The Influence of Iron Chelators on the Accumulation of Protoporphyrin IX in 5-aminolaevulinic Acid-treated Cells

Human adenocarcinoma cells of the line WiDr and Chinese hamster lung fibroblasts of the line V79 were treated with 5-aminolaevulinic acid (5-ALA) and exposed to light. The effects of the iron chelators ethylenediaminetetraacetic acid (EDTA) and desferrioxamine (DEF) were assessed. Both cell lines were treated with various concentrations of 5-ALA in the presence or absence of the iron chelators for 4 h in serum-free medium. The accumulation of protoporphyrin IX (PpIX) reached a maximum level at 1 mM 5-ALA in WiDr cells [280 ng PpIX (mg protein x 4 h-1] and at 0.1 mM 5-ALA in V79 cells [55 ng PpIX (mg protein x 4 h)-1]. PpIX was the only fluorescing porphyrin in these cells after 5-ALA treatment alone or in combination with the chelators. The iron chelators did not influence the intracellular localisation pattern of PpIX in 5-ALA-treated cells. While both chelators enhanced the accumulation of PpIX in 5-ALA-treated cells, DEF was found to be superior at equal concentrations. A linear relationship between the applied concentration of DEF and the DEF-induced increase in PpIX accumulation was observed in double-reciprocal plots. The intercepts of the regression lines with the ordinate indicate that the ferrochelatase is saturated with PpIX when the 5-ALA concentration exceeds 0.3 mM and 0.05 mM in WiDr and V79 cells respectively. The DEF-induced enhancement of PpIX accumulation in 5-ALA-treated cells was cell line and 5-ALA concentration dependent. At a 5-ALA concentration inducing a maximum level of PpIX accumulation, inhibition of ferrochelatase activity enhanced the PpIX accumulation 3- and 1.4-fold in V79 and WiDr cells respectively. The relative gain in PpIX accumulation increased with decreasing concentration of 5-ALA. In cells treated with the lowest concentrations of 5-ALA used in this study, DEF enhanced PpIX accumulation 44- and 3.5-fold in V79 and WiDr cells respectively. The iron chelator-induced increase in cellular PpIX accumulation was followed by a similar increase in sensitivity to photoinactivation. The ferrochelatase inhibitor dihydropyridine 3,5-diethoxycarbonyl-1,4-dihydrocollidine reduced the accumulation of PpIX in both cell lines.