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.

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 (Bruch's 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 accumulation in the human retina: effects of age, gender, and cellular toxicity.

Tobacco smoking and aging are among the few factors linked to age-related macular degeneration (AMD), a major cause of blindness in the elderly. Recent studies indicate that cadmium (Cd), an environmental toxic trace metal, is approximately four-fold higher in the retinas of smokers compared to non-smokers. In this study, we determined the effects of age and gender on Cd accumulation in human retinal tissues, specifically the neural retina, retinal pigment epithelium (RPE), and choroid. Cadmium levels in cultured RPE cells or retinal tissues isolated from frozen donor eyes were measured using inductively coupled plasma mass spectrometry (ICP-MS) and graphite furnace atomic absorption spectrophotometry (GF-AAS). Cadmium uptake in cultured human RPE cells (ARPE-19) was also assessed using GF-AAS. Toxic effects of cadmium were determined from cell loss (measured as a decrease in cell density) and lactate dehydrogenase release (an indicator of membrane disruption). In "young" eyes (< 55 years) Cd was highest in the retinal pigment epithelium and lowest in the neural retina. Cd was higher in all tissues in aged eyes (>or=55 years) and was significantly higher in the neural retina and RPE in older females. Cultured RPE cells exposed to Cd showed altered cell morphology, decreased cell survival, elevated ROS levels and concentration-dependent disruption of membrane integrity. We conclude that cadmium is accumulated differently in the neural retinal and RPE of older men and women. The deleterious effects of Cd on RPE cells indicate that this environmental toxin is a potentially important factor in age-related retinal disease.

Macular pigment in the human retina: histological evaluation of localization and distribution.

PURPOSE: Clinical investigations have demonstrated variation in both the peak optical density and the spatial distribution of macular pigment. To confirm these impressions histologically, the present study examined the distribution of macular pigment in the human retina. MATERIALS AND METHODS: The macular retina of 11 donor eyes of different ages (28-91 years) were examined histologically on 100 microm vibratome sections directly, without further staining. Measurements were made in two dimensions: (1) adding the number of macular sections with visible macular pigment, and (2) direct measurement of the extension of macular pigment in the foveolar section, which visibly contained the most macular pigment. RESULTS: The measurements with two methods demonstrated good correlation. The macula demonstrated a variation in the spatial extension of the visible macular pigment between 200 and 900 microm diameter around the centre of the fovea, which was also found when direct measurements were taken. There was no correlation with the donor age. The main location of macular pigment was in the layer of the fibres of Henle in the fovea and in the inner nuclear layer at the parafoveal site. CONCLUSIONS: Histologically, a wide variation of the spatial distribution of macular pigment was found that confirms clinical observations. The primary localization of human macular pigment is in the inner retinal layers.

Toxicity and detoxification of lipid-derived aldehydes in cultured retinal pigmented epithelial cells.

Age-related macular degeneration (ARMD) is the leading cause of blindness in the developed world and yet its pathogenesis remains poorly understood. Retina has high levels of polyunsaturated fatty acids (PUFAs) and functions under conditions of oxidative stress. To investigate whether peroxidative products of PUFAs induce apoptosis in retinal pigmented epithelial (RPE) cells and possibly contribute to ARMD, human retinal pigmented epithelial cells (ARPE-19) were exposed to micromolar concentrations of H2O2, 4-hydroxynonenal (HNE) and 4-hydroxyhexenal (HHE). A concentration- and time-dependent increase in H2O2-, HNE-, and HHE-induced apoptosis was observed when monitored by quantifying DNA fragmentation as determined by ELISA, flow cytometry, and Hoechst staining. The broad-spectrum inhibitor of apoptosis Z-VAD inhibited apoptosis. Treatment of RPE cells with a thionein peptide prior to exposure to H2O2 or HNE reduced the formation of protein-HNE adducts as well as alteration in mitochondrial membrane potential and apoptosis. Using 3H-HNE, various metabolic pathways to detoxify HNE by ARPE-19 cells were studied. The metabolites were separated by HPLC and characterized by ElectroSpray Ionization-Mass Spectrometry (ESI-MS) and gas chromatography-MS. Three main metabolic routes of HNE detoxification were detected: (1) conjugation with glutathione (GSH) to form GS-HNE, catalyzed by glutathione-S-transferase (GST), (2) reduction of GS-HNE catalyzed by aldose reductase, and (3) oxidation of HNE catalyzed by aldehyde dehydrogenase (ALDH). Preventing HNE formation by a combined strategy of antioxidants, scavenging HNE by thionein peptide, and inhibiting apoptosis by caspase inhibitors may offer a potential therapy to limit retinal degeneration in ARMD.