Photobinding of 8-methoxypsoralen, 4,6,4'-trimethylangelicin and chlorpromazine to Wistar rat epidermal biomacromolecules in vivo.

Photoinduced binding of drugs to endogenous biomacromolecules may cause both toxic and therapeutic effects. For example, photobinding of certain phenothiazines to biomolecules possibly underlies their phototoxic and photoallergic potential, whereas photobinding of furocoumarins to epidermal DNA is held responsible for their advantageous effects in the photochemotherapy of psoriasis. Usually, the in vitro photobinding of drugs is investigated. However, under in vivo conditions, the metabolism and distribution of the drug and the light absorption by endogenous compounds will significantly affect the photobinding of drugs to biomolecules. Therefore, in the present study, the photobinding of 8-methoxypsoralen (8-MOP), 4,6,4'-trimethylangelicin (TMA) (two therapeutically used furocoumarins) and chlorpromazine (CPZ) (a member of the phenothiazines) was investigated in vivo. The compounds were applied topically on the shaven skin of Wistar rats; one group was exposed to UVA and the other was kept in a dimly lit environment. Immediately, and at certain time intervals after UVA exposure, members of the two groups were sacrificed. By separating epidermal lipids, DNA/RNA and proteins by a selective extraction method, irreversible binding of 8-MOP, TMA or CPZ to each of these biomacromolecules was determined. In contrast with in vitro experiments, photobinding of CPZ to epidermal DNA/RNA was not found in vivo; apparently the bioavailability in the nucleus is very low. Compared with TMA, 8-MOP was observed to bind more extensively to epidermal DNA/RNA (again in contrast with findings from in vitro experiments) and proteins, but less extensively to lipids. The rates of removal of photobound 8-MOP and TMA were comparable. Photobound CPZ was more slowly removed from epidermal proteins and lipids than the furocoumarins. The observed in vivo photobinding is discussed with respect to the UVA-induced (side) effects of these drugs.

Effect of alpha-tocopherol and di-butyl-hydroxytoluene (BHT) on UV-A-induced photobinding of 8-methoxypsoralen to Wistar rat epidermal biomacromolecules in vivo.

The possible formation of singlet oxygen via photoexcited psoralens has been associated with the occurrence of, amongst others, erythema. Therefore it has been suggested to combine PUVA with the topical or systemic administration of antioxidants. However, the effect of these antioxidants on the photobinding of psoralens to DNA, which is held responsible for the anti-proliferative effect, should be taken into account. In the present study the effect of two phenolic antioxidants, alpha-tocopherol (AT) and butylated hydroxytoluene (BHT), on the in vivo photobinding of 8-methoxypsoralen (8-MOP) to not only epidermal DNA, but also proteins and lipids was determined. After topical application of an ethanolic antioxidant solution onto the shaven skin of Wistar rats, labeled 8-MOP was applied using the same solvent. After this the rats were exposed to UV-A. By separating epidermal lipids, DNA/RNA and proteins by a selective extraction method, irreversible binding of 8-MOP to each of these biomacromolecules was determined. Both AT and BHT caused a decrease in the photobinding of 8-MOP to epidermal DNA and proteins. To investigate the underlying mechanism of this protection, the effect of AT was compared with that of AT-acetate. It also proved helpful to study the effects of the antioxidants on the photobinding of another photosensitizer, namely chlorpromazine. From these experiments it was concluded that AT and BHT affect 8-MOP photobinding by quenching reactive 8-MOP intermediates, involving the phenolic hydroxyl group of the antioxidants. BHT offered protection against lipid binding of 8-MOP but AT, especially at high concentrations, enhanced the UV-A-induced binding of 8-MOP to lipids.(ABSTRACT TRUNCATED AT 250 WORDS)

UVA-induced genetic effects of thioridazine, mesoridazine and sulforidazine: an in vitro study.

This in vitro study focuses on the UVA-induced reactions with DNA of thioridazine (TRZ), and two of its major metabolites (TRZ-2-sulfoxide or mesoridazine, MRZ; and TRZ-2-sulfone or sulforidazine, SRZ). TRZ binds covalently to DNA upon UVA-irradiation. Under comparable irradiation conditions, MRZ binds to a lesser extent and almost no binding was observed with SRZ. Besides, photo-induced genetic effects were investigated by means of a differential DNA repair test in E. coli. The photo-induced genetic effects in E. coli decreased from TRZ, MRZ to SRZ, which corresponds with their capacity for UVA-induced binding to DNA. TRZ, MRZ and SRZ differed in their rate of photodecomposition rather than in the intrinsic reactivity towards DNA of the instable intermediates formed. Irreversible binding to DNA was also observed upon treatment with peroxidase, which is known to oxidize phenothiazines via the formation of reactive radical cation species. As both the colour of the intermediate and its reactivity towards DNA were comparable for peroxidase treatment and exposure to UVA, we assume that the radical cation is the reactive species in the latter case as well.

In vivo photodegradation of chlorpromazine.

The in vivo photodegradation of chlorpromazine (CPZ) in the skin was investigated after systemic administration of 3H-CPZ to shaven Wistar rats and exposure to UV-A. Promazine (PZ) and 2-hydroxy-promazine (2-OH-PZ) appeared to be formed in irradiated rats, but not in the skin of rats kept in the dark. This indicates that upon irradiation with UV-A the PZ-radical is formed which can be held responsible for the photobinding to eye and skin constituents as observed earlier [Schoonderwoerd and Beijersbergen von Henegouwen (1987) Photochem. Photobiol. 46, 501-505]. Chlorpromazine-sulfoxide (CPZSO) is a major metabolite of CPZ. Less CPZSO was found in the skin of irradiated rats compared to those kept in the dark. As this appeared not to be caused by photobinding or photodegradation of CPZSO it can be concluded that CPZSO is not a photoproduct of CPZ under these experimental conditions. This study shows that the in vivo photodegradation of CPZ proceeds via the promazinyl radical rather than via the radical cation.

Singlet oxygen mediated photobinding of 8-methoxypsoralen to DNA and genotoxicity in E. coli.

Although oxygen dependent photoreactions of 8-methoxypsoralen (8-MOP) are known, damage to DNA is mostly considered to proceed via photocycloaddition to this biomacromolecule. In this study the survival of colony forming ability of E. coli K12/343, which is deficient in DNA-repair capability, appeared to be lower in D2O than in H2O after exposure to the combination of 8-MOP and UV-A. Photobinding to bacterial DNA was approximately 40% higher in D2O than in H2O. However this last difference was found only when the bacteria were kept in the reaction medium for 1 h at 37 degrees C after the irradiation was stopped and not when they were plated out immediately afterwards. The results indicate that in this bacterial test system 1O2 mediated photobinding of 8-MOP to DNA contributes to the genotoxic effect observed.