A. Moysan

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.

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.

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

Summary UVA (320–400nm) 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 20 J/cm2). Overnight incubation of epidermal cells with vitamin E (400 μmol/1) 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 cellpresenting 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.

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.

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 cytotocixity with a higher antioxidant status.

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.

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.

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.

DETECTION OF PYRIMIDINE DIMERS AND MONOADDUCTSINDUCED BY 7‐METHYLPYRIDO(3,4‐c) PSORALEN AND UVA IN CHINESE HAMSTER V79 CELLS BY ENZYMATIC CLEAVAGE AND HIGH‐PRESSURE LIQUID CHROMATOGRAPHY

Abstract 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.