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Effects of Selenium on Apoptosis Induced by Methyl Mercury Chloride in RAW 264.7 Cells
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Keun Snag Kwon, Dai Ha Koh, Jung Ho Youm, Wook Hee Yoon
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Korean Journal of Occupational and Environmental Medicine 2003;15(3):237-251. Published online September 30, 2003
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DOI: https://doi.org/10.35371/kjoem.2003.15.3.237
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 Abstract
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- OBJECTIVE: This study was performed to evaluate the protective effects of selenium against the methyl mercury chloride (MeHgCl) induced cell apoptosis.
METHODS
The effect of selenium on the MeHgCl induced cell apoptosis was observed in mouse macrophage-derived RAW 264.7 cells, in vitro. The cells were cultured in Dulbecco's modified Eagle's medium (DMEM).
RESULTS
MeHgCl exerted a dose dependent cytotoxicity, as demonstrated by the MTT assay, an assay dependent, in part, on mitochondrial function. Concurrent exposure to selenium provided complete protective effects against the cytotoxicity induced by MeHgCl. Pretreatment with selenium increased the protective effects of subsquent administrations of selenium in conjunction with MeHgCl, but pretreatment of selenium alone did not provide protection against MeHgCl when given alone. Selenium administered after exposure to MeHgCl did not repair the existing MeHgCl induced cytotoxicity.Furthermore, the apoptosis induced by MeHgCl was revealed by the DNA fragmentation, using the terminal deoxynucleotidyl transferase Biotin-dUTP nick end labeling (TUNEL) assay, alterations to the nuclear morphology, by nuclei staining, and the plasma membrane lipid organization, as shown by cell flow cytometry. The apoptosis induced by MeHgCl was prevented by the concurrent exposure to selenium, or pretreatment with selenium, prior to the administration of selenium in conjunction with MeHgCl. However, no inhibittion of the MeHgCl induced apoptosis was observed with selenium pretreatment prior to exposure to MeHgCl alone, or with the administration of selenium after exposure to MeHgCl.
CONCLUSIONS
These results suggest that the coexistence of selenium and MeHgCl are essential for the protective effects of selenium against the MeHgCl-induced apoptosis, and the cytotoxicity, in RAW 264.7 cells, and may involve selenium-MeHgCl binding.
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Effect of Glutathione on Apoptosis Induced by Methyl Mercury Chloride
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Jung Ho Youm, Dai Ha Koh, Keun Sang Kwon, Me Yae Lee
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Korean Journal of Occupational and Environmental Medicine 2002;14(4):377-391. Published online December 31, 2002
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DOI: https://doi.org/10.35371/kjoem.2002.14.4.377
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Abstract
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- OBJECTIVES
This study was performed to evaluate the critical role of glutathione(GSH) in methyl mercury chloride(MeHgCl)induced cell apoptosis.
METHODS
The effect of GSH in MeHgCl induced cell apoptosis was observed in mouse macrophage-derived RAW 264.7 cells in vitro. The cells were cultured in Dulbecco's modified Eagle's medium(DMEM).
RESULTS
MeHgCl exerted a dose dependent cytotoxicity,as demonstrated by the MTT assay, which is an assay dependent partially on the mitochondrial function. Moreover, in the presence of NAC, a GSH precursor, the MeHgCl induced cytotoxicity was significantly decreased whereas BSO, a specific GSH synthesis inhibitor,increased the MeHgCl induced cytotoxicity.The MeHgCl induced DNA fragmentation and chromatin condensation was consistent with the morphological alterations. The MeHgCl treated cells exhibited increasing annexin V-FITC binding to the phos-phatidylserine(PS)translocated from the inner to the outer leaflet of the plasma membrane and those cells with NAC pretreatment significantly exhibited decreasing annexin V-FITC binding compared to the cells treated with MeHgCl only. However BSO pretreatment markedly exhibited the increasing annexin V-FITC binding. The MeHgCl treated cells generated ROS, which was evidenced by the oxidation of dihydroethidine and the generation of the fluorescent product, ethidium. In addition, BSO pretreatment further enhanced the extent of ROS generation caused by MeHgCl whereas NAC pretreatment decreased the amount of ROS generation. MeHgCl led to a dose dependent decrease in the GSH content. Although MeHgCl exposure significantly reduced the GSH level, those cells that had a NAC pretreatment contained a higher level of GSH compared to the cells treated with MeHgCl only. In contrast, BSO pretreatment futher enhanced the extent of GSH depletion caused by MeHgCl.
CONCLUSIONS
These results indicate that MeHgCl reduced the GSH content and impaired the defense against oxidative damage caused by ROS formation in RAW 264.7 cells. It is possible that these factors leads to the activation of the apoptosis signaling pathway. Ultimately these results suggest that GSH plays a crucial role in protecting the activity against MeHgCl induced apoptosis.
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Cadmium-induced Apoptosis in HL-60 Cells Via Signal Transduction
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Nam Song Kim, Gyung Jae Oh, Kwang Ho Cho, Mee Sun Hyun, Yoo Chang Kim, Tae Ho Sung, Jung Ho Youm, Keun Sang Kwon
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Korean Journal of Occupational and Environmental Medicine 2002;14(1):1-12. Published online March 31, 2002
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DOI: https://doi.org/10.35371/kjoem.2002.14.1.1
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Abstract
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- OBJECTIVES
Apoptosis is a process of active cell death, distinct from necrosis and characterized by specific morphological and biochemical features. Apoptosis induced by metals and metal-related deleterious conditions has only recently been studied. Although the toxic effects of heavy metals are well described, little is known about the mechanism of apoptosis via cadmium toxicity. Therefore, this study is designed to define the induction mechanism of apoptosis by which cadmium exerts its cytotoxic effect on human promyelocytic leukemic HL-60 cells. The cytotoxic effects of cadmium on HL-60 cells are studied in regards to apoptotic signal transduction pathways.
METHODS
The mode of cadmium-induced apoptosis was investigated in HL-60 cells. HL-60 cells were treated with various concentrations of cadmium and antioxidants after which the viability of the cells were measured by MTT assay. The morphological features of cadmium- induced apoptosis were evaluated by fluoromicroscopy and the DNA fragmentation was analyzed using 1.5% agarose gel electrophorosis. Kinase activity was assayed by autoradiography and activity of NF-kappaB and nuclear proteins were measured by EMSA.
RESULTS
Cadmium (125 microM) induces the characteristic morphological features of apoptosis, which are characterized by a shrinkage of the cytoplasm and a condensation of chromatin. In addition, cadmium induced the ladder pattern of DNA fragmentation. Antioxidants(Sodium nitroprusside, glutathione and N-acethylcysteine), which were not toxic to the cells, did not suppress apoptosis induced by cadmium. Cadmium enhances the expression of several classes of genes at elevated cytotoxic concentrations. Poly(ADP-ribose) polymerase(PARP) was predominantly in the fragmented form when doses of 125 microM were used. Since PARP is cleaved by CPP32 (caspase-3), we next determined if cadmium was capable of effecting changes in CPP32 activity. The results of these experiments showed that cadmium increased caspase-3 activity in a time dependent manner, corresponding to the time of appearance of fragmented PARP. Cadmium also increased the phosphotransferase activities of c-JUN N-terminal kinase (JNK). Furthermore, cadmium increased the activation of transcriptional factors including the activation of protein-1 (AP-1) and NF-kappaB .
CONCLUSIONS
These results suggest that cadmium induces the apoptotic death of HL-60 cells via the activation of a DEVD-specific caspase, JNK and transcriptional factors such as AP-1 and NF-kappaB .
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Citations
Citations to this article as recorded by  - The association between blood cadmium level, frequency and amount of gejang (marinated crab) intake
Chang Yul Choi, Gun Il Park, Young Seok Byun, Man Joong Jeon, Kwang Hae Choi, Joon Sakong
Annals of Occupational and Environmental Medicine.2016;[Epub] CrossRef
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Role of Nitric Oxide in the Nickel and Cobalt Induced Cytotoxicity in RAW 264.7 cell
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Jung Ho Youm, Gyung Jae Oh, Young Cheun Yoo
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Korean Journal of Occupational and Environmental Medicine 2001;13(3):274-285. Published online September 30, 2001
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DOI: https://doi.org/10.35371/kjoem.2001.13.3.274
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Abstract
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- OBJECTIVES
The nickel and cobalt present in many industrial working environments and consumer products. They are two of the leading causes of allergic contact dermatitis, which is a typical delayed(type IV) hypersensitivity reaction. However, the mechanism by which nickel and cobalt causes this pathology is not well known. The nickel and cobalt induced contact dermatitis is mediated primarily through macrophages. This mechanism is similar to the autotoxicity procedure for NO. Therefore, this study was designed to examine whether the metals could modulate NO production and how the metals may affect ATP production and cell viability. In summary, the purpose of this study was to elucidate the role of NO in the nickel and cobalt induced cytotoxicity.
METHODS
This study is based on observations of cultures of RAW 264.7 cells which are originated from a tumor of Balb/c mouse that was induced by Abelson murine leukemia virus. RAW 264.7 cells were treated with either Ni, Co, Ni plus Co, or Nmonomethyl-L- arginine(NMLA) for 24-72 h. The cytotoxicity of the nickel and cobalt was measured by cell viability and NO2-, and mitochondrial function was evaluated by adenosine triphosphate(ATP) production in RAW 264.7 cells. In addition, the morphology of cells was observed using an inverted microsope.
RESULTS
The NO2- synthesis of RAW 264.7 cells increased with increasing concentrations of Ni and Co up to 50 microM after 24 and 48 h of exposure to Ni and Co but then decreased if the concentration was greater than 50 microM and the time period was greater than 48 h. However, the viability of cells was decreased by Ni and Co exposure in a dose and time dependent manner. Therefore, 50 microM Ni or Co and 48 h of treatment were used in this study. A complete inhibition of NO2- synthesis by Ni or/and Co occurred when iNOS inhibitor, NMLA, were pretreated prior to addition of Ni or/and Co, whereas Ni or/and Co induced decrease of synthesis of ATP and viability completely recovered when NMLA were pretreated prior to addition of Ni or/and Co. Ni or/and Co(50 microM) induced the characteristic morphological features of cytotoxicity which is characterized by a shrinkage of cytoplasm and irregular shape of the cells, but the pretreatment of NMLA resulted in a recovered morphological change of the cells to their normal appearance.
CONCLUSIONS
These results suggest that NO plays an important role in the pathogenesis of the cytotoxicity of nickel and cobalt, and nickel and cobalt may exert their toxicities by means of modulation of NO production. The results from this study may facilitate further understanding the role of NO on nickel and cobalt induced immune and inflammatory processes.
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Induction of Apoptosis by Heavy Metals in HL-60 Cells
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Nam Song Kim, Tae Ho Seong, Kwang Ho Cho, Jung Ho Youm, Dai Ha Koh
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Korean Journal of Occupational and Environmental Medicine 1999;11(4):557-568. Published online December 31, 1999
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DOI: https://doi.org/10.35371/kjoem.1999.11.4.557
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Abstract
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- OBJECTIVES
Apotosis induced by metals and metal-related deleterious conditions has only recently been studied. Although the toxic effects of heavy metal are well described, little is known about the mechanism of apoptosis by heavy metal toxicity. This study is designed to define the induction of apoptosis by which heavy metals exert the cytotoxic effect on human promyelocytic leukemic HL-60 cells.
Methods
After the incubation with CdC12, Na2SeO3 and HgC12, viability of the cells were measured by MTT assay. DNA fragmentation was analyzed by electrophoresis. For measurement of caspase 1 and 3-like proteases activity, the whole lysates were subjected to the proteolytic cleavage and then measured by using fluorospectrometry. c-JUN N-terminal kinase (JNK) activity was detected by an in vitro kinase assay. Transcriptional activities of activating protein-1 (AP-1) and nuclear factor-kB (NF-kB) were measured by elec trophoresis mobility shift assay (EMSA).
RESULTS
Cadmium (l2OuiN/I) and selenium (30,iM) induce the apoptosis of HL-60 cells which is characterized by the ladder pattern of DNA fragmentation. Cadmium and selenium induce the activation of caspase-3 in a time dependent manner. They also increase the phosphotransferase activities of c-JUN N-terminal kinase (JNK) in cadmium and selenium treated HL-60 cells. Furthermore, cadmium and selenium increase the activation of transcriptional factors including AP-i and NF-kB.
CONCLUSIONS
These results suggest that cadmium and selenium induce the apoptotic death of HL-60 cells via activation of DEVD-specific caspase, JNK and transcriptional factors such as AP-1 and NF-kB.
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A Study of Protective Effect of Selenium Against Cytotoxicity of Methylmercury Chloride
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Dai Ha Koh, Jung Ho Youm, Young Sang Koh, Sun Hwan Joh, Tak Soon Oh
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Korean Journal of Occupational and Environmental Medicine 1998;10(3):310-319. Published online August 31, 1998
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DOI: https://doi.org/10.35371/kjoem.1998.10.3.310
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Abstract
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- The purpose of the present study was to elucidate the cytotoxical influence of mercurial compounds and the protective effect of selenium against mercurial compounds. The effects of mercury compounds and selenium on the syntheses of nitrite(NO2-) and ATP were observed in the cell cultures of EMT-6 cells and peritoneal macrophages from Balb/c mouse. The viabilities of EMT-6 cells and peritoneal macrophages at the end of culture were significantly decreased in dose-dependent manner by methylmercury chloride (CH3HgCl) added into the media. NO2- and ATP syntheses of the cells were dose-dependently inhibited by CH3HgCl. Simultaneous addition of the equimolar dose of selenium completely prevented mercury-induced inhibitions of NO2- and ATP syntheses, which were observed in both of EMT-6 cells and peritoneal macrophages. But these effects of selenium were not appeared in the new medium containing mercurials only which were removed the selenium after the pretreatment of selenium for 6 hours. These results suggest that protective effect of selenium against mercurial compounds was archived by the formation of a complex consisting of Se-Hg or Se-Hg-protein. Though its mechanism was not clear, the protective role of selemium against the mercury toxicity would be exhibited in the immunological system.
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Cytotoxic Influence of Mercurial Compounds and the Protective Effect of Selenium in the EMT-6 Cells
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Jung Ho Youm, Dai Ha Koh, Byoung Yul Soh
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Korean Journal of Occupational and Environmental Medicine 1997;9(3):469-477. Published online October 31, 1997
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DOI: https://doi.org/10.35371/kjoem.1997.9.3.469
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Abstract
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- No abstract available.
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A Study on Factors Related to NO Synthesis by Mercurial Compounds in the EMT-6 cell
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Jung Ho Youm
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Korean Journal of Occupational and Environmental Medicine 1997;9(1):122-130. Published online February 28, 1997
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DOI: https://doi.org/10.35371/kjoem.1997.9.1.122
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Abstract
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- The effects of several factors on the nitrite synthesis were observed in the culture of EMT-6 cells which are originated from mammary adenocarcinoma of Balb/c mouse. The cells were cultured in Dulbecco's Modified Eagle's Medium containing 10% fetal calf serum. Amounts of nitrite in the culture media after 24 and 36 hours of culture were about 2 fold, and 3-fold of those measured after 12 hours respectively. There were very close associations between the amounts of nitrite measured in the culture media. A significant nitrite synthesis by EMT-6 cells occurred when IL-1 was added to the culture medium with other cytokines as IFN gamma or TNF alpha . One of each cytokines were less effective as an inducer of nitrite than the combinations of cytokines. When mercury chloride or cinnamate was added in the culture medium, the nitrite synthesis was dose-dependently decreased by the concentration of these materials. The viability of EMT-6 cells were kept on 95% or above in 36 hours after beginning of culture without any specific additives except cytokines. While after 48 hours it went down to 85% or less. These viability were decreased by the prolongation of culture time (48 hours or more), the addition of TNF alpha to cytokine mixture, and the higher concentrations of mercury chloride or cinnamate to culture medium. Simultaneous addition of the equimolar dose of selenium completely prevented mercurial compounds-induced inhibitions of nitrite syntheses. But the single addition of selenium neither influenced the viability of cells nor the productions of nitrite. These results suggests that the disorder of cell mediated immunity by mercurial compounds could be related to the inhibition of nitric oxide synthesis and selenium decreased the cytotoxicity of mercurial compounds.
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Effect of Mercury Chloride on Peritoneal Macrophage or EMT-6 cell from Balb/c mice
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Dai Ha Koh, Jung Ho Youm, No Suk Ki, Gyung Jae Oh, Kuen Sang Kwon, Sung Yeup Kim, Nam Song Kim
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Korean Journal of Occupational and Environmental Medicine 1996;8(2):201-209. Published online September 30, 1996
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DOI: https://doi.org/10.35371/kjoem.1996.8.2.201
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Abstract
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- The effect of treatment of mercury chloride on the nitrite and nitrate synthesis was observed in peritoneal macrophages from Balb/c mice and EMT-6 cells in vitro. The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with cytokines. Amounts of nitrite and nitrate in the culture media after 24 and 36 hours of culture were about 2-fold, and 3-fold of those measured after 12 hours respectively. There were very close associations between the amounts of nitrite and nitrate measured in the culture media, according to culture time. The survival rate of peritoneal macrophages was significantly decreased by mercury chloride added into the media in dose-dependent manner, however the survivals of EMT-6 cells were not influenced by mercury chloride concentration in media. Nitrite and nitrate syntheses were dose-dependently decreased by mercury chloride added in culture media. These results reported here suggest that the disorder of cell mediated immunity by mercurials could be related to the inhibition of nitric oxide synthesis which seems to be caused by the inhibition of metabolism of cells.
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A Study on Working Environment and Health Status of Workers in Some Dyeing Factories
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No Suk Ki, Chung Ja Ahn, Dai Ha Koh, Jung Sang Lee, Yoo Yong Lee, Jae Hyung Lee, Jung Ho Youm, Yong Il Shin
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Korean Journal of Occupational and Environmental Medicine 1994;6(1):3-16. Published online February 28, 1994
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DOI: https://doi.org/10.35371/kjoem.1994.6.1.3
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Abstract
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- This study was carried out to evaluated the actual conditions of working environment and health status of workers and search more effective health management method of workers in some dyeing factories. This study was conducted from April 1 to October 30, 1992, for 426 workers in two dyeing factories and an electric wire making factories.
Among 324 workers in two dyeing factories, 57.5% were male and 42.5% were female. Most of the engaged workers had less than 2 years of working carrier and aged 30 years or below. The used chemical substances exceeding 1 ton per a month were sodium hydroxide(NaOH), hydrogen peroxide(H2O2), sodium chlorite(NaClO2), sodium sulfate(Ma2SO4), sodium carbonate(Na2CO3), and sodium hydrosulfite(Na2S2O4). The used chemical dyes exceeding 100kg per a month were suncion blue H-ERD, levafix brilliant red E-4BA, suncion yellow E-3G, and remazol black B. As the allowable exposure time by governmental threshold limit valuses to industrial noise levels in 90 dBA for 8 hours. Average noise levels of the individual plants were ranged from 75 to 95 dB (A). The TLV for total cotton dust in 2.0mg/m3. Average cotton-dust concentration in these working environmental air were ranging from 0.2-1.3mg/m3. The TLV chlorine, acetic acid and formic acid are 1 ppm, 10 ppm & 5ppm, respectively. The range of chlorine, and acetic and formic acid concentration in these working environmental air were detected 0.2-1.6 ppm and at trace level. The accident by chemical substances and dyes was not found on these working environment.
From the physical examination and Todai Health Index scores results, there was no significant correlation between the used chemical substances and the diseases, such as bronchial asthma, other hyperreactive respiratory diseases and contact dermatitis. It was suggested that long term survey should be performed to detect the occupational health problem on these working environment.
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