MECHANISM OF PHOTOTOXICITY OF PROTOPORPHYRIN AND UROPORPHYRIN ON BOVINE CORNEAL ENDOTHELIUM

Porphyrins are potent photosensitizers. This paper reports the mechani sms of phototoxicity produced by protoporphyrin (PP) and uroporphyrin (UP) in corneal endothelium. Corneas from fresh bovine eyes were expos ed to ultraviolet- (UV) visible radiation in the presence of PP or UP. The control corneas exposed were either irradiated in the absence of porphyrins or incubated in the dark in the presence of PP or UP. After incubation in the dark or irradiation of the corneas, damage to the e ndothelium was assessed by morphologic examination after staining with trypan blue and alizarin red S, increase in corneal thickness measure d using ultrasound pachymetry, and by determining the quantity of lact ate dehydrogenase (LDH) released. The results presented in this paper demonstrate that UV-visible irradiation of bovine corneas in the prese nce of PP and UP induces endothelial damage. Larger numbers of dead en dothelial cells per unit area and larger areas of damage were observed in the corneas irradiated with PP or UP. However the concentration of PP necessary to induce phototoxicity (2-5 mu M) was lower than that o f UP (5-10 mu M). Catalase inhibited the phototoxic effect of UP more than that of PP at the same concentrations. Morphometric analyses of e xperimental and control corneal endothelium supported our qualitative observation with the vital staining of the corneas. Pachymetric studie s show that the corneal thickness increases after irradiation in the p resence of PP or UP. However, in contrast to the endothelial damage ob served by vital staining and morphometry, the increase in corneal thic kness produced by PP or UP was of the same order. Corneal thickness wa s increased after irradiation in the presence of different concentrati ons of PP or UP. As the concentration of PP or UP increased, the corne al thickness was proportionately increased. At 5 mu M PP or UP the thi ckness was increased by 27.9% and 37.1%, respectively. Incubation of c orneas in the dark with the 5 mu M of PP or UP increased the corneal t hickness only by 12.3% and 11.8%, respectively. The LDH leakage into t he media increased as the concentration of the porphyrin was increased . Addition of catalase to the medium did not affect the LDH release wh en 1 or 2 mu M PP was added. It decreased the LDH release when the PP concentration was 5 mu M. However, the LDH release at 5 or 10 mu M UP was completely abolished by catalase. Therefore, it seems that the UP phototoxicity in cornea is largely due to H2O2, whereas the PP phototo xicity is more due to O-1(2).