[Photohemolysis sensitized by psoralen: dependence on pH].

The effect of pH on the hemolysis of erythrocytes photosensitized (366 nm, 23 Wt/m2) by psoralen has been studied. The dependence of the photohemolysis rate (V) on irradiation dose (D) was described by the equation V = Vo + kD, where Vo is the rate of hemolysis without irradiation (dark), and k is the constant. The index of the power at dose x was approximately equal to 2, and its value did not change as the pH of the erythrocyte suspension was changed. It was found that changes in pH led to a sharp change in the value of coefficient k and correspondingly V. The lowest rate of photohemolysis was observed in the pH range from 8.0 to 8.4. As pH was changed from 3.4 to 9.0 or from 8.0 to 7.4, the V value increased approximately twofold. At pH below 7.4, an abrupt increase (approximately fourfold) in V was observed, with the pK value being equal to 7.3. The psoralen molecule lacks titratable acidic and basic groups; therefore, the effects of pH can hardly be assigned to changes in the photophysical properties of the sensitizer. The increase in V in the alkaline region is prohably related to the acceleration of photooxidation of reduced glutathione, whereas the jump of V at pH of about 7.3 may be due to the titration of the product of psoralen photooxidation. The latter assumption is confirmed by the data of hign performance liquid chromatography. In these experiments, psoralen was oxidized in ethanol and mixed with the phosphate buffer at different pH values followed by a qualitative and quantitative analysis by high performance liquid chromatography of photoproducts. Several photoproducts of psoralen have been identified whose content depended on pH. The curve of titration of one photoproduct was similar in shape to the pH dependence of psoralen-photosensitized hemolysis.

Dietary eicosapentaenoic acid prevents systemic immunosuppression in mice induced by UVB radiation.

Moison, R. M. W. and Beijersbergen van Henegouwen, G. M. J. Dietary Eicosapentaenoic Acid Prevents Systemic Immunosuppression in Mice Induced by UVB Radiation. Radiat. Res. 156, 36-44 (2001). Reactive oxygen species (ROS) contribute to the immunosuppression induced by UVB radiation. Omega-3 fatty acids in fish oil, e.g. eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), can modulate immunoresponsiveness, but because of their susceptibility to ROS-induced damage, they can also challenge the epidermal antioxidant defense system. The influence of dietary supplementation with different omega-3 fatty acids on systemic immunosuppression induced in mice by UVB radiation was studied using the contact hypersensitivity response to trinitrochlorobenzene. In an attempt to study the mechanisms involved, UVB-radiation-induced changes in epidermal antioxidant status were also studied. Mice received high-fat (25% w/w) diets enriched with either oleic acid (control diet), EPA, DHA, or EPA + DHA (MaxEPA). Immunosuppression induced by UVB radiation was 53% in mice fed the oleic acid diet and 69% in mice fed the DHA diet. In contrast, immunosuppression was only 4% and 24% in mice fed the EPA and MaxEPA diets, respectively. Increased lipid peroxidation and decreased vitamin E levels (P < 0.05) were found in unirradiated mice fed the MaxEPA and DHA diets. For all diets, exposure to UVB radiation increased lipid peroxidation (P < 0.05), but levels of glutathione (P < 0.05) and vitamin C (P > 0.05) decreased only in the mice given fish oil. UVB irradiation did not influence vitamin E levels. In conclusion, dietary EPA, but not DHA, protects against UVB-radiation-induced immunosuppression in mice. The degree of protection appears to be related to the amount of EPA incorporated and the ability of the epidermis to maintain an adequate antioxidant level after irradiation.

Cysteine derivatives protect against UV-induced reactive intermediates in human keratinocytes: the role of glutathione synthesis.

Several recent studies have shown cysteine derivatives can protect against negative effects of UV exposure. In this study, an attempt was made to correlate cellular bioavailability and metabolism of cysteine derivatives with protection against UV-induced reactive intermediates. Human keratinocytes were treated with cysteine, N-acetylcysteine (NAC), cysteine-ethylester (CYSET) and N-acetylcysteine-ethylester. The uptake of the compounds and their metabolism to cysteine and eventually to glutathione (GSH) was measured. Large differences in uptake were observed, with CYSET resulting in the highest and NAC in the lowest intracellular thiol levels. The increase in intracellular GSH was similar for all derivatives with a maximum of 23-54% over the control level. Protective efficacy of the derivatives was measured as the inhibition of binding of UV-induced reactive intermediates from 8-methoxypsoralen. There was only a small difference between the compounds, with maximum protection of 25-31%. No relation was found between total intracellular thiol and protection. However, for NAC, there was a linear relation between GSH level and protective efficacy (r = 0.94). Even though this was not clear for the other derivatives (r = 0.55 for CYS; r = 0.60 for CYSET; r = 0.70 for NACET), it indicates that GSH synthesis is an important factor. This was confirmed by experiments using cells with irreversibly inhibited GSH synthesis. Even though the total intracellular thiol level was comparable to uninhibited cells, protection was decreased. We conclude that the intracellular GSH increase is the most important factor in photoprotection by cysteine derivatives.

The protective effect of N-acetylcysteine on UVB-induced immunosuppression by inhibition of the action of cis-urocanic acid.

A recent study has shown that N-acetylcysteine (NAC) not only has sun-protective properties but also inhibits the UVB-induced suppression of contact hypersensitivity (CHS) in mice. Because NAC does not absorb any UVA (320-400 nm radiation) or UVB (290-320 nm radiation) we have studied the underlying mechanism of protection. Irradiation of solutions of plasmid DNA with UVC (200-290 nm radiation) (10 J m-2) resulted in the formation of cyclobutane pyrimidine dimers, but the extent to which this occurred was not affected by the presence of NAC as was determined by an in vitro T4 endonuclease assay. N-acetylcysteine proved not to have any effect on the photoisomerization of trans-urocanic acid (UCA) to its cis-form in vitro; at equilibrium, approximately 55% cis-UCA was formed. The same percentage was also found in vivo on exposure of mice to UVB (15 kJ m-2). Topical application of NAC 30 min prior to irradiation did not have any influence as well on the photoisomerization of trans- to cis-UCA. These in vivo experiments were performed under the same conditions used previously to show the protective effect of NAC against UVB-induced suppression of CHS. We conclude that this protection of NAC is at least partly based on interference in the role of cis-UCA in UVB-induced suppression of CHS. This conclusion is supported by the observation that NAC completely inhibits the suppression of CHS by cis-UCA administered to mice that were always kept in the dark. In the same range of doses as used in the present study, it was shown in our previous study that NAC alone does not affect the CHS response.

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.

Protection against UV-induced reactive intermediates in human cells and mouse skin by glutathione precursors: a comparison of N-acetylcysteine and glutathione ethylester.

Because glutathione (GSH) plays a central part in the endogenous defense against UV radiation, an increase in GSH might provide photoprotection. Two agents that increase GSH levels were investigated. Cultured human cells and mouse skin were treated with N-acetylcysteine (NAC) and glutathione ethylester (GSH-Et). After 30 min, the GSH level was determined by HPLC. Photoprotection was assessed by testing the ability of the thiols to scavenge UV-induced reactive intermediates in the same models. As compared to control cells, NAC and GSH-Et increased intracellular GSH in vitro to maximally 144% and 174% respectively. In vitro protection (maximum 23% for NAC and 21% for GSH-Et) did not correlate to the intracellular GSH level but to the concentration of the thiols in the medium. In vivo, epidermal GSH was increased to maximally 163% of the control level by NAC and 1234% by GSH-Et. The maximum in vivo photoprotection provided by GSH-Et was 55%, similar to what was found previously for NAC. Again, the protection seemed more closely correlated to the thiol dose than to the GSH level. The study showed that the protection against UV-induced reactive intermediates depends on a general antioxidant action of these thiols, rather than only on their role as GSH precursors.

Topically applied eicosapentaenoic acid protects against local immunosuppression induced by UVB irradiation, cis-urocanic acid and thymidine dinucleotides.

UVB-induced immunosuppression, a promoter of photocarcinogenesis, involves the formation of pyrimidine dimers and cis-urocanic acid (cis-UCA), but reactive oxygen species (ROS) also plays an important role. Eicosapentaenoic acid (EPA) can inhibit photocarcinogenesis, but due to its polyunsaturated nature it is susceptible to oxidative damage by ROS. The antioxidant defense system may therefore be challenged upon ultraviolet-B (UVB) irradiation in the presence of EPA. We investigated whether topically applied EPA in mice could protect against local immunosuppression (contact hypersensitivity response to dinitrofluorobenzene) induced by UVB radiation (1.5 J/cm2), or topically applied cis-UCA (150 nmol/cm2) or thymidine dinucleotides (pTpT) (5 nmol/cm2). The influence of EPA on epidermal lipid peroxidation and antioxidant status was also measured. UVB irradiation, cis-UCA and pTpT all caused 70% immunosuppression. Topical pretreatment of mice with EPA partially protected against immunosuppression; the EPA dose needed to accomplish this was 10 nmol/cm2 for UVB irradiation, 100 nmol/cm2 for cis-UCA and 1000 nmol/cm2 for pTpT. Higher EPA doses caused higher UVB-induced lipid peroxidation and lower vitamin C levels. Glutathione only decreased with the highest EPA dose whereas vitamin E was not decreased after UVB irradiation. In conclusion, topically applied EPA protects against UVB-, cis-UCA- and pTpT-induced immunosuppression and maintenance of an adequate antioxidant defense seems to be an important prerequisite for the protective action by EPA.

An animal model and new photosensitizers for photopheresis.

Recently, photopheresis was introduced as a therapy for several T cell-mediated disorders. The treatment results in a specific immune response against the pathogenic clone of T cells involved. However, although promising there is controversy concerning the use of photopheresis in some diseases, e.g. systemic sclerosis. One of the problems is that there is not yet sufficient insight into the mechanism underlying the therapy. This lack of knowledge is partly caused by the fact that there are no easy-to-handle animal models available for photopheresis. This report describes such a model--a Wistar-derived rat with contact hypersensitivity (CHS); a T cell-mediated immune response. White blood cells from CHS rats were simultaneously exposed to 8-methoxypsoralen (8-MOP) and ultraviolet A radiation (UVA) and subsequently intravenously injected into other syngeneic rats suffering from the same disorder. This treatment appears to be very efficacious in suppressing the immunological response against the applied contact allergen, 2,4-dinitrofluorobenzene (DNFB). In addition, the generated suppression of CHS is highly specific and transferable. Furthermore, drugs other than 8-MOP (chlordiazepoxide, nitrofurantoin and chlorpromazine) also appear to be active in our model.

Time and dose-related ultraviolet B damage in viable pig skin explants held in a newly developed organ culture system.

For facilitating photochemical and toxicological studies an ex vivo skin model was developed in our laboratory using skin from domestic pigs. The model comprised the use of a complete skin piece, including the dermis and stratum corneum, of bigger areas to make future topical applications easier. Fully differentiated skin explants (5 x 50 mm, thickness 5 mm) were irradiated with ultraviolet B (UVB; 1-10 kJ/m2; 6 W/m2). Directly thereafter they were brought in culture (Dulbeccos modified Eagles medium containing hydrocortisone; air/liquid interface) for a maximum of 144 h. In nonirradiated skin explants, signs of tissue degeneration were observed after 48 h in culture (hematoxylin and eosin, light microscope). However, keratinocytes, isolated enzymatically (thermolysin and trypsin) at different time intervals in culture from nonirradiated skin explants showed negligible loss in viability (trypan blue exclusion) and increased apoptosis (terminal deoxynucleotidyl transferase-mediated deoxy uridine triphosphatase nick end labeling assay) for up to 72 h. Explants irradiated with a single dose of UVB showed a clear and reproducible dose- and time-dependent tissue degeneration, loss in keratinocyte viability and increase in apoptosis compared with nonirradiated explants at the same time interval. In conclusion, the presently designed ex vivo pig skin model can be a useful and cheap tool for future investigations of short-term UV-induced effects in combination with phototoxic and photoprotective compounds.

Derivatives of 5-aminolevulinic acid for photodynamic therapy: enzymatic conversion into protoporphyrin.

In recent years, 5-aminolevulinic acid (ALA) has become a widespread agent for photodynamic therapy (PDT). In nucleated cells, ALA is converted into the endogenous photosensitizer protoporphyrin IX (PpIX). A major drawback of ALA is its low bioavailability. As a result, high doses of ALA must be administered in order to reach clinically relevant levels of PpIX. Moreover, only superficially located lesions can be treated as a result of the poor penetration of ALA into tissues. A possible solution for this problem may be provided by the prodrug concept. In the present study, prodrugs of ALA have been synthesized. These ALA prodrugs are shown to result in higher PpIX levels in cells than does ALA itself. Of a range of ester prodrugs of ALA, the ALA-pentyl ester elicits the highest fluorescence. Furthermore, the enzymatic conversion of the derivatives into ALA and PpIX has been studied in lysed cells. Under these circumstances, the esters with the shorter alkyl chains induce the highest fluorescence. The alcohols that arise as side products from enzymatic conversion of the prodrugs are shown to have no influence on the experiments.

In vitro fluorescence, toxicity and phototoxicity induced by delta-aminolevulinic acid (ALA) or ALA-esters.

Synthesis of delta-aminolevulinic acid (ALA) derivatives is a promising way to improve the therapeutic properties of ALA, particularly cell uptake or homogeneity of protoporphyrin IX (PpIX) synthesis. The fluorescence emission kinetics and phototoxic properties of ALA-n-pentyl ester (E1) and R,S-ALA-2-(hydroxymethyl) tetrahydrofuranyl ester (E2) were compared with those of ALA and assessed on C6 glioma cells. ALA (100 micrograms/mL), E1 and E2 (10 micrograms/mL) induced similar PpIX-fluorescence kinetics (maximum between 5 and 7 h incubation), fluorescence being limited to the cytoplasm. The 50% lethal dose occurred after 6 h with 45, 4 and 8 micrograms/mL of ALA, E1 and E2, respectively. ALA, E1 and E2 induced no dark toxicity when drugs were removed after 5 min of incubation. However, light (25 J/cm2) applied 6 h after 5 min incubation with 168 micrograms/mL of each compound induced 85% survival with ALA, 27% with E1 and 41% with E2. Increasing the incubation time with ALA, E1 and E2 before washing increased the phototoxicity, but E1 and E2 remained more efficient than ALA, regardless of incubation time. ALA-esters were more efficient than ALA in inducing phototoxicity after short incubation times, probably through an increase of the amount of PpIX synthesized by C6 cells.

Protoporphyrin IX fluorescence kinetics and localization after topical application of ALA pentyl ester and ALA on hairless mouse skin with UVB-induced early skin cancer.

In order to improve the efficacy of 5-aminolevulinic acid-based (ALA) photodynamic therapy (PDT), different ALA derivatives are presently being investigated. ALA esters are more lipophilic and therefore may have better skin penetration properties than ALA, possibly resulting in enhanced protoporphyrin IX (PpIX) production. In previous studies it was shown that ALA pentyl ester (ALAPE) does considerably enhance the PpIX production in cells in vitro compared with ALA. We investigated the in vivo PpIX fluorescence kinetics after application of ALA and ALAPE to hairless mice with and without UVB-induced early skin cancer. ALA and ALAPE (20% wt/wt) were applied topically to the mouse skin and after 30 min, the solvent was wiped off and PpIX fluorescence was followed in time with in vivo fluorescence spectroscopy and imaging. At 6 and 12 h after the 30 min application, skin samples of visible lesions and adjacent altered skin (UVB-exposed mouse skin) and normal mouse skin were collected for fluorescence microscopy. From each sample, frozen sections were made and phase contrast images and fluorescence images were recorded. The in vivo fluorescence kinetics showed that ALAPE induced more PpIX in visible lesions and altered skin of the UVB-exposed mouse skin, but not in the normal mouse skin. In the microscopic fluorescence images, higher ALAPE-induced PpIX levels were measured in the stratum corneum, but not in the dysplastic layer of the epidermis. In deeper layers of the skin, PpIX levels were the same after ALA and ALAPE application. In conclusion, ALAPE does induce higher PpIX fluorescence levels in vivo in our early skin cancer model, but these higher PpIX levels are not located in the dysplastic layer of the epidermis.

Urocanic acid does not photobind to DNA in mice irradiated with immunosuppressive doses of UVB.

Ultraviolet B (UVB, 290-320 nm) radiation initiates in vivo a dose- and wavelength-dependent down regulation of cell-mediated immunity. An action spectrum for UV-induced immunosuppression indicated that the photoreceptor for this effect is urocanic acid (UCA), which undergoes a trans to cis isomerization in the stratum corneum on UV exposure. An accumulation of evidence has supported this conclusion. However, evidence has also been presented that formation of thymine dimers in DNA is responsible for initiation of UV-induced immunosuppression. Because photobinding of UCA to DNA in vitro forming cyclobutane-type adducts has been shown, we sought to resolve this dilemma by investigating if UCA photobinds to DNA in vivo. The [14C]cis-UCA, [14C]trans-UCA or [3H]8-MOP (8-methoxypsoralen) was applied topically to BALB/c mice that were then irradiated with a dose of UV previously shown to cause systemic suppression of contact hypersensitivity. The DNA was prepared from epidermal cells by phenol extraction immediately after in vivo irradiation and bound radioactivity determined. Although photobinding of [3H]8-MOP was readily demonstrable under these conditions (0.9 nmol/mg DNA), no significant binding of either isomer of UCA to DNA (between 1.2 x 10(-3) and 2.1 x 10(-3) ng/mg DNA) could be detected. Uptake studies in keratinocytes prepared from epidermis of untreated animals indicated that [3H]8-MOP was taken up with a rate constant of 4.2 x 10(-3) pmol/s/mg protein/mumol/L. In contrast, uptake of [14C]cis-UCA was not statistically significant from zero and uptake of [14C]trans-UCA was negligible (0.8 x 10(-3) +/- 0.08 x 10(-3) pmol/s/mg protein/mumol/L). There was no significant difference between uptake of UCA isomers, but uptake of [3H]8-MOP was significantly greater than that of either UCA isomer (P < 0.01). These studies indicate that the photobinding of UCA to DNA does not play a role in UV-induced immunosuppression.

Cytotoxicity and induction of repairable DNA damage by photoactivated 5-nitrofurfural.

5-Nitrofurfural (NFA) an important photodecomposition product and metabolite of medicinal nitrofurans is phototoxic in bacterial test systems. Its major photodecomposition product 5-hydroxymethylene-2(5H)-furanone (HMF) appears to be responsible for this. Furthermore HMF and photoactivated nitrofurfural can induce repairable DNA damage in Escherichia coli bacteria. These effects may be important with respect to skin and allergic reactions and carcinogenicity appearing in nitrofuran therapy.

Formation of methemoglobin by photoactivation of nitrofurantoin or of 5-nitrofurfural in rats exposed to UV-A light.

The antibacterial drug nitrofurantoin (NFT) is notorious for causing hemolytic anemia, which may be related to the methemoglobinemia, another side-effect of NFT. As NFT is photolabile, and nitrite, well known as a MetHb generator, is an important photoproduct of NFT, it seems not unlikely that light is a cause of NFT-induced MetHb formation. When rats were irradiated with UV-A immediately after oral NFT administration, the amount of MetHb significantly increased: 0.97 +/- 0.37% n = 36 (P less than 0.001 Student's t-test, control value: 0.5%). An increase in MetHb was also observed with rats simultaneously exposed to UV-A and the major photodecomposition product of NFT, viz. 5-nitrofurfural. In addition in vitro experiments proved the formation of MetHb as a result of photoactivation of NFT. Nitrite, photochemically formed from nitrofurfural and from the metabolite nitrofuroic acid, plays an important role. A dark reaction of the other photoproduct, nitrofurfural, with hemoglobin also appeared to cause a considerable amount of MetHb in vitro. However, because of rapid deactivation of nitrofurfural by either photodecomposition or metabolism, this dark reaction is not expected to contribute to the in vivo MetHb formation.

Mestranol: the assignment of its 1H and 13C NMR spectra by means of two-dimensional NMR spectroscopy, and its photochemical decomposition.

The 1H- and 13C-nmr spectra of mestranol were assigned with the help of a 2 D-J-resolved, a 2D spin echo J-correlated (SECSY) and a 2D 1H-13C hetero-shift correlation experiment. The analysis of the spectra facilitated the identification of some of the photodecomposition products of mestranol. It was shown that, upon irradiation with UV-B light in water-ethanol (1:1, v/v), products are formed by oxidation of rings B and C of the steroid.

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), N,S-diacetylcysteinamide (SNACA), N,S-diacetylcysteine (SNAC) and N,S-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 < SNACET,SNACA,SACET < NACET, SNAC,NAC. The results of this study are discussed in view of susceptibility to oxidation, epidermal bioavailability and metabolic activation. With respect to the viable epidermis we postulate that NACET and SNAC have the most promising properties as UV protective agents.

Interaction of the photosensitization products of oestrone with DNA: comparison with the horseradish peroxidase catalyzed reaction.

The interaction with DNA of [4-14C]oestrone upon photosensitization with hematoporphyrin (HP) as a photosensitizer has been investigated. By means of Sephadex LH-20 gel filtration and extraction with dichloromethane it was found that, after irradiation (lambda greater than 425 nm) of a solution of HP, DNA and [4-14C]oestrone 21% of the radiolabel was associated with DNA. If DNA was added after irradiation 23% was bound to DNA, whereas 25% of the oestrone remained after photoreaction under the conditions applied. The binding occurs via the reactive 10 beta-hydroperoxy-1,4-estradien-3,17-dione, which is the only product after photosensitization of oestrone. The hydroperoxide has a strong interaction with DNA compared with that of other steroids. By repeated precipitation with 5 M NaCl and ethanol the association can be broken. It is reported, that binding of oestrone to protein induced by both photosensitization and horseradish peroxidase (HRPO)/H2O2 is irreversible, but that the amount of binding to DNA is dependent on the method of determination. However, neither the hydroperoxide nor its reduced product, a p-quinol, is intermediate or product in the HRPO catalyzed reaction of oestrogens. The tight association of the hydroperoxide product of oestrone with DNA, which may proceed via hydrogen bonding between the -OOH group and oxygen atoms of the backbone phosphate groups or of the furanose ring, might be a cause of chemical modification of DNA and of mutagenic effects.

Investigation into the effects of the photodecomposition of norethisterone by UV-B light on Salmonella typhimurium TA98 and the rat.

In view of the light-induced side-effects of the oral contraceptive pill and the development of injectable and subdermal contraceptive devices, the effects of norethisterone irradiated with UV-B light (280-320 nm) on the microorganism Salmonella typhimurium TA98 and male Wistar rats have been investigated. The observed cytotoxic effect of the photo-products on Salmonella typhimurium TA98 may in part be caused by the irreversible binding of steroid to protein from this bacterium, of which binding is in line with previous experiments. However, after intraperitoneal or percutaneous administration of 4-14C-norethisterone to rats, followed by irradiation with UV-B light, no significantly higher level of radioactivity was observed in blood, organ material of the skin, kidney or liver, on extraction or dialysis of the samples.

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.