The impact of skin colour on human photobiological responses

Terrestrial solar ultraviolet radiation (UVR) exerts both beneficial and adverse effects on human skin. Epidemiological studies show a lower incidence of skin cancer in people with pigmented skins compared to fair skins. This is attributed to photoprotection by epidermal melanin, as is the poorer vitamin D status of those with darker skins. We summarize a wide range of photobiological responses across different skin colours including DNA damage and immunosuppression. Some studies show the generally modest photoprotective properties of melanin, but others show little or no effect. DNA photodamage initiates non‐melanoma skin cancer and is reduced by a factor of about 3 in pigmented skin compared with white skin. This suggests that if such a modest reduction in DNA damage can result in the significantly lower skin cancer incidence in black skin, the use of sunscreen protection might be extremely beneficial for susceptible population. Many contradictory results may be explained by protocol differences, including differences in UVR spectra and exposure protocols. We recommend that skin type comparisons be done with solar‐simulated radiation and standard erythema doses or physical doses (J/m²) rather than those based solely on clinical endpoints such as minimal erythema dose (MED).     

The potential value of the neutral comet assay and the expression of genes associated with DNA damage in assessing the radiosensitivity of tumor cells

The assessment of tumor radiosensitivity would be particularly useful in optimizing the radiation dose during radiotherapy. Therefore, the degree of correlation between radiation-induced DNA damage, as measured by the alkaline and the neutral comet assays, and the clonogenic survival of different human tumor cells was studied. Further, tumor radiosensitivity was compared with the expression of genes associated with the cellular response to radiation damage. Five different human tumor cell lines were chosen and the radiosensitivity of these cells was established by clonogenic assay. Alkaline and neutral comet assays were performed in γ-irradiated cells (2-8Gy; either acute or fractionated). Quantitative PCR was performed to evaluate the expression of DNA damage response genes in control and irradiated cells. The relative radiosensitivity of the cell lines assessed by the extent of DNA damage (neutral comet assay) immediately after irradiation (4Gy or 6Gy) was in agreement with radiosensitivity pattern obtained by the clonogenic assay. The survival fraction of irradiated cells showed a better correlation with the magnitude of DNA damage measured by the neutral comet assay (r=-0.9; P<0.05; 6Gy) than evaluated by alkaline comet assay (r=-0.73; P<0.05; 6Gy). Further, a significant correlation between the clonogenic survival and DNA damage was observed in cells exposed to fractionated doses of radiation. Of 15 genes investigated in the gene expression study, HSP70, KU80 and RAD51 all showed significant positive correlations (r=0.9; P<0.05) with tumor radiosensitivity. Our study clearly demonstrated that the neutral comet assay was better than alkaline comet assay for assessment of radiosensitivities of tumor cells after acute or fractionated doses of irradiation.     

Evaluation of DNA damage in a population of bats (Chiroptera) residing in an abandoned monazite mine

Ionising radiation has the ability to induce DNA damage. While the effects of high doses of radiation of short duration have been well documented, the biological effects of long-term exposure to low doses are poorly understood. This study evaluated the clastogenic effects of low dose ionising radiation on a population of bats (Chiroptera) residing in an abandoned monazite mine. Bats were sampled from two chambers in the mine, where external radiation levels measured around 20 microSv/h (low dose) and 100 microSv/h (higher dose), respectively. A control group of bats was sampled from a cave with no detectable radiation above normal background levels. The micronucleus assay was used to evaluate residual radiation damage in binucleated lymphocytes and showed that the micronucleus frequency per 500 binucleated lymphocytes was increased in the lower radiation-exposed group (17.7) and the higher radiation-exposed group (27.1) compared to the control group (5.3). This study also showed that bats exposed to radiation presented with an increased number of micronuclei per one thousand reticulocytes (2.88 and 10.75 in the lower and high radiation-exposed groups respectively) when compared to the control group (1.7). The single-cell gel electrophoresis (comet) assay was used as a means of evaluating clastogenecity of exposure to radiation at the level of individual cells. Bats exposed to radiation demonstrated increased DNA damage as shown by the length of the comet tails and showed an increase in cumulative damage. The results of the micronucleus and the comet assays indicated not only a statistically significant difference between test and control groups (P<0.001), but also a dose-dependent increase in DNA damage (P<0.001). These assays may thus be useful in evaluating the potential clastogenecity of exposure to continuous low doses of ionising radiation.     

Comet assay to sense neutron 'fingerprint'

The suitability of comet assay to identify DNA damage induced by neutrons of varying energy was tested. For this purpose, monoenergetic neutrons from Hiroshima University Radiobiological Research Accelerator (HIRRAC) were used to induce DNA damage in irradiated human peripheral blood lymphocytes. The level of damage was computed as tail moment for different doses (0.125-1 Gy) and compared with the effects resulting from irradiation with (60)Co gamma. The neutron-irradiated cells exhibited longer comet tails consisting of tiny pieces of broken DNA in contrast to the streaking tails generated by (60)Co gamma. The peak biological effectiveness occurred at 0.37 and 0.57 MeV; a further increase or decrease in neutron energy led to a reduced RBE value. The RBE values, as measured by the comet assay, were 6.3, 5.4, 4.7, 4.3, 2.6, and 1.7 for 0.37, 0.57, 0.79, 0.186, 1, and 2.3 MeV neutrons. The lower RBE value obtained by the comet assay when compared to that for other biological end points is discussed. This study reports the usefulness of the alkaline comet assay for identifying DNA damage induced by neutrons of the same radiation weighting factor. The comet assay is a potential tool for use in neutron therapy, as well as a method for the rapid screening of samples from individuals accidentally exposed to radiation.     

DNA damage evaluated by the comet assay in lymphocytes of children with 137Cs internal contamination caused by the Chernobyl accident

The comet assay is one of the most versatile and popular tools for evaluating DNA damage. Its sensitivity to low dose radiation has been tested in vitro, but there are limited data showing its application and sensitivity in chronic exposure situations. The influence of the internal contamination caused by the Chernobyl accident on the level of DNA damage was evaluated by the comet assay on lymphocytes of 56 Ukrainian children. The study was performed during 2003 on children with demonstrable 137Cs internal contamination caused by food consumption. The children were selected for the study immediately after a 137Cs whole body counter measurement of internal contamination. The minimal detectable amount of 137Cs was 75 Bq. The control group included 29 children without detectable internal contamination, while in the exposed group 27 children with measured activity between 80 and 4037 Bq and committed effective dose between 54 and 3155 microSv were included. Blood samples were taken by a finger prick. The alkaline version of the comet assay was used, in combination with silver stained comets and arbitrary units (AU), for comet measurement. Factors such as disease, medical treatment, surface contamination of children's living location, etc., were considered in the study. Non-significant differences (p > 0.05) in DNA damage in control (9.0 +/- 5.7 AU) versus exposed (8.5 +/- 4.8 AU) groups were found. These results suggest that low doses of 137Cs internal contamination are not able to produce detectable DNA damage under the conditions used for the comet assay in this study. Further studies considering effects of high exposure should be performed on chronically exposed people using this assay.     

Zinc protects against ultraviolet A1-induced DNA damage and apoptosis in cultured human fibroblasts

Ultraviolet Al (UVA1) radiation generates reactive oxygen species and the oxidative stress is known as a mediator of DNA damage and of apoptosis. We exposed cultured human cutaneous fibroblasts to UVA1 radiation (wavelengths in the 340–450-nm range with emission peak at 365 nm) and, using the alkaline unwinding method, we showed an immediate significant increase of DNA strand breaks in exposed cells. Apoptosis was determined by detecting cytoplasmic nucleosomes (enzyme-linked immunosorbent assay method) at different time points in fibroblasts exposed to different irradiation doses. In our conditions, UVA1 radiation induced an early (8 h) and a delayed (18 h) apoptosis. Delayed apoptosis increased in a UVA dosedependent manner. Zinc is an important metal for DNA protection and has been shown to have inhibitory effects on apoptosis. The addition of zinc (6.5 mg/L) as zinc chloride to the culture medium significantly decreased immediate DNA strand breaks in human skin fibroblasts. Moreover, zinc chloride significantly decreased UVA1-induced early and delayed apoptosis. Thus, these data show for the first time in normal cutaneous cultured cells that UVA1 radiation induces apoptosis. This apoptosis is biphasic and appears higher 18 h after the stress. Zinc supplementation can prevent both immediate DNA strand breakage and early and delayed apoptosis, suggesting that this metal could be of interest for skin cell protection against UVA1 irradiation.     

The Impact of UV Radiation on Paramecium Populations from Alpine Lakes

Paramecium populations from a clear and a glacier‐fed turbid alpine lake were exposed to solar simulated ultraviolet (UVR) and photosynthetically active radiation (PAR) at 8 and 15 °C. The ciliates were tested for DNA damage (comet assay), behavioral changes, and mortality after UVR + PAR exposure. High DNA damage levels (~58% tail DNA) and abnormal swimming behavior were observed, although no significant changes in cell numbers were found irrespective of the lake origin (clear, turbid), and temperatures. We conclude that environmental stressors such as UVR and their effects may influence the adaptation of ciliates living in alpine lakes.     

Effect of UVA fluence rate on indicators of oxidative stress in human dermal fibroblasts

During the course of a day human skin is exposed to solar UV radiation that fluctuates in fluence rate within the UVA (290-315 nm) and UVB (315-400 nm) spectrum. Variables affecting the fluence rate reaching skin cells include differences in UVA and UVB penetrating ability, presence or absence of sunscreens, atmospheric conditions, and season and geographical location where the exposure occurs. Our study determined the effect of UVA fluence rate in solar-simulated (SSR) and tanning-bed radiation (TBR) on four indicators of oxidative stress---protein oxidation, glutathione, heme oxygenase-1, and reactive oxygen species--in human dermal fibroblasts after receiving equivalent UVA and UVB doses. Our results show that the higher UVA fluence rate in TBR increases the level of all four indicators of oxidative stress. In sequential exposures when cells are exposed first to SSR, the lower UVA fluence rate in SSR induces a protective response that protects against oxidative stress following a second exposure to a higher UVA fluence rate. Our studies underscore the important role of UVA fluence rate in determining how human skin cells respond to a given dose of radiation containing both UVA and UVB radiation.     

Biochemical alterations in human cells irradiated with alpha particles delivered by macro- or microbeams

Irradiation of individual cell nuclei with charged-particle microbeams requires accurate identification and localization of cells using Hoechst staining and UV illumination before computer-monitored localization of each cell. Using Fourier-transform infrared microspectroscopy (FT-IRM), we investigated whether the experimental conditions used for cell recognition induce cellular changes prior to irradiation and compared biochemical changes and DNA damage after targeted and nontargeted irradiation with alpha particles delivered by macro- or microbeams, using gamma radiation as a reference. Molecular damage in single HaCaT cells was studied by means of FT-IRM and comet assay (Gault et al., Int. J. Radiat. Biol. 81, 767-779, 2005). Hoechst 33342-stained HaCaT cells were exposed to single doses of 2 Gy (239)Pu alpha particles from a broad-beam irradiator, five impacted alpha particles from a microbeam irradiator, or 6 Gy gamma rays from (137)Cs, each of which resulted in about 5% clonogenic survival. FT-IRM of control cells indicated that Hoechst binding to nuclear DNA induced subtle changes in DNA conformation, and its excitation under UV illumination induced a dramatic shift of the DNA conformation from A to B as well as major DNA damage as measured by the comet assay. Comparison of the FT-IRM spectra of cells exposed to gamma rays or alpha particles specifically targeted to the nucleus, alpha particles from a broad-beam irradiator revealed spectral changes corresponding to all changes in constitutive bases in nucleic acids, suggesting oxidative damage in these bases, as well as structural damage in the deoxyribose-phosphate backbone of DNA and the osidic structure of nucleic acids. Concomitantly, spectral changes specific to protein suggested structural modifications. Striking differences in IR spectra between targeted microbeam- and nontargeted macrobeam-irradiated cells indicated greater residual unrepaired or misrepaired damage after microbeam irradiation. This was confirmed by the comet assay data. These results show that FT-IRM, together with the comet assay, is useful for assessing direct radiation-induced damage to nucleic acids and proteins in single cells and for investigating the effects of radiation quality. Significantly, FT-IRM revealed that Hoechst 33342 binding to DNA and exposure to UV light induce a dramatic change in DNA conformation as well as DNA damage. These findings suggest that fluorochrome staining should be avoided in studies of ionizing radiation-induced bystander effects based on charged-particle microbeam irradiation. An alternative cell nucleus recognition system that avoids nuclear matrix damage and its possible contribution to propagation of biological effects from irradiated cells to neighboring nontargeted cells needs to be developed.     

Quantitation and Visualization of Ultraviolet‐Induced DNA Damage Using Specific Antibodies: Application to Pigment Cell Biology

The major types of DNA damage induced by sunlight in the skin are DNA photoproducts, such as cyclobutane pyrimidine dimers (CPDs), (6‐4)photoproducts (6‐4PPs) and Dewar isomers of 6‐4PPs. A sensitive method for quantitating and visualizing each type of DNA photoproduct induced by biologically relevant doses of ultraviolet (UV) or sunlight is essential to characterize DNA photoproducts and their biological effects. We have established monoclonal antibodies specific for CPDs, 6‐4PPs or Dewar isomers. Those antibodies allow one to quantitate photoproducts in DNA purified from cultured cells or from the skin epidermis using an enzyme‐linked immunosorbent assay. One can also use those specific antibodies with in situ laser cytometry to visualize and measure DNA photoproducts in cultured cells or in the skin, using indirect immunofluorescence and a laser‐scanning confocal microscope. This latter method allows us to reconstruct three‐dimensional images of nuclei containing DNA photoproducts and to simultaneously examine DNA photoproducts and histology in multilayered epidermis. Using those techniques, one can determine the induction and repair of these three distinct types of DNA photoproducts in cultured cells and in the skin exposed to sublethal or suberythematous doses of UV or solar simulated radiation. As examples of the utility of these techniques and antibodies, we describe the DNA repair kinetics following irradiation of human cell nuclei and the photoprotective effect of melanin against DNA photoproducts in cultured pigmented cells and in human epidermis.     

Quantitation and visualization of ultraviolet-induced DNA damage using specific antibodies: application to pigment cell biology

The major types of DNA damage induced by sunlight in the skin are DNA photoproducts, such as cyclobutane pyrimidine dimers (CPDs), (6-4)photoproducts (6-4PPs) and Dewar isomers of 6-4PPs. A sensitive method for quantitating and visualizing each type of DNA photoproduct induced by biologically relevant doses of ultraviolet (UV) or sunlight is essential to characterize DNA photoproducts and their biological effects. We have established monoclonal antibodies specific for CPDs, 6-4PPs or Dewar isomers. Those antibodies allow one to quantitate photoproducts in DNA purified from cultured cells or from the skin epidermis using an enzyme-linked immunosorbent assay. One can also use those specific antibodies with in situ laser cytometry to visualize and measure DNA photoproducts in cultured cells or in the skin, using indirect immunofluorescence and a laser-scanning confocal microscope. This latter method allows us to reconstruct three-dimensional images of nuclei containing DNA photoproducts and to simultaneously examine DNA photoproducts and histology in multilayered epidermis. Using those techniques, one can determine the induction and repair of these three distinct types of DNA photoproducts in cultured cells and in the skin exposed to sublethal or suberythematous doses of UV or solar simulated radiation. As examples of the utility of these techniques and antibodies, we describe the DNA repair kinetics following irradiation of human cell nuclei and the photoprotective effect of melanin against DNA photoproducts in cultured pigmented cells and in human epidermis.     

Larger yield of cyclobutane dimers than 8-oxo-7,8-dihydroguanine in the DNA of UVA-irradiated human skin cells

Exposure to solar ultraviolet light is the major cause of most skin cancers. While the genotoxic properties of UVB radiation are now well understood, the DNA damaging processes triggered by less energetic but more abundant UVA photons remain to be elucidated. Evidence has been provided for the induction of oxidative lesions to cellular DNA including strand breaks and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo). Formation of cyclobutane pyrimidine dimers (CPDs) has also been reported, mostly in rodent cells. In order to gain insights into the relevance of the latter photoproducts in UVA-mutagenesis of human skin, we quantified the level of 8-oxodGuo and CPDs within primary cultures of normal fibroblasts and keratinocytes using specific chromatographic assays. The yield of formation of CPDs was found to be higher than that of 8-oxodGuo in both cell types. In addition, CPDs were mostly TT derivatives, and neither (6-4) photoproducts nor Dewar valence isomers were detected. These observations are reminiscent of results obtained in rodent cells and suggest that a photosensitized triplet energy transfer occurs and that this reaction is more efficient than photooxidation of DNA components. The predominant formation of CPDs with respect to oxidative damage within normal human skin cells exposed to UVA radiation should be taken into account in photoprotection strategies.     

PCR-based methodology for the determination of the photoprotection afforded by sunscreen application

We have applied a PCR-based methodology to study the DNA damage induced by UV-A, UV-B and sunlight itself. Our results, employing a cell-free system, indicate that UV-B (310 nm) is approximately 30-fold more potent at inhibiting DNA synthesis than UV-A (365 nm). We were also able to show that 20 min of sunlight exposure on a summer day induced DNA damage capable of inhibiting DNA synthesis. Hence, this methodology has a sensitivity suitable to detect biologically relevant doses of UV light. In addition, we propose that this technique may be suitable to assess the relative photoprotection of commercially available sunscreens. We present here preliminary data on the photoprotection afforded by the topical application of sunscreen. This photoprotection was measured by a reduction in the subsequent UV-B- and UV-A-induced DNA damage when sunscreen was applied. Our results demonstrate that the particular sunscreen tested was effective against both UV-B and UV-A. However, the estimated photoprotective factor of the sunscreen (against both UV-A and UV-B) was approximately tenfold less than the stated Sun Protection Factor (SPF) of 25. This methodology may also be useful in identifying new photoprotective agents by assessing their relative value as UV-B and UV-A absorbing agents.     

The evaluation of protective effect of lycopene against genotoxic influence of X-irradiation in human blood lymphocytes

Many studies suggest that exogenous antioxidants may protect cells against DNA damage caused with ionizing radiation. One of the most powerful antioxidants is lycopene (LYC), a carotenoid derived from tomatoes. The aim of this study was to investigate, using the comet assay, whether LYC can act as protectors/modifiers and prevent DNA damage induced in human blood lymphocytes, as well as to mitigate the effects of radiation exposure. In this project, LYC, dissolved in DMSO at a concentration of 10, 20 or 40 μM/ml of cell suspension, was added to the isolated lymphocytes from human blood at appropriate intervals before or after the X-irradiation at doses of 0.5, 1 and 2 Gy. Cell viability in all groups was maintained at above 70%. The results showed the decrease of DNA damage in cells treated with various concentrations of LYC directly and 1 h before exposure to X-rays compared to the control group exposed to irradiation alone. Contrary results were observed in cells exposed to LYC immediately after exposure to ionizing radiation. The studies confirmed the protective effect of LYC against DNA damage induced by ionizing radiation, but after irradiation the carotenoid did not stimulate of DNA repair and cannot act as modifier. However, supplementation with LYC, especially at lower doses, may be useful in protection from radiation-induced oxidative damage.     

Gamma ray induced genetic changes in different organs of chick embryo using peripheral blood micronucleus test and comet assay

Ionizing radiation is known to produce a variety of cellular and sub cellular damage in both prokaryotic and eukaryotic cells. Present studies were undertaken to assess gamma ray induced DNA damage in different organs of the chick embryo using alkaline comet assay and peripheral blood micronucleus test. Further the suitability of chick embryo, as an alternative model for genotoxicity evaluation of environmental agents was assessed. Fertilized eggs of Rhode island red strain were exposed to 0.5, 1 and 2 Gy of gamma rays delivered at a dose rate of 0.316 Gy/min using a ⁶⁰Co teletherapy machine. Peripheral blood smears were prepared from 8- to 11-day-old chick embryos for micronucleus test. Alkaline comet assay was performed on 11-day-old chick embryos in different organs such as the heart, liver, lung, blood, bone marrow, brain and kidney.Analysis of the data revealed a significant increase in the frequency of micronucleated polychromatic erythrocytes, micronucleated normochromatic erythrocytes and total micronucleated erythrocytes in the peripheral blood of gamma irradiated chick embryos at all the doses tested as compared to the respective controls. The polychromatic to normochromatic erythrocytes ratio which is an indicator of proliferation rate of hematopoetic tissue, decreased in the irradiated groups as compared to the controls. Data obtained from comet assay, clearly demonstrated a significant increase in DNA strand breaks in all the organs of irradiated chick embryos as compared to the respective controls. However, maximum damage was observed in the heart tissue on all the doses tested, followed by kidney, brain, lung, blood and liver. The lowest damage was observed in the bone marrow tissue. Both micronucleus test and comet assay were found to be suitable biomarkers for the evaluation of genotoxicity of gamma radiation in the chick embryo.     

DNA damage evaluated by the comet assay in lymphocytes of children with Cs internal contamination caused by the Chernobyl accident

The comet assay is one of the most versatile and popular tools for evaluating DNA damage. Its sensitivity to low dose radiation has been tested in vitro, but there are limited data showing its application and sensitivity in chronic exposure situations. The influence of the internal contamination caused by the Chernobyl accident on the level of DNA damage was evaluated by the comet assay on lymphocytes of 56 Ukrainian children. The study was performed during 2003 on children with demonstrable ¹³⁷Cs internal contamination caused by food consumption. The children were selected for the study immediately after a ¹³⁷Cs whole body counter measurement of internal contamination. The minimal detectable amount of ¹³⁷Cs was 75 Bq. The control group included 29 children without detectable internal contamination, while in the exposed group 27 children with measured activity between 80 and 4037 Bq and committed effective dose between 54 and 3155 μSv were included. Blood samples were taken by a finger prick. The alkaline version of the comet assay was used, in combination with silver stained comets and arbitrary units (AU), for comet measurement. Factors such as disease, medical treatment, surface contamination of children's living location, etc., were considered in the study. Non-significant differences (p > 0.05) in DNA damage in control (9.0 ± 5.7 AU) versus exposed (8.5 ± 4.8 AU) groups were found. These results suggest that low doses of ¹³⁷Cs internal contamination are not able to produce detectable DNA damage under the conditions used for the comet assay in this study. Further studies considering effects of high exposure should be performed on chronically exposed people using this assay.     

Silymarin protects epidermal keratinocytes from ultraviolet radiation-induced apoptosis and DNA damage by nucleotide excision repair mechanism

Solar ultraviolet (UV) radiation is a well recognized epidemiologic risk factor for melanoma and non-melanoma skin cancers. This observation has been linked to the accumulation of UVB radiation-induced DNA lesions in cells, and that finally lead to the development of skin cancers. Earlier, we have shown that topical treatment of skin with silymarin, a plant flavanoid from milk thistle (Silybum marianum), inhibits photocarcinogenesis in mice; however it is less understood whether chemopreventive effect of silymarin is mediated through the repair of DNA lesions in skin cells and that protect the cells from apoptosis. Here, we show that treatment of normal human epidermal keratinocytes (NHEK) with silymarin blocks UVB-induced apoptosis of NHEK in vitro. Silymarin reduces the amount of UVB radiation-induced DNA damage as demonstrated by reduced amounts of cyclobutane pyrimidine dimers (CPDs) and as measured by comet assay, and that ultimately may lead to reduced apoptosis of NHEK. The reduction of UV radiation-induced DNA damage by silymarin appears to be related with induction of nucleotide excision repair (NER) genes, because UV radiation-induced apoptosis was not blocked by silymarin in NER-deficient human fibroblasts. Cytostaining and dot-blot analysis revealed that silymarin repaired UV-induced CPDs in NER-proficient fibroblasts from a healthy individual but did not repair UV-induced CPD-positive cells in NER-deficient fibroblasts from patients suffering from xeroderma pigmentosum complementation-A disease. Similarly, immunohistochemical analysis revealed that silymarin did not reduce the number of UVB-induced sunburn/apoptotic cells in the skin of NER-deficient mice, but reduced the number of sunburn cells in their wild-type counterparts. Together, these results suggest that silymarin exert the capacity to reduce UV radiation-induced DNA damage and, thus, prevent the harmful effects of UV radiation on the genomic stability of epidermal cells.     

New Approach to Develop Optimized Sunscreens that Enable Cutaneous Vitamin D Formation with Minimal Erythema Risk

Sunscreens protect the skin against erythemal radiation (E_er). But at the same time they reduce the effective radiation dose (E_VD) responsible for the formation of previtamin D in the skin. The paper describes a calculation method for optimizing the ratio E_VD/E_er behind sunscreens e.g. with SPF 5, 15 and 30 respectively. Taking into account that a majority of people in industrialized countries suffer from a shortage in vitamin D even in summer time, the ratio E_vd/E_er is a new and important criterion for the quality of sunscreens. Furthermore the exposure time t_vd needed per day for forming the equivalent of the recommended amount of 2000 IU of vitamin D per day for skin type 2 is estimated when sunscreens with different filter compositions are used. In vitro experiments show a significant increase of the conversion of 7-dehydrocholesterol (7-DHC) to previtamin D when exposed to artificial solar radiation behind an experimental sunscreen optimized for previtamin D production compared to a commercial sunscreen having the same SPF.     

Vanadate induces DNA strand breaks in cultured human fibroblasts at doses relevant to occupational exposure

To study possible genotoxic effects of occupational exposure to vanadium pentoxide, we determined DNA strand breaks (with alkaline comet assay), 8-hydroxy-2'deoxyguanosine (8-OHdG) and the frequency of sister chromatid exchange (SCE) in whole blood leukocytes or lymphocytes of 49 male workers employed in a vanadium factory in comparison to 12 non-exposed controls. In addition, vanadate has been tested in vitro to induce DNA strand breaks in whole blood cells, isolated lymphocytes and cultured human fibroblasts of healthy donors at concentrations comparable to the observed levels of vanadium in vivo. To investigate the impact of vanadate on the repair of damaged DNA, co-exposure to UV or bleomycin was used in fibroblasts, and DNA migration in the alkaline and neutral comet assay was determined. Although, exposed workers showed a significant vanadium uptake (serum: median 5.38microg/l, range 2.18-46.35microg/l) no increase in cytogenetic effects or oxidative DNA damage in leukocytes could be demonstrated. This was consistent with the observation that in vitro exposure of whole blood leukocytes and lymphocytes to vanadate caused no significant changes in DNA strand breaks below concentrations of 1microM (50microg/l). In contrast, vanadate clearly induced DNA fragmentation in cultured fibroblasts at relevant concentrations. Combined exposure of fibroblasts to vanadate/UV or vanadate/bleomycin resulted in non-repairable DNA double strand breaks (DSBs) as seen in the neutral comet assay. We conclude that exposure of human fibroblasts to vanadate effectively causes DNA strand breaks, and co-exposure of cells to other genotoxic agents may result in persistent DNA damage.     

Modulation of DNA damage by pentoxifylline and alpha-tocopherol in skin fibroblasts exposed to Gamma rays

Previous in vivo studies showed the combination pentoxifylline (PTX) and alpha-tocopherol was highly efficient in reducing late radiation-induced skin damage. The present work aimed at investigating the molecular and cellular mechanisms involved in the effects of this combination. Primary cultures of confluent dermal fibroblasts were gamma-irradiated in the presence of PTX and trolox (Tx), the water-soluble analogue of alpha-tocopherol. Drugs were added either before or after radiation exposure and were maintained over time. Their antioxidant capacity and their effect on radiation-induced ROS production was assessed together with cell viability and clonogenicity. DNA damage formation was assessed by the alkaline comet assay and by the micronucleus (MN) test. Cell cycle distribution was also determined. The combination of PTX/ Tx was shown to reduce both immediate and late ROS formation observed in cells after irradiation. Surprisingly, decrease in DNA strand breaks measured by the comet assay was observed any time drugs were added. In addition, the micronucleus test revealed that for cells irradiated with 10 Gy, a late significant increase in MN formation occurred. The combination of PTX/Tx was shown to be antioxidant and to decrease radiation-induced ROS production. The observed effects on DNA damage at any time the drugs were added suggest that PTX/Tx could interfere with the DNA repair process.