The Role of Photo-Activated Melanosomes and Cellular Antioxidant ...

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Oct 15, 2001 - Physical and optical properties of RPE melanosomes contributing to these .... determining photophysical properties of the melanosome that ...
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Final Report, 1 Apr 1998 to 31 Mar 2001

15Oct2001

5. FUNDING NUMBERS

4. TITLE AND SUBTITLE

The role of photo-activated melanosomes and cellular antioxidant defenses in the response of RPE cells to laser radiation

G F49620-98-1-210

6. AUTHOR(S)

Randolph D. Glickman, Ph.D. 7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES)

Department of Ophthalmology University of Texas Health Science Center 7703 Floyd Curl Drive San Antonio, Texas 78229-3900

8. PERFORMING ORGANIZATION REPORT NUMBER

UTHSCSA-OPH-01-02

9. SPONSORING / MONITORING AGENCY NAME(S) AND ADDRESS(ES)

AFOSR/NL 801 North Randolph Street, Rm. 732 Arlington, VA 22203-1977

20011126 088

II. SUPPLEMENTARY NOTES

The views, opinions and/or findings contained in this report are those of the author(s) and should not be construed as an official Department of the Air Force position, policy or decision, unless so designated by other documentation. 12 a. DISTRIBUTION / AVAILABILITY STATEMENT

12 b. DISTRIBUTION CODE

Approved for public release; distribution unlimited. 13. ABSTRACT (Maximum 200 words)

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The pigments of the retinal pigment epithelium, i.e. the intracellular granules of melanm,?M$c|^rJfearjp jnej SE shown to catalyze free radical activity, especially when illuminated with visible or ultraviolet hgT— . — „^ ,'*\tfhetl these Ulcse reactions ieacuuiis are aic sufficient suiiiuicin to iu produce piuuiK/C oxidative UAiuauvc damage uamage in 111 the uic eye, for IUI GAauijjic, example, lAjinjwmg following laser KBH ^Apusuit exposure belo urauw lusjjmCTiuamwjU««», threshold. To address this question, the relative photoreactivity of isolated RPE pigment granules towards cellular components has* been determined, including the dark as well as the light-stimulated reactions. Hydroperoxide derivatives of docosahexaenoic acid were produced by irradiation with short wavelength ( i

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Retention Time (min) Figure 12. HPLC analysis of DCFH components. Left panel: DCFH-DA; Center panel: showing the separation of DCFH-DA, DCFH, and DCF simultaneously injected into the analytical column; Right panel: DCF alone (hydrolyzed and oxidized).

hydrolyzed-reduced form, DCFH, elutes at about 5 min, between DCF and DCFH-DA. Note that all forms of DCFH have some degree of fluorescence, which facilitated the detection of each moiety by HPLC, but the quantal efficiency of the oxidized form is much greater than that of the reduced form.

HPLC analysis demonstrated that, following uptake, the probe is hydrolyzed to DCFH inside the RPE cells. Cells were incubated with a 10 [iM solution of DCFH-DA, and aliquots were removed for assay at 20 minute intervals, beginning at 0 min of incubation. The sample was extracted using SPE, chemically oxidized to enhance detection of the compounds, and analyzed by HPLC with fluorescence detection. The peaks in the resulting chromatograms were identified by comparison to the known retention times of the identified chemical species shown in Figure 12. The 25

chromatograms obtained at t = 0', 15', 20', and 60' are shown in Figure 13. At time = 0', all of the probe is present as DCFH-DA. By 15', probe has accumulated in the cells and is starting to be hydrolyzed. (Note that because these samples have been manually oxidized prior to HPLC analysis, little or no DCFH is found, because all DCFH produced by the action of cellular esterases is oxidized to DCF by our procedure). By 20', most accumulated probe has been hydrolyzed, and by 60'. virtually all detectable probe in the cells has been hydrolyzed. Apparently, the total probe content in the cells declines by 60' either because of efflux from the cell, or metabolic breakdown to a form that is either non-fluorescent or not captured by our isolation procedure.

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