| BackgroundChronic arsenic poisoning is now a major worldwide public health problem. One of the clinical manifestations of arsenicosis is skin lesions including depigmentation or pigmentation on trunk and hyperkeratosis on palms and soles. Some cases demonstrated with arsenic-related Bowen's disease and skin cancer. Up to now, mechanism of arsenic-related skin pigmentation or depigmentation is still uncertain.People in the epidemic areas of arsenicosis expose to not only arsenic but also many other factors among which ultraviolet is the most important one. On one hand, most of the epidemic areas of arsenicosis are villages, thus, the main patients of arsenicosis are peasants. They expose to arsenic by drinking water containing high level of arsenic and expose to ultraviolet by everyday farm work at the same time. On the other hand, ultraviolet exposure can induce skin pigmentation. Therefore, as far as the research of metabolism of melanin in melanocyte is concerned, the most influential factor is ultraviolet.The metabolism of melanin is a complicated process. The main route is: phenylalanine changed to tyrosine under the action of phenylalanine hydroxylase (PAH) unreversed. Tyrosine then is hydroxylated into DOPA by tyrosinase (TYR). DOPA finally is changed to benzpyrrole-5,6-quinone by a series of action including oxidation and decarboxylation. Melanin is the polymer of benzpyrrole-5,6-quinone. The most important step of this process is changing of DOPA from tyrosine. TYR is the rate-limited enzyme of this process.In this research, we will discuss the mechanism of arsenic-related pigmentation and depigmentation in human melanoma cell and explore whether there are differences of the effects induced between UV exposure and co-exposure of UV and arsenic on melanin metabolism.ObjectivesIn epidemiological investigation research, the level of arsenic in the drinking water, hair, urine and blood of patients will be detected. Skin lesions especially changes of melanocyte will be detected by pathological examination.In the in vitro study, we will make research on the mechanism of arsenic-related pigmentation and depigmentation in human melanoma cell and explore whether there are differences of the effects induced between UV exposure and co-exposure of UV and arsenic on melanin metabolism.Methods1. epidemiological investigationResearch of this part was carried out in Wuyuan, Inner Mogolia. According to the diagnosis standards (WS/T211-2001) of arsenicosis, 26 patients (male 17, female 9) and 10 persons without arsenic exposure (male 5, female 5) were chosen from volunteers.2. detection of arsenic levels in drinking water, hair, urine and blood of patients Arsenic levels in drinking water were detected by high performance liquid chromatography. Arsenic levels in hair, urine and blood of patients were detected by HPLC-HG-AAS.3. pathological examinationSkin samples were collected from patients and stained by HE method.4. culture of human melanoma cell line of G361 and A375Cells were cultured in DMEM supplemented with 10% FBS, 100 U of penicillin/ml, and 100μg of streptomycin/ml. Cultures were maintained at 37℃in a humidified 5% CO2 atmosphere.5. detection of cell viabilityCell viability were detected by CellTiter 96 AQueous One Solution Cell Proliferation Assay.6. detection of melanin106 cells were collected by centrifuge after treated by arsenic or ultraviolet or arsenic plus ultraviolet. Cell pellets for melanin determination was dissolved in 0.5 ml of lmol/L NaOH/10%DMSO by vigorous vortexing, incubated at 100℃for 30min, then optical densities were measured at 400 nm using an ELISA reader. Standard curves of synthetic melanin (0-50μg/ml) were prepared in triplicate for each experiment. Melanin levels were demonstrated by pg/cell.7. TYR activity measurement106 cells were collected by centrifuge after treated by arsenic or ultraviolet or arsenic plus ultraviolet. Cell pellets was dissolved in 0.5 ml PBS (containing 0.1mmol/L PMSF, 0.5%Triton X-100, PH 6.8), then homogenated by ultrasonic. After centrifuge (1000g, 10min), 50μl of supernatant were added into PBS of 500μl (containing 0.1mmol/L tyrosine, 2μCi/ml[3H]tyrosine, 0.1mmol/L L-DOPA, 0.1mmol/L PMSF, PH6.8). After incubation for 2h at 37℃, 1ml charcoal (10%w/v in HC1) were added into the mixture, then vortex for 30s. After centrifuge (2000g, 10min), 100μl of supernatant were added into 5ml scintillation fluid, and TYR activity were measured by TM scintillation spectrometer. Standard curves of mushroom tyrosinase were prepared in triplicate for each experiment. Results were demonstrated by the percentage of control.8. detection of mRNA expression of TYRCells were collected by centrifuge after treated by arsenic or ultraviolet or arsenic plus ultraviolet. Total RNA were extracted by Trizol, then mRNA expression of TYR were detected according to the guide book of real-time PCR kit (Roche, USA, Cat. 04 735 536 001).9. apoptosis rate measurementCells were collected by centrifuge after treated by arsenic or ultraviolet or arsenic plus ultraviolet. Apoptosis rate were measured by MEBCYTO Apoptosis Kit.10. statisticsData were analyzed by SPSS13.0 statistic soft ware. Significant difference were demonstrated by p＜0.05.ResultsLevels of AsⅢ, AsV, organic arsenicals and tAs in water consumed by patients were significantly higher than those of control. Levels of iAs, DMA, and tAs in hair, iAs, MA, DMA and tAs in urine, MA and DMA in blood of patients were significantly higher than those of non-exposure persons.Increased melanin production and apoptosis of melanocyte were found in the pathological samples of melanocyte, Other skin lesions include decreased dermal papilla, thickened basal layer, heterocaryon appeared, cuticular layer disorder, liquid degeneration of basal layer, edema of dermis, infiltration of hypodermal cell, and nucleus of keratinocyte condensed.Cell viability was increased by exposed to NaAsO2 (0.01 and 0.1μmol/L) in both G361 and A375, while no significant differences were found between arsenic treated cells and control. Cell viability decreased as long as arsenic concentration increased. Cell viabilities of 5, 10 and 25μmol/L for 24h exposure and 0.5, 1, 5, 10 and 25μmol/L for 48h exposure were significantly lower than those of control.No significant differences were found between melanin levels of cells treated by NaAsO2 (0.1and 1μmol/L, 72h) and control, as well as TYR activity. No significant differences of mRNA expression were found when cells were exposed to NaAsO2 for short period at low concentration(0.1 and 1μmol/L); mRNA expression of group 12h-10μmol/L, 24h-5μmol/L, 24h-20μmol/L, 48h-5μmol/L and 48h-10μmol/L exposed to NaAsO2 for short period at high concentration (5, 10 and 20μmol/L)were significantly lower than that of control; when cells were exposed to NaAsO2 for long period at low concentration(0.1μmol/L, 16w), mRNA expression at the 8th week was significantly higher than that of control.Apoptosis rates were increased as long as NaAsO2 concentration increased. Apoptosis rate of 20 and 40μmol/L were significantly higher than that of control in both G361 and A375.Melanin levels of 10mJ/cm2 UV group, 0.1μmol/L+10mJ/cm2 group and 1μmol/L+10mJ/cm2 group were significantly higher than those of 5mJ/cm2 UV group, 0.1μmol/L+5mJ/cm2 group and 1μmol/L+5mJ/cm2 group and control. No significant differences have been found between 10mJ/cm2 UV group, 0.1μmol/L+10mJ/cm2 group and 1μmol/L+10mJ/cm2 group. No significant differences have been found in TYR activity and the expression of mRNA compared with control.Melanin levels of G361 in the group of control was significantly higher than that of A375 (p=0.0009). TYR acitivity of G361 (3273.5±12.9 CPM 3[H]) in the group of control was also markedly higher than that of A375 (2520.2±12.4 CPM3[H]) (p=0.0003). Apoptosis rate induced at the same condition was different between G361 and A375. Apoptosis rates of G361 exposed to NaAsO2 of 20 and 40μmol/L was significantly higher than those of A375.ConclusionsArsenic-related skin lesions of arsenicosis patients may have some relationship with high level of MA in blood. Depigmentation induced by arsenic exposure may be resulted by apoptosis of melanocytes.Human melanoma G361 cell line exposed to sodium arsenite for long period at low concentration will lead to significant increase of mRNA expression of TYR, which may result in the increase of melanin production. This might be the reason of arsenic-related pigmentation.NaAsO2 exposure can induce significant increase of apoptosis rate in human melanoma cell line of G361 and A375. increased apoptosis rate may have some relationship with depigmentation.The basal level of melanin production, TYR activity, and apoptosis rate induced by NaAsO2 exposure at the same concentration was different with those of A375. This indicates that individual differences at melanin metabolism may play an important role in the variation of symptoms among those who exposed to arsenic in almost the same environment.No significant differences have been found in the effects induced between UV exposure and co-exposure of UV and arsenic. The increase production of melanin might not be induced by TYR, but some other signal pathways. Arsenic has no synergistic effect with UV on melanin metabolism.
|
PDF Full Text Request