Modulation of ultraviolet light-induced oxidative stress in mice skin related to dietary vitamin A and selenium intake.

Weanling female SKH1 hairless mice were randomly divided into 4 groups. Each group was fed a particular regimen for 20 weeks: 1) normal basal diet with 5 IU vitamin A/g and 0.45 microgram selenium/g, 2) vitamin A deficient, 3) selenium deficient, and 4) vitamin A plus selenium deficient. Three hours before being sacrificed, half of the animals were subjected to UV A + B irradiation (3 J/cm2). Superoxide dismutases (SOD), catalase, Se glutathione peroxidase activities were determined in dorsal skin homogenates, as well as the concentrations of GSH and of thiobarbituric acid-reactive substances (TBARS), the latter being an index of lipid peroxidation. Ultraviolet light altered the antioxidant defense of mouse skin tissue: GSH level, catalase and Se glutathione peroxidase activities were lowered and SOD was unequally enhanced according to the nutritional status. Vitamin A and Se deficiencies did not perceptibly aggravate the UV-induced oxidative stress, although the former enhanced the decline of catalase expression induced by the irradiation. Probably through an adaptive mechanism, both dietary deficiencies increased the skin reserve of antioxidant GSH and thus appear to modulate the effects caused by reactive oxygen species.

Vitamin A status and metabolism of cutaneous polyamines in the hairless mouse after UV irradiation: action of beta-carotene and astaxanthin.

Solar radiations (UV A and B) can cause epidermis photoaging and skin cancers. These frequently irreversible effects result from the in situ generation of free radicals. However, it has been noted that nutritional factors can modulate photochemical damage, in particular the common carotenoids present in food, which can be considered as potential prophylactic agents against carcinogenesis. We investigated the effect of UV A and B radiations on the skin of the SKH1 hairless mouse fed a diet either lacking in vitamin A or supplemented with retinol, beta-carotene or astaxanthin. The latter is an oxygenated carotenoid (like canthaxanthin) without provitamin A activity and with strong singlet oxygen quenching ability. After analysing of vitamin status of each group (plasma retinol concentrations and hepatic reserves), we searched for UV-induced modifications of polyamine metabolism by measuring epidermal ornithine decarboxylase (ODC) activity and free polyamines concentration (putrescine, spermidine and spermine). In the basal state without irradiation, differences in ODC activity between groups were nonsignificant; but after UV stimulation, ODC increased markedly in the skin of vitamin A-deficient animals, much more than in other groups. Curiously, the addition of astaxanthin or beta-carotene to the regimen containing retinol reduced the protective effect of retinol alone. Regarding polyamines after irradiation, putrescine was significantly increased in the skin of deficient animals, in parallel with ODC activity. However, astaxanthin had a stronger inhibitory effect on putrescine accumulation than retinol, and decreased spermidine and spermine concentrations: this suggests a specific action on transglutaminases.

Malondialdehyde dosimetry in laser-irradiated tissues sensitized by hematoporphyrin derivative.

Photodynamic therapy (PDT) is based on the selective retention of a photosensitizer (hematoporphyrin derivative [HPD]) by tumor tissue and the subsequent irradiation of this tissue with light. Unsaturated phospholipids are important targets of membrane photodamage, and malondialdehyde (MDA), an ultimate marker of lipid peroxidation, can easily be measured by the fluorometric thiobarbituric acid (TBA) assay. To determine whether MDA content represents a reference for PDT intensity, tissue content in 7-week-old male nude mice was studied on biopsy samples after PDT (5 mg/kg HPD injected intravenously 24 hr before irradiation at 632 nm) with or without intraperitoneal injection of WR-2721 40 min before treatment. The FeCl3 method requiring only 15 min of incubation in a 95 degrees C waterbath proved most effective compared with the method involving phosphotungstic acid or the MDA-TBA determination using a commercially available kit. Whatever the method used, the best results were found using a 60-min interval between treatment and freezing. MDA concentration in HPD-PDT-treated samples was significantly higher than in HPD-tested controls (P < .01) and increased at laser irradiation doses ranging from 0 to 50 J/cm2. Administration of WR-2721 intraperitoneally significantly reduced MDA concentration (from 70% to 38%). A maximal effect was obtained with 100 mg/kg for brain (-70%) and 200 mg/kg for muscle (-47%). Higher doses produced no additional changes. The MDA assay is a simple tool for indirect evaluation of PDT, and WR-2721 can decrease MDA content in normal tissue, suggesting the possibility of good protection for normal tissue during PDT.

Modulation of colonic cancer cell adhesiveness by haematoporphyrin derivative photodynamic therapy.

Haematoporphyrin derivative photodynamic therapy (HPD-PDT) induces damage of plasma membranes and other cellular targets. This damage could modify the adhesiveness of cancer cells, which is an important parameter in cancer metastasis. We studied the effect of HPD alone and HPD incubation followed by argon laser light on the adhesiveness of progressive (PROb) or regressive (REGb) cancer cells of the same colonic origin. Adhesiveness was studied on plastic or endothelial cell monolayers (ECMs). In the absence of treatment, both PROb and REGb cells adhered better on plastic than on ECs. HPD alone and HPD-PDT induced toxicity proportional to the HPD dose. HPD-PDT increased the adhesiveness rate of both cell lines on plastic and decreased adhesiveness to ECs. HPD-PDT of ECMs increased adhesiveness but only for HPD doses giving at least 50% cell death. HPD alone and HPD-PDT of culture media led to an insignificant decrease in the cell adhesiveness to ECMs. As cells which are more metastatic are also more adherent, a decreased adhesiveness to ECMs after HPD-PDT suggests that PDT is a safe treatment considering metastasis.

Effects of high glucose concentration on survival and respiration of human endothelial cells.

To determine whether or not endothelial cell survival was decreased after incubation with high glucose concentrations in culture media, we studied the influence of D-glucose or L-glucose (a non-metabolizable stereoisomer of D-glucose) on cell survival using the trypan-blue exclusion test. Simultaneously, the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide) assay was used to measure both the mitochondrial respiratory chain activity and cell viability. Respiratory chain activity per cell increased when D-glucose concentrations rose but at the same time trypan-blue excluded cells were decreased. Comparison with data in the literature showed that the MTT assay was not reliable for studies involving endothelial cell survival when glucose reduction was affected on these cells. It seems important to check MTT assay reliability carefully when it is used for drugs affecting glucose metabolism, or with other cell types.

Photosensitization of L1210 leukaemic cells by argon laser irradiation after incubation with haematoporphyrin derivative and rhodamine 123.

The selectivity and efficacy of photodynamic therapy (PDT) may be improved by the combined use of photosensitizers in a similar manner to the combined use of drugs in cancer chemotherapy. Two photosensitizers (haematoporphyrin derivative (HPD) and rhodamine 123 (Rh-123) were analysed which can be irradiated at the same wavelength (514 nm), are preferentially taken up by tumour tissue and are not specific for the same target (membrane for HPD, mitochondria for Rh-123). The analysis of the phototoxic effects in surviving fractions showed a dependence on dose for both products and a dependence on incubation time for HPD but not Rh-123. The lethal dose for 50% cell death (LD50) for HPD increased from 25 to 56 J cm-2 when the HPD dose was reduced from 2.5 to 1 micrograms ml-1 for the same incubation time. When the incubation time was increased from 15 to 45 min, the surviving fraction decreased by 37% and 17% for doses of 1 and 2.5 micrograms ml-1 respectively. For low doses (0.5 and 1 microgram ml-1), the toxicity of the two photosensitizers added simultaneously was weaker than for Rh-123 alone, whereas for high doses (2.5 micrograms ml-1) the surviving fraction was less than that obtained with Rh-123 alone. These results were compared with the light energy absorbed, the quantum yield of singlet oxygen and Rh-123 uptake as determined by flow cytometry analysis.

Photoprotective effect of vitamins A and E on polyamine and oxygenated free radical metabolism in hairless mouse epidermis.

The purpose of this study was to confirm the photoprotective effect on skin of vitamins A and E, due to inhibition of polyamine synthesis and production of free radicals. These variables were measured in the lumbar epidermis of the female hairless mouse subjected to UVA + B irradiation. Polyamines were assayed in epidermal homogenate by HPLC, and production of oxygenated free radicals was determined by spectrofluorometric assay of malonyl dialdehyde. It was determined that butyl-hydroxy-toluene and vitamin E inhibited production of free radicals (56% and 60%, respectively) and caused a significant reduction in polyamine biosynthesis (P less than 0.01), whereas the inhibitory effect of malonyl dialdehyde induced by vitamin A (30%) had no associated effect on polyamine metabolism.

Comparison of the effects of pentobarbital and chloralose on the cardiac rhythms of the dog in chronic atrio-ventricular heart block.

In the dog in chronic atrio-ventricular dissociation, pentobarbital at a dose of 25 mg/kg causes atrial tachycardia, but does not change the ventricular frequency. The vagolytic effect of pentobarbital explains this phenomenon. The pronounced vagal tonus seen in the Keith-Flack node, is not seen in the ventricle. Chloralose at a dose of 80 mg/kg i.v. does not alter atrial frequency but depresses ventricular frequency, in a significant way, for 40 min.