Spectroscopic detection of photoproducts in lecithin model system after 8-methoxypsoralen plus UV-A treatment.

Photoreactions of 8-methoxypsoralen (8-MOP) in the presence of synthetic lecithins esterified at the beta-position with linoleic/oleic and gamma-palmitic acid (PCd2/d1pal) have been studied. Following UV-A (320-400 nm) irradiation, the photoproducts separated by thin-layer chromatography are analysed by UV absorption spectroscopy, nuclear magnetic resonance and mass spectroscopy. The new isolated products are lecithin double cyclobutane adducts, PC-(8-MOP)2, fatty acid-8-MOP split adducts from phosphatidylcholine and lecithin adducts with photo-oxidized 8-MOP. The photolysis performed in the presence of 8-MOP is related to the previously reported lecithin cyclobutane adducts with psoralen. A hypothetical scheme of lecithin photolysis under PUVA (psoralen + UV-A) treatment is proposed. We suggest that photolysis of lecithin may have a significant role in the chain of reactions triggered in cell membrane submitted to PUVA treatment.

PUVA (psoralen + UVA) photochemotherapy: processes triggered in the cells.

Photochemotherapy using psoralens and UVA is a treatment used widely in some skin diseases, in cutaneous lymphomas and in autoimmune diseases. This review has selected recent publications dealing with the photochemical processes triggered in the cells by UVA radiation and psoralen treatment. The photochemical changes initiated in the cell membranes were described.

Photoreactions of psoralens with lecithins.

The formation of cyclobutane (cb) photoadducts of psoralen with a model lecithin has been shown. The adducts are formed both in ethanol solution and in micellar suspension in water. In spite of their sensitivity to various factors such as light, temperature, air, etc., they are isolated by thin-layer chromatography (TLC) and high-pressure liquid chromatography (HPLC) and characterized by mass spectrometry, NMR and UV absorption spectroscopy. The NMR analysis indicates the similarity of isomeric forms of cb adducts in lecithin to those formed with free oleic acid.

Psoralen photosensitization: damages to nucleic acid and membrane lipid components.

For decades the therapeutic properties of psoralens were related to their photochemical reactions with DNA and RNA. Such approach, although fruitful in treatments, did not explain satisfactorily the way of healing of the multitude of diseases manifested through skin disorders. The new research field presented in our review is directed to another target: the lipid components of the cell. The studies on the photobiology of phospholipids may help to elucidate the phototoxic and pigment inducing activity of psoralens.

Cell membrane, a target for PUVA therapy.

The photochemical reactions occurring in the cell membranes, sensitized photo-oxidation and psoralen photoaddition to lipids, are briefly reviewed. Phospholipid dynamics in the membrane structure, based on erythrocyte lipid organization, are described. Evidence for alterations of cell membrane functions under psoralen plus UVA radiation (PUVA) treatment in a variety of mammalian cells is presented. Cell receptor dysfunctions under PUVA treatment are demonstrated in a number of biological investigations. The purpose of this survey is to illustrate the feasibility of studying psoralen photobiology with phospholipids. The reaction of psoralens with phospholipids is considered to be one of the triggering mechanisms of the subsequent physiological responses, which may be relevant to PUVA photochemotherapy.

Two subpopulations of patients with early and late onset of psoriasis differ regarding alpha 1-proteinase inhibitor activity.

Two subpopulations of patients with psoriasis were identified when activity of alpha 1-proteinase inhibitor was analysed according to the patient's age at onset of the disease. Patients with early onset had more active lesions, high incidence of familial psoriasis, and reduced alpha 1-proteinase inhibitor during flare similar to that activity in remission. In contrast, alpha 1-proteinase activity increased significantly during flare in patients with late onset (above 21 yrs of age) in response to the inflammatory process in the psoriatic skin.

Alpha 1-proteinase inhibitor in psoriasis: reduced activity in symptom-free patients and during flare.

The aim of this study was to quantitate the active fraction of the alpha 1-proteinase inhibitor (alpha 1-PI) in psoriasis. Serum proteinase inhibitory capacity was measured vs porcine pancreatic elastase of a known active fraction against its specific substrate (Suc-Ala3-pNA). The inhibitory capacity was determined in 21 symptom-free patients, 134 patients with skin lesions, and 23 healthy volunteers. Alpha 1-PI was found to be significantly decreased in symptom-free patients and in those with stationary lesions, in a manner similar to the reduced activity of neutrophil proteinases, elastase, and cathepsin G. The synthesis of alpha 1-PI was stimulated during the appearance of active psoriatic lesions, but to a much lesser degree in patients with early onset (less than or equal to 21 years) than in patients with late onset of psoriasis (greater than 21 years). The early onset subgroup differed by a more frequent familial occurrence of psoriasis and a more severe course of the disease. The data indicate that the regulation of the proteinase-alpha 1-PI system in psoriasis is abnormal and this may contribute to the pathogenesis of the disease. The decreased alpha 1-PI during flare may be responsible for the disease activity, at least in patients with early onset of psoriasis.

Repair of UV-damaged DNA in mammalian skin followed by the immunohistochemical method.

DNA repair in murine and guinea pig skin has been studied by the immunohistochemical method. For the detection of DNA photolesions in situ by the indirect immunofluorescence (IF) method two antisera directed against DNA-pyrimidine-dimers and DNA-psoralen-photoadducts have been applied. The IF assay enabled to detect the DNA photodamage induced by high UV-doses, exceeding more than fivefold minimal phototoxic response of the skin. It was found that IF staining gradually disappeared due to DNA repair, and at 48 h after UV-exposure both types of the DNA photolesions were no more detectable. Importantly, the IF method revealed that the persistence of DNA-pyrimidine-dimers could be traced for a longer time than that detectable by UV-endonuclease incision method.

Detection of DNA-psoralen photoadducts in mammalian skin.

An immunofluorescence (IF) method for the detection of 8-methoxypsoralen (8-MOP) photoadducts to DNA has been developed to assess nuclear damage in keratinocytes and melanocytes after psoralen plus UVA (PUVA) treatment, both under in vitro and in vivo conditions. Cryostat sections of the albino and pigmented guinea pig and human skin were used for in vitro studies to establish minimal and maximal drug concentration and UVA dosimetry for the detection of DNA-8-MOP photoadducts. Limits of detection were as low as 10 ng/cm2 8-MOP and 1 J/cm2 UVA for skin sections and sodium bromide-split epidermal sheets. Guinea pigs treated with topical PUVA revealed positive IF stain in epidermal cell nuclei at a threshold dose of 100 micrograms/cm2 8-MOP and 13 J/cm2 UVA. Pretreatments of cryostat cuts with ethanol and alkali before IF test enhanced the sensitivity of detection in vivo about 10-fold and enabled us to follow the repair of DNA damage after treating normal guinea pig skin with a dose of 50 micrograms/cm2 8-MOP plus 6 J/cm2 UVA. The most interesting findings were as follows: A sensitive method to detect PUVA-induced nuclear damage in epidermal and dermal cells was developed. PUVA treatment induced nuclear DNA damage to melanocytes as well as to adjacent keratinocytes, and melanocytes appeared to be 10 times less vulnerable to photo-damage than keratinocytes. There was a greater propensity for the proliferative cells to be damaged by PUVA. PUVA induced nuclear damage up to 700 micron depth in the dermis. The usefulness of the IF test in detecting DNA damage in microgram and ng amounts in vivo and in following the repair of damaged DNA induced by PUVA.

Leukopheresis for treatment of psoriasis: is therapeutical benefit related to reduced activities of neutral proteinases of polymorphonuclear leukocytes?

Ten patients were treated with repeated leukophereses performed one to three times per week for 2-5 weeks. Two of the patients was cleared completely, four exhibited regression of more than one-half of the lesions, and four showed only a slight improvement. The therapy did not markedly affect the granulocyte count in peripheral blood, and the beneficial clinical response was not related to the number of polymorphonuclear leukocytes (PMNs) removed by leukophereses. During therapy, the activities of elastase, cathepsin G, lysozyme, and myeloperoxidase in PMNs were determined by spectrophotometry. PMNs isolated using a Haemonetics 30 blood-cell separator were about 50% deficient in these activities in comparison to cells obtained directly from peripheral blood. Thus, leukopheresis induces a marked degranulation of PMNs. Repeated leukophereses were found to generate significant variations in the activities of circulating PMN granule enzymes and in the levels of acid-soluble proteins. Remission or great improvement were observed in patients who, during therapy, exhibited decreased PMN elastase and cathepsin G activities, whereas a poor clinical response was accompanied by high enzymatic activities.

Neutral proteinases and other neutrophil enzymes in psoriasis, and their relation to disease activity.

The activities of elastase, cathepsin G, lysozyme and myeloperoxidase of polymorphonuclear leukocytes were determined by spectrophotometry in thirty-six patients with psoriatic lesions, twelve symptom-free patients with psoriasis and fifteen normal controls. The mean activities of cathepsin G, elastase and lysozyme were found to be increased by 55 to 70% in patients with actively spreading plaque lesions compared with healthy controls (P less than 0.01). Most patients with guttate lesions had total enzyme activities within the normal range. Those with stationary plaque psoriasis had activities of both neutral proteinases (cathepsin G and elastase) which were about 40% lower than normal controls (P less than 0.05). In the lesion-free psoriatics, the activities of neutral proteinases were about 70% of control values. Our findings emphasize the importance of assessment of disease activity in this sort of investigation. The present data may help to resolve much of the confusion regarding PMN function in psoriasis.

Immune serum against anti-DNA-8-methoxypsoralen photoadduct.

Specific immune serum against photo-damaged DNA with the photoadducts of 8-methoxypsoralen (8-MOP) was obtained. The antigenic determinant is a polynucleotide chain with a mono-photoadduct: a coumarin moiety of psoralen linked through a cyclobutane ring to the 5,6-bond of thymine. The specific rabbit antiserum was used in the immunofluorescence method, for the detection of photodamaged DNA in the kinetoplasts and nuclei of unicellular organisms Crithidia luciliae, in the nuclei of snap-frozen tissue of mammals, and in human blood lymphocytes. Detection by the immunofluorescence method was limited by psoralen availability in situ; the psoralen concentration should be in the range 0.05-0.2 microgram/cm2 on specimens submitted to a topical application or about 1 microgram/ml in cell suspension. The long-wave ultraviolet light (UV-A) exposure dose applied to the nuclei should exceed 1 J/cm2.

The activity of polymorphonuclear leukocyte neutral proteinases and their inhibitors in patients with psoriasis treated with a continuous peritoneal dialysis.

3 of 16 patients with extensive psoriasis have been completely cleared of skin lesions within 2-3 weeks of continuous peritoneal dialysis, and 2 of them up to 2 mo after termination of therapy. In 5 cases there was a great improvement of psoriatic lesions and in 6 remaining cases only a slight improvement was found. The remission of psoriasis was correlated with extremely high polymorphonuclear leukocyte depletion through the peritoneal cavity in a short time. Neutral serine proteinases were extracted from polymorphonuclear leukocytes and quantitated. The quantity of enzymes in the cells recovered from peritoneal dialysates was found to decrease with duration of treatment, and it was 2-5 times lower than amounts of neutral proteinases extracted from peripheral blood polymorphonuclear leukocytes of psoriatics and normals. The enzyme content per polymorphonuclear leukocyte of patients with active psoriasis was significantly higher (2-fold) than that in inactive psoriasis and in normal controls. Proteinase activity was also found in the sera of psoriatics and normals, as well as in the peritoneal dialysates. However, this activity appeared to be about 30-50 times lower than serum inhibitory activity against neutral proteinases. The concentration of neutral proteinase inhibitors in 5 of 17 sera of patients with psoriasis was significantly lower than that in normal sera. These data indicate that the depletion of activated PMNL with increased amounts of neutral proteinases may account for the beneficial effect of peritoneal dialysis in the clearing of psoriatic lesions.

The DNA polymerase beta reaction with ultraviolet-irradiated DNA incised by correndonuclease.

Covalently closed circular Col E1 DNA was ultraviolet-irradiated with a dose of 60 J/m2, thus introducing about 3.2 pyrimidine dimers per DNA molecule. Treatment of irradiated Col E1 DNA with Micrococcus luteus correndonuclease resulted, in the vicinity of pyrimidine dimers, in an average of 3.3 incisions per DNA molecule, and converted DNA to the open circular form. Incised Col E1 DNA stimulated no reaction with calf thymus DNA polymerase alpha but was recognized as a template by DNA polymerase beta. The latter enzyme incorporated about 1.6 molecules of dTMP (corresponding to 6 molecules od dNMP) per one correndonuclease incision. The length of the DNA polymerase beta product was comparable to the anticipated length of the DNA region within which the hydrogen bonds were disrupted owing to dimer formation. The enzyme required Mg(2)=nd four dNTPs for reaction and was resistant to N-ethylmaleimide or p-mercuribenzoate. The average numbers of deoxynucleotides incorporated per one DNAase I incision or per one nonspecific break, measured in control samples, were equal, amounting to 0.3 dTMP molecule. This value corresponded to 1.2 dNMP molecule; in our opinion, this reflects contaminating nuclease activity of the system used. The present results testify to the ability of DNA polymerase beta to repair synthesis by the "patch and cut' mechanism.

Immunological phenomena induced by UV rays.

Antiserum against UV-irradiated DNA was prepared and used as a specific reagent in an indirect immunofluorescence (IF) technique for the detection of photochemically damaged DNA in the epidermis of hairless mice. Fluorescence of cell nuclei was found in sections of dorsal epidermis of mice immediately after the irradiation. It persisted for 24 hours. The IF reaction became negative after 48 hours, irrespective of the duration of UV exposure to which the mice were subjected. This may indicate: either (a) the occurrence of DNA repair processes, or (b) the relation of DNA repair to the life cycle of the epidermal cell. Mice exposed to UV radiation similar to the erythema spectrum of sunlight do not show any changes in the cellular DNA, even after a dose of 40 MED.