[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.

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.

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. OBJECTIVES: The pharmacokinetics of ALA- and ALA-n-pentylester-induced PpIX, together with the phototoxicity after PDT, were compared in human skin in vivo, using iontophoresis as a quantitative drug delivery system. METHODS: A series of six increasing doses of equimolar prodrug solutions was iontophoresed into normal skin of the upper inner arms of 20 healthy subjects. The kinetics of PpIX metabolism in skin (n = 4) and the response to light exposure, performed at 4.5 h (n = 6) and 6 h (n = 10) after application, were assessed by skin surface PpIX fluorescence and postirradiation erythema. RESULTS: ALA and ALA-n-pentylester showed a linear correlation between logarithm of dose and PpIX fluorescence (P < 0.005), and logarithm of dose and skin phototoxicity with irradiation at 4.5 h (P < 0.001 and P < 0.005, respectively) and 6 h (P < 0.05 and P < 0.0001, respectively) after iontophoresis. Higher phototoxicity was observed with ALA-n-pentylester than with ALA when sites were irradiated at 6 h, as indicated by the significantly lower theoretical threshold dose for erythema (P < 0.05) and the shift of the PpIX fluorescence/phototoxicity curve towards greater skin erythema at equal PpIX fluorescence levels. Depth of PpIX fluorescence in skin, as determined by fluorescence microscopy, was similar for both prodrugs, but a more homogeneous distribution of PpIX was seen with the more lipophilic ALA-n-pentylester. CONCLUSIONS: The observed greater phototoxicity of ALA-n-pentylester relative to ALA may be attributable to a more favourable PpIX localization in tissue and/or greater intrinsic toxicity.

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.

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.

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.

The mechanism of N-acetylcysteine photoprotection is not related to dipyrimidine photoproducts.

Topical application of N-acetylcysteine prior to UVB irradiation of BALB/c mice has previously been shown to inhibit systemic suppression of the contact hypersensitivity response. Formation of cis-urocanic acid, however, is not affected. Besides urocanic acid, UV-induced DNA damage has been held responsible for the initiation of suppression of the contact hypersensitivity response. Therefore, the possible inhibitory effect of N-acetylcysteine on UVB-induced cyclobutane pyrimidine dimers and pyrimidine (6-4) pyrimidone photoproducts has been investigated. No effect on the photoproducts studied is observed, suggesting that N-acetylcysteine exerts its photoprotective effect during the post-initiation phase of photoimmunosuppression.

Photoreactivity of nifedipine in vitro and in vivo.

Side effects of nitroaromatics used as drugs are often assumed to be caused by incomplete enzymatic reduction of the nitro group. However, photoactivation, although usually not considered as a cause of the toxic effects of nitroaromatics, can play a considerable role. Nifedipine, a nitroaromatic as well, is an important drug used in the treatment of myocardial ischaemia and hypertension. It is extremely sensitive to ultraviolet and visible light up to 450 nm and forms a nitroso derivative in vitro. In the present study it is shown that the nitroso compound is a short-lived intermediate in blood and plasma. It reacts with other constituents to form a stable lactam. In vivo, this lactam proves to be rapidly clear from the blood of rats and is excreted almost quantitatively via the bile. The first photoproduct of nifedipine, nitroso nifedipine, is shown to be converted into the lactam mentioned. Beside the lactam, two other products, which are considered to be derivatives of the lactam, are found. One of these two products is also found in the bile of a rat that was exposed to UVA light after intravenous nifedipine administration. This shows that in an in vivo situation, photoproducts can reach organs other than those exposed to the light. In vitro about 45 to 50% of the original amount of nifedipine is not recovered after exposure of nifedipine to UVA in the presence of bovine serum albumin or after incubation of nitroso nifedipine with bovine serum albumin in the dark. As complex binding of nifedipine of plasma proteins is high, the latter finding may have important implications for the situation in vivo.

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.

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.

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.

Clinical and mechanistic aspects of photopheresis.

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.

Singlet oxygen producing photosensitizers in photophoresis.

In this study, the immunosuppressive properties of two photosensitizers (benzoporphyrin derivative monoacid ring A (BPD) and Photofrin (HPD)), used for the photodynamic therapy of cancer, were investigated. The investigations were performed in our rat model for photophoresis. The validity of this model has been amply demonstrated. It enables a distinction to be made between antigen-specific and antigen non-specific immune suppression. With this model, the immune response which can be specifically suppressed is the contact hypersensitivity (CHS). CHS is induced by 2,4-dinitrofluorobenzene (DNFB). Both BPD and HPD are able to suppress CHS induced by DNFB. Furthermore, this generated suppression is transferable by the spleen cells of treated animals and is antigen non-specific.

Photopheresis, a possible therapy for airway hyperreactivity?

Airway hyperreactivity is an almost universal feature of asthma. The exact origin of this phenomenon is poorly understood. However, there is increasing evidence that T cells play an important role in the pathogenesis of this disorder. This fact makes it challenging to photopheresis to suppress the pulmonar hyperreactivity response. Photopheresis is a therapy for T cell mediated diseases aiming at specific suppression of the pathogenic clone of T cells involved. The use of photopheresis for the treatment of airway hyperreactivity was investigated in this study. We performed experiments in a murine model for airway hyperreactivity. In short, mice were sensitized by cutaneous application of 2,4,6-trinitrochlorobenzene. The immune system was challenged by an intratracheal injection of 2,4,6-trinitrobenzenesulfonic acid and a bronchoalveolar lavage was performed. In this lavage the total number of leukocytes was established and the number of macrophages was determined. It was found that photopheresis treatment was capable to suppress the airway hyperreactivity response for about 80%. In addition, the generated suppression proved to be transferable by splenocytes of treated animals. We conclude that photopheresis can be an interesting therapy for airway hyperreactivity (and perhaps also for asthma) especially when one takes into account that photopheresis induces specific immune suppression and has hardly any adverse effects.

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.

Photopheresis; the risk of photoallergy.

Photopheresis is a therapy for several T-cell-mediated disorders, aiming at a specific immune response against the pathogenic clone of T cells involved. With photopheresis, a mixture of patients' buffy coat and plasma, which contains 8-methoxypsoralen (8-MOP), is diluted with phosphate-buffered saline (PBS) and exposed to ultraviolet A radiation (UVA). After the irradiation the treated fraction is reinfused. As photomodification of biomacromolecules is considered to be crucial in photopheresis, the presence of plasma during irradiation can pose a problem. The fact is that photomodification of plasma proteins is supposed to be the causal step in the occurrence of photoallergy. Whether the presence of plasma during photopheresis is a risk for photoallergy was investigated with 8-MOP and chlorpromazine (CPZ; well-established photoallergen). It proved that both sensitizers can induce photoallergy, although the concentration of 8-MOP needed to induce photoallergy is 4.5 times higher than that of CPZ. It is concluded that the presence of plasma during irradiation should be avoided in order to prevent the risk of induction of photoallergy.

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.

Treatment with 8-MOP and UVA enhances MHC class I synthesis in RMA cells: preliminary results.

The response of psoriasis and cutaneous T-cell lymphoma to treatment with 8-methoxypsoralen (8-MOP) and long wavelength ultraviolet light (UVA) is only partly understood. Psoralens form photoadducts within the DNA after activation by UVA and this damage leads to the inhibition of DNA synthesis. Additionally, it has been shown that different forms of DNA damage can induce a stress response, leading to upregulation of selected products. Among these are the major histocompatibility complex (MHC) class I genes. Thus the aim of the present study was to assess the rate of synthesis of MHC class I proteins in murine T-cell lymphoma cells (RMA) after treatment with 8-MOP and UVA. RMA cells were treated with 8-MOP (50-200 ng ml-1) and UVA (1 J.cm-2) and metabolically labelled with 35S-methionine 4 and 24 h after treatment. MHC class I synthesis was determined by immunoprecipitation of the cell lysates with an anti-Kb monoclonal antibody, Y3. After 4 h, treated and untreated cells demonstrated no differences in the rate of MHC class I synthesis. However, after 24 h a dose-dependent increase in MHC class I synthesis was observed. This increase in MHC class I expression could be responsible, at least partly, for the responses observed in patients treated with photopheresis.

Increased surface expression of class I MHC molecules on immunogenic cells derived from the xenogenization of P815 mastocytoma cells with 8-methoxypsoralen and long-wavelength ultraviolet radiation.

In a previous study we demonstrated that the treatment of the highly tumorigenic cell line, P815, with 8-methoxypsoralen and long-wavelength ultraviolet radiation resulted in the production of several immunogenic clones (tum-). Mice inoculated with the tum- cells survived much longer than mice inoculated with the original tumorigenic cells (tum+). It was suggested that the increased survival of mice treated with the tum- clones arose as a result of an increased antigenicity derived from the phototreatment. In this report we show that the tum- cells have a greater density of class I MHC molecules on their surface (50-157% compared to P815). Class I MHC density on the cell surface is required to elicit targeted cytotoxic responses. These results can be considered in terms of human class I MHC assays which show that many human tumor cells have a reduced expression of class I MHC. Because other DNA damaging agents have also been shown to enhance class I expression, it is suggested that in addition to the cytotoxic effects of these agents, other pleiotropic effects must be considered. Photochemotherapy may phenotypically alter cells so that the enhanced expression of class I MHC molecules on the surface of phototreated cells may be associated with the clinical responses observed in cutaneous T cell lymphoma patients.