HYPOCRELLIN-A PHOTOSENSITIZATION INVOLVES AN INTRACELLULAR PH DECREASE IN 3T3 CELLS

The fluorescent pH probe carboxy-seminaphtorhoda-fluor-1 (C-Snarf-1) h as been used for laser microspectrofluorometric assays of intracellula r pH in 3T3 mouse fibroblasts treated with hypocrellin A. These result s are compared to those previously obtained with the structurally rela ted hydroxylated polycyclic quinone, hypericin (Sureau et al., J, Am, Chem, Sec. 118, 9484-9487, 1996), A mean local intracellular pH drop o f 0.6 units has been observed in the presence of 1 mu M hypocrellin A after 90 s of exposure to 0.1 mu W Of laser irradiation at 514.5 mm, T he time evolution of the cytoplasm acidification for hypocrellin A-tre ated cells is faster than that for cells treated by hypericin, Thus, r elease of protons from an excited state of hypocrellin A appears to be more efficient than that from hypericin, In addition, the pH dependen ce of the quenching of C-Snarf-1 fluorescence in 3T3 cells under conti nuous irradiation has been observed. It is shown here that under conti nuous illumination, a pH decrease is able to induce a modification of the intracellular binding equilibrium of C-Snarf-1 that results in an increase of C-Snarf-1 fluorescence intensity. This latter observation suggests that the protons generated upon the photoexcitation of hyperi cin or its analogs may be involved in the production of other photorea ctive species. Finally, we suggest that, just as for hypericin, this p H drop may be involved in the antiviral and antitumor activity of hypo crellin A.