Divergence between the high rate of p53 mutations in skin carcinomas and the low prevalence of anti-p53 antibodies.

Circulating anti-p53 antibodies have been described and used as tumoural markers in patients with various cancers and strongly correlate with the p53 mutated status of the tumours. No study has yet looked at the prevalence of such antibodies in skin carcinoma patients although these tumours have been shown to be frequently p53 mutated. Most skin carcinoma can be diagnosed by examination or biopsy, but aggressive, recurrent and/or non-surgical cases' follow up would be helped by a biological marker of residual disease. We performed a prospective study looking at the prevalence of anti-p53 antibodies using an ELISA technique in a series of 105 skin carcinoma patients in comparison with a sex- and age-matched control skin carcinoma-free group (n = 130). Additionally, p53 accumulation was studied by immunohistochemistry to confirm p53 protein altered expression in a sample of tumours. Anti-p53 antibodies were detected in 2.9% of the cases, with a higher prevalence in patients suffering from the more aggressive squamous cell type (SCC) of skin carcinoma (8%) than for the more common and slowly growing basal cell carcinoma type or BCC (1.5%). p53 protein stabilization could be confirmed in 80% of tumours studied by IHC. This low level of anti-p53 antibody detection contrasts with the high rate of p53 mutations reported in these tumours. This observation shows that the anti-p53 humoral response is a complex and tissue-specific mechanism.

Analyses of cutaneous fluoroquinolones photoreactivity using the integrated model for the differentiation of skin reactions.

Currently available test models for the differentiation of photoallergic and photoirritant reactions are extremely time consuming and the protocols are very heterogeneous. In vitro tests are of proven value in predicting irritant or toxic effects, but these tests fail to predict chemical-induced allergic side effects. We developed test systems for this endpoint which is not easily detected by existing assays. In a previous publication we were able to discriminate between a contact sensitizer and a skin irritant with a combination of primary ear swelling analysis and cell counting of the ear-draining lymph nodes [Toxicol. Appl. Pharm. 153 (1998) 83; Arch. Toxicol. 73 (2000) 501]. This combination of tests was called the Integrated Model for the Differentiation of chemical-induced allergic and irritant Skin reactions (IMDS). In addition, it had been shown before that inclusion of UV irradiation in the local lymph node assay enables discrimination of photoallergic from photoirritant reactions after dermal application [Photodermatol. Photoimmunol. Photomed. 10 (1994) 57]. Because of the fact that fluoroquinolones are known to induce photoreactions after oral but not dermal treatment, the aim of the present study was to apply the IMDS for the fast and reliable differentiation of photoreactions due to fluoroquinolones after oral treatment. Enoxacin, lomefloxacin, ofloxacin, sparfloxacin and BAY y 3118 were tested in this system. We found a good correlation between the results of UV light-irradiated IMDS and a guinea pig model with the quinolones as far as photoirritancy was concerned. This holds true also for the photoallergic standard olaquindox and the photoirritant standard 8-methoxypsoralen. However, in contrast to the guinea pig assays the IMDS is fast and extremely predictive for the risk of both photosensitization and photoirritancy depending on the route of exposure. Thus, the UV light-irradiated IMDS turned out to be a good tool for the preclinical risk assessment procedure in terms of discriminating photoreactions. In addition, flow cytometric analyses were used to underline the fact that antigen-independent activation occurred after the induction of photoirritant reactions.

UVA-induced immune suppression in human skin: protective effect of vitamin E in human epidermal cells in vitro.

UVA (320-400 nm) radiation damage to membranes, proteins, DNA and other cellular targets is predominantly related to oxidative processes. In the present study, we demonstrated that cutaneous UVA-induced immunosuppression can be related, at least in part, to the appearance of these oxidative processes. The UVA-induced oxidative processes in freshly isolated epidermal cells were monitored by measuring the thiobarbituric acid reactive substances (TBARS) as an index of peroxidation. The in vitro immunosuppressive effects of UVA were demonstrated by measuring the allogeneic lymphocyte proliferation induced by epidermal cells or purified Langerhans cells in the mixed epidermal cell-lymphocyte reaction (MECLR). In addition, the effects of a potent antioxidant (vitamin E) on these two UVA-induced processes were analysed. Our results showed that the antigen-presenting function of Langerhans cells measured in the MECLR is dose-dependently decreased by UVA radiation (up to 20J/cm2). Overnight incubation of epidermal cells with vitamin E (400 mumol/l) before irradiation partially protected epidermal cells from the immunosuppressive effects of UVA radiation, and decreased TBARS release into the supernatant (a decrease of 35% compared with a control without vitamin E). Our results suggest that UVA radiation may alter cell-presenting antigen function partly via the generation of reactive oxygen species which trigger peroxidative processes, and these data contribute to the understanding of the role of oxidative mechanisms in immune suppression induced by UVA radiation. Our in vitro model can be used to quantify UV-mediated epidermal cell damage and the degree of immune photoprotection provided by various agents.

Action spectrum for UV-induced lipid peroxidation in cultured human skin fibroblasts.

Lipid peroxidation was measured by release of thiobarbituric acid-reactive substances (TBARS) into the supernatant of cultured human skin fibroblasts. This process is triggered by ultraviolet A (UVA) and ultraviolet B (UVB) radiations. For UVA irradiances and irradiation times up to 40 W.m-2 and 90 min, respectively, the peroxidation response is linear and obeys the reciprocity law. Corresponding values for UVB are 12 W.m-2 and 30 min, respectively. The action spectrum of the peroxidation process shows a continuously increasing response from about 425 to 275 nm. Whereas the UVB to UVA effectiveness ratio lies in the range of 10(3) to 10(4) for most in vitro or in vivo UV-induced responses, the ratio is only 10 to 100 for the peroxidation process. Given the solar spectral distribution, solar UVA radiation is by far the most effective in triggering the peroxidation response.

Effects of selenium on UVA-induced lipid peroxidation in cultured human skin fibroblasts.

The effect of selenium on the lethal action of ultraviolet radiations and on the lipid peroxidation induced by exposures to ultraviolet A (320-400 nm; 360 kJ.m-2) and ultraviolet B (290-320 nm; 2 kJ.m-2) have been measured in cultured human skin fibroblasts. The experiments have been performed with either pure selenium or a spring water containing selenium and other trace elements (zinc and strontium). For cells cultured in a standard medium containing 10% fetal calf serum, no effect of selenium or spring water addition to the culture medium was observed on the lethality or on the peroxidative process induced by ultraviolet A and B radiations. Concurrently, there was no detectable increase of the seleno-dependent glutathione peroxidase activity. For cells previously depleted in selenium by a culture in a medium containing only 2% serum, a protective effect of selenium can be detected. Depending on the fibroblast donor, we observed (1) a protective effect on lethality of dividing fibroblasts induced by ultraviolet A radiations, (2) a protective effect on lipid peroxidation induced by ultraviolet A radiations on dividing or quiescent fibroblasts and (3) an increase in glutathione peroxidase activity in fibroblasts.

Effects of ultraviolet A and antioxidant defense in cultured fibroblasts and keratinocytes.

Lipid peroxidation, measured by the thiobarbituric acid-reactive substances assay, was evaluated for cultured human skin fibroblasts and keratinocytes exposed to ultraviolet A radiation (320-400 nm, UVA). Peroxidation increases with increasing UVA doses and is much lower for keratinocytes than for fibroblasts. Immediate UVA-induced cytotoxicity, monitored by the trypan blue exclusion assay, is also lower for keratinocytes. Thus, cultured human skin keratinocytes are less sensitive than fibroblasts to the immediate deleterious effects of UVA with respect to membrane damage and lipid peroxidation. As a first attempt to understand this lower sensitivity of keratinocytes, basal levels of antioxidant defenses including total glutathione, superoxide dismutase, glutathione peroxidase and catalase were evaluated in both keratinocytes and fibroblasts from the same donors. We failed to correlate this lower susceptibility of keratinocyte to UVA-induced lipid peroxidation and cytotoxicity with a higher antioxidant status.

Detection of pyrimidine dimers and monoadducts induced by 7-methylpyrido(3,4-c) psoralen and UVA in Chinese hamster V79 cells by enzymatic cleavage and high-pressure liquid chromatography.

The photochemotherapeutically active psoralen derivative 7-methylpyrido(3,4-c) psoralen (MePyPs) has been recently shown to be able to photoinduce monoadducts of the C4-cycloaddition type as well as pyrimidine dimers in DNA in vitro. In the present study, we report on the induction of these two types of photolesions in mammalian cells in culture. The MePyPs photocycloadducts were quantified in V79 Chinese hamster cells after treatment with MePyPs plus UVA following enzymatic hydrolysis of the DNA by DNase I, S1 nuclease and acidic phosphatase treatments. Concomitantly induced pyrimidine dimers were determined by two methods, high-pressure liquid chromatography and alkaline gel electrophoresis after dimer-specific endonucleolytic cleavage. The results show that, in Chinese hamster cells treated with MePyPs plus UVA, the yield of pyrimidine dimers is approximately 5-10% that of MePyPs-DNA photocycloadducts. Because psoralen monoadditions to DNA alone are generally not considered as being very phototoxic, a synergistic interaction of monoadditions with pyrimidine dimers may be expected to occur in order to explain the high photobiological effectiveness of this psoralen derivative.

Membrane damage induced in cultured human skin fibroblasts by UVA irradiation.

Irradiation of cultured human skin fibroblasts with ultraviolet light from 320 to 400 nm (UVA) leads to a decrease in the membrane fluidity exemplified by an enhanced fluorescence anisotropy of the lipophilic fluorescent probe 1-[4-trimethylamino)-phenyl]-6-phenylhexa-1,3,5-triene. This UVA-induced decrease in fluidity is associated with lactate dehydrogenase leakage in the supernatant. Vitamin E, an inhibitor of lipid peroxidation, exerts a protective effect on both phenomena. Therefore, this UVA-induced damage in membrane properties may be related to lipid peroxidation processes. Moreover, exponentially growing cells are more sensitive to these UVA-induced alterations than confluent cells.

Ultraviolet A-induced lipid peroxidation and antioxidant defense systems in cultured human skin fibroblasts.

Cultured human skin fibroblasts from healthy donors were irradiated with 180 kJ.m-2 ultraviolet (UV) A (320-400 nm) and assayed for thiobarbituric acid-reactive substances (TBARS), taken as an indicator of lipid peroxidation. Antioxidant defenses, including total glutathione (GSH) levels, superoxide dismutase (SOD), glutathione peroxidase (GSHPx), and catalase (Cat) activities were simultaneously assayed before and after irradiation. For the various donors, with different activities of these antioxidant systems before irradiation, TBARS correlated positively with SOD activity and negatively with Cat activity, whereas no correlation with GSH level or GSHPx activity was found. These data support the view that O2- is generated by UVA irradiation. They also suggest that H2O2, arising from O2- dismutation by SOD is not completely removed by Cat. Thus, the sensitivity of human fibroblasts to UVA-induced lipid peroxidation depends on a balance between SOD and Cat activities. After UVA irradiation, Cat activity was strongly inhibited, whereas GSH level was slightly decreased. By contrast, GSHPx and SOD activity remained unchanged after UVA irradiation.

Repair of the two diastereoisomer photoadducts formed between 7-methylpyrido(3,4-c)psoralen (MePyPs) and thymidine in yeast cells: a chemical approach.

When analysing the repair of psoralen plus UVA-induced photoadducts in DNA, it must be realized that, in most cases, different isomers are formed. The monofunctional psoralen derivative 7-methylpyrido(3,4-c)psoralen (MePyPs) is known for its high antiproliferative activity at the cellular level and interesting photochemotherapeutic properties. To understand its photobiological efficiency in more detail, the induction of specific photoadducts in DNA and their repair were analysed in a eukaryotic cell system, the yeast Saccharomyces cerevisiae. After photoaddition of MePyPs, two main diastereoisomers were characterized after enzymatic hydrolysis of the DNA and analysis by high performance liquid chromatography. One diastereoisomer was more effectively repaired in yeast than the other during post-treatment incubation, suggesting that the two diastereoisomers may be recognized differently by cellular enzymatic repair systems.

Furan-side pyridopsoralens monoadducts to the thymine moiety of DNA.

Isolation of the main cycloadducts formed in DNA by the UV-A (ultraviolet light of class A) photoreaction of 7-methyl-pyrido[3,4-c]psoralen (MePyPs) and 7-methyl-pyrido[4,3- c]psoralen (2N-MePyPs) was achieved by HPLC separation subsequent to enzymatic hydrolysis of DNA. The photoadducts have been quantified and their chemical structure assigned on the basis of spectroscopic measurements, including absorption and fluorescence spectroscopy, and circular dichroism as well as mass spectrometry analysis. They all present characteristics which are consistent with furan-side monoadducts resulting from a C4-cycloaddition of the psoralens to thymidine. The two major MePyPs-thymidine monoadducts formed in DNA exhibit a diastereoisomeric relationship and are likely to have a cis-syn stereochemistry.

Evaluation of phototoxic and photogenotoxic risk associated with the use of photosensitizers in suntan preparations: application to tanning preparations containing bergamot oil.

Bases for the elaboration of a standardized protocol are proposed for studying phototoxic effects of skin tanning preparations containing photosensitizing agents. The experimental procedure includes in vivo phototoxicity tests, evaluation of the photogenotoxic risk and determination of the photosensitizer concentration in plasma after topical application. This procedure was carried out with tanning preparations containing a well-known photosensitizer, 5-methoxypsoralen, as a component of bergamot oil. The whole study has been performed using topical application of the commercial suntan product, i.e. containing the sunscreens and all other components. Whereas the exposure to solar simulated radiation never triggered any phototoxic response, a photosensitizing effect was observed for skin type I volunteers exposed to high doses of ultraviolet A. The transepidermal penetration resulted in a 5-methoxypsoralen concentration of 1-4 ng/ml in the suction blister fluid. The photogenotoxicity of this suction blister fluid containing 5-methoxypsoralen and also other ingredients of the tanning preparation was assayed on yeast cells and was found to be rather low. 5-Methoxypsoralen was also detected in plasma after repeated applications but at low concentrations (about 1 ng/ml) which do not present a potential risk for systemic ocular effects.

[Ultraviolet A radiation and the skin. Implications of activated forms of oxygen. Current trends and newest results]. / Ultraviolet A et peau. Implications d'espèces activées de l'oxygène. Tendances actuelles et résultats récents.

Involvement of activated oxygen species in the responses of skin to solar ultraviolet radiations, especially ultraviolet A radiations, is being addressed by an increasing number of studies. The aim of this review is to outline the concept of "photooxidative stress". The various activated oxygen species, mechanisms involved in their formation, potential cellular targets, and cell defense mechanisms are discussed. Recently published findings are briefly described to illustrate this fast developing line of research.

Formation of cyclobutane thymine dimers photosensitized by pyridopsoralens: quantitative and qualitative distribution within DNA.

As after irradiation with 254-nm UV light, exposure of thymidine and three isomeric pyridopsoralen derivatives to UVA radiation, in the dry state, leads to the formation of the six diastereomers of cyclobutadithymidine as the predominant reaction. This unexpected photosensitized reaction, which also gives rise to both 5R* and 5S* diastereomers of 5,6-dihydro-5-(alpha-thymidylyl)thymidine (or "spore" photoproduct), is selective since [2 + 2] dimerization of 2'-deoxycytidine was not detected under the same experimental conditions. The cis-syn isomer of cyclobutadithymine was also found to be produced within isolated DNA following UVA irradiation in aqueous solutions containing 7-methylpyrido[3,4-c]psoralen. Quantitatively, this photoproduct represents about one-fifth of the overall yield of the furan-side pyridopsoralen [2 + 2] photocycloadducts to thymine. DNA sequencing methodology was used to demonstrate that pyridopsoralen-photosensitized DNA is a substrate for T4 endonuclease V and Escherichia coli photoreactivating enzyme, two enzymes acting specifically on cyclobutane pyrimidine dimers. Furthermore, the dimerization reaction of thymine is sequence dependent, with a different specificity from that mediated by far-UV irradiation as inferred from gel sequencing experiments. Interestingly, adjacent thymine residues are excellent targets for 7-methylpyrido[3,4-c]psoralen-mediated formation of cyclobutadithymine in TTTTA and TTAAT sites, which are also the strongest sites for photoaddition. The formation of cyclobutane thymine dimers concomitant to that of thymine-furocoumarin photoadducts and their eventual implication in the photobiological effects of the pyridopsoralens are discussed.

UVA-induced lipid peroxidation in cultured human fibroblasts.

The UVA irradiation of cultured human fibroblasts leads to the formation and to the release of thiobarbituric acid-reactive substances in the supernatant. The major thiobarbituric acid-reactive substance is identified by fluorescence spectroscopy and HPLC, as malondialdehyde or malondialdehyde-forming substances under the thiobarbituric acid assay conditions. Malondialdehyde formation strongly suggests a UVA-induced lipid peroxidation. Lipid peroxidation is also supported by the inhibitory effect of D,L-alpha-tocopherol, the well-known chain breaking antioxidant, by the additional malondialdehyde formation in the dark after the photooxidative stress and by membrane damage revealed by lactate dehydrogenase leakage.

Formation of cyclobutane thymine dimers photosensitized by pyridopsoralens: a triplet-triplet energy transfer mechanism.

The 365 nm irradiation of thymine thin films in the presence of pyridopsoralens is shown to induce the formation of cyclobutane thymine dimers, in contrast to other compounds such as 8- and 5-methoxypsoralen. In order to elucidate the mechanism of such a photosensitized reaction, we have determined the energy of the lowest triplet state (T1) of these compounds, using phosphorescence spectroscopy and CNDO/S quantum chemistry calculations. The T1 energy values were found to be significantly higher for pyridopsoralens--up to 0.3 eV--than for 8- and 5-methoxypsoralen (approximately 2.8 eV), which are not able to photoinduce cyclobutane thymine dimers. The determination of the relative efficiency of cyclobutane thymine dimer formation was performed using chromatographic analysis. A good correlation was found between the energy of the T1 state of the psoralen derivatives and the related cyclobutane thymine dimer formation. Moreover, the photosensitized cyclobutane thymine dimer formation appeared to be temperature-dependent. Our results are consistent with a mechanism involving a triplet energy transfer from the pyridopsoralen to thymine.

Chemical structure of 3-carbethoxypsoralen-DNA photoadducts.

The enzymatic digest from salmon sperm DNA photochemically modified by the monofunctional 3-carbethoxypsoralen was analyzed by high-performance liquid chromatography. The modified nucleosides extracted from DNA were compared with model compounds obtained from irradiation in the dry state of mixtures of 3-carbethoxypsoralen with 2'-deoxyribonucleosides whose chemical structures had previously been characterized. The main photoadducts formed in DNA are two cis-syn diastereoisomers formed via a C4-cycloaddition reaction involving the 4', 5' double bond of 3-carbethoxypsoralen and the 5,6 double bond of 2'-deoxythymidine. Among them, the most polar one accounts for 72%. Under the same conditions, photoadducts formed between 3-carbethoxypsoralen and 2'deoxycytidine account for less than 1%.