Protective effects of antioxidants against UVA-induced DNA damage in human skin fibroblasts in culture

Ultraviolet A radiation (UVA, 320–400 nm) is mutagenic and induces genomic damage to skin cells. N-acetyl-cysteine (NAC), selenium and zinc have been shown to have antioxidant properties and to exhibit protective effects against UVA cytotoxicity. The present work attempts to delineate the effect of these compounds on genomic integrity of human skin fibroblasts exposed to UVA radiation using the single cell gel electrophoresis (SCGE) or Comet assay. The cells were incubated with NAC (5 mM), sodium selenite (0.6 μM) or zinc chloride (100 μM). Then cells were embedded in low melting point agarose, and immediately submitted to UVA fluences ranging from 1 to 6J/cm². In the Comet assay, the tail moment increased by 45% (1J/cm²) to 89% (6J/cm²) in non-supplemented cells (p<0.01). DNA damage was significantly prevented by NAC, Se and Zn, with a similar efficiency from 1 to 4J/cm² (p<0.05). For the highest UVA dose (6J/cm²), Se and Zn were more effective than NAC (p<0.01).     

Baicalin protects human skin fibroblasts from ultraviolet A radiation-induced oxidative damage and apoptosis

Reactive oxygen species (ROS) are an important factor in the development of skin photodamage after ultraviolet A (UVA) radiation. A flavonoid antioxidant, baicalin, can selectively neutralize super-oxide anion (O₂^?) while having no significant effect on ^?OH. Fibroblasts are a key component of skin dermis. In the present study, we investigated the protective effects of baicalin on human skin fibroblasts (HSFs) under UVA induced oxidative stress. Fluorescence microscopy and flow cytometry were used to assay the intracellular O₂^?, NO, ROS concentrations and the mitochondrial membrane potential. Cell viability was determined using the Cell Counting Kit-8 (CCK-8). The concentrations of cellular MDA, SOD, GSH, T-AOC, and 8-oxo-dG were also measured. Cellular apoptosis was measured by flow cytometry and caspase-3 detection. The results revealed that UVA radiation could cause oxidative stress and apoptosis in HSFs. Interestingly, the use of baicalin after UVA radiation signi?cantly reduced the level of intracellular O₂^?, NO, and ROS, stabilized the mitochondrial membrane potential, and attenuated production of MDA and 8-oxo-dG. These ef?ciently enhanced the antioxidative defense system and protected the HSFs from subsequent oxidative stress damage and apoptosis. In other words, baicalin decreased the excessive generation of intracellular ROS and NO, and elevated the cellular antioxidative defense, which eventually mitigate the UVA-induced apoptosis. Based on our results, baicalin may have applications in the treatment of skin photodamage caused by UVA irradiation.     

Estrogen prevents oxidative damage to the mitochondria in Friedreich's ataxia skin fibroblasts

Estrogen and estrogen-related compounds have been shown to have very potent cytoprotective properties in a wide range of disease models, including an in vitro model of Friedreich's ataxia (FRDA). This study describes a potential estrogen receptor (ER)-independent mechanism by which estrogens act to protect human FRDA skin fibroblasts from a BSO-induced oxidative insult resulting from inhibition of de novo glutathione (GSH) synthesis. We demonstrate that phenolic estrogens, independent of any known ER, are able to prevent lipid peroxidation and mitochondrial membrane potential (ΔΨm) collapse, maintain ATP at near control levels, increase oxidative phosphorylation and maintain activity of aconitase. Estrogens did not, however, prevent BSO from depleting GSH or induce an increased expression level of GSH. The cytoprotective effects of estrogen appear to be due to a direct overall reduction in oxidative damage to the mitochondria, enabling the FRDA fibroblast mitochondria to generate sufficient ATP for energy requirements and better survive oxidative stress. These data support the hypothesis that phenol ring containing estrogens are possible candidate drugs for the delay and/or prevention of FRDA symptoms.     

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.     

2,4,6-trinitrotoluene-induced reproductive toxicity via oxidative DNA damage by its metabolite

Several epidemiological studies and animal experiments showed that 2,4,6-trinitrotoluene (TNT), a commonly used explosive, induced reproductive toxicity. To clarify whether the toxicity results from the interference of endocrine systems or direct damage to reproductive organs, we examined the effects of TNT on the male reproductive system in Fischer 344 rats. TNT administration induced germ cell degeneration, the disappearance of spermatozoa in seminiferous tubules, and a dramatic decrease in the sperm number in both the testis and epididymis. TNT increased the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in sperm whereas plasma testosterone levels did not decrease. These results suggest that TNT-induced toxicity is derived from direct damage to spermatozoa rather than testosterone-dependent mechanisms. To determine the mechanism of 8-oxodG formation in vivo , we examined DNA damage induced by TNT and its metabolic products in vitro . 4-Hydroxylamino-2,6-dinitrotoluene, a TNT metabolite, induced Cu(II)-mediated damage to 32 P-labeled DNA fragments and increased 8-oxodG formation in calf thymus DNA, although TNT itself did not. DNA damage was enhanced by NADH, suggesting that NADH-mediated redox reactions involving TNT metabolites enhanced toxicity. Catalase and bathocuproine inhibited DNA damage, indicating the involvement of H 2 O 2 and Cu(I). These findings suggest that TNT induces reproductive toxicity through oxidative DNA damage mediated by its metabolite. We propose that oxidative DNA damage in the testis plays a role in reproductive toxicity induced by TNT and other nitroaromatic compounds.     

Antioxidant prenylated phenols of Artocarpus plants attenuate ultraviolet radiation-induced damage on human keratinocytes and fibroblasts

Two novel prenylated phenols, cyclocomunoindenol (1) and cyclocomunohexanol (2) were isolated from the cortex of roots of Artocarpus communis. Their structures were determined by spectroscopic methods. Compounds 2 and 6 revealed significant DPPH-scavenging activity with an IC₅₀ values of 435.48 ± 0.93 and 53.55 ± 8.73 μM, respectively. Compounds 1, 2, and 6 displayed significant ABTS⁺ scavenging activity with an IC₅₀ values of 164.26 ± 2.44, 227.01 ± 3.64, and 44.09 ± 0.88 μM, respectively. Compound 6 exhibited an inhibitory effect on XO activity with an IC₅₀ value of 10.91 ± 1.77 μM and the relative oxygen radical absorbance capacity (ORAC) values of 1–6, using ORAC-pyrogallol red (PGR) assay, were determined to be 0.28 ± 0.06, 0.31 ± 0.02, 0.55 ± 0.25, 0.84 ± 0.36, 0.35 ± 0.15, and 0.70 ± 0.09, respectively. Antioxidants 5 and 6 significantly attenuate UVA radiation-induced damage on human HaCaT keratinocytes and Hs68 fibroblasts. These finding showed that 1–6 may be used as antioxidants and 5 and 6 may protect skin against the adverse effects of UV radiation.     

Glycosylation of human LDL and its metabolism in human skin fibroblasts

Extrachromosomal DNA of heterogeneous size has been isolated from bursal lymphocytes and splenocytes of five week old chickens, and from splenocytes of mice. This DNA contains covalently closed circles, open circles, and open circles with tails. Open circular molecules with and without tails are more frequent than covalently closed species, and the total number of small circular DNA molecules per cell is in the range of 100–200 copies.     

The specificity of UVA-induced DNA damage in human melanocytes

Exposure to solar UV radiation is the origin of most skin cancers, including deadly melanomas. Melanomas are quite different from keratinocyte-derived tumours and exhibit a different mutation spectrum in the activated oncogenes, possibly arising from a different class of DNA damage. In addition, some data suggest a role for UVA radiation in melanomagenesis. To get further insight into the molecular mechanisms underlying induction of melanoma, we quantified a series of UV-induced DNA damage in primary cultures of normal human melanocytes. The results were compared with those obtained in keratinocytes from the same donors. In the UVB range, the frequency and the distribution of pyrimidine dimers was the same in melanocytes and keratinocytes. UVA was also found to produce thymine cyclobutane dimer as the major DNA lesion with an equal efficiency in both cell types. In contrast, following UVA-irradiation a large difference was found for the yield of 8-oxo-7,8-dihydroguanine; the level of this product was 2.2-fold higher in melanocytes than in keratinocytes. The comet assay showed that the induction of strand breaks was equally efficient in both cell types but that the yield of Fpg-sensitive sites was larger in melanocytes. Our data show that, upon UVA irradiation, oxidative lesions contribute to a larger extent to DNA damage in melanocytes than in keratinocytes. We also observed that the basal level of oxidative lesions was higher in the melanocytes, in agreement with a higher oxidative stress that may be due to the production of melanin. The bulk of these results, combined with qPCR and cell survival data, may explain some of the differences in mutation spectrum and target genes between melanomas and carcinomas arising from keratinocytes.     

3,3'-Dihydroxyisorenieratene and isorenieratene prevent UV-induced DNA damage in human skin fibroblasts

Skin cancer is among the most frequent neoplastic malignancies and exposure to UV irradiation is a major risk factor. In addition to topical sunscreens, photoprotection by dietary antioxidants such as carotenoids or polyphenols has been suggested as a means of prevention. Isorenieratene (IR) and dihydroxyisorenieratene (DHIR) are aromatic carotenoids with particular antioxidant properties produced by Brevibacterium linens. The aim of this study was to investigate the photoprotective and antioxidant activities of DHIR and IR in comparison to the nonaromatic carotenoid lutein in human dermal fibroblasts. Incubation of the cells with DHIR and IR significantly decreased the UV-induced formation of cyclobutane pyrimidine dimers and formation of DNA strand breaks. Lipid oxidation was lowered as determined by the formation of malondialdehyde as a biomarker. Both aromatic carotenoids also prevented oxidatively generated damage to DNA as demonstrated by a decrease in DNA strand breaks associated with the formation of oxidized DNA bases. These data highlight the multifunctional photoprotective properties of aromatic carotenoids, which may be suitable natural compounds for the prevention of skin cancer.     

UVA-induced oxidative damage to rat liver nuclei: reduction of iron ions and the relationship between lipid peroxidation and DNA damage

Lipid peroxidation and DNA damage and the relationship between the two events were studied in rat liver nuclei irradiated with low dose UVA. Lipid peroxidation was measured as thiobarbituric acid-reactive substances (TBARS) by spectrophotometric method and as malondialdehyde-TBA adduct by HPLC, and DNA damage was measured as 8-hydroxy-deoxyguanosine (8-OH-dGu) and strand breakage (or loss of double-stranded DNA) by a fluorometric analysis of alkaline DNA unwinding method. The results show that UVA irradiation by itself increased nuclear lipid peroxidation but caused little or no DNA strand breakage or 8-OH-dGu. When 0.5 mM ferric (Fe+3) or ferrous (Fe+2) ions were added to the nuclei during UVA irradiation, lipid peroxidation and DNA damage, measured both as 8-OH-dGu and loss of double-stranded DNA, were strongly enhanced. Lipid peroxidation occurred concurrently with the appearance of 8-OH-dGu. Fe3+ ions were reduced to Fe2+ in this UVA/Fe2+/nuclei system. Lipid peroxidation and DNA damage were neither inhibited by scavengers of hydroxyl radical and singlet oxygen nor inhibited by superoxide dismutase and catalase. Inclusion of EDTA or chain-breaking antioxidants, butylated hydroxytoluene (BHT) and diphenylamine (an alkoxy radical scavenger), inhibited lipid peroxidation but not the level of 8-OH-dGu. BHT also did not inhibit the loss of double-stranded DNA in this system. This study demonstrates the reduction of exogenous Fe+3 by UVA when added to rat liver nuclei, and, as a result, oxidative damage is strongly enhanced. In addition, the results show that DNA damage is not a result of lipid peroxidation in this UVA/Fe2+/nuclei system.     

Lignin-derived lignophenols attenuate oxidative and inflammatory damage to the kidney in streptozotocin-induced diabetic rats

Abstract

This study investigated the effects of lignin-derived lignophenols (LPs) on the oxidative stress and infiltration of macrophages in the kidney of streptozotocin (STZ)-induced diabetic rats. The diabetic rats were divided into four groups with 0%, 0.11%, 0.33% and 1.0% LP diets. The vehicle-injected controls were given a commercial diet. At 5 weeks, superoxide (O₂^?) production, macrophage kinetics, the degree of fibrosis in glomeruli and mRNA expression for monocyte chemoattractant protein-1 (MCP-1) were examined. The NADPH-stimulated O₂^? levels in the kidney of the diabetic rats treated with 1.0% LP were significantly lower than those in untreated diabetic rats. The number of macrophages, levels of MCP-1 mRNA expression and degree of glomerular fibrosis increased in untreated LP and these levels were significantly lower in 1.0%LP-treated rats. The results suggested that LPs suppress the excess oxidative stress, the infiltration and activation of macrophages and the glomerular expansion in STZ-induced diabetic kidneys.     

Juglans mandshurica leaf extract protects skin fibroblasts from damage by regulating the oxidative defense system

Skin is mainly damaged by genetic and environmental factors such as ultraviolet light, xenobiotics, hormonal changes, heat, and smoking. ROS production is commonly involved in the pathogenesis of skin damage induced by these factors, causing skin aging, including wrinkling, by activating the metalloproteinases (MMP-1) that break down type I collagen (COL1A1). The walnut tree Juglans mandshurica MAX. (JM) is found in China, Siberia and Korea. JM has been reported to have various pharmacological activities, such as anti-tumor, anti-oxidative, and anti-bacterial effects. In the present study, we investigated the protective effect of JM leaf extract (JME) against oxidative stress in HS68 human skin fibroblasts. JME significantly and dose-dependently protected HS68 cells against H₂O₂-induced damage, as assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and lactate dehydrogenase assay. Other assays demonstrated that JME protected HS68 cells by regulating ROS production and increasing levels of glutathione, heme oxygenase-1, and activated NF-E2-related factor 2. JME additionally prevented the elevation of MMP-1 and reduction of COL1A1 induced by H₂O₂. It also inhibited H₂O₂-induced phosphorylation of ERK, p38, and JNK. These results indicate that JME protects human skin fibroblasts from H₂O₂-induced damage by regulating the oxidative defense system.     

Deep senescent human fibroblasts show diminished DNA damage foci but retain checkpoint capacity to oxidative stress

Critically shortened telomeres trigger a DNA damage response in replicatively senescent cells. Here we report that while DNA damage foci can be detected in newly senescent cells, these foci eventually diminished in deep senescent cells. However, DNA checkpoint signalling and repair machinery in response to oxidative stress remain uncompromised in these deep senescent cells. Activation of p53 by oxidative stress is unaffected despite a marked decrease in expression of platelet-derived growth factor alpha-receptor. These findings suggest that cellular senescence is not a static process hence care must be taken in the selection of biomarkers of senescence in studies of ageing.     

Effect of antioxidant supplementation on the adaptive response of human skin fibroblasts to UV-induced oxidative stress

The effect of supplementation with substances having antioxidant properties on the adaptive responses of human skin fibroblasts to UV-induced oxidative stress was studied in vitro. UVR was found to induce a substantial oxidative stress in fibroblasts, resulting in an increased release of superoxide anions and an increase in lipid peroxidation (shown by an elevated malonaldehyde content). Sub-lethal doses of UVR were also found to induce adaptive responses in the fibroblast antioxidant defences, with a transient rise in catalase and superoxide dismutase activities followed by a slower, large increase in cellular glutathione content. Supplementation of the fibroblasts with the antioxidants, Trolox (a water soluble analogue of alpha-tocopherol), ascorbic acid or beta-carotene, had differential effects on these responses. Trolox supplementation reduced the UVR-induced cellular oxidative stress and adaptive response in a predictable concentration-dependent manner. This was in contrast to ascorbic acid which increased superoxide release from fibroblasts. At low doses, ascorbate supplements also reduced the magnitude of the adaptive increases in catalase and superoxide dismutase activities and increase in glutathione content. Beta-carotene had a similar effect to ascorbic acid, reducing the extent of the adaptations to UVR at lower doses while simultaneously increasing superoxide release and malonaldehyde content. These in vitro data indicate that only the vitamin E analogue suppressed UVR-induced oxidative stress in a predictable manner and suggest that common dietary antioxidants may not be equally effective in reducing the potential deleterious effects of UVR-induced oxidative stress in skin.     

Effect of antioxidant supplementation on the adaptive response of human skin fibroblasts to UV-induced oxidative stress

Abstract

The effect of supplementation with substances having antioxidant properties on the adaptive responses of human skin fibroblasts to UV-induced oxidative stress was studied in vitro. UVR was found to induce a substantial oxidative stress in fibroblasts, resulting in an increased release of superoxide anions and an increase in lipid peroxidation (shown by an elevated malonaldehyde content). Sub-lethal doses of UVR were also found to induce adaptive responses in the fibroblast antioxidant defences, with a transient rise in catalase and superoxide dismutase activities followed by a slower, large increase in cellular glutathione content. Supplementation of the fibroblasts with the antioxidants, Trolox (a water soluble analogue of α-tocopherol), ascorbic acid or β-carotene, had differential effects on these responses. Trolox supplementation reduced the UVR-induced cellular oxidative stress and adaptive response in a predictable concentration-dependant manner. This was in contrast to ascorbic acid which increased superoxide release from fibroblasts. At low doses, ascorbate supplements also reduced the magnitude of the adaptive increases in catalase and superoxide dismutase activities and increase in glutathione content. β-Carotene had a similar effect to ascorbic acid, reducing the extent of the adaptations to UVR at lower doses while simultaneously increasing superoxide release and malonaldehyde content. These in vitro data indicate that only the vitamin E analogue suppressed UVR-induced oxidative stress in a predictable manner and suggest that common dietary antioxidants may not be equally effective in reducing the potential deleterious effects of UVR-induced oxidative stress in skin.     

Nonenzymatic galactosylation of human LDL decreases its metabolism by human skin fibroblasts

Incubation of human LDL with galactose in vitro resulted in a glycosylated-LDL containing radiolabel covalently attached to apo B. The rate of radiolabel incorporation was proportional to the time of incubation and concentration of carbohydrate. The rate of incorporation of galactose into apo B was higher than with glucose or mannose. The nonenzymatic glycosylation of LDL decreased its uptake and metabolism by the high affinity, receptor dependent process for LDL in normal human skin fibroblasts.     

Repair response of human fibroblasts to bleomycin damage

The ability of human fibroblasts to repair the specific types of DNA damage caused by bleomycin (BLM) was examined in whole-cell experiments. The method utilized for analysis was alkaline sucrose-gradient centrifugation of DNA. The results of these studies show that a repair pathway exists for the damage produced in DNA by bleomycin. DNA from BLM-treated cells shows a decrease in molecular weight, caused by chemical or enzymatic incision at sites of drug action. If the drug is removed, the DNA rapidly returns to high molecular weight, demonstrating reformation of damaged DNA. This repair response to BLM-damage was also confirmed in fibroblasts isolated from patients with putative DNA-repair defects. We observed that the response (to BLM) of cells from patients with Fanconi anemia was altered in that the fall in molecular weight of DNA from treated cells was not as great as that observed in other cell strains after drug treatment.     

Bisphenol A and its methylated congeners inhibit growth and interfere with microtubules in human fibroblasts in vitro

Bisphenol A (BPA), a monomer of polycarbonate plastics and epoxy resins, has previously been reported to induce micronuclei containing whole chromosomes in Chinese hamster V79 cells. In the present study, the aneuploidogenic potential of BPA was investigated in cultured human AG01522C fibroblasts. In contrast to the known aneugens diethylstilbestrol (DES) and 17beta-estradiol, which caused mitotic arrest and the induction of kinetochore-positive micronuclei, BPA did not induce micronuclei and inhibited the proliferation of AG01522C cells in G2 phase and probably also in G1 phase. Fluorescence microscopy of the BPA-treated cells after immunofluorescent staining of microtubules revealed structural abnormalities of the cytoplasmic microtubule complex (CMTC): densely stained rings and loops of tubulin were observed, which increased in number with increasing BPA concentration and were more stable against low temperature than normal microtubules. The mechanisms of the growth inhibition and the interference with microtubules elicited by BPA in AG01522C cells are presently unknown. The formation of rings and loops in the CMTC of AG01522C cells was also observed with two congeners of BPA carrying one and two, respectively, additional methyl groups in ortho-position to the phenolic hydroxyl group at each aromatic ring. However, in contrast to BPA itself, these congeners of BPA behaved "DES-like" by inducing mitotic arrest and kinetochore-positive micronuclei in AG01522C cells.     

Potentially lethal damage repair in cell lines of radioresistant human tumours and normal skin fibroblasts

Radiation cell survival data were obtained in vitro for three cell lines isolated from human tumours traditionally considered to be radioresistant--two melanomas and one osteosarcoma--as well as from a diploid skin fibroblast cell line. One melanoma cell line was much more radioresistant than the other, while the osteosarcoma and fibroblast cell lines were more radiosensitive than either. For cells growing exponentially, little potentially lethal damage repair (PLDR) could be demonstrated by comparing survival data for cells in which subculture was delayed by 6 h with those sub-cultured immediately after treatment. For the malignant cells in plateau phase, which in these cells might be better termed 'slowed growth phase', since an appreciable fraction of the cells are still cycling, a small amount of PLDR was observed, but not as much as reported by other investigators in the literature. The normal fibroblasts, which achieved a truer plateau phase in terms of noncycling cells, showed a significantly larger amount of PLDR than the tumour cells.     

Humic acid induces oxidative DNA damage,growth retardation,and apoptosis in human primary fibroblasts

Humic acid (HA) has been implicated as an etiological factor of Blackfoot disease endemic in the southwest coast of Taiwan. Dysfunction of endothelial cells and vasculopathy have been proposed to explain the onset of ulcerous changes at extremities. However, little is known about the effect of HA on activities of cells in these nonhealing wounds. In the present study, we demonstrate that HA adversely affects the growth properties of fibroblasts, one of the key players in wound repair. HA treatment caused growth arrest and apoptosis in human foreskin fibroblasts (HFF). This was accompanied by a significant increase in the level of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in cellular DNA. The increased fluorescence in dichlorofluorescin (H2DCF)-stained and HA-treated cells suggests the involvement of reactive oxygen species (ROS) in HA-induced biological effects. Conversely, vitamin E pretreatment, which significantly reduced the 8-OHdG formation in HA-treated cells, alleviated the growth-inhibitory and apoptosis-inducing effects of HA. These results indicate that HA initiates oxidative damages to fibroblasts, and leads to their dwindling growth potential and survival. The present study suggests that HA-induced growth retardation and apoptosis of fibroblasts may play a role in the pathogenesis of Blackfoot disease.