DNA-protein cross-linking by ultraviolet radiation in normal human and xeroderma pigmentosum fibroblasts.

DNA-protein cross-linking by ultraviolet radiation was measured in human fibroblasts by an adaptation of the method of DNA alkaline elution. To measure cross-linking, a controlled frequency of DNA single-strand breaks was introduced by exposing the cells to a low dose of X-ray at 0 degrees C prior to analysis by alkaline elution. The effect of prior exposure of the cells to ultraviolet radiation was to reduce the rate and/or extent of DNA elution from X-irradiated cells. This effect was attributed to DNA-protein cross-linking, since the effect was reversed by treatment of the cell lysates with proteinase-K. Cross-linking in normal human fibroblasts occurred immediately after ultraviolet irradiation, prior to the appearance of DNA single-strand breaks due to excision repair. Upon incubation of normal cells after exposure, to ultraviolet radiation, the cross-linking was partially repaired. In xeroderma pigmentosum cells, cross-links appeared as in normal cells, but there was no repair. Instead, the extent of cross-linking appeared to increase upon incubation after ultraviolet irradiation.     

Enhancement of radiation-induced DNA-protein crosslinking by N-methylformamide

The effects of the differentiating agent N-methylformamide (NMF) on radiation-induced DNA damage and repair in vitro were investigated using the alkaline elution assay. Two tumor cell lines were examined: Clone A, a human colon adenocarcinoma, and HCA-1, a murine hepatocarcinoma. Both cell lines showed changes suggestive of a better differentiated phenotype when exposed to NMF. Treatment with NMF enhanced the radiation sensitivity of Clone A cells but had no effect on the radiation response of HCA-1 cells. Irradiation of NMF-treated cells, both Clone A and HCA-1, induced the formation of DNA-protein crosslinks (DPCs). The level of DPCs induced increased linearly as a function of increasing gamma-ray dose. The DPCs did not seem to be the result of NMF exposure alone, but rather an NMF-mediated modification of the spectrum of gamma-ray-induced DNA lesions. When the DPCs were removed by proteolytic digestion, no NMF effect was observed on either strand-break formation or repair.     

DNA-protein crosslinks as a biomarker of exposure to solar radiation: a preliminary study in brick-kiln workers.

In India, fired clay bricks are produced in small-scale factories. There are 60, 000 active brick kilns, providing employment to nearly 12 million people in different suboccupations. This industry is largely non-mechanized and operates from November to June. Almost all the workers are exposed to direct sunlight for 8-10 h a day. Cellular DNA-protein crosslinks (DPCs) are the biologically active nucleoprotein complexes formed between DNA and proteins. Ultraviolet light and gamma-rays, and other suspected carcinogens in humans, induce DPC formation in blood cells. DPCs have therefore been identified as a biomarker for monitoring exposure to these hazardous agents. Here we report steady-state levels of DPCs in human peripheral lymphocytes from 46 brick-kiln workers exposed occupationally for 8-10 h a day to solar radiation in brickfields and 25 unexposed controls. A significant increase (p <0.05) in DPC content and DPC coefficients in peripheral lymphocytes was observed in the brick-kiln workers compared with the controls. The data suggest that the DPC content of lymphocytes could be a possible biomarker of exposure to solar radiation. However, further work is necessary to confirm this.     

Role of light in human skin color viariation.

The major source of color in human skin derives from the presence within the epidermis of specialized melanin-bearing organelles, the melanosomes. Tanning of human skin on exposure to ultraviolet light results from increased amounts of melanin within the epidermis. Melanosomes synthesized by melanocytes are acquired by keratinocytes and transported within them to the epidermal surface. In some cases, the melanosomes are catobolized en route. New information indicates that the multicellular epidermal melanin unit (melanocyte and associated pool of keratinocytes) rather than the melanocyte alone is the focal point for the control of melanin metabolism within mammalian epidermis. Gross human skin color derives from the visual impact of the summed melanin pigmentation of the many epidermal melanin units. In theory, constitutive skin color in man designates the genetically-determined levels of melanin pigmentation developed in the absence of exposure to solar radiation or other environmental influences; facultative skin color or "tan" characterizes the increases in melanin pigmentation above the constitutive level induced by ultraviolet light. The details of genetic regulation of pigment metabolism within the epidermal melanin units are being clarified. In some mammals at least, the function of epidermal melanin units is significantly influenced by hormones which may be regulated by radiations received through the eyes. Based on an evolutionary history of the human family which exceeds ten million years, it is proposed that melanin pigmentation may have played a number of roles in human adaptions to changing biologic and physical environments.     

Motility and orientation of a dinoflagellate, Gymnodinium, impaired by solar and ultraviolet radiation

Abstract The effects of solar and artificial ultraviolet radiation on the motility and orientation of the dinoflagellate Y-100 were studied. The cells show a weak photokinesis but a pronounced phototaxis which is consistently positive between 1 and 100 klx (= 4 mW m⁻² to 400 mW m⁻²); the precision of orientation increases with the fluence rate. Unfiltered solar radiation as well as artificial ultraviolet radiation reduce the percentage of motile cells increasingly with exposure time but the velocity of the still motile cells is less affected. Unirradiated control cells show a negative gravitaxis. After short exposure to solar or artificial ultraviolet radiation the precision of gravitaxis decreases and after prolonged exposure the cells start to actively move downward in the water column (positive gravitaxis). Phototaxis is also strongly impaired by ultraviolet radiation.     

Protective action of bacterial melanin against DNA damage in full UV spectrums by a sensitive plasmid-based noncellular system

The purpose of this study was to introduce a simple and sensitive plasmid-based noncellular system to evaluate the photoprotection of bacterial melanin on DNA damage against ultraviolet (UV) radiation. Plasmid DNA was used to assess the role of melanin in different ranges of UV using a series of in vitro assays. Fluorometric measurements suggested that melanin could efficiently scavenge reactive oxygen species (ROS) generated by UVA irradiation in solution, and the scavenging capability was proportional to the pigment concentration. The protective effect of melanin on plasmid DNA under UVB irradiation was confirmed by the transformation efficiency of the protected DNA, which was at least 10-fold higher than that of the non melanin protected DNA. After the UVC irradiation, the DNA damage of strand breaks was quantified by laser-induced fluorescence capillary electrophoresis. The percentage of supercoiled plasmid was reduced from 80% to less than 5% without melanin protection. In contrast, the percentage of supercoiled DNA only decreased to about 40% in the presence of melanin under the same radiation conditions. All these results demonstrated that bacterial melanin did protect DNA from being damaged throughout full UV irradiation. This system, avoiding the potential interference by cellular DNA repair machinery and intracellular substances, may provide a sensitive in vitro means to evaluate the functions of melanin and other photoprotective compounds from different sources.     

Effects of PGF2alpha on human melanocytes and regulation of the FP receptor by ultraviolet radiation

Prostaglandins are potent lipid hormones that activate multiple signaling pathways resulting in regulation of cellular growth, differentiation, and apoptosis. In the skin, prostaglandins are rapidly released by keratinocytes following ultraviolet radiation and are chronically present in inflammatory skin lesions. We have shown previously that melanocytes, which provide photoprotection to keratinocytes through the production of melanin, express several receptors for prostaglandins, including the PGE2 receptors EP1 and EP3 and the PGF2alpha receptor FP, and that PGF2alpha stimulates melanocyte dendricity. We now show that PGF2alpha stimulates the activity and expression of tyrosinase, the rate-limiting enzyme in melanin synthesis. Analysis of FP receptor regulation showed that the FP receptor is regulated by ultraviolet radiation in melanocytes in vitro and in human skin in vivo. We also show that ultraviolet irradiation stimulates production of PGF2alpha by melanocytes. These results show that PGF2alpha binding to the FP receptor activates signals that stimulate a differentiated phenotype (dendricity and pigmentation) in melanocytes. The regulation of the FP receptor and the stimulation of production of PGF2alpha in melanocytes in response to ultraviolet radiation suggest that PGF2alpha could act as an autocrine factor for melanocyte differentiation.     

Ionizing radiation enhances IL-6 and IL-8 production by human endothelial cells

Irradiation exposure is known to induce an inflammatory reaction. Endothelial cells play a crucial role both in the inflammatory process and in radiation damage. Therefore, supernatants and cell lysates of (60)Co-irradiated human umbilical vein endothelial cells (HUVEC) have been assessed for the presence of pro-inflammatory cytokines. After gamma irradiation, interleukin (IL)-1alpha, IL-1beta and tumor necrosis factor (TNF)-alpha remained undetectable in both cell supernatants and cell lysates. However, a dose-dependent increase in the production of IL-6 and IL-8 has been demonstrated up to 6 days after exposure. These data indicate that the pro-inflammatory cytokines IL-6 and IL-8 may be involved in the inflammatory response of vascular endothelium induced by exposure to ionizing radiation.     

The Role of Alpha-Synuclein in Melanin Synthesis in Melanoma and Dopaminergic Neuronal Cells

The relatively high co-occurrence of Parkinson’s disease (PD) and melanoma has been established by a large number of epidemiological studies. However, a clear biological explanation for this finding is still lacking. Ultra-violet radiation (UVR)-induced skin melanin synthesis is a defense mechanism against UVR-induced damage relevant to the initiation of melanoma, whereas, increased neuromelanin (NM), the melanin synthesized in dopaminergic neurons, may enhance the susceptibility to oxidative stress-induced neuronal injury relevant to PD. SNCA is a PD-causing gene coding for alpha-Synuclein (α-Syn) that expresses not only in brain, but also in skin as well as in tumors, such as melanoma. The findings that α-Syn can interact with tyrosinase (TYR) and inhibit tyrosine hydroxylase (TH), both of which are enzymes involved in the biosynthesis of melanin and dopamine (DA), led us to propose that α-Syn may participate in the regulation of melanin synthesis. In this study, by applying ultraviolet B (UVB) light, a physiologically relevant stimulus of melanogenesis, we detected melanin synthesis in A375 and SK-MEL-28 melanoma cells and in SH-SY5Y and PC12 dopaminergic neuronal cells and determined effects of α-Syn on melanin synthesis. Our results showed that UVB light exposure increased melanin synthesis in all 4 cell lines. However, we found that α-Syn expression reduced UVB light-induced increase of melanin synthesis and that melanin content was lower when melanoma cells were expressed with α-Syn, indicating that α-Syn may have inhibitory effects on melanin synthesis in melanoma cells. Different from melanoma cells, the melanin content was higher in α-Syn-over-expressed dopaminergic neuronal SH-SY5Y and PC12 cells, cellular models of PD, than that in non-α-Syn-expressed control cells. We concluded that α-Syn could be one of the points responsible for the positive association between PD and melanoma via its differential roles in melanin synthesis in melanoma cells and in dopaminergic neuronal cells.     

The role of melanocytes in protection against photooxidative stress Krystyna Stepień

Excessive exposure to solar ultraviolet radiation is an essential etiological factor for skin cancer. UV radiation, directly or indirectly through the generation of reactive oxygen species (ROS), causes damage to DNA, proteins and lipids, and induces inflammation and immunosuppression. Cutaneous pigmentation afforded by melanocytes is the main photoprotective mechanism in human skin. In response to UV, melanocytes produce melanin pigments and transfer them to adjacent keratinocytes. This review describes: (i) the photoprotective action of melanin; (ii) the regulation of UV-induced melanogenesis and the role of p53 in this process; (iii) the relation between melanogenic and antioxidant activities in melanocytes. The possible involvement of UV-induced ROS in the stimulation of melanin synthesis is also discussed.     

Formation of bone-like tissue by dental follicle cells co-cultured with dental papilla cells

During tooth root formation, dental follicle cells (DFCs) differentiate into osteoblasts/cementoblasts when they are in contact with pre-existing dentin. Since some factors of dentin matrix were also produced by dental papilla cells (DPCs) and could induce DFCs differentiation, we hypothesized that DPCs can directly promote DFCs differentiation and that differentiation could occur in a co-culture model. To test this hypothesis, we investigated the characteristics of DFCs that are influenced by DPCs in an in vitro co-culture and in vivo heterotopic transplant model. One week into the co-culture, there were significant increases in the mRNA level of bone morphogenetic protein 2 (BMP2), osteoprotegerin (OPG), bone sialoprotein (BSP) and osteocalcin (OCN), and a decrease of the receptor activator of nuclear factor κB ligand (RANKL). Additionally, the number of BMP2-, OPG-, BSP- and OCN-positive DFCs increased whereas RANKL-positive DFCs decreased. Three weeks after co-culture, DFCs produced calcified nodules, accompanied with increased sub-cellular organelles for protein synthesis and secretion. In the heterotopic transplant model, the adult male rats were used as hosts, DFCs were transplanted into the omentum. In vivo 5-week growth of DFCs in the presence of DPCs led to the formation of bone-like tissues, positive for BSP, OCN and BMP2. In contrast, DFCs alone led to fibrous-like tissues. These results indicated that in the absence of pre-existing dentin, DPCs can stimulate osteogenesis and inhibit osteoclastogenesis in DFCs and suggested a novel strategy to promote DFCs differentiation.     

Melanin decreases clastogenic effects of ionizing radiation in human and mouse somatic cells and modifies the radioadaptive response

Melanin’s influence on the chromosome aberration frequency induced by radiation in human lymphocytes and mouse bone marrow cells has been studied. We revealed earlier that melanin significantly decreases the frequencies of different radiation-induced mutations in animal germ cells. Melanin protection in somatic cells has been found to be less effective. The melanin effect in somatic cells depends on radiation dose: the lower the damage level, the better the melanin protection. In order to determine the influence of melanin at low radiation doses, the adaptive response was investigated in mouse bone marrow cells in vivo. The level of chromosome aberrations in these cells after fractionated irradiation of 0.2 Gy+1.5 Gy with a 4-h interval was about half that after a single dose of 1.7 Gy. If melanin was injected prior to irradiation, the aberration level decreased by a factor of about two in both cases. This observed result may be due to the potential radioprotective effect of melanin and to the absence of any adaptive response, whereas in the case of melanin application between the priming and challenge doses, the combined effect of the adaptive response as well as melanin protection resulted in a 4-fold decrease of chromosome aberrations. These results allow us to draw the following conclusions: adaptive response can be prevented by a radioprotector such as melanin, and melanin is capable of completely removing low-dose radiation effects. Received: 2 December 1998 / Accepted in revised form: 15 September 1999     

Melanosomal dynamics assessed with a live-cell fluorescent melanosomal marker

Melanocytes present in skin and other organs synthesize and store melanin pigment within membrane-delimited organelles called melanosomes. Exposure of human skin to ultraviolet radiation (UV) stimulates melanin production in melanosomes, followed by transfer of melanosomes from melanocytes to neighboring keratinocytes. Melanosomal function is critical for protecting skin against UV radiation, but the mechanisms underlying melanosomal movement and transfer are not well understood. Here we report a novel fluorescent melanosomal marker, which we used to measure real-time melanosomal dynamics in live human epidermal melanocytes (HEMs) and transfer in melanocyte-keratinocyte co-cultures. A fluorescent fusion protein of Ocular Albinism 1 (OA1) localized to melanosomes in both B16-F1 cells and HEMs, and its expression did not significantly alter melanosomal distribution. Live-cell tracking of OA1-GFP-tagged melanosomes revealed a bimodal kinetic profile, with melanosomes exhibiting combinations of slow and fast movement. We also found that exposure to UV radiation increased the fraction of melanosomes exhibiting fast versus slow movement. In addition, using OA1-GFP in live co-cultures, we monitored melanosomal transfer using time-lapse microscopy. These results highlight OA1-GFP as a specific and effective melanosomal marker for live-cell studies, reveal new aspects of melanosomal dynamics and transfer, and are relevant to understanding the skin's physiological response to UV radiation.     

光谱法研究甲醇和盐酸对短梗霉黑色素的分离效果

应用紫外和红外光谱对甲醇和盐酸分离的短梗霉黑色素进行了分析。分析结果表明:甲醇和盐酸法分离的黑色素在紫外图谱215 nm处都有最大吸收峰,而甲醇法分离的黑色素其紫外图谱在260、280 nm处无吸收峰表明此法分离的黑色素不含核酸及蛋白质;红外图谱中,在3340 cm-1、1637 cm-1处有很强的吸收峰表现为黑色素的典型特征;同时在对照中发现盐酸多次处理后的黑色素显示较少的结构信息,说明盐酸沉降法有可能破坏黑色素的结构。由此选用甲醇作为沉淀剂有利于黑色素的纯化及确保其结构信息的完整,为进一步分析短梗霉黑色素的结构表征奠定了基础。     

Isolation of a novel strain of Aeromonas media producing high levels of DOPA-melanin and assessment of the photoprotective role of the melanin in bioinsecticide applications

AIMS: To isolate a bacterium that produces high yield of melanin and to examine the effect of this bacterial pigment on the efficacy of a bioinsecticide. METHODS AND RESULTS: A novel melanin-producing bacterium, designated as strain WS, was isolated from the East Lake, Wuhan, China. Taxonomic studies of this strain indicate that it belongs to Aeromonas media. Physicochemical analysis of the pigment produced by strain WS (melanin WS) suggests that it is the authentic 3,4-dihydroxyphenylalanine (DOPA)-melanin. This melanin and that produced by Pseudomonas maltophilia P28 (melanin P28) share many biophysical properties, but the yield of the melanin WS is significantly higher than that of the melanin P28. In addition, the melanin WS appears to be more effective in the protection of a bioinsecticide against ultraviolet (UV) or solar radiation. At the concentration of 10 ppm, the melanin P28 exhibited no photoprotective effect on the bioinsecticide against UV radiation; in contrast, 5 ppm of melanin WS displayed an obvious protective effect. Similarly, the melanin WS displayed more protective effect on the bioinsecticide against solar radiation than the melanin P28 did over a 4-day period, with the effect being more dramatic for the last 2 days. CONCLUSIONS: We have isolated a novel bacterial strain of A. media that produces high levels of melanin. The melanin produced by this strain offers effective photoprotection of a commercial bioinsecticide BTI against UV and solar radiation. SIGNIFICANCE AND IMPACT OF THE STUDY: Our study suggests that the melanin produced by our newly isolated A. media strain has the potential to be used as a general photoprotective agent for bioinsecticides.     

Ultraviolet stimulated melanogenesis by human melanocytes is augmented by di-acyl glycerol but not TPA

Epidermal melanocytes (MC) synthesise melanin in response to ultraviolet radiation (UVR). The mechanisms mediating the UV-induced activation of melano-genesis are unknown but since UVR induces turnover of membrane phospholipids generating prostaglandins (PGs) and other products, it is possible that one of these might provide the activating signal. We have examined the effects of prostaglandins (PGs) E₁, E₂, D₂, F_2α, and di-acyl glycerol upon the UV-induced responses of cultured human MC and the Cloudman S91 melanoma cell line. The PGs had little effect on unirradliated cells and did not alter the response to UVR in either human MC or S91 melanoma cells. However, a synthetic analogue of di-acyl glycerol, 1-oleyl 2-acetyl glycerol (OAG), caused a significant (P<0.0001), dose-related augmentation of melanin content both in human MC (seven-fold) and S91 cells (three-fold). UVR caused a significant augmentation of the OAG-induced melanognesis of both human MC and S91 cells. Since OAG is known to activate protein kinase C, it was possible that the observed modulation of the UVR signal could be via that pathway. Di-octanoyl glycerol, another di-acyl glycerol, which activates kinase C, caused a small (70%) increase in melanogenesis in MC which was not altered by UVR. However, 12-0 tetradecanoyl phorbol 13-acetate (TPA), a potent activator of protein kinase C, had no significant effect on either basal or UV-induced melanin synthesis in either cell type. These data suggest that the UV-induced signal activating melanogenesis could be mediated by di-acyl glycerol. Furthermore, they imply that the signal is transduced via an alternative, pathway that might be independent of protein kinase C.     

Loss of heterozygosity in yeast can occur by ultraviolet irradiation during the S phase of the cell cycle

A CAN1/can1Δ heterozygous allele that determines loss of heterozygosity (LOH) was used to study recombination in Saccharomyces cerevisiae cells exposed to ultraviolet (UV) light at different points in the cell cycle. With this allele, recombination events can be detected as canavanine-resistant mutations after exposure of cells to UV radiation, since a significant fraction of LOH events appear to arise from recombination between homologous chromosomes. The radiation caused a higher level of LOH in cells that were in the S phase of the cell cycle relative to either cells at other points in the cell cycle or unsynchronized cells. In contrast, the inactivation of nucleotide excision repair abolished the cell cycle-specific induction by UV of LOH. We hypothesize that DNA lesions, if not repaired, were converted into double-strand breaks during stalled replication and these breaks could be repaired through recombination using a non-sister chromatid and probably also the sister chromatid. We argue that LOH may be an outcome used by yeast cells to recover from stalled replication at a lesion.