Tryosinase Oxidation Of L–dopa Induces Resistance To Oxidative Stress In Human Retinal Pigment Epithelial Cells

Abstract

Abstract: : Purpose: Oxidative stress is likely to be involved in AMD. Development of drugs or therapies effective in reducing oxidative damage to the retinal pigment epithelium is important to preventing or delaying onset of AMD. Methods: Human ARPE–19 cell cultures were exposed to cytotoxic and/or DNA damaging levels of either hydrogen peroxide (H2O2) or tert–butylhydroperoxide (tBHP). Cytotoxicity was measured by MTT assay and Trypan blue exclusion. Cellular damage was measured by quantitating intracellular levels of GSH, effectiveness of choline transport and thymidine and rubidium uptake. DNA damage (single strand break formation) was assayed by alkaline elution. Cell cultures were pretreated with tryosinase and 100 µM L–Dopa in mem or dPBS for up to to 4 hrs prior to washing and exposure to 70–90 µM of either peroxide. Results: As little as 20 min pre–incubation of RPE cells with tyrosinase and L–Dopa protects from tBHP or H2O2 induced cytotoxicity, loss of biochemical integrity and DNA damage. Similar protection is seen when dopamine or tyrosine is substituted for L–Dopa. Protection is evident even when cells are challenged 4 hrs after removal of Dopa and tyrosinase from the medium. No protection is seen by incubation with any of the four compounds alone. Dopa/Tyrosinase treatment does not affect long–term cell survival. Preincubation of tyrosinase with L–Dopa from 1–24 hrs prior to addition to cell cultures results in loss of protection. Conclusions:L–Dopa is known to be the precursor to dopamine, a neurotransmitter active in neural tissue, including RPE cells. Tyrosinase is one of the key enzymes involved in intracellular production of melanin, utilizing tyrosine, Dopa or dopamine. The rapidity with which the protection is generated, its persistence even after the reactants are removed from the medium and the ability to protect against oxidative stress even when relatively low levels of Dopa are used, suggest that the reaction product(s) induces intracellular signaling or processes leading to protection. It is less likely that the protective compound(s) directly detoxify peroxides or other reactive oxygen intermediates. Efforts to identify whether protection is afforded against other types of oxidative stress likely to be relevant to human retinal disease (e.g., photoxidative stress or lipofuscin) are underway as are attempts to identify the protective component(s).

Keywords: dopamine • oxidation/oxidative or free radical damage