J. Decuyper

DAMAGES INDUCED IN NUCLEIC ACIDS BY PHOTOSENSITIZATION

The topic of nucleic acids alteration induced by photosensitization was discussed in this journal three years ago as a part in the area of photosensitization (Girotti, 1983). During the intervening years, a substantial amount of new papers have appeared. This review will cover selected developments concerning nucleic acids photosensitization which were published from approximately December 1984 to June 1986. This paper is divided into three sections dealing with the following classes of nucleic atids damages: (1) base alteration, (2) DNA breakag,e and (3) miscellaneous alterations (including i.e. photoaffinity labeling, DNA protein crosslinks, etc).

Induction of breaks in deoxyribonucleic acid by photoexcited promazine derivatives.

Near-u.v. photoexcited promazine and three of its derivatives are shown to induce single-strand breaks in phi X174-DNA replicative form. The mechanisms of this DNA breakage depend upon the various photochemical properties of the promazine derivatives. Chlorpromazine is shown to act predominantly via the photodechlorination reaction both in aerobic and anaerobic conditions. The three other promazine derivatives (promazine, trifluopromazine and methoxypromazine) display two mechanisms for DNA breakage. One of them occurs through the cation radical, which is formed during near-u.v. irradiation of promazine derivatives. The second mechanism is demonstrated to act via an hydroxyl radical-dependent pathway. Acepromazine is without photoactivated action. EPR-spin-trapping studies of irradiated mixtures, containing the drugs and 5,5-dimethyl-1-pyrroline-N-oxide (as spin trap), suggest the production of superoxide radical by photoexcited promazines. When DNA is present in the irradiation mixture, this superoxide radical is converted into hydroxyl radical probably via a Haber Weiss-type reaction, catalysed by DNA-iron complexes.

Phototoxicity of phenothiazine derivatives. II. Photosensitized cross-linking of erythrocyte membrane proteins

Irradiation in the presence of O2, with near-UV light of five promazine (PZ) derivatives added to erythrocyte ghost membranes, causes covalent cross-linking between proteins as revealed by a progressive decrease in the amounts of proteins separable by electrophoresis after denaturation. The induction of cross-links in the two spectrin subunits is a single-hit process as a function of the irradiation time; relatively the rate constants (in min-1) of the photoreactions were 0.060 with chlorpromazine (CPZ), 0.039 with methoxypromazine (MTPZ), 0.031 with PZ, 0.029 with triflupromazine (TFPZ) and 0.006 with acepromazine (ACPZ). A main photochemical intermediate implicated in the spectrin aggregation seems to be the cation radical of the PZ derivatives. Indeed, (i) the chemically generated cation radicals can induce the reaction in the dark; (ii) the photoaggregation is regularly reduced upon addition of increasing concentrations of NaN3; (iii) NaN3 similarly affects the amount of cross-links induced by the isolated cation radicals. Hydroxyl radicals are also involved in the photocross-linking when the reaction is initiated only by MTPZ and not by the other sensitizers. In the absence of oxygen during irradiation, PZ, MTPZ and ACPZ completely loose their cross-linking activities whereas CPZ and TFPZ remain as efficient as in the presence of oxygen.

Biomolecular photoalterations mediated by phenothiazine derivatives.

This survey focuses on recent developments in the field of the ultraviolet photochemistry and photobiology of phenothiazine derivatives. One of the major alterations introduced by this kind of photosensitized reaction is a covalent addition of the photosensitizer or one of its photoproducts onto the macromolecular target. This reaction has been observed with soluble and membrane proteins, lipids and DNA. In the latter case, the addition occurs at the level of guanine residues and leads to inhibition of DNA replication.

Lack of singlet oxygen formation by photoexcited promazine derivatives in aqueous and ethanolic solutions

SummaryThe EPR detection of nitroxide formation and the observation by thin layer chromatography of the specific1O2 oxidation product of cholesterol, have been used to appreciate singlet oxygen production by promazine and four of its derivatives during irradiation with near-UV light of ethanolic and aqueous solutions. Within the range of sensitivity of the methods, no1O2 has been detected.

Phototoxicity of phenothiazine derivatives. I. Inactivating and mutagenic effects on bacteriophage φX174

Inactivation of øX174 bacteriophages as a function of the irradiation time in the near-UV and in the presence of triflupromazine (TFPZ), promazine (PZ), chlorpromazine (CPZ) or methoxypromazine (MTPZ) proceeds according to single hit kinetics. Acepromazine (ACPZ) has no significant activity. At low concentrations (0.1 mM) TFPZ and PZ are the most active compounds. Higher concentrations (up to 5 mM) result in a protective effect by these two compounds but cause increased inactivation rates in the case of MTPZ or CPZ. Photoinactivation mediated by TFPZ or CPZ increases the reversion frequency of a øXamber mutant. Neither MTPZ nor PZ sensitization induces mutagenesis. The effect of NaN3 on the phage inactivation rate varies depending upon both the sensitizer and the concentration of the quencher. Phage inactivation in an N2 atmosphere is measurable only in the presence of high concentrations of CPZ and MTPZ. The drugs do not show any selectivity for calf thymus DNA or bovine serum albumin, at least as measured by dialysis equilibrium experiments.

EVIDENCE FOR THE PRODUCTION OF SINGLET OXYGEN BY PHOTOEXCITED ACRIDINE ORANGE

Abstract In contrast to previous results obtained with the nitroxide radical detection technique, generation of the specific 1O2 oxidation product of cholesterol shows that photoexcited acridine orange produces singlet oxygen.

Activated oxygen species produced by photoexcited furocoumarin derivatives

Furocoumarin derivatives (FCD) are investigated in order to determine their ability to photosensitize the production of activated oxygen species. Using the method based on the specific singlet oxygen (1O2) oxydation of cholesterol, all FCD except bergaten appeared to be able to produce 1O2 with various efficiencies. EPR spin trapping experiments show that photoexcited FCD produce hydroxyl radicals as detected by the formation of a DMPO-OH signal which can be abolished when the photosensitization reaction is carried out in the presence of specific OH scavengers. Moreover, the photo-ejection of hydrated electron (e-) by FCD is demonstrated by the loss of paramagnetic absorption of nitroxide free radicals as e- trap.

Phototoxic potentialities of tartrazine: screening tests.

Abstract Tartrazine (TRT) a cosmetics, food and drug additive, was tested with respect to phototoxic potentialities. Although, using the cholesterol technique, TRT did not appear to produce any 1O2 upon visible light irradiation, the EPR spin trapping experiments with 5,5‐dimethyl‐1‐pyrroline‐N‐oxide, as spin trap, were in favour of activated oxygen species (O2˙, OH˙) and hydrated electron production by photoexcited TRT.

Photolysis of chlorpromazine: hydroxyl radical detection using 2-methyl-2-nitrosopropane as a spin trap

Abstract The spin trapping method was used to detect hydroxyl radicals during the photolysis of chlorpromazine. Two spin traps, the nitrone compound 5,5-dimethyl-1-pyrroline- N -oxide (DMPO) and the nitroso compound 2-methyl-2-nitrosopropane (MNP), were used. The minor drawbacks associated with nitrone spin traps are discussed. It was shown that a hydroxyl radical scavenger such as ethanol also inhibits the production of spin adducts initiated by the oxidation of DMPO in aqueous solution. Another strategy employing MNP and a hydroxyl radical scavenger such as dimethyl sulphoxide or alanine is described. The alkyl free radicals resulting from the reaction of the hydroxyl radicals with these scavengergs are trapped by MNP. The corresponding spin adduct is more easily identified when the nitroso spin trap is used.