Metallothionein Shows an Age-Related Decrease in Human. Macular Retinal Pigment Epithelium. David J. Tate,Jr.,* David A. Neiusome,*-f and Peter D. Olivef*' ...
Metallothionein Shows an Age-Related Decrease in Human Macular Retinal Pigment Epithelium David J. Tate,Jr.,* David A. Neiusome,*-f and Peter D. Olivef*'X
Purpose. To investigate the possible role of zinc-metallothionein in human retinal pigment epithelium with regard to age-related changes. Methods. A cadmium/heme assay was used to quantitate metallothionein in isolated macular and peripheral retinal pigment epithelium from donors ranging in age from 28 to 91 yr (n = 1 6, mean age = 68.6 yr). Results. It was found that peripheral retinal pigment epithelium contained significantly more metallothionein and zinc than macular retinal pigment epithelium. Macular retinal pigment epithelium cells contained 17.6 ± 2.2 jug metallothionein/mg cytosolic protein in donors younger than 70 yr, compared to 5.6 ± 0.9 in macular retinal pigment epithelium from donors older than 70 yr, a 68% decline (P = 0.0007). In cultured retinal pigment epithelium, when we lowered the zinc concentration in the medium, metallothionein was reduced by 72% after 1 wk of incubation. Conclusions. It is suggested that lower levels of metallothionein in retinal pigment epithelium are caused by reduced metallothionein gene activity or a faster rate of protein degradation, both of which are known to be regulated, at least partly, by bioavailable zinc. Invest Ophthalmol Vis Sci 1993;34:2348-2351.
W e recently reported the presence and inducibility of metallothionein (MT) in human retinal pigment epithelium (RPE) in addition to focusing on possible roles of this protein in the RPE.1 One essential property of MT is the binding and releasing of zinc. A reduction or increase in bioavailable zinc affects intracellular MT content, which may provide an index of the intracellular zinc status. For example, in zinc-deficient rats, liver MT is undetectable.2 Conversely, dietary zinc supplementation in rats causes an increase in cytosolic zinc caused by greater amounts of zinc-MT.3 From the *Sensory and Eleclrophysiology Research Unit, Tmiro Infirmary, New Orleans. Umisimm, and the Departments of ^Ophthalmology and %Anatomy, Tultme University School of Medicine, New Orleans, Louisiana. Supported by grant EY-006677 (D.A.N.)from the National Institutes of Health, Bethesda, Maryland. Support of the f. Willard Marriott Foundation, Washington, D.C. is also acknowledged. Submitted for publication: April 24, 1992; accepted August 31, 1992. Proprietary Interest Category: N. Reprint requests: Peter D. Oliver, Sensory and Eleclrophysiology Research Unit, Touro Infirmary, HOI Voucher Street. New Orleans, LA 70115.
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Zinc and MT are also believed to protect cells from oxidative damage. Normal exposure of RPE cells to oxidative processes may be exacerbated by additional sources of reactive oxygen intermediates generated during phagocytosis and degradation of photoreceptor outer segments.4 We previously reported an observed age-related decline in detectable activity of one specific RPE antioxidant enzyme, catalase, whereas superoxide dismutase did not show a significant decline.5 In the current study, we extended our studies on age-associated changes in macular RPE by measuring MT in RPE isolated from human donors of various ages.
MATERIALS AND METHODS Isolation of Ocular Tissues Human donor eyes were obtained from the National Disease Research Interchange (Philadelphia, PA) by ;stigative Ophthalmology & Visual Sc Copyright © Association for Kesear
1993, Vol. 34, No. 7 ii and Ophthalmology
Age-Related Decrease in Human RPE Metallothionein
2349 Micrograms of MT/mg Protein
24 hr postmortem. RPE cells were isolated by scraping with a small spatula (Moria, Paris, France), as previously described.5 The macular region was extended to include a circular area with its radius extending from the fovea to the optic nerve. Because of limitations of tissue, zinc analysis and metallothionein assays were not run on the same series of donors.
^ 1 Peripheral ^M Macular
Preparation of Cellular Protein Tissue samples were sonicated (VirSonic 50; Virtis Co, Inc., Gardiner, NY) at 30 sec in 100 p\ ice-cold 20 mM Tris-HCl buffer (pH 7.8). All chemicals were obtained from Sigma Chemical Company (St. Louis, MO) unless otherwise specified. After centrifugation (Eppendorf Centrifuge 5415, Brinkmann, Westbury, NY) at 13,000 rpm for 10 min, the supernatants were collected for protein determination based on the method of Bradford (Biorad, Richmond, CA). For measuring zinc content, ocular tissue was placed in 0.5 M NaOH overnight, and a sample was taken for protein determination. The remainder was neutralized with 100 fi\ of 0.5 M HC1, dried, and treated with 100 /x\ of H 2 O 2 (65°C). Samples were analyzed for total zinc content by flame atomic absorption spectroscopy (laboratory of Dr. Nancy W. Alcock, Galveston, TX). Cadmium/Hemoglobin Assay for Metallothionein The MT was determined using the procedure of Eaton and Toal.6 The protein concentration in each sample was adjusted to 50 /itg/200 /xl After heating (100°C, 2 min), the supernatant was added to 200 til of a solution containing 1 /iCi/ml cadmium-109 (NEN, Boston, MA) and 1 jug/ml total cadmium. Statistical analyses were performed using the Student's t test and linear regression analysis on the Statistical Analysis Software program (SAS Institute Inc., Cary, NC).
Culture of RPE Cells RPE cell cultures were established and maintained as previously described.1 Zinc depletion studies on cultured cells were done in Dulbecco's Modified Eagle's medium (DMEM). Control cells were cultured in
l. Zinc and Metallothionein in Peripheral and Macular Retinal Pigment Epithelium
TABLE
Tissue
Zinc (ng/mg Protein)
Metallothionein (f-g/mg Protein)
Peripheral Macular P/M ralio
1.73 ±0.21* 0.89 ± 0.07* 1.94
20.0 ± 1.90f 11.6 ± 1.90t 1.72
Values are mean ± SEM. * P = 0.0001, mean age 70.3 yr, n = 12. fP= 0.0008, mean age 68.6 yr, n = 16.
5
^^^^^^^^H Under 70
Above 70
Age Group FIGURE l. MT in RPE assessed by Cd/heme assay. The values for MT in macular and peripheral RPE were separated by age, younger than 70 (mean age 58 yr) and older than 70 (mean age 80 yr). DMEM supplemented with fetal bovine serum (2.5% FBS, Hyclone, Logan, UT), GASP (glutamine, ascorbate, penicillin G, and streptomycin) and epidermal growth factor (EGF, Intergen, Purchase, NY). To eliminate zinc in the medium, 100 ml of complete medium was incubated overnight in two changes of 30 g of Chelex-100.7 To reconstitute possible loss of other cations, the medium was supplemented with CaCl2 and MgCl2 (1 mM), CuSO4-5H2O (125 pg/ml), MnCl2 (50 pg/ml), Na2SeO3 (5 ng/ml). The zinc content of complete DMEM, determined by flame atomic absorption spectroscopy, was 2 juM and 0.6 IAM before and after chelation, respectively. The control medium consisted of the zinc-deficient medium (0.6 nM zinc) supplemented with an additional 15 /uM ZnCl2. After 1 wk of culture, some of the cells in zinc-deficient medium were incubated 24 hr with ZnCl2 (30 and 100 fiM) before collection.
RESULTS Zinc content of peripheral and macular RPE was measured and expressed as Mg/mg cytosolic protein (Table 1). Zinc content in the peripheral RPE (1.73 ± 0.21) was significantly greater than in macular RPE (0.89 ± 0.07), with a ratio of 1.94. There was no significant decline in the total zinc content with regard to age. We also found significantly higher levels of MT in peripheral RPE (20 ± 1.90 jig/ing protein) than in macular RPE (11.6 ± 1.90), with a ratio of 1.72 (Table 1). The differences in MT levels between peripheral and macular RPE were further analyzed by dividing the subjects into two equal age groups, younger and older than 70 (n = 8, mean age = 58 yr and n = 8, mean age = 80 yr, respectively). In the group younger than 70 yr, the levels of MT in the peripheral and macular RPE were 24.2 ± 2.6 and 17.6 ± 2.2, respec-
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Investigative Ophthalmology & Visual Science, June 1993, Vol. 34, No. 7
tively (Fig. 1). In comparison, MT in RPE of the older group was lower in peripheral (15.9 ± 2.1) and macular RPE samples (5.6 ± 0.9), a 2.8-fold difference (p = 0.001). In comparing the younger with the older age category, there was a 34% decrease in MT of peripheral RPE (p = 0.03) and a 68% decrease in the macular RPE (P = 0.0007). The age-related decrease in macular MT was further evidenced by plotting the MT levels against the age of each subject (ages 28-91 yr). These data generated a linear regression curve showing a significant correlation between the decline of MT and increasing age (R = -0.87, P < 0.0001; Fig. 2). To test the possibility that reduced RPE MT levels could be partly attributable to lowered levels of bioavailable zinc concentrations, we cultured cells in medium that had been pretreated with Chelex-100 to reduce the zinc content as described in Methods. After 1 wk in culture, the cells in the control medium (15.6 /uM ZnCl2) contained MT levels of 8.4 ± 0.5 Mg/mg P ro ~ tein. RPE cells cultured in the zinc-deficient medium (0.6 fiM ZnCl2) for 7 days showed 72% less than control values {P = 0.0001). The addition of 30 /M ZnCl2 to the zinc-deficient medium for 24 hr before collection of cells increased MT levels to that of control (Fig. 3). Addition of 100 ^M ZnCl2 to the zinc-deficient group for 24 hr caused an increase to 178% of the control value (P = 0.0001), representing a sixfold increase over the zinc-deficient cells.
DISCUSSION We found both zinc and MT content to be greater in peripheral RPE than in macular RPE. Although we found no significant decline in zinc with regard to age, our procedures did not allow fractionation of the cells to distinguish between zinc in pigment granules from that in the soluble cytoplasmic protein fraction, where Micrograms of MT/mg Protein
a
—
30
50
60 Age
70
80
90
100
FIGURE 2. Decline of MT in macular RPE as a function of age. Ages of subjects ranged from 28 to 91 yr (P = 0.0001, R = - 0 . 8 7 , n = 16).
% Control
Zn-Deflcient
30 uM Zn
100 uM Zn
FIGURE 3. MT levels in RPE cells cultured in media with various levels of zinc presented as % control (8.4 /xg/mg protein). 30 iM and 100 fiM zinc were added to zinc-reduced medium 24 hr before collection of cells.
one would expect to find MT. Human and nonhuman pigmented ocular tissues contain high levels of zinc, much of which may be associated with melanin.8 The difference between the macular and peripheral MT levels was greater in the over-70 age group. In addition, the age-related decline in MT levels in RPE was more pronounced in the macula, which showed a 68% decrease in MT in the over-70 age group. Because our results are expressed in jug MT/mg protein, one must consider the possibility that the relative amount of MT could be reduced by an age-related accumulation of cellular proteins. One possible source of protein accumulation is in lipofuscin. In a recent study about age-related changes in lipofuscin-associated protein isolated from human RPE, it was shown that an older age group (mean age = 71 yr) had 33 jig/eye of recoverable lipofuscin associated protein, compared with 8 fJ-g/eye in a younger age group (mean age = 25 yr).9 If one estimates that the RPE layer of an eye contains 500 jug of total protein, a 25 n-g increase in lipofuscin-associated protein represents only 5% of total cellular protein. This would not account for the 68% decrease in macular RPE MT we have observed in our study when comparing the younger (mean = 58 yr) and the older (mean = 80 yr) age groups. In a previous study, we also observed an age-related decline in catalase activity in RPE, but found no difference in activity between the macular and peripheral regions.5 Conversely, superoxide dismutase activity/mg protein showed neither a decrease with age, nor a difference between macular and peripheral RPE.5 Initial studies in our laboratory measuring glutathione peroxidase activity/mg protein also reveal no decline with respect to age. High levels of MT gene expression in human RPE may be a normal physiologic response to stress-in-
Age-Related Decrease in Human RPE Metallothionein duced biologic factors. The lower levels of MT in the RPE may result from decreased gene activity or an increased rate of degradation, both of which can be influenced by zinc levels. Zinc has been shown to stimulate MT mRNA production by direct interaction with the promoter region of the MT gene.10 Conversely, when extracellular zinc concentrations are reduced, zinc dissociates from MT, resulting in a faster rate of degradation of the MT.7 Our studies on cultured cells show that MT levels in human RPE cells increase or decrease substantially as zinc levels in the medium are increased or decreased. Zinc deficiency can cause certain visual defects, e.g., night blindness, which can be reversed by dietary zinc supplementation.11 In the case of other diseases with multiple causes, e.g., macular degeneration, it is not clear to what extent zinc deficiency can contribute to the disease processes.12 Zinc has an essential function in numerous enzyme and molecular interactions, and zinc-MT may protect cells from damage resulting from exposure to heavy metal toxicity, alkylating agents, and oxygen-generated free radicals.13 MT is believed to act as an intracellular storage site capable of donating zinc to other molecules that require zinc to function. 710 It should be pointed out that zinc itself is toxic if taken in excessive doses and may substantially interfere with the utilization of copper and iron.14 More studies are needed to determine how MT levels are regulated in human RPE, and to elucidate the importance of MT and zinc in maintaining RPE functional integrity. Key Words metallothionein, human retinal pigment epithelium, zinc, and aging References 1. Oliver PD, Tate DJ, Newsome DA. Metallothionein in human retinal pigment epithelial cells: expression, induction and zinc uptake. Curr Eye Res. 1992; 11:183188.
2351 2. Sato M, Mehra RK, Bremner I. Measurement of plasma metallothionein-I in the assessment of zinc status of zinc deficient and stressed rats. J Nutr. 1984;114:1683-1689. 3. McCormick CC, Menard MP, Cousins RJ. Induction of hepatic metallothionein by feeding zinc to rats of depleted zinc status. Am J Physiol. 1981;240:E414E421. 4. Liles MR, Miceli MV, Oliver PD, Newsome DA. Catalase protects against intracellular oxidative damage during phagocytosis of bovine ROS by RPE. ARVO Abstracts. Invest Ophthalmol Vis Sci. ]992; 33(suppl):912A. 5. Liles MR, Newsome DA, PD Oliver. Antioxidant enzymes in the aging human retinal pigment epithelium. Arch Ophthalmol. 1991; 109:1285-1288. 6. Eaton DL, Toal BF. Evaluation of the Cd/hemoglobin affinity assay for the rapid determination of metallothionein in biological tissues. Toxicol Appl Pharmacol. 1982;66:134-142. 7. Krezoski SK, Villalobos J, Shaw III CF, Petering DH. Kinetic lability of zinc bound to metallothionein in Ehrlich cells. BiochemJ. 1984;255:483-491. 8. Newsome DA, Oliver PD, Deupree DM, Miceli MV, Diamond JG. Zinc uptake by primate retinal pigment epithelium and choroid. Curr Eye Res. 1992; 11:213217. 9. Bazan HEP, Bazan NG, Feeney-Burns L, Berman ER. Lipids in human lipofuscin-enriched subcellular fractions of two age populations. Comparison with rod outer segments and neural retina. Invest Ophthalmol Vis Sci. 1990;31:1433-1443. 10. Richards RI, Heguy A, Karin M. Structural and functional analysis of the human metallothionein-IA gene: differential induction by metal ions and glucocorticoids. Cell. 1984;37:263-272. 11. McClain CJ, Kasarskis Jr EJ, Allen JJ. Functional consequences of zinc deficiency. Prog Food Nutr Sci. 1985;9:185-226. 12. Newsome DA, Swartz M, Leone NC, Elston RC, Miller E. Oral zinc in macular degeneration. Arch Ophthalmol. 1988; 106:192-198. 13. Prasad AS. Discovery of human zinc deficiency and studies in an experimental human model. Am J Clin Nulr. I991;53:403-412. 14. Fosmire GJ. Zinc toxicity. Am J Clin Nutr. 1990;51:225-227.
