxidant therapy may ameliorate visual complications. Preclinical studies with experimental animals and cultured cells demonstrate significant anti-inflammatory effects of a number of promising antioxidant agents. Many of these antioxidants are under clinical trial for various inflammatory diseases other than uveitis such as cardiovascular, rheumatoid arthritis and cancer. Well planned interventional clinical studies in the field of ocular inflammation will be necessary to sufficiently investigate the potential medical benefits of antioxidant therapies for uveitis. This review summarizes the recent investigations of novel antioxidant agents for ocular inflammation, with selected studies focused on uveitis.

Human retinal cadmium accumulation as a factor in the etiology of age-related macular degeneration

Cadmium is a naturally occurring, highly toxic, metallic element. It pollutes the environment as a result of industrial activity and accumulates in human tissues with a long biological half-life. Cadmium content has been demonstrated to increase in human retinal tissues as a function of age and tobacco smokers have approximately twice as much cadmium in retinal tissues than non-smokers. Smoking is also a key environmental risk factor for the retinal disease age-related macular degeneration (AMD). Recent studies have shown that urinary cadmium levels (a measure of Cd body burden) are higher in smokers who have AMD. We now report the Cd measurements in human retinal tissues from eyes afflicted with AMD compared to non-diseased eyes (controls) from age-matched donors. Human donor eyes frozen under argon gas were assessed for AMD severity using color stereoscopic fundus photographs and the Minnesota Grading System. Cadmium, zinc and, copper levels were measured in retinal tissues (neural retina, retinal pigment epithelium and choroid) using inductively coupled plasma mass spectrometry and graphite furnace spectrophotometry and values were normalized to tissue protein levels. Higher Cd levels were found in the neural retina and RPE for eyes afflicted with AMD compared to controls in males, differences were not statistically significant in females. The results indicate that higher retinal cadmium burdens are associated with the presence of AMD at least in males and suggest possible gender differences in the metabolism of metals in the human retina.

Prevention of Endotoxin-Induced Uveitis in Rats by Plant Sterol Guggulsterone

PURPOSE To investigate the anti-inflammatory effects of guggulsterone, an antioxidant and antitumor agent, in endotoxin-induced uveitis (EIU) in rats and to elucidate the underlying molecular mechanism or mechanisms related to ocular inflammation. METHODS EIU was induced by subcutaneous injection of lipopolysaccharide (LPS; 150 μg) into Lewis rats treated with guggulsterone (30 mg/kg body weight, intraperitoneally) or its carrier. After 24 hours the rats were killed, eyes were enucleated, and aqueous humor (AqH) was collected. Numbers of infiltrating cells and levels of matrix metalloproteinase-2 (MMP-2), nitric oxide (NO), and prostaglandin E(2) (PGE(2)) were determined in AqH by specific ELISAs. An antibody array was used to measure the expression of various inflammatory cytokines in AqH. The expression of MMP-2, iNOS, Cox-2, phospho-IκB, and phospho-NF-κB was determined immunohistochemically. Human primary nonpigment ciliary epithelial cells (HNPECs) were used to determine the in vitro efficacy of guggulsterone on the LPS-induced inflammatory response. RESULTS Compared with control, the EIU rat eye AqH had a significantly higher number of infiltrating cells, total protein, and inflammatory markers, such as MMP-2, NO, and PGE(2), and the treatment of guggulsterone prevented EIU-induced increases. Guggulsterone also prevented the expression of MMP-2, iNOS, and Cox-2 proteins and of IκB and NF-κB in various eye tissues. Moreover, in cultured HNPECs, guggulsterone inhibited LPS-induced expression of inflammatory proteins. CONCLUSIONS These results for the first time demonstrate that the plant sterol guggulsterone suppresses ocular inflammation in EIU, suggesting that the supplementation of guggulsterone could be a novel approach for the treatment of ocular inflammation.

Genotoxic effects of carotenoid breakdown products in human retinal pigment epithelial cells

Purpose: To investigate the genotoxic effects of lutein (LBP) and β -carotene breakdown products (β -apo-8-carotenal, BA8C) and the preventive role of GSH in human retinal pigment epithelial cells (ARPE-19). Methods: LBP- and BA8C-induced DNA damage in human retinal pigment epithelial cells (ARPE-19) was determined by comet assay. The DNA damage was quantified by the image analysis system using Comet Score™ software. ARPE-19 cell viability was determined by CellTiter 96 AQueous one-solution cell proliferation assay kit. Intracellular GSH levels were measured by Ellmans reagent. Results: Incubation of serum-starved ARPE-19 cells with LBP and BA8C caused significant DNA damage in a dose- and time-dependent manner. The DNA damage and cell death incurred by LBP and BA8C were significantly prevented by N-acetylcysteine (NAC) but not by α -tocopherol + ascorbic acid (T + AA). Furthermore, BSO-induced GSH depletion in ARPE-19 cells caused a significant elevation in LBP- and BA8C-induced DNA damage, whereas increased GSH levels in ARPE-19 cells prevented it. Conclusions: Our results suggest that breakdown products of dietary carotenoids could be genotoxic in ARPE-19 cells. LBP-induced genotoxic effects could worsen oxidative stress. The intracellular GSH pool in ARPE-19 cells might play a critical role in carotenoid breakdown products-induced genotoxicity.

Protective role of benfotiamine, a fat-soluble vitamin B1 analogue, in lipopolysaccharide-induced cytotoxic signals in murine macrophages.

This study was designed to investigate the molecular mechanisms by which benfotiamine, a lipid-soluble analogue of vitamin B1, affects lipopolysaccharide (LPS)-induced inflammatory signals leading to cytotoxicity in the mouse macrophage cell line RAW264.7. Benfotiamine prevented LPS-induced apoptosis, expression of the Bcl-2 family of proapoptotic proteins, caspase-3 activation, and PARP cleavage and altered mitochondrial membrane potential and release of cytochrome c and apoptosis-inducing factor and phosphorylation and subsequent activation of p38-MAPK, stress-activated kinases (SAPK/JNK), protein kinase C, and cytoplasmic phospholipase A2 in RAW cells. Further, phosphorylation and degradation of inhibitory kappaB and consequent activation and nuclear translocation of the redox-sensitive transcription factor NF-kappaB were significantly prevented by benfotiamine. The LPS-induced increased expression of cytokines and chemokines and the inflammatory marker proteins iNOS and COX-2 and their metabolic products NO and PGE(2) was also blocked significantly. Thus, our results elucidate the molecular mechanism of the anti-inflammatory action of benfotiamine in LPS-induced inflammation in murine macrophages. Benfotiamine suppresses oxidative stress-induced NF-kappaB activation and prevents bacterial endotoxin-induced inflammation, indicating that vitamin B1 supplementation could be beneficial in the treatment of inflammatory diseases.

Original ContributionProtective role of benfotiamine, a fat-soluble vitamin B1 analogue, in lipopolysaccharide-induced cytotoxic signals in murine macrophages

This study was designed to investigate the molecular mechanisms by which benfotiamine, a lipid-soluble analogue of vitamin B1, affects lipopolysaccharide (LPS)-induced inflammatory signals leading to cytotoxicity in the mouse macrophage cell line RAW264.7. Benfotiamine prevented LPS-induced apoptosis, expression of the Bcl-2 family of proapoptotic proteins, caspase-3 activation, and PARP cleavage and altered mitochondrial membrane potential and release of cytochrome c and apoptosis-inducing factor and phosphorylation and subsequent activation of p38-MAPK, stress-activated kinases (SAPK/JNK), protein kinase C, and cytoplasmic phospholipase A2 in RAW cells. Further, phosphorylation and degradation of inhibitory kappaB and consequent activation and nuclear translocation of the redox-sensitive transcription factor NF-kappaB were significantly prevented by benfotiamine. The LPS-induced increased expression of cytokines and chemokines and the inflammatory marker proteins iNOS and COX-2 and their metabolic products NO and PGE(2) was also blocked significantly. Thus, our results elucidate the molecular mechanism of the anti-inflammatory action of benfotiamine in LPS-induced inflammation in murine macrophages. Benfotiamine suppresses oxidative stress-induced NF-kappaB activation and prevents bacterial endotoxin-induced inflammation, indicating that vitamin B1 supplementation could be beneficial in the treatment of inflammatory diseases.