Turnover of phospholipids in HUT 102 lymphoblasts and chromatographic characterization of purified lecithins after their exposure to long-wave UV light, psoralen, and UV light and psoralen.

The turnover of 32P-labeled phospholipids in HUT 102 lymphoblasts was determined after a 2 h interaction of lymphoblasts with 8-methoxypsoralen (8-MOP) (15 micrograms ml-1), longwave UV light (UVA) irradiation and PUVA (8-MOP and UVA). In parallel experiments, micellar suspensions of lyso-phosphatidylcholine (PtdC), dipalmitoyl-PtdC and dilinoleoyl-PtdC, treated in a similar manner, served for the correlative assessments of cellular lipid changes. The dark reaction, UVA irradiation and PUVA all depressed total phospholipid levels in HUT 102 cells, although only PUVA induced a statistically significant decline. Thin layer chromatography (TLC) analysis revealed that neither UVA nor 8-MOP alone triggered any significant changes in the cellular content of phosphatidylinositol (PtdI), phosphatidylinositol 4-monophosphate (PtdIP) and phosphatidylinositol 4,5-bisphosphate (PtdIP2), whereas the lyso-PtdC and PtdI content of lymphoblasts showed a two-fold increase after PUVA. The TLC analysis of lyso-PtdC and micelles of dipalmitoyl-PtdC did not reveal any detectable changes after the dark reaction with 8-MOP, UVA irradiation and PUVA. In contrast, the derivatives of dark and UVA mediated reactions of 8-MOP with dilinoleoyl-PtdC were detected by TLC. These results suggest that the formation of 8-MOP derivatives of cellular phospholipids effected by PUVA, modulates the turnover of phosphoinositides and the rate of cellular proliferation.     

8-Methoxypsoralen and long-wave ultraviolet A inhibit the release of proinflammatory mediators and cytokines from human Fc epsilon RI+ cells: an in vitro study

The in vitro effects of 8-MOP (concentrations of 20, 100 and 500 ng/ml) alone or in combination with UVA on mediator release from human basophils and skin mast cells (HSMC), activated with immunological and non-immunological stimuli, were investigated. With respect to basophils activated with anti-IgE serum, the results of this study show that: (i) 8-MOP alone inhibits histamine, LTC(4), IL-4 and IL-13 release concentration dependently with a maximal effect at 500 ng/ml (a concentration not reached in vivo); and (ii) UVA irradiation (5 J/cm(2)), after 8-MOP incubation, enhances this inhibitory effect on all released mediators, but for IL-4 and IL-13 the percentage inhibition is also significant for the 8-MOP concentrations (20-100 ng/ml) employed in vivo during PUVA treatment. Moreover, histamine release from basophils activated with non-immunological stimuli (FMLP and A23187) is inhibited by 8-MOP, alone or in combination with UVA. With respect to the HSMC activated with anti-IgE serum, the results show that: (i) 8-MOP alone reduces histamine release concentration dependently; and (ii) this inhibitory effect is enhanced by UVA irradiation (5 J/cm(2)). Histamine release from HSMC activated with A23187 is not modified either by 8-MOP alone or by 8-MOP plus UVA.     

Psoralen derivatives and longwave ultraviolet irradiation are active in vitro against human melanoma cell line

Cutaneous malignant melanoma is a very serious form of skin cancer that arises from melanocytes. Currently there is no effective treatment for metastatic melanoma so intense clinical trials are evaluating new drugs for this human malignancy. Psoralens are a group of compounds that bind to DNA in rapidly dividing cells and with ultraviolet light in the A band (UVA) cause DNA crosslinking, thereby preventing cellular division. They are used in the treatment of psoriasis and cutaneous T-cell lymphoma among other skin and blood diseases. We have investigated the cytotoxic potential of three psoralen derivatives plus UVA exposure (PUVA) on a established cell line of human melanoma. Cells were treated with different concentrations of 8-methoxypsoralen (8-MOP), 4,5',8-trimethylpsoralen (TMP) and 7-methylpyridopsoralen (MPP), for 1 h and after exposure to UVA light (0.3 J/cm(2)) were allowed to recover over a 24-72 h period. Viability was assessed by the microculture 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. Cisplatin, one of the most important drugs in the chemotherapy of melanoma, was included for comparative studies. All the psoralen derivatives tested were markedly cytotoxic in a dose and post-exposure-time dependent manner. The IC(50) values for 72 h of post-exposure time were as follows: MPP=0.05+/-0.01, TMP=0.13+/-0.003 and 8-MOP=10.79+/-1.85 micromol/L. Regardless of the limitations of the in vitro model, our results suggested that the lower IC(50) values of TMP and MPP might be of clinical importance.     

Simple equilibrium dialysis-high-performance liquid chromatographic method for the in vitro assessment of 5-methoxypsoralen bound to human albumin

Increasingly used in therapeutics, 5-methoxypsoralen (5-MOP), a linear furocoumarin, associated with UVA irradiation (PUVA), is now an established treatment for skin diseases such as vitiligo, mycosis funcoides and particularly psoriasis. Successful PUVA therapy depends on a sufficiently high peak 5-MOP plasma concentration coinciding with the UVA irradiation. However, as with most drugs, only the free plasma fraction is able to enter the target cells and has a pharmacological effect. In this work, the binding of 5-MOP to human albumin was studied in vitro, using a dialysis chamber. Bound and free 5-MOP fractions were quantified by a modification of Stolk's high-performance liquid chromatographic method. Dialysis was performed at 37 degrees C and pH 7.4 for 2 h, against a 4% albumin solution in phosphate buffer. The 5-MOP concentrations used were from 5 x 10(-5) to 5 x 10(-2) g/l in 1 x 10(-1) g/l steps. The 5-MOP bound strongly to human albumin in an unsaturable way. The mean 5-MOP binding to albumin was 95.3%. These results are in accordance with those published by Artuc et al. and not with those of Veronese et al., who found a lower saturable fixation (91%). These two research groups used tritiated 5-MOP. The technique used in this work is simple and inexpensive. It can be employed easily in vivo, e.g., for the assessment of 5-MOP free fractions in different therapeutic conditions.     

Effects of butylated hydroxytoluene upon PUVA-tumorigenesis and induction of ornithine decarboxylase activity in the mouse

Psoralen photochemotherapy (PUVA) is widely used in the treatment of psoriasis. Some therapy regimen have been associated with increased risk of skin cancer. Free radical species are thought to play a role in psoralen phototoxicity and photocarcinogenesis. It has been reported that the antioxidant butylated hydroxytoluene (BHT) inhibits acute phototoxicity by PUVA but does not reduce therapeutic efficacy. It has also been shown that BHT inhibits UVB-induced erythema, tumorigenesis and induction of ornithine decarboxylase (ODC) activity--ODC activity is thought by some to be associated with tumor promotion. Therefore, we have investigated the effect of BHT on psoralen tumorigenesis and PUVA-induced epidermal ODC activity. SKH-Hr-1 hairless albino mice were treated with topically applied 8-MOP and exposed to UVA (3X weekly) for 31 weeks with and without BHT administered either in the diet or topically. Induction of ODC activity was determined in similar experimental groups 24 h after a single exposure to UVA. Neither route of BHT administration had any effect on 8-MOP phototumorigenesis. However, BHT when administered in the diet reduced induction of ODC activity by 40% (p less than 0.05). These data indicate different mechanisms for UVB- and PUVA-induced carcinogenesis and again bring into question the relationship between induction of ODC activity and photocarcinogenesis.     

PUVA treatment of the nasal cavity improves the clinical symptoms of allergic rhinitis and inhibits the immediate-type hypersensitivity reaction in the skin

We earlier reported that intranasal irradiation with the 308 nm xenon chloride (XeCl) ultraviolet-B laser and irradiation with a combination of ultraviolet-B (UVB), ultraviolet-A (UVA) and visible light (VIS) is highly effective in the treatment of allergic rhinitis and inhibit the immediate-type hypersensitivity reaction in the skin. Since photochemotherapy with 8-methoxypsoralen (8-MOP) plus UVA light (PUVA) is widely used in the treatment of different inflammatory skin disorders due to its immunosuppressive effect, in the present study we investigated the efficacy of intranasal PUVA treatment in allergic rhinitis and the effect of PUVA treatment on the skin prick test (SPT) reaction. An open study was performed in 17 patients with hay fever. Intranasal PUVA therapy was given four times weekly for 3 weeks. The treatment was started with a fluence of 0.5x of the individual minimal phototoxic dose (MPD) and the dosages were gradually increased. Evaluation was based on the symptom scores. The effect of PUVA treatment on the allergen-induced wheal formation was also studied in the SPT. PUVA treatment of the nasal cavity significantly decreased the nasal symptoms of the patients with allergic rhinitis. Treatment of the skin with PUVA also significantly suppressed the allergen-induced wheal formation in the SPT reaction. These data suggest that intranasal PUVA phototherapy is also an effective modality in the treatment of allergic rhinitis.     

PROPHAGE INDUCTION, MUTAGENESIS AND CELL SURVIVAL OF AMES' MUTAGEN TESTER STRAINS AFTER 8-METHOXYPSORALEN PLUS ULTRAVIOLET LIGHT-A

Abstract— The furocoumarin 8-methoxypsoralen (8-MOP) is not detected as a mutagen in the standard Ames test either in the presence or absence of S9-mix and/or ultraviolet light-A (320–400 nm). The Ames strains have recently been shown to harbor bacteriophages that are inducible by carcinogens and mutagens. Psoralen (8-MOP) plus UVA (PUVA) was found to be a potent prophage inducing treatment. Induction was observed in TA1535, TA1538, TA98, TA100, TA1978 and TA1975 with 0.1 μg/ml of 8-MOP and 2.5 kJ/m² of UVA. PUVA is a potent bactericidal treatment at concentrations of 8-MOP above 0.5 μg/ml and 2.5 kJ/m² in tester strains TA1535, TA1538, TA98, and TA100. PUVA is known to be bactericidal, but the cytotoxicity observed in this study was unique in that the frameshift tester strains (TA1538 and TA98) were more sensitive to the lethal effects of PUVA than the base pair tester strains (TA1535 and TA100). The differential cytotoxicity in such closely related strains led to the examination of some of the strains from which the Ames strains were derived. The data suggest mutations introduced into the Ames strains to make them more sensitive to carcinogens and mutagens (Λ uvrB ) have resulted in an altered response to PUVA. It is postulated that TA1535 retains a DNA repair function that is lost by TA1538 during the selection for uvrB deficient strains.
PUVA is also unique in that plasmid pKM101 does not confer enhanced survival of TA100 compared to TA1535 in contrast to many other carcinogens and mutagens. In conclusion, these data show that PUVA induces a pathway leading to prophage induction and demonstrates the potential use of prophage induction assays to detect mutagens and carcinogens that may otherwise be lethal to the Ames tester strains.     

Tracing the Photoaddition of Pharmaceutical Psoralens to DNA

The psoralens 8-methoxypsoralen (8-MOP), 4,5′,8-trimethylpsoralen (TMP) and 5-methoxypsoralen (5-MOP) find clinical application in PUVA (psoralen + UVA) therapy. PUVA treats skin diseases like psoriasis and atopic eczema. Psoralens target the DNA of cells. Upon photo-excitation psoralens bind to the DNA base thymine. This photo-binding was studied using steady-state UV/Vis and IR spectroscopy as well as nanosecond transient UV/Vis absorption. The experiments show that the photo-addition of 8-MOP and TMP involve the psoralen triplet state and a biradical intermediate. 5-MOP forms a structurally different photo-product. Its formation could not be traced by the present spectroscopic technique.     

The Lack of Efficacy of 4,6,4'-Trimethylangelicin to Induce Immune Suppression in an Animal Model for Photopheresis: a Comparison with 8-MOP

Photopheresis is an extracorporeal form of photochemo-therapy with 8-methoxypsoralen (8-MOP) and UVA (PUVA). Patients ingest 8-MOP and then a psoralen-rich buffy coat is obtained by centrifugation and mixed with saline. This mixture is recirculated through a UVA radiation field and then reinfused. Photopheresis appears to be effective for several T cell-mediated disorders, because the treatment results in a specific immune response against the pathogenic clone of T cells involved. With PUVA therapy, the whole body of the patient is exposed to UVA, after ingestion of 8-MOP. Upon UVA exposure 8-MOP binds to, amongst others, DNA and induces DNA monoadducts and interstrand cross-links. As a result of these photoadducts photocarcinogenicity is a risk in PUVA. In PUVA for psoriasis, it proved that angular furocoumarins, although almost incapable of inducing DNA cross-links (less carcinogenic), are still effective. In order to determine if monoadducts induced by photopheresis could also be effective we used, specifically, 4,6,4'-trimethylangelicin (TMA). In this report, we compare the photodegradation of both TMA and 8-MOP under conditions relevant to the in vivo situation, as well as the effect both compounds have on the viability of rat lymphocytes as measured with the 3–(4,5-dimethylthia-zol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. We show that TMA did not induce immunosuppression in vivo , even after extensive irradiation. In addition a dose dependency of 8-MOPNVA versus the induced immune suppression was carried out. It was shown that there is a log doselresponse correlation of r = 0.9205.     

ACTIVATION OF THE HUMAN IMMUNODEFICIENCY VIRUS PROMOTER BY UVA RADIATION IN COMBINATION WITH PSORALENS OR ANGELICINS

Abstract— The effects of mono- and bifunctional furocoumarins plus UVA radiation (PUVA and related treatments) on the human immunodeficiency virus-1 (HIV-1) promoter were studied using HeLa cells stably transfected with the chloramphenicol acetyl transferase gene under the control of the HIV-1 promoter. The experiments were performed with three psoralens (5-methoxypsoralen, 5-MOP; 8-methoxypsoralen, 8-MOP; and 4′-aminomethyl-4,8,5′-trimethyl-psoralen, AMT) and four angelicins (angelicin; 4,5′-diniethylangclicin, 4,5′-DMA; 6,4′-dimethylangelicin, 6,4′-DMA; and 4,6,4′-trimethylangelicin, TMA). The drugs alone and UVA radiation alone showed no erect on the HIV promoter. However, when the cells were incubated with the furocoumarins at 0.1–40 μg/mL and then irradiated. the HIV promoter was activated in distinct fluence ranges, i.e. (1) no promoter activity was discernible at low fluences (e.g. at 0.1 μg/mL of 8-MOP up to 100 kJ/m²), (2) as the fluence was increased, the promoter activity increased to reach a maximum (10–50-fold with respect to the unexposcd samples), and (3) as the fluence was further increased, the promoter activity decreased. Similar (although shifted on the fluence scale) pattcrns were observed with either > 340-nm UVA radiation or with UVA radiation contaminated with a small amount of UVB radiation (typical for PUVA lamps). The effective fluences were inversely related to the drug concentration. Experiments with 5-MOP and 8-MOP indicated reciprocity of the drug concentration and radiation hence. The HIV promoter response patterns were similar for monofunctional angelicins and bifunctional psoralens. This indicated that the furocoumarin-DNA crosslinks are not a prerequisite for the promoter activation and that the monoadducts suffice to elicit the HIV promoter response. The HIV promoter-activating effectiveness of diKcrent drugs correlated with their photosensitizing potential. Thus, among psoralens the effectiveness order was AMT >. 5-MOP >8-MOP, and among angelicins: TMA > 6,4′-DMA > 4,5′-DMA > angelicin. The ektiveness did not vary substantially for 5-MOP, 8-MOP, 4,5′-DMA, and 6,4′-DMA. The combined drug and UVA radiation doses were higher than those that elicit cellular responses or those that may be received by the human white blood cells during cxtracorporeal PUVA therapy (photopheresis).     

MUTAGENIC EFFECTS PHOTOINDUCED IN MAMMALIAN CELLS IN VITRO BY TWO MONOFUNCTIONAL PYRIDOPSORALENS

Abstract— The photobiological activity of the two monofunctional pyridopsoralens pyrido (3,4-c) psoralen (PyPs) and 7-methyl pyrido (3,4-c) psoralen (MePyPs) was studied in mammalian cells in vitro taking 8-methoxypsoralen (8-MOP) as a reference compound.
In the presence of 365-nm irradiation (UVA) MePyPs was found to be more effective than 8-MOP in terms of DNA photobinding capacity and inhibition of cell cloning ability in Chinese hamsterV–79 cells. As a function of UVA dose and of the number of total photoadducts induced MePyPs produced a higher frequency of 6-thioguanine resistant mutants than 8-MOP. PyPs showed an intermediate response for cell killing and mutation induction. At equal cytotoxic levels both monofunctional pyridopsoralens exhibited the same mutagenic activity as the Afunctional furocoumarin 8-MOP.
The antiproliferative effect being taken as indicative for an efficient photochemotherapeutic activity against psoriasis, the inhibition of cloning capacity induced by MePyPs plus UVA was studied in parallel on human skin fibroblasts. Such cells were more sensitive to 8-MOP photoadditions thanV–79 cells and even more so to MePyPs photoadditions. Data obtained on the rate of DNA semi conservative synthesis on both cell lines following treatments with the two compounds are in line with these observations.     

ANTIGENICITY OF DNA INDUCED BY PHOTOADDITION OF 8-METHOXYPSORALEN

Abstract— Rabbits immunized with sonicated DNA UV-irradiated (365 nm) in the presence of 8-methoxypsoralen (8-MOP) produced a specific antiserum directed against DNA-8-MOP-photoadduct. None of the long chain DNA preparations modified photochemically in the same way elicited an immunological response, although all of them gave a positive immunodiffusion test on agarose with the antiserum directed against DNA-8-MOP-photoadduct. A positive reaction has also been demonstrated in the indirect immunofluorescence test, using as a source of cellular native DNA Crithidia luciliae cells treated with 8-MOP and irradiated at 365 nm (UVA). A possible use of the specific antiserum for detecting the formation of DNA-8-MOP-photoadduct, in the skin and/or lymphocytes of patients with psoriasis treated by combination of 8-MOP and UVA irradiation, is suggested.     

Photoinduced cutaneous inflammatory response by psoralens.

Our studies describe the inflammatory response in rabbit skin induced by topical application of 8-methoxypsoralen (8-MOP) and UVA-visible irradiation (320-700 nm). Increase in vascular permeability (iVP) and accumulation of polymorphonuclear leucocytes (aPMN) at the test sites were quantitated using 125I-albumin and 51Cr-labelled PMNs respectively. Erythema was graded visually. 8-MOP cream was applied topically and irradiated. The erythemal response, aPMN and iVP at the test sites were quantitated at 6, 24, 48 and 72 h post-irradiation. The iVP and aPMN were maximal at 24 h; the erythemal response was the same at 24-48 h. The responses were dependent on 8-MOP concentration and irradiation dose. Topical application of 200 micrograms 8-MOP cream followed by irradiation for 2 h (9.4 J cm-2) produced 3-7 times iVP, 2-4 times aPMN and intense erythema at the test sites after 24 h. Neither aPMN nor iVP was detected before 6 h and erythemal response was not observable up to 16 h after irradiation. The aPMN and iVP gradually subsided in 72 h, although the erythemal response was still present. The repeated exposure of 8-MOP-treated sites for three consecutive days 24 h apart did not produce appreciable iVP or aPMN at 72 h or 24 h after the last exposure; however, erythema persisted. The 8-MOP-treated sites previously exposed for three consecutive days on reapplication of 8-MOP cream plus irradiation showed significantly less response compared with non-pretreated sites. Our results suggest that the erythemal response is not directly related to either iVP or aPMN.     

Kinetic Analysis of Apoptosis Induction in Human Cell Lines by UVA and 8-MOP

Whereas previous studies have indicated that DNA damage as a result of 8-methoxypsoralen (8-MOP) and UVA treatment leads to cell death, this study establishes the minimum concentrations of 8-MOP and UVA necessary to induce apoptosis in human T-lymphocytic and mono-cytic cell lines. In order to assess apoptosis, we used fluorescent microscopy to examine changes in light scattering as well as internucleosomal DNA fragmentation. Generation of a dose response curve showed that the minimum combination of UVA and 8-MOP that was necessary to induce greater than background levels of apoptosis within 24 h of treatment was 0.5 J/cm² UVA and 12.5 ng/mL of 8-MOP. A striking observation was that UVA alone at doses 1.0 J/cm², but not 8-MOP alone (6300 ng/mL), induced significant apoptosis in the Sup-T1 cell line within 24 h. Although the percentage of apoptotic Sup-T1 cells induced by UVA alone was not as great as that of 8-MOP and UVA in combination, a highly significant correlation between the product of the concentration of 8-MOP (ng/mL) times the dose of UVA (J/ cm²) and the percentage of apoptotic cells was observed. This correlation provides an important tool for studying the relationship of UVA-induced DNA damage to apoptosis induction. Moreover, it will provide a means by which early events in the apoptotic pathway can be dissected.     

The evolution of photochemotherapy with psoralens and UVA (PUVA): 2000 BC to 1992 AD.

The therapeutic uses of naturally occurring psoralens in modern-day medicine (8-methoxypsoralen (8-MOP), 5-methoxypsoralen (5-MOP), 4,5',8-trimethylpsoralen, and a few other synthetic psoralens) have evolved through five stages of development. (1) In the historical period (2000 BC to 1930 AD), the pigment-stimulating properties of naturally occurring plants containing psoralens were described anecdotally. (2) The second period (1930-1960) dealing with the chemistry of psoralens involved extraction, identification of their structure, synthesis, and the relationship between chemical structure and their photoreactivity and pigment-stimulating properties. The treatment of vitiligo with oral and topical 8-MOP became popular. (3) In the third period (1960-1974), we witnessed a new beginning and the growth of basic science studies and clinical investigations into various biological properties of psoralens including action spectrum studies, mutagenesis and carcinogenesis studies, in vitro and in vivo photoreactivity studies of various psoralens with DNA, RNA, proteins, and pharmacological and toxicological studies in vitiligo patients undergoing long-term therapy for repigmentation. (4) The fourth period (1974-1988) is recognized as the period of photochemotherapy and the development of the science of photomedicine which established the therapeutic effectiveness of psoralens in combination with newly developed UV irradiation systems that emitted high-intensity UVA radiation in the treatment of severe psoriasis, mycosis fungoides, and over 16 other skin diseases. The effectiveness of PUVA (psoralen + UVA) was confirmed by well controlled clinical trials in thousands of patients, both in the USA and in European countries. Combination therapy with oral retinoids and PUVA contributed to greater effectiveness and long-term safety of psoralen photochemotherapy. (5) In the fifth period (1989 and beyond), psoralens are now emerging as photochemoprotective agents against non-melanoma skin cancers and as immunologic modifiers in the management of certain patients with disorders of circulating T-cells using new techniques of photopheresis. In the final analysis, perhaps the application of pharmacological and therapeutic concepts and principles of using psoralens in combination with UVA has contributed to the development of a new science of photomedicine in which the interaction between basic scientists, photobiologists, and physicians has produced both basic and new clinical knowledge for the care and control of human suffering.     

8-METHOXYPSORALEN-PHOTOINDUCED DNA CROSSLINKS AS DETERMINED IN YEAST BY ALKALINE STEP ELUTION UNDER DIFFERENT REIRRADIATION CONDITIONS. RELATION WITH GENETIC EFFECTS

Abstract— DNA damage induced by 8-methoxypsoralen (8-MOP) plus near UV light (UVA) was analyzed in diploid yeast using the alkaline step elution technique. The presence of 8-MOP and UVA induced DNA interstrand cross-links was revealed by the increase of DNA retained on elution filters as compared to untreated controls. The fraction of DNA retained on filters increased linearly with UVA dose. The amount of cross-links was estimated from the fraction of DNA retained on filters using a dose of -radiation leading to a number of DNA strand breaks at least equivalent to the number of 8-MOP induced photoadducts.
When 8-MOP treated cells were exposed to monochromatic light, 365 nm light induced monoadducts and cross-links whereas 405 nm light induced only monoadducts. When submitting 8-MOP plus 405 nm light treated cells to 365 nm irradiation, after removal of unbound 8-MOP by washing, a portion of 8-MOP plus 405 nm light induced monoadducts was converted into cross-links. The amount of monoadducts transformed into cross-links was dependent on the dose of 365 nm irradiation up to a maximum likely to correspond to the number of suitably positioned furan-side monoadducts that could be converted into biadducts. When 8-MOP plus 365 nm light treated cells were reirradiated with 365 nm light, following the same protocol, the maximum level of cross-linking obtainable in yeast was lower than that obtained with 8-MOP in a 405 nm plus 365 nm reirradiation protocol.
In the presence of 8-MOP single exposures to 405 nm light were found to be only slightly genotoxic. However, when followed by second exposures to 365 nm light, a dose-dependent increase in genetic effects, i.e. mutation and gene conversion, was observed in parallel to the induction of DNA crosslinks. These results stress again the prominent role of DNA cross-links in the genotoxicity of 8-MOP.     

INHIBITORY EFFECT OF 8-METHOXYPSORALEN PLUS ULTRAVIOLET-A ON INTERLEUKIN-1 PRODUCTION BY MURINE KERATINOCYTES

We report the effects of 8-methoxypsoralen (8-MOP) plus ultraviolet-A (UV-A) irradiation on interleukin-1 (IL-1) production by murine epidermal keratinocytes, correlating its effect on IL-1 with cell viability, DNA synthesis, and 8-MOP-DNA photoadduct formation. Freshly isolated murine keratinocytes were treated with various doses of 8-MOP (5-100 ng/mL; incubation time, 30 min) plus 1 J/cm2 UV-A and cultured for 1-3 days. The IL-1/epidermal cell-derived thymocyte-activating factor (ETAF) activity in both supernatant and cell extract was reduced proportionately with increasing doses of 8-MOP/UV-A. Interleukin-1 inhibitors induced by 8-MOP plus UV-A were not detected in either supernatant or cell extract. A clear reduction of the IL-1 production was induced by the treatment as low as 15 ng/mL 8-MOP plus 1 J/cm2 UV-A, which led to the formation of 0.52 8-MOP photoadducts per million DNa bases and affected neither cell viability nor DNA synthesis of the treated cells. Cells treated with 100 ng/mL 8-MOP and 1 J/cm2 UV-A exhibited 57% suppression of IL-1 production in both 2- and 3-day culture samples. This treatment resulted in the formation of 3.8 photoadducts per million bases as well as significant abrogation of DNA synthesis although cell viability was unchanged. These observations provide some insights into the phototoxicity mechanisms of 8-MOP and the effect of PUVA therapy on the cytokine regulation in keratinocytes.     

MUTAGENIC AND RECOMBINOGENIC ACTION OF DNA MONOADDUCTS PHOTOINDUCED BY THE BIFUNCTIONAL FUROCOUMARIN 8-METHOXYPSORALEN IN YEAST (Saccharomyces cerevisiae)

Abstract— For the same furocoumarin 8-MOP and the same total number of photoadditions, the genetic activity of DNA monoadducts and a mixture of mono- and biadducts photoinduced by the bifunctional furocoumarin 8-methoxypsoralen (8-MOP) is compared in the yeast Saccharomyces cerevisiae. In the presence of 8-MOP, 405 nm irradiation induces only monoadducts, whereas 365 nm irradiation induces mono- and biadducts (interstrand cross-links) in DNA. This is shown by heat denaturation-renaturation experiments on calf thymus DNA treated in vitro and by alkaline step elution analysis of DNA from treated yeast cells. For the same photobinding of tritiated 8-MOP to DNA in diploid yeast, about 20 times higher doses are needed with 405 nm than with 365 nm irradiation. Re-irradiation experiments reveal that part of the monoadducts induced by 8-MOP and 405 nm irradiation can be effectively converted into DNA interstrand cross-links by exposures to 365 nm radiation after washing-out of unbound 8-MOP molecules. 8-MOP and 405 nm irradiation induce per lethal hit cytoplasmic "petite" mutations in yeast as efficiently as the monofunctional furocoumarin 3-carbethoxypsoralen (3-CPs) and 365 nm irradiation, both treatments being much more efficient than 8-MOP and 365 nm irradiation. At equal survival, treatments with 8-MOP and 405 nm radiation are clearly less efficient than treatments with 8-MOP and 365 nm radiation for the induction of forward ( CAN *) and reverse ( HIS +) mutations in haploïd yeast and for the induction of mutations ( ILV +) and genetically aberrant colonies including mitotic crossing-over in diploid yeast. The two treatments are equally efficient for the induction of mitotic gene conversion. At equal photobinding of 8-MOP, the monoadducts induced by 405 nm irradiation are found less effective than the mixture of mono-and biadducts induced by 365 nm irradiation for the induction of cell killing, mutations and mitotic recombination.     

DETECTION OF DNA-PSORALEN PHOTOADDUCTS in situ

Abstract— An immunological method, with the use of specific immune serum, has been developed for detection of 8-methoxypsoralen (8-MOP) photoadducts to DNA, formed in situ in cell nuclei, after combined treatment with 8MOP and UV-A irradiation (Zarçbska et al. , 1978). Lymphocytes fixed on slides or in suspension, and cryostat sections of different mammalian tissues, served as antigenic substrate, after treatment with 8-MOP and UV-A in vitro. Specific fluorescence in these substrates was detected in the nuclei after treatment with 30 ˜ 140 kJ/m² UV-A in the presence of 0.1-0.3 μg/cm² 8-MOP. PHA-stimulated-lymphocytes appeared to be the most sensitive substrate.
However, hairless mice treated with high doses of UV-A in vivo , 70 ˜ 360 kJ/m² did not reveal a specific fluorescence of epidermal nuclei, unless a high local concentration of 8-MOP was attained.
The apparent discrepancy in the level of photoadduct detection between the in vitro and in vivo treated specimens was explained by the low number of DNA-8-MOP-photoadducts formed in vivo under these experimental conditions. The relevance of these findings to the role of DNA-8-MOP-photoadducts formed during PUVA photochemotherapy is discussed.