PHOTODYNAMIC EFFECTS INDUCED BY FUROCOUMARINS ON A MEMBRANE SYSTEM. COMPARISON WITH HEMATOPORPHYRIN

Abstract Isolated rat liver mitochondria have been used as a model to investigate the photodynamic action of psoralen (Ps) and 4,5',8-trimethylpsoralen (TMP) on cellular membrane systems in comparison with hematoporphyrin (Hp). Oxidation was detected by the consumption of free oxygen as measured polarographically in the respiratory chamber when irradiated with UV light (320-380 nm). In the presence of Ps, singlet oxygen was produced in the respiratory medium but neither the respiration nor the oxidative phosphorylation were affected. On the contrary the hydrophobic derivative TMP impaired the respiration with rapid uncoupling of oxidative phosphorylation as did Hp. The ineffectiveness of Ps as well as the effectiveness of TMP vs. Hp is explained on the basis of photophysical properties of the molecules and their partition coefficient. These results may indicate that, in the photochemiotherapy of skin diseases, furocoumarins can drive photodynamic reactions at various subcellular levels according to their hydrophobicity.     

PHOTOSENSITIZATION OF MITOCHONDRIA BY LIPOSOME-BOUND PORPHYRINS

We have compared the photodynamic activities of hematoporphyrin (HP) and protoporphyrin (PP) on isolated rat liver mitochondria by measuring the decline of the respiratory control ratio (RCR) after irradiation at 365 nm. Before addition to the respiratory mcdium, the dyes were dissolved in phosphate-buffered saline (PBS) or incorporated into unilamellar liposomes of dipalmitoyl-phosphatidylcholine (DPPC), sometimes enriched with cholesterol (Chol) or cardiolipin (Card), which are naturally present in mitochondrial membranes. Chol and especially Card strongly increase the porphyrin uptake by mitochondria. In all experimental conditions, PP is taken up by mitochondria to a higher extent than HP. Nevertheless, under conditions giving the same amount of mitochondriabound dye, HP is a morc efficient photosensitizer than PP. As the efficiency of singlet oxygen production has been shown to be equivalent for the two porphyrins in monomeric state, the resulting photobiological effects are explained in terms of different localization of HP and PP in the mitochondrial membrancs. In particular, HP preferentially localizes in the protein-rich polar domains of the inner mitochondrial membrane, whereas PP dissolvcs in the lipid regions of the mcmbrancs.     

4-THIOURIDINE INCORPORATION INTO THE RNA OF MONKEY KIDNEY CELLS (CV-1) TRIGGERS NEAR-UV LIGHT LONG-TERM INHIBITION OF DNA, RNA AND PROTEIN SYNTHESIS

Monkey kidney cells (CV-1) grown for 4 h in the presence of 0.1 m M 4-thiouridine (s⁴Urd) incorporate this photoactivable uridine analog in their RNA. A minor, 5–8%, thiolated RNA fraction can be isolated from bulk RNA by affinity chromatography. This RNA fraction contains 1.5–2.5 s⁴Urd residues per 100 nucleotides and exhibits a broad chain length distribution ranging from 700 to 7000 nucleotides. It is essentially of nuclear origin and amounts to 30% of the RNA synthesized during exposure of cells to s⁴Urd. Under the same s⁴Urd labeling conditions, no thiolated pyrimidine residues have been detected in DNA.
Irradiation with 365 nm light (45 kJ/m²) of the cells immediately after s⁴Urd exposure triggers long-term inhibition of DNA, RNA and protein synthesis accompanied by a linear decline (50% in 2 days) in the total cell mass of cultured cells. In contrast, exposure to s⁴Urd alone results in moderate but reversible inhibitory effects. The available data suggest that s⁴Urd-induced photolesions in newly synthesized RNA such as RNA-RNA cross-links as well as RNA-protein bridges are directly involved in impairment of essential cellular functions.     

Factors influencing the distribution pattern of porphyrins in cell membranes

The mechanism of the sensitizer-membrane interactions has been studied by following the distribution properties of selected porphyrins, including haematoporphyrin (HP) and protoporphyrin (PP), into unilamellar liposomes of dipalmitoyl phosphatidylcholine (DPPC). The endomembrane distribution of HP and PP has been checked as a function of the membrane fluidity and composition by fluorescence polarization and quenching techniques. At porphyrin concentrations below 0.5 microM, HP and PP exclusively localize in the inner phospholipid monolayer; at higher concentrations, the outer monolayer also becomes populated. The porphyrin binding sites in liposomes, however, are different for HP and PP: HP preferentially distributes into water-accessible lipid regions, while PP localizes in the most hydrophobic loci of the lipid matrix. A porphyrin redistribution occurs when the fluidity properties of the liposomes are changed by addition of cholesterol or cardiolipin. In DPPC-cholesterol vesicles, all HP molecules dissolve in DPPC-rich regions while all PP molecules partition in cholesterol-rich environments. In DPPC-cardiolipin vesicles both porphyrins preferentially localize in regions accessible to the external medium. The effect of the nature of the carrier on porphyrin distribution in membranes has been studied by following the uptake and photosensitization properties of free and DPPC-incorporated PP and HP with rat liver mitochondria. The porphyrin photosensitizing efficiency has been checked by following the impairment of the respiratory function of mitochondria upon irradiation. Liposome-bound HP is less active than aqueous HP in determining membrane photodamage in mitochondria. On the contrary, aqueous PP is a very poor sensitizer as compared to a DPPC liposome-entrapped drug.(ABSTRACT TRUNCATED AT 250 WORDS)