Nitroxide radicals protect DNA from damage when illuminated in vitro in the presence of dibenzoylmethane and a common sunscreen ingredient

Indolinonic nitroxide radicals efficiently scavenge oxygen- and carbon-centered radicals. They protect lipid and protein systems against oxidative stress, but little is known about their capacity to protect DNA against radical-mediated damage. We compare indolinonic nitroxides and the piperidines TEMPO and TEMPOL for their ability to inhibit strand breaks inflicted on DNA when it is illuminated in vitro in the presence of dibenzoylmethane (DBM) and a relative, Parsol 1789, used as a UVA-absorbing sunscreen. We used spin-trapping EPR to examine the formation of radicals and plasmid nicking assays to evaluate DNA strand breakage. The results have a two-fold interest. First, they show that all the nitroxides tested efficiently prevent DNA damage in a dose-dependent fashion. Vitamin E had no effect under the conditions used. Second, they show that carbon-centered radicals are produced on illumination of DBM and its relative and that their formation is probably responsible for the direct strand breaks found when naked DNA is illuminated in vitro in their presence. Additional work on the ability of sunscreens to enter human cells and their response to the light that penetrates sunscreen-protected skin would be necessary before any conclusion could be drawn as to whether the results reported here are relevant to human use of sunscreens.     

The effect of MC1R variants and sunscreen on the response of human melanocytes in vivo to ultraviolet radiation and implications for melanoma

We conducted a clinical trial to compare the molecular and cellular responses of human melanocytes and keratinocytes in vivo to solar‐simulated ultraviolet radiation (SSUVR) in 57 Caucasian participants grouped according to MC1R genotype. We found that, on average, the density of epidermal melanocytes 14 days after exposure to 2 minimal erythemal dose (MED) SSUVR was twofold higher than baseline (unirradiated) skin. However, the change in epidermal melanocyte counts among people carrying germline MC1R variants (97% increase) was significantly less than those with wild‐type MC1R (164% increase; P = 0.01). We also found that sunscreen applied to the skin before exposure to 2 MED SSUVR completely blocked the effects of DNA damage, p53 induction, and cellular proliferation in both melanocytes and keratinocytes.     

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.     

Melanin protects melanocytes and keratinocytes against H2O2-induced DNA strand breaks through its ability to bind Ca2+

Reactive oxygen species (ROS) such as hydrogen peroxide (H(2)O(2)) are produced in the skin under the influence of UV radiation. These compounds are highly reactive and can induce DNA lesions in epidermal cells. Melanin is considered to protect human skin against DNA damage by absorbing UV radiation. We have investigated whether melanin can, in addition, offer protection against the effects of H(2)O(2) in human melanocytes and HaCaT keratinocytes. In the present study, it was shown that 40 and 100 microM H(2)O(2) increased the number of DNA strand breaks as measured using the comet assay, in melanocytes of Caucasian origin. In melanocytes of the same origin in which melanin levels were increased by culturing in presence of 10 mM NH(4)Cl and elevated l-tyrosine, H(2)O(2)-induced DNA damage was reduced compared to that in control melanocytes. Similarly, HaCaT cells that were loaded with melanin were better protected against H(2)O(2)-induced DNA strand breaks than control HaCaT cells. These protective effects of melanin were mimicked by the intracellular Ca(2+)-chelator BAPTA. Thus, BAPTA reduced the level of H(2)O(2)-induced DNA strand breaks in melanocytes. Like BAPTA, melanin is known to be a potent chelator of Ca(2+) and this was confirmed in the present study. It was shown that melanin levels in melanocytic cells correlated directly with intracellular Ca(2+) binding capacity and, in addition, correlated inversely with H(2)O(2)-induced increases in intracellular Ca(2+). Our results show that melanin may have an important role in regulating intracellular Ca(2+) homeostasis and it is suggested that melanin protects against H(2)O(2)-induced DNA strand breaks in both melanocytes and keratinocytes and through its ability to bind Ca(2+).     

Radiation-induced DNA base damage detected in individual aerobic and hypoxic cells with endonuclease III and formamidopyrimidine-glycosylase.

X-ray-induced DNA base damage can be detected using endonuclease III and formamidopyrimidine-glycosylase, which create DNA strand breaks at enzyme-sensitive sites. Strand breaks can then be measured with excellent sensitivity using the alkaline comet assay, a single-cell gel electrophoresis method that detects DNA damage in individual cells. In using this approach to measure the oxygen enhancement ratio (OER) for radiation-induced base damage, we observed that the number of enzyme-sensitive sites increased with dose up to 4 Gy in air and 12 Gy in hypoxic WIL2NS cells. After rejoining of radiation-induced strand breaks, base damage was detected more easily after higher doses. The number of radiation-induced enzyme-sensitive sites was similar under both air and nitrogen. Base damage produced by hydrogen peroxide and 4-nitroquinoline-N-oxide (4NQO) was also measured. Results with hydrogen peroxide (20 min at 4 degrees C) were similar to those observed for X rays, indicating that enzyme-sensitive sites could be detected most efficiently when few direct strand breaks were present. Removing DNA-associated proteins before irradiation did not affect the ability to detect base damage. Base damage produced by 4NQO (30 min at 37 degrees C) was readily apparent after treatment with low concentrations of the drug when few 4NQO-induced strand breaks were present, but the detection sensitivity decreased rapidly as direct strand breaks increased after treatment with higher concentrations. We conclude that: (1) the OER for base damage is approximately 1.0, and (2) the presence of direct DNA strand breaks (>2000-4000 per cell) prevents accurate detection of base damage measured as enzyme-sensitive sites with the alkaline comet method.     

Increased oxidative modification of albumin when illuminated in vitro in the presence of a common sunscreen ingredient: protection by nitroxide radicals

We previously reported on the ability of dibenzoylmethane (DBM) and a relative, Parsol 1789, used as a ultraviolet A (UVA)-absorbing sunscreen, to generate free radicals upon illumination, and as a consequence, to inflict strand breaks in plasmid DNA in vitro. This study has now been extended to determine the effects of Parsol 1789 and DBM on proteins, under UVA illumination, with the sole purpose of gaining more knowledge on the photobiological effects of sunscreen chemicals. Parsol 1789 (100 microM) caused a 2-fold increase in protein carbonyl formation (an index of oxidative damage) in bovine serum albumin (BSA) when exposed to illumination, and this damage was both concentration- and time-dependent. The degree of protein damage was markedly reduced by the presence of free radical scavengers, namely piperidinic and indolinonic nitroxide radicals, in accordance with our previous study. Vitamin E had no effect under the conditions used. The results obtained corroborate the fact that Parsol 1789 generates free radicals upon illumination and that these are, most probably, responsible for the protein damage observed under the conditions used in our system. However, at present, we cannot extrapolate from these results the relevance to human use of sunscreens; therefore, further studies should be necessary to determine the efficacy at the molecular and cellular level of this UVA-absorber in order to ascertain protection against photocarcinogenic risk.     

Importance of UVA photoprotection as shown by genotoxic related endpoints: DNA damage and p53 status

In order to demonstrate the importance of photoprotection in the UVA range (320-400 nm), an in vitro approach where sun formulations are spread on a quartz slide, and placed over human keratinocytes in culture is proposed as a convenient test for photoprotection assessment at the DNA level. Using the comet assay, DNA strand breaks, oxidative DNA damage or drug-induced DNA breaks were assessed. Accumulation of p53 protein was also studied as a marker for UV-induced genotoxic stress. Such a method was used to compare two formulations with different photostability. Spectroradiometry showed that a photounstable formulation lost its effectiveness in UVA screening when pre-irradiated by simulated sunlight (UVB+UVA). As a consequence, it was also shown that this formulation was not as protective as the photostable one at the genomic level. These data demonstrate that the loss of absorbing efficiency within UVA wavelengths due to photounstability may have detrimental consequences leading to impairments implicated in genotoxic events.     

Role of sunscreen formulation and photostability to protect the biomechanical barrier function of skin

The impact of sunscreen formulations on the barrier properties of human skin are often overlooked leading to formulations with components whose effects on barrier mechanical integrity are poorly understood. The aim of this study is to demonstrate the relevance of carrier selection and sunscreen photostability when designing sunscreen formulations to protect the biomechanical barrier properties of human stratum corneum (SC) from solar ultraviolet (UV) damage. Biomechanical properties of SC samples were assayed after accelerated UVB damage through measurements of the SC's mechanical stress profile and corneocyte cohesion. A narrowband UVB (305–315 nm) lamp was used to expose SC samples to 5, 30, 125, and 265 J cm^?2 in order to magnify damage to the mechanical properties of the tissue and characterize the UV degradation dose response such that effects from smaller UV dosages can be extrapolated. Stresses in the SC decreased when treated with sunscreen components, highlighting their effect on the skin prior to UV exposure. Stresses increased with UVB exposure and in specimens treated with different sunscreens stresses varied dramatically at high UVB dosages. Specimens treated with sunscreen components without UVB exposure exhibited altered corneocyte cohesion. Both sunscreens studied prevented alteration of corneocyte cohesion by low UVB dosages, but differences in protection were observed at higher UVB dosages indicating UV degradation of one sunscreen. These results indicate the protection of individual sunscreen components vary over a range of UVB dosages, and components can even cause alteration of the biomechanical barrier properties of human SC before UV exposure. Therefore, detailed characterization of sunscreen formulation components is required to design robust protection from UV damage.     

Inhibition of UV radiation-induced DNA damage by a 5-methoxypsoralen tan in human skin

Previously untanned buttock skin of 4 volunteers (skin type II; tan with difficulty as they sunburn easily) was treated with various sunscreen preparations and solar--simulated radiation (SSR) or SSR alone for 2 weeks. One week later, the treatment sites were challenged with a DNA-damaging dose of SSR--twice the minimal erythema dose (2 MED). Skin biopsy samples were assayed for the levels of unscheduled DNA synthesis (a measure of DNA damage), melanin distribution, and skin thickening. 5-Methoxypsoralen-containing sunscreen preparations plus SSR or SSR alone induced melanogenesis and increased the stratum corneum thickness, but only the former regimen afforded a high degree of protection against subsequent SSR-induced DNA damage. 5-Methoxypsoralen-free sunscreen preparations plus SSR induced negligible tanning, skin thickening, and photoprotection. These findings are relevant to the risk-benefit analysis of sunscreen preparations, especially in skin type II, as they provide evidence that a 5-methoxypsoralen-induced tan is protective against the DNA-damaging effects of solar UV radiation, and thus has the potential to reduce the carcinogenic risk of exposure to such radiation.     

Synthesis and application of a novel sunscreen-antioxidant

Background information on the inefficacy of sunscreens to provide free radical protection in skin, despite their usefulness in preventing sunburn/erythema, prompted us to synthesize a compound which would display in the same molecule both UV-absorbing and antioxidant capacities. For this purpose, the UVB absorber, 2-ethylhexyl-4-methoxycinnamate (OMC) was combined with the piperidine nitroxide TEMPOL, which has antioxidant properties. The spectral properties of the new nitroxide-based sunscreen (MC-NO) as well as its efficacy to prevent photo-oxidative damage to lipids induced by UVA, natural sunlight and 4-tert-butyl-4-methoxydibenzoylmethane (BMDBM), a photo-unstable sunscreen which generates free radicals upon UV radiation, was studied. The results obtained demonstrate that MC-NO: (a) absorbs in the UVB region even after UVA irradiation; (b) acts as free radical scavenger as demonstrated by EPR experiments; (c) strongly reduces both UVA-, sunlight- and BMDBM-induced lipid peroxidation in liposomes, measured as reduced TBARS levels; and (d) has comparable antioxidant activity to that of commonly used vitamin E and BHT in skin care formulations. These results suggest that the use of the novel sunscreen-antioxidant or of other nitroxide-based sunscreens in formulations aimed at reducing photoinduced skin damage may be envisaged.     

DNA damage by carbonyl stress in human skin cells

Reactive carbonyl species (RCS) are potent mediators of cellular carbonyl stress originating from endogenous chemical processes such as lipid peroxidation and glycation. Skin deterioration as observed in photoaging and diabetes has been linked to accumulative protein damage from glycation, but the effects of carbonyl stress on skin cell genomic integrity are ill defined. In this study, the genotoxic effects of acute carbonyl stress on HaCaT keratinocytes and CF3 fibroblasts were assessed. Administration of the alpha-dicarbonyl compounds glyoxal and methylglyoxal as physiologically relevant RCS inhibited skin cell proliferation, led to intra-cellular protein glycation as evidenced by the accumulation of N(epsilon)-(carboxymethyl)-L-lysine (CML) in histones, and caused extensive DNA strand cleavage as assessed by the comet assay. These effects were prevented by treatment with the carbonyl scavenger D-penicillamine. Both glyoxal and methylglyoxal damaged DNA in intact cells. Glyoxal caused DNA strand breaks while methylglyoxal produced extensive DNA-protein cross-linking as evidenced by pronounced nuclear condensation and total suppression of comet formation. Glycation by glyoxal and methylglyoxal resulted in histone cross-linking in vitro and induced oxygen-dependent cleavage of plasmid DNA, which was partly suppressed by the hydroxyl scavenger mannitol. We suggest that a chemical mechanism of cellular DNA damage by carbonyl stress occurs in which histone glycoxidation is followed by reactive oxygen induced DNA stand breaks. The genotoxic potential of RCS in cultured skin cells and its suppression by a carbonyl scavenger as described in this study have implications for skin damage and carcinogenesis and its prevention by agents selective for carbonyl stress.     

A possible role of Ku in mediating sequential repair of closely opposed lesions

One of the hallmarks of ionizing radiation exposure is the formation of clustered damage that includes closely opposed lesions. We show that the Ku70/80 complex (Ku) has a role in the repair of closely opposed lesions in DNA. DNA containing a dihydrouracil (DHU) close to an opposing single strand break was used as a model substrate. It was found that Ku has no effect on the enzymatic activity of human endonuclease III when the substrate DNA contains only DHU. However, with DNA containing a DHU that is closely opposed to a single strand break, Ku inhibited the nicking activity of human endonuclease III as well as the amount of free double strand breaks induced by the enzyme. The inhibition on the formation of a free double strand break by Ku was found to be much greater than the inhibition of human endonuclease III-nicking activity by Ku. Furthermore, there was a concomitant increase in the formation of Ku-DNA complexes when endonuclease III was present. Similar results were also observed with Escherichia coli endonuclease III. These results suggest that Ku reduces the formation of endonuclease III-induced free double strand breaks by sequestering the double strand breaks formed as a Ku-DNA complex. In doing so, Ku helps to avoid the formation of the intermediary free double strand breaks, possibly helping to reduce the mutagenic event that might result from the misjoining of frank double strand breaks.     

Deletions at short direct repeats and base substitutions are characteristic mutations for bleomycin-induced double- and single-strand breaks, respectively, in a human shuttle vector system.

Using the radiomimetic drug, bleomycin, we have determined the mutagenic potential of DNA strand breaks in the shuttle vector pZ189 in human fibroblasts. The bleomycin treatment conditions used produce strand breaks with 3'-phosphoglycolate termini as > 95% of the detectable dose-dependent lesions. Breaks with this end group represent 50% of the strand break damage produced by ionizing radiation. We report that such strand breaks are mutagenic lesions. The type of mutation produced is largely determined by the type of strand break on the plasmid (i.e. single versus double). Mutagenesis studies with purified DNA forms showed that nicked plasmids (i.e. those containing single-strand breaks) predominantly produce base substitutions, the majority of which are multiples, which presumably originate from error-prone polymerase activity at strand break sites. In contrast, repair of linear plasmids (i.e. those containing double-strand breaks) mainly results in deletions at short direct repeat sequences, indicating the involvement of illegitimate recombination. The data characterize the nature of mutations produced by single- and double-strand breaks in human cells, and suggests that deletions at direct repeats may be a 'signature' mutation for the processing of DNA double-strand breaks.     

Detection of DNA damage and repair by alkaline elution using human lymphocytes

The repair of DNA alkylation damage in human cells is poorly understood. We have adapted the alkaline elution technique for use with human peripheral blood lymphocytes in culture. We have also established conditions necessary for short-term culture of human lymphocytes. Lymphocyte growth which can be maintained for up to 30 days is dependent upon irradiated TK6 feeder cells and T-cell growth factor (crude TCGF). The amount of damage induced by a given concentration of methyl methane-sulfonate (MMS) is dependent upon cell number per ml of growth medium. The DNA damage measured, in lymphocytes, by alkaline elution is a composite of single strand breaks and alkali-labile lesions. Repair of this damage after appropriate recovery periods is also detectable. The irradiated feeder TK6 cells do not contribute to the number of strand breaks detected or the amount of recovery after treatment. This method offers a quick and reproducible means of detecting DNA damage and repair in human T-lymphocytes.     

Assessment of radiation-induced DNA strand breaks and their repair in unlabeled cells.

Radiation induced damage, i.e., the induction of DNA strand breaks, was studied on the level of single, unlabeled cells. DNA strand breaks were determined by direct partial alkaline unwinding in intact cell nuclei followed by staining with acridine orange, a development of a proposal first described by B. Rydberg (Int J Radiat Biol 46:521-527, 1984). The ratio of green fluorescence (double-stranded DNA) to red fluorescence (single-stranded DNA) in single cells was taken as a measure of DNA strand breaks. CHO-K1 and M3-1 cells irradiated with X-rays show a dose dependent induction of DNA strand breaks. Incubation at 37 degrees C after irradiation leads to repair of breaks. A repair halflife of about 10-11 min can be determined. Cell cycle specific differences in the induction of DNA strand breaks or repair behavior are not detectable at the resolution achieved so far. This new method offers two major advantages: the resolution of DNA damage and repair on the level of single cells and no need for labeling, thereby allowing for DNA damage and repair to be assessed in biopsy material from tumor patients.     

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.     

Cytoprotective effects of 6′-O-galloylpaeoniflorin against ultraviolet B radiation-induced cell damage in human keratinocytes

The cytoprotective effects of 6′-O-galloylpaeoniflorin against injury and death of human HaCaT keratinocytes resulting from ultraviolet B radiation were investigated. 6′-O-galloylpaeoniflorin exhibited the capacity to scavenge intracellular reactive oxygen species (ROS) generated by ultraviolet B radiation. 6′-O-galloylpaeoniflorin also attenuated ultraviolet B-induced oxidative macromolecular damage to DNA, lipids, and proteins, decreasing the number of DNA strand breaks, the level of 8-isoprostane (a biomarker of lipid peroxidation), and the level of protein carbonylation. Moreover, 6′-O-galloylpaeoniflorin rescued HaCaT cells from ultraviolet induced cell death, by downregulating the mitochondrial apoptotic pathway. Taken together, these results indicate that 6′-O-galloylpaeoniflorin has the potential to be developed as a medical agent against ROS-mediated skin diseases.     

A new healthy sunscreen system for human: solid lipid nanoparticles as carrier for 3,4,5-trimethoxybenzoylchitin and the improvement by adding Vitamin E

The aim of this study was to find safer cosmetics for human through application chitin and natural solid lipsomes to sunscreen. Pure chitin is not solved completely in majority chemical agents and traditional chemical UV blockers are potentially harmful to human skin. So the combination of chemical UV absorber and chitin formed 3,4,5-trimethoxybenzoylchitin (TMBC), which was proven as an active chemical sunscreen by Dr. Clausen, and can overcome their inherent defects. Lipophilic ability of the new material can be increased while harmfulness can be decreased due to incorporation natural material chitin. Then solid lipid nanoparticles (SLN) loaded with TMBC to act both as physical sunscreens themselves and as carriers in order to enhance the effect of UVB protection. The improvement of the system can been observed when tocopherol was added.     

Nature of DNA lesions induced in human hepatoma cells, human colonic cells and human embryonic lung fibroblasts by the antiretroviral drug 3'-azido-3'-deoxythymidine

This study tried to clarify the question if nuclear genotoxicity played a role in 3'-azido-3'-deoxythymidine (AZT) toxicity. We investigated cytotoxic and DNA-damaging effects of AZT on human hepatoma HepG2 and human colonic CaCo-2 cells as well as on human diploid lung fibroblasts HEL. The amount of induced DNA damage was measured by standard alkaline single cell gel electrophoresis (SCGE). The nature of induced DNA lesions was evaluated (1) by modified SCGE, which includes treatment of lysed cells with DNA repair enzymes Endo III and Fpg and enables to recognize oxidized bases of DNA, and (2) by SCGE processed in parallel at pH 13.0 (standard technique) and pH 12.1, which enables to recognize alkali labile DNA lesions and direct DNA strand breaks. Cytotoxicity of AZT was evaluated by the trypan blue exclusion technique. Our findings showed that 3-h treatment of cells with AZT decreased the viability of all cell lines studied. SCGE performed in the presence of DNA repair enzymes proved that AZT induced oxidative lesions to DNA in all cell types. In hepatoma HepG2 cells and embryonic lung fibroblasts HEL the majority of AZT-induced DNA strand breaks were pH-independent, i.e. they were identified at both pH values (12.1 and 13.0). These DNA lesions represented direct DNA breaks. In colonic Caco-2 cells DNA lesions were converted to DNA strand breaks particularly under strong alkaline conditions (pH>13.0), which is characteristic for alkali-labile sites of DNA. DNA strand break rejoining was investigated by the standard comet assay technique during 48 h of post-AZT-treatment in HepG2 and Caco-2 cells. The kinetics of DNA rejoining, considered an indicator of DNA repair, revealed that AZT-induced DNA breaks were repaired in both cell types slowly, though HepG2 cells seemed to be more repair proficient with respect to AZT-induced DNA lesions.     

Quantifying clustered DNA damage induction and repair by gel electrophoresis, electronic imaging and number average length analysis

Assessing DNA damage induction, repair and consequences of such damages requires measurement of specific DNA lesions by methods that are independent of biological responses to such lesions. Lesions affecting one DNA strand (altered bases, abasic sites, single strand breaks (SSB)) as well as damages affecting both strands (clustered damages, double strand breaks) can be quantified by direct measurement of DNA using gel electrophoresis, gel imaging and number average length analysis. Damage frequencies as low as a few sites per gigabase pair (10(9)bp) can be quantified by this approach in about 50ng of non-radioactive DNA, and single molecule methods may allow such measurements in DNA from single cells. This review presents the theoretical basis, biochemical requirements and practical aspects of this approach, and shows examples of their applications in identification and quantitation of complex clustered damages.