G. M. J. Beijersbergen van Henegouwen

FORMATION OF SINGLET MOLECULAR OXYGEN BY 8‐METHOXYPSORALEN*

Abstract— The formation of singlet molecular oxygen (lO2) by energy transfer from the excited 8‐meth‐oxypsoralen (8‐MOP) molecule was investigated. This was done in several ways: (a) In the reaction of irradiated 8‐MOP with the 1O2 acceptor 2‐methyl‐2‐pentene, the characteristic oxidation products were identified. (b) The rate of the 8‐MOP sensitized photooxidation of 3, 4‐dihydroxy phenylalanine (dopa), which appeared to be also a useful 1O2 acceptor, was larger in D2O than in H2O. (c) The β‐values for reaction of 1O2with dopa in the presence of 8‐MOP or of methylene blue as 1O2 generators were in accordance with each other. The consequences of 1O2 formation by 8‐MOP sensitization is discussed for the clinical use of this compound.

Relation between some photobiological properties of furocoumarins and their extent of singlet oxygen production.

Abstract— The production of singlet oxygen (1O2) by a series of furocoumarins with different skin sensitizing abilities has been investigated with methods already proven to be suitable to establish the ability of 8‐methoxypsoralen (8‐MOP) to generate 1O2.

On the involvement of singlet oxygen in mutation induction by 8-methoxypsoralen and UVA irradiation in Escherichia coli K-12

The possible mutagenic effects induced by single oxygen, which is formed during UVA irradiation of bacterial cells pretreated with 8-methoxypsoralen (8-MOP), were investigated. As genetic endpoint, black mutation from arg-56 to arg+ was assayed in strain Escherichia coli K-12/343/113/uvrB; this system, in preliminary experiments, was rather sensitive to 8-MOP-induced photodynamic effects. To assess the involvement of singlet oxygen (1O2) in the mutation induction process, 2 tests were applied, namely, comparative mutation induction in D2O and H2O media (pH 7.0) and quenching of 1O2 with 1,4-diazabicyclo[2.2.2]octane (DABCO). When photodynamy was performed with the indicator cells suspended in D2O buffer, the mutagenic effect was substantially higher than that obtained with cells suspended in H2O buffer; this increase was even more pronounced when the incubation mixtures were thoroughly oxygenated before irradiation. D2O itself was not mutagenic under th present experimental conditions. Addition of DABCO in concentrations of 0.1--10 mM to the irradiation mixtures effectively reduced the number of 8-MOP-induced mutant yields by about 40%. DABCO itself had no effect on cell viability or on spontaneous mutation frequency under our experimental conditions. From these 2 sets of results, and from the preliminary findings that the photomutagenic effect of 8-MOP is higher in the uvrB derivative than in the corresponding excision-repair-proficient parent strain, which is in concordance with previous observations in other E coli strains, it can be concluded that 1O2 generated upon UVA irradiation of 8-MOP solutions is probably responsible for part of the observed genetic effects.

SINGLET OXYGEN FORMATION BY SENSITIZATION OF FUROCOUMARINS COMPLEXED WITH, OR BOUND COVALENTLY TO DNA

Abstract— The formation of singlet molecular oxygen (1O2) by sensitization of the furocoumarins 5‐methoxypsoralen (5‐MOP), 8‐methoxypsoralen (8‐MOP) and psoralen complexed with DNA was investigated. From the results it is concluded that 5‐MOP complexed with native DNA is able to generate 1O2, even in a larger extent than 5‐MOP free in solution. Also, with 8‐MOP and especially with psoralen, 1O2 formation by the complexed compound could be observed. The 1O2 formation sensitized by covalently bound furocoumarin was demonstrated with psoralen as a model compound. 4′,5′‐Dihydropsoralen, a model compound for the UVA light absorbing 4′,5’monoadducts of furocoumarins to DNA, is also able to generate 1O2.

Protection against UV-induced systemic immunosuppression in mice by a single topical application of the antioxidant vitamins C and E

PURPOSE Reactive oxygen species are involved in UV-induced suppression of the immune system. Topical treatment with the antioxidant vitamins C (L-ascorbic acid, ASC) and E (D-alpha-tocopherol, TOC) can support the endogenous antioxidant defence system and prevent immunosuppression. MATERIALS AND METHODS Mice were topically treated with a single dose of ASC, TOC or a combination and irradiated with UVB. Then systemic immunosuppression was measured using a model based on the induction of a contact hypersensitivity response to dinitrofluorobenzene. To investigate the mechanism of protection, cis-urocanic acid-induced immunosuppression was investigated in a different contact hypersensitivity model measuring local immunosuppression. The levels of ASC and TOC in the epidermis were determined by HPLC. RESULTS Both ASC and TOC prevented UV-induced suppression of the contact hypersensitivity response. TOC was effective at doses of 2.5 to 10 nmol/cm2 and ASC at 0.5 to 5 micromol/cm2. At the highest dose, the response in the ASC-treated mice was no longer significantly different from that in the positive control group. Contrary to expectations, combinations of the two compounds did not provide additional protection. The experiments with ASC or TOC against immunosuppression by cis-urocanic acid also yielded protection, but this was less efficient than against UV. The concentrations of ASC and TOC in the epidermis were so low that UVB absorption could be excluded as the cause of the protection. CONCLUSIONS ASC and TOC can be used to prevent systemic UV-induced immunosuppression. They are effective at relatively low doses after a single topical application prior to the irradiation.

A quantitative assessment of protoporphyrin IX metabolism and phototoxicity in human skin following dose-controlled delivery of the prodrugs 5-aminolaevulinic acid and 5-aminolaevulinic acid-n-pentylester

Background Topical 5‐aminolaevulinic acid (ALA) is widely used in photodynamic therapy (PDT) to generate protoporphyrin IX (PpIX) in the skin. However, other prodrugs may be more effective.

PHOTOPHARMACOLOGY OF THE TRANQUILIZER CHLORDIAZEPOXIDE IN RELATION TO ITS PHOTOTOXICITY

Abstract— This study demonstrates that photobiological effects by exogenous molecules are not per se restricted to the skin. As an example the photopharmacology of chlordiazepoxide (e.g. LibriumR) was studied. Rats being exposed to UV‐A after administration of 14C2 labeled chlordiazepoxide (CDZ) showed a marked change in the pattern of urinary metabolites; the quantity of metabolites without N4‐oxide function increased, whereas that of compounds with N4‐oxide function decreased. Covalent binding to organ tissue of especially liver and skin of the irradiated rats was found. This would mean that in the irradiated rats oxaziridines which may also react with tissue of the inner organs are formed from CDZ and its N4‐oxymetabolites.

Clinical and mechanistic aspects of photopheresis

Abstract Photopheresis is an extracorporeal form of photochemotherapy with 8-methoxypsoralen (8-MOP) and ultraviolet A (UVA) radiation. Photopheresis is used for the management of T-cell-mediated diseases, and such treatment leads to the induction of antigen-specific immune suppression directed to the pathogenic clone of T cells. Photopheresis is used to treat a wide variety of diseases—such as cutaneous T-cell lymphoma, systemic sclerosis, rheumatoid arthritis, lupus erythematosus—and is also successfully applied in the suppression of graft rejection. In addition to the clinical achievements, attention will be paid to results from animal studies. An important outcome of these studies is that photopheresis can be used to treat airway hyperreactivity. Furthermore, it was shown that the therapeutic strategy can be changed drastically: the presence of plasma during irradiation should be avoided and the amount of blood that must be treated to obtain the desired antigen-specific immunosuppression can be greatly decreased. Also, results from cellular experiments are discussed. An example of this is the increase in the major histocompatibility complex expression on the surface of cells found after treatment. The mechanism that underlies photopheresis has not yet been elucidated, but progress has been made. The following related points will be reviewed: models for investigation; and mechanistic aspects, with the emphasis on cellular biomacromolecules and on photosensitizers (drugs) other than 8-MOP.

UV Radiation Protecting Efficacy of Cysteine Derivatives, Studies with UVA-induced Binding of 8-MOP and CPZ to Rat Epidermal Biomacromolecules in Vivo

With the aim of optimizing the UV radiation protecting efficacy of N-acetylcysteine (NAC), the following topically applied cysteine derivatives were investigated: N-acetylcysteine ethylester (NACET), S-acetylcysteine ethylester (SACET), cysteine ethylester (CYSET), NS-diacetylcysteinamide (SNACA), NS-diacetylcysteine (SNAC) and NS-diacetylcysteine ethylester (SNACET). As a measure for protection the inhibition of in vivo irreversible photobinding of the labelled phototoxic drugs chlorpromazine (CPZ) and 8-methoxypsoralen (8-MOP) to rat epidermal biomacromolecules was used. The duration of protection of the cysteine derivatives was shortened by S-acetylation, N-acetylation and carboxyl derivatization. Compounds with a free thiol group showed a long-lasting presence in the stratum corneum, probably by the formation of mixed disulphides with proteins. The intrinsic protecting efficacy with respect to the total epidermis increased in the order CYSET < SANACET,SNACA,SACET < NACET,SNAC,NAC. The results of this ...

In vivo PHOTODEGRADATION OF CHLORPROMAZINE

Abstract— 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 [Schoonder‐woerd and Beijersbergen von Henegouwen (1987) Photochem. Photobiol. 46, 501–505]. Chlorpromaz‐ine‐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.