Pub Date : 2001-01-01DOI: 10.1089/109793301753407993
D. Yang, X. Lu, W. Zhang, F. He
To investigate the molecular mechanism of intermediate myasthenia syndrome (IMS), we analyzed the toxic effects of the representative organophosphate dimethoate on the function and expression of the nicotinic acetylcholine receptor (nAChR) in primary skeletal muscle cell culture. The results showed that the expression of nAChR on the muscle cell membrane was significantly increased after cells were exposed to dimethoate (130 microM). AChR function measured by carbachol-induced (22)Na+ influx demonstrated that dimethoate may inhibit the nAChR function either by binding to a noncompetitive site and changing the conformational state of nAChR or by blocking the nAChR channel directly. This study also demonstrated that dimethoate could rapidly induce the expression of c-fos, with a maximal effect at about 40 min, and c-fos might act as a transcriptional factor in regulating the expression of nAChR in the primary skeletal muscle cell culture following organophosphate exposure.
{"title":"Effect of dimethoate on the function and expression of nicotinic acetylcholine receptor in primary skeletal muscle cell culture.","authors":"D. Yang, X. Lu, W. Zhang, F. He","doi":"10.1089/109793301753407993","DOIUrl":"https://doi.org/10.1089/109793301753407993","url":null,"abstract":"To investigate the molecular mechanism of intermediate myasthenia syndrome (IMS), we analyzed the toxic effects of the representative organophosphate dimethoate on the function and expression of the nicotinic acetylcholine receptor (nAChR) in primary skeletal muscle cell culture. The results showed that the expression of nAChR on the muscle cell membrane was significantly increased after cells were exposed to dimethoate (130 microM). AChR function measured by carbachol-induced (22)Na+ influx demonstrated that dimethoate may inhibit the nAChR function either by binding to a noncompetitive site and changing the conformational state of nAChR or by blocking the nAChR channel directly. This study also demonstrated that dimethoate could rapidly induce the expression of c-fos, with a maximal effect at about 40 min, and c-fos might act as a transcriptional factor in regulating the expression of nAChR in the primary skeletal muscle cell culture following organophosphate exposure.","PeriodicalId":80284,"journal":{"name":"In vitro & molecular toxicology","volume":"14 3 1","pages":"241-5"},"PeriodicalIF":0.0,"publicationDate":"2001-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1089/109793301753407993","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"60636654","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2001-01-01DOI: 10.1089/10979330152560496
H. Pae, G. Oh, N. Kim, M. Shin, H. S. Lee, Y. Yun, H. Oh, Y. M. Kim, H. Chung
The mitogen-activated protein kinase (MAPK) family members have been implicated in cell survival. We have previously demonstrated that cytotoxic lectin-II isolated from Korean mistletoe induces apoptotic cell death in the human monoblastic leukemia cell line, U937, via the activation of the stress-activated protein kinases/c-Jun N-terminal kinase (SAPK/JNK). In the present study, the roles of extracellular signal-regulated kinases (ERK1/2) and p38 MAPK in lectin-II-induced apoptosis have been investigated. Treatment of U937 cells with lectin-II resulted in apoptotic DNA fragmentation, which was preceded by the activation of ERK1/2, p38 MAPK and SAPK/JNK. This lectin-II-induced DNA fragmentation was significantly enhanced when ERK1/2 activation was selectively inhibited by PD098059. 12-O-tetradecanoylphorbol-13-acetate, which stimulates ERK activity in U937 cells, markedly reduced lectin-II-induced DNA fragmentation. Inhibition of p38 MAPK activity with p38-specific inhibitor, SB203580, partially inhibited lectin-II-induced DNA fragmentation. These results suggest that ERK1/2 and p38 MAPK may have opposite effects on cell survival in response to cytotoxic mistletoe lectin-II, which may contribute to the modulation of lectin-II-mediated cytotoxic activity.
{"title":"Roles of extracellular signal-regulated kinase and p38 mitogen-activated protein kinase in apoptosis of human monoblastic leukemia U937 cells by lectin-II isolated from Korean mistletoe.","authors":"H. Pae, G. Oh, N. Kim, M. Shin, H. S. Lee, Y. Yun, H. Oh, Y. M. Kim, H. Chung","doi":"10.1089/10979330152560496","DOIUrl":"https://doi.org/10.1089/10979330152560496","url":null,"abstract":"The mitogen-activated protein kinase (MAPK) family members have been implicated in cell survival. We have previously demonstrated that cytotoxic lectin-II isolated from Korean mistletoe induces apoptotic cell death in the human monoblastic leukemia cell line, U937, via the activation of the stress-activated protein kinases/c-Jun N-terminal kinase (SAPK/JNK). In the present study, the roles of extracellular signal-regulated kinases (ERK1/2) and p38 MAPK in lectin-II-induced apoptosis have been investigated. Treatment of U937 cells with lectin-II resulted in apoptotic DNA fragmentation, which was preceded by the activation of ERK1/2, p38 MAPK and SAPK/JNK. This lectin-II-induced DNA fragmentation was significantly enhanced when ERK1/2 activation was selectively inhibited by PD098059. 12-O-tetradecanoylphorbol-13-acetate, which stimulates ERK activity in U937 cells, markedly reduced lectin-II-induced DNA fragmentation. Inhibition of p38 MAPK activity with p38-specific inhibitor, SB203580, partially inhibited lectin-II-induced DNA fragmentation. These results suggest that ERK1/2 and p38 MAPK may have opposite effects on cell survival in response to cytotoxic mistletoe lectin-II, which may contribute to the modulation of lectin-II-mediated cytotoxic activity.","PeriodicalId":80284,"journal":{"name":"In vitro & molecular toxicology","volume":"14 2 1","pages":"99-106"},"PeriodicalIF":0.0,"publicationDate":"2001-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1089/10979330152560496","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"60635614","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2001-01-01DOI: 10.1089/10979330152560504
C. Steinebach, G. Bauer
Transforming growth factor (TGF)-beta pretreated nontransformed fibroblasts induce apoptosis selectively in transformed fibroblasts. This potential control step during oncogenesis has been termed intercellular induction of apoptosis. Selectivity and efficiency of intercellular induction of apoptosis depend on transformed target cell-derived superoxide anions that drive two intercellular signaling pathways--the HOCl/hydroxyl radical and the nitric oxide (NO)/peroxynitrite pathway. Other natural antitumor systems like macrophages or cells of the granulocyte lineage seem to utilize the same signaling chemistry. Our data demonstrate the existence of an alternative signaling pathway in these systems. This pathway depends on the presence of nitrite and is still effective when the two conventional signaling pathways are blocked by superoxide dismutase (SOD). Nitrite-dependent apoptosis induction is neither blocked by SOD nor by the hydroxyl radical scavenger terephthalate, but it is inhibited by the peroxidase inhibitor aminobenzoyl hydrazide and by the hypochlorous acid (HOCl) scavenger taurine. Therefore, nitrite, that is nontoxic for our cells, seems to interact with HOCl to form the apoptosis inducer nitryl chloride. Nitryl chloride-mediated apoptosis induction might be relevant for apoptosis induction in tumor cells that release SOD and thus escape the two classical signaling pathways.
{"title":"An alternative signaling pathway based on nitryl chloride during intercellular induction of apoptosis.","authors":"C. Steinebach, G. Bauer","doi":"10.1089/10979330152560504","DOIUrl":"https://doi.org/10.1089/10979330152560504","url":null,"abstract":"Transforming growth factor (TGF)-beta pretreated nontransformed fibroblasts induce apoptosis selectively in transformed fibroblasts. This potential control step during oncogenesis has been termed intercellular induction of apoptosis. Selectivity and efficiency of intercellular induction of apoptosis depend on transformed target cell-derived superoxide anions that drive two intercellular signaling pathways--the HOCl/hydroxyl radical and the nitric oxide (NO)/peroxynitrite pathway. Other natural antitumor systems like macrophages or cells of the granulocyte lineage seem to utilize the same signaling chemistry. Our data demonstrate the existence of an alternative signaling pathway in these systems. This pathway depends on the presence of nitrite and is still effective when the two conventional signaling pathways are blocked by superoxide dismutase (SOD). Nitrite-dependent apoptosis induction is neither blocked by SOD nor by the hydroxyl radical scavenger terephthalate, but it is inhibited by the peroxidase inhibitor aminobenzoyl hydrazide and by the hypochlorous acid (HOCl) scavenger taurine. Therefore, nitrite, that is nontoxic for our cells, seems to interact with HOCl to form the apoptosis inducer nitryl chloride. Nitryl chloride-mediated apoptosis induction might be relevant for apoptosis induction in tumor cells that release SOD and thus escape the two classical signaling pathways.","PeriodicalId":80284,"journal":{"name":"In vitro & molecular toxicology","volume":"14 2 1","pages":"107-20"},"PeriodicalIF":0.0,"publicationDate":"2001-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1089/10979330152560504","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"60635659","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2001-01-01DOI: 10.1089/109793301753407920
J. Medina, C. Elsaesser, V. Picarles, O. Grenet, M. Kolopp, S. Chibout, A. de Brugerolle de Fraissinette
The goal of this study was to design a model system for the assessment of phototoxic potential using a human reconstructed epidermis (HRE, SkinEthic Laboratories, Nice, France), by testing some representative phototoxic (P) and non-phototoxic (NP) compounds and finished topical products. The tissue response to 24-h application of 5-5000 microg/mL of the test agents in the presence and absence of UVA light was analyzed in terms of viability (Lactate Dehydrogenase release), pro-inflammatory activity (IL-8 release and mRNA expression) and morphology (histopathology). 8-Methoxypsoralen (P) and promethazin (P), but not sodium lauryl sulfate (NP) produced cytotoxicity concentration-response curves significantly different between irradiated and nonirradiated tissues. Only irradiated tissues showed morphological damage. Application of tetracyclin (P) in the culture medium, but not topically, induced similar signs of phototoxicity. 6-Methylcoumarine (weak P) was not cytotoxic, yet it increased IL-8 release and mRNA expression only following UVA irradiation. PUVA therapy creams containing 1% 8-Methoxy-psoralen (P) or coal tar (P) decreased viability and induced histologic damage in UVA-exposed tissues. In conclusion, the phototoxic potential of the tested agents was correctly predicted by using a tiered strategy that involves determining cytotoxicity, production of IL-8, and morphological damage following exposure of the HRE to the compounds and UVA light.
{"title":"Assessment of the phototoxic potential of compounds and finished topical products using a human reconstructed epidermis.","authors":"J. Medina, C. Elsaesser, V. Picarles, O. Grenet, M. Kolopp, S. Chibout, A. de Brugerolle de Fraissinette","doi":"10.1089/109793301753407920","DOIUrl":"https://doi.org/10.1089/109793301753407920","url":null,"abstract":"The goal of this study was to design a model system for the assessment of phototoxic potential using a human reconstructed epidermis (HRE, SkinEthic Laboratories, Nice, France), by testing some representative phototoxic (P) and non-phototoxic (NP) compounds and finished topical products. The tissue response to 24-h application of 5-5000 microg/mL of the test agents in the presence and absence of UVA light was analyzed in terms of viability (Lactate Dehydrogenase release), pro-inflammatory activity (IL-8 release and mRNA expression) and morphology (histopathology). 8-Methoxypsoralen (P) and promethazin (P), but not sodium lauryl sulfate (NP) produced cytotoxicity concentration-response curves significantly different between irradiated and nonirradiated tissues. Only irradiated tissues showed morphological damage. Application of tetracyclin (P) in the culture medium, but not topically, induced similar signs of phototoxicity. 6-Methylcoumarine (weak P) was not cytotoxic, yet it increased IL-8 release and mRNA expression only following UVA irradiation. PUVA therapy creams containing 1% 8-Methoxy-psoralen (P) or coal tar (P) decreased viability and induced histologic damage in UVA-exposed tissues. In conclusion, the phototoxic potential of the tested agents was correctly predicted by using a tiered strategy that involves determining cytotoxicity, production of IL-8, and morphological damage following exposure of the HRE to the compounds and UVA light.","PeriodicalId":80284,"journal":{"name":"In vitro & molecular toxicology","volume":"48 1","pages":"157-68"},"PeriodicalIF":0.0,"publicationDate":"2001-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1089/109793301753407920","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"60636308","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2001-01-01DOI: 10.1089/109793301316882504
M. Berry, D. Jeffreys
We undertook a study of patients with nonsevere ocular injuries from chemicals used in the home to (1) establish the frequency of presentation to the Accident and Emergency (A&E) Department; (2) assess and grade any common symptoms and signs of injury; and (3) evaluate cytokine concentrations in the preocular fluid as markers of toxicity. Of the 216 reviewed chemical injuries, 85% were sustained by adults (twice as many men than women). Chemicals implicated were: alkalis, cleaners, organic solvents, personal hygiene products, contact lens solutions, and disinfectants. Conjunctival redness occurred in 80% of cases, irrespective of chemical. Low correlations were obtained for the extent, type, and degree of epithelial damage in different areas of the lower lid and bulbar conjunctiva. With one exception, interleukin (IL)1beta and IL10 levels were not different in control and injured eyes, whereas IL6 was significantly elevated above uninjured levels. We have shown that interleukins, as representatives of signal chemicals, can be noninvasively sampled and reliably measured in tears after chemical injury. An indication of injury is obtained clearly from IL6 levels in tears, and there is a hint that the pattern of IL1beta/IL10 might help discriminate between levels of severity. A larger study is needed to verify these results.
{"title":"Ocular injuries from household chemicals: early signs as predictors of recovery.","authors":"M. Berry, D. Jeffreys","doi":"10.1089/109793301316882504","DOIUrl":"https://doi.org/10.1089/109793301316882504","url":null,"abstract":"We undertook a study of patients with nonsevere ocular injuries from chemicals used in the home to (1) establish the frequency of presentation to the Accident and Emergency (A&E) Department; (2) assess and grade any common symptoms and signs of injury; and (3) evaluate cytokine concentrations in the preocular fluid as markers of toxicity. Of the 216 reviewed chemical injuries, 85% were sustained by adults (twice as many men than women). Chemicals implicated were: alkalis, cleaners, organic solvents, personal hygiene products, contact lens solutions, and disinfectants. Conjunctival redness occurred in 80% of cases, irrespective of chemical. Low correlations were obtained for the extent, type, and degree of epithelial damage in different areas of the lower lid and bulbar conjunctiva. With one exception, interleukin (IL)1beta and IL10 levels were not different in control and injured eyes, whereas IL6 was significantly elevated above uninjured levels. We have shown that interleukins, as representatives of signal chemicals, can be noninvasively sampled and reliably measured in tears after chemical injury. An indication of injury is obtained clearly from IL6 levels in tears, and there is a hint that the pattern of IL1beta/IL10 might help discriminate between levels of severity. A larger study is needed to verify these results.","PeriodicalId":80284,"journal":{"name":"In vitro & molecular toxicology","volume":"14 1 1","pages":"5-13"},"PeriodicalIF":0.0,"publicationDate":"2001-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1089/109793301316882504","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"60635274","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2001-01-01DOI: 10.1089/109793301753407939
A. Lucas
In situ polymers are used by mixing two or more compounds that are then placed directly in tissues to form a unique product. This type of reaction can generate heat, reactive oxygen species, free radicals, and other by-products of unknown toxicities, but the polymer itself is biocompatible. Many regulatory agencies require in vitro testing, however, standard guidelines (ASTM, ISO, AAMI) test polymers in a final form prior to use as a medical device. To better estimate the cytotoxicity of these in situ polymers, various means of introducing the reacting material to cells in culture were explored. Coating the material on a sterile glass cover slip then adding the cover slip to the in vitro test system immediately provided reasonable cytotoxicity data that reflected actual use conditions. For in situ polymeric devices that are more viscous, such as dental materials and bone cements, a mold was used that was placed directly into cell culture. This approach in testing in situ polymers generated in vitro toxicity data that reflects the actual use of the material.
{"title":"Strategies for the in vitro testing of in situ polymers.","authors":"A. Lucas","doi":"10.1089/109793301753407939","DOIUrl":"https://doi.org/10.1089/109793301753407939","url":null,"abstract":"In situ polymers are used by mixing two or more compounds that are then placed directly in tissues to form a unique product. This type of reaction can generate heat, reactive oxygen species, free radicals, and other by-products of unknown toxicities, but the polymer itself is biocompatible. Many regulatory agencies require in vitro testing, however, standard guidelines (ASTM, ISO, AAMI) test polymers in a final form prior to use as a medical device. To better estimate the cytotoxicity of these in situ polymers, various means of introducing the reacting material to cells in culture were explored. Coating the material on a sterile glass cover slip then adding the cover slip to the in vitro test system immediately provided reasonable cytotoxicity data that reflected actual use conditions. For in situ polymeric devices that are more viscous, such as dental materials and bone cements, a mold was used that was placed directly into cell culture. This approach in testing in situ polymers generated in vitro toxicity data that reflects the actual use of the material.","PeriodicalId":80284,"journal":{"name":"In vitro & molecular toxicology","volume":"14 3 1","pages":"169-75"},"PeriodicalIF":0.0,"publicationDate":"2001-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1089/109793301753407939","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"60636355","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2001-01-01DOI: 10.1089/109793301753407957
E. Elmore, Thanh-Thuy Luc, V. Steele, J. Redpath
Comparative toxicity was determined for twenty potential chemopreventive agents in the Human Epithelial Cell Cytotoxicity (HECC) Assay using epithelial cell cultures from eight different tissues including: skin, kidney, breast, bronchus, cervix, prostate, oral cavity, and liver. The endpoints assessed were inhibition of: growth at 3 and 5 days; mitochondrial function; and proliferating cell nuclear antigen or albumin expression. Difluoromethylornithine (DFMO), s-allylcysteine, dehydroepiandrosterone (DHEA) analogue 8543, l-selenomethionine, and vitamin E acetate were not toxic or only produced mild toxicity with all endpoints in all eight cell types. N-acetyl-l-cysteine, calcium chloride, DHEA, genistein, ibuprofen, indole-3-carbinol, 4-hydroxyphenylretinamide (4-HPR), oltipraz, piroxicam, phenylethyl isothiocyanate, 9-cis-retinoic acid, and p-xylylselenocyanate each showed at least a 10-fold decrease in their TC(50) (toxic concentration that inhibited growth by 50%) for at least one endpoint with one or more cell types. For some agents such as DHEA and piroxicam, the TC(50)s for growth inhibition were 10-fold lower after 5 days compared with 3 days. Unique tissue-specific toxicity was observed for each toxic agent suggesting that tissue-specific effects are the rule rather than the exception. The HECC Assay is effective in identifying tissue-specific toxicity for chemopreventive agents and may help to identify potential toxicity problems in phase I human clinical trials.
{"title":"Comparative tissue-specific toxicities of 20 cancer preventive agents using cultured cells from 8 different normal human epithelia.","authors":"E. Elmore, Thanh-Thuy Luc, V. Steele, J. Redpath","doi":"10.1089/109793301753407957","DOIUrl":"https://doi.org/10.1089/109793301753407957","url":null,"abstract":"Comparative toxicity was determined for twenty potential chemopreventive agents in the Human Epithelial Cell Cytotoxicity (HECC) Assay using epithelial cell cultures from eight different tissues including: skin, kidney, breast, bronchus, cervix, prostate, oral cavity, and liver. The endpoints assessed were inhibition of: growth at 3 and 5 days; mitochondrial function; and proliferating cell nuclear antigen or albumin expression. Difluoromethylornithine (DFMO), s-allylcysteine, dehydroepiandrosterone (DHEA) analogue 8543, l-selenomethionine, and vitamin E acetate were not toxic or only produced mild toxicity with all endpoints in all eight cell types. N-acetyl-l-cysteine, calcium chloride, DHEA, genistein, ibuprofen, indole-3-carbinol, 4-hydroxyphenylretinamide (4-HPR), oltipraz, piroxicam, phenylethyl isothiocyanate, 9-cis-retinoic acid, and p-xylylselenocyanate each showed at least a 10-fold decrease in their TC(50) (toxic concentration that inhibited growth by 50%) for at least one endpoint with one or more cell types. For some agents such as DHEA and piroxicam, the TC(50)s for growth inhibition were 10-fold lower after 5 days compared with 3 days. Unique tissue-specific toxicity was observed for each toxic agent suggesting that tissue-specific effects are the rule rather than the exception. The HECC Assay is effective in identifying tissue-specific toxicity for chemopreventive agents and may help to identify potential toxicity problems in phase I human clinical trials.","PeriodicalId":80284,"journal":{"name":"In vitro & molecular toxicology","volume":"14 3 1","pages":"191-207"},"PeriodicalIF":0.0,"publicationDate":"2001-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1089/109793301753407957","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"60636521","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2001-01-01DOI: 10.1089/109793301753407966
J. I. Ram, L. Hiebert
Previous studies produced models of oxygen-derived free radical (OFR) injury, using H(2)O(2) or xanthine/xanthine oxidase (X/XO), in cultured porcine aortic endothelium (PAE) and rat coronary endothelium. H(2)O(2) at 0.1 mM resulted in 50% viability in both cell types. To determine if comparable H(2)O(2) or X/XO concentrations have the same injurious effect on endothelium from other sources, models of OFR injury were developed for bovine aortic endothelium (BAE) and bovine brain microvessel endothelium (BBME). Varying concentrations of H(2)O(2) (0.01 to 6 mM) or X/XO (10 microM/0.1 to 0.3 U/mL) were added to medium 24 h prior to evaluating cell damage. Injury was assessed using the Trypan blue exclusion test (% viability) and by measuring the release of lactate dehydrogenase into medium. H(2)O(2) concentrations required to produce 50% viability were >6 mM in BAE and BBME versus 1 mM in PAE when cells were grown in Dulbecco's modified Eagle's medium (DMEM). Similarly, BAE and BBME were less sensitive than PAE to damage by X/XO. Cells from both species were more sensitive to H(2)O(2) or X/XO injury when grown in Medium 199 (M199) versus DMEM. The most profound difference was observed with PAE where 50% viability was obtained with 0.12 versus 1.05 mM H(2)O(2) in M199 versus DMEM. These results indicate that bovine endothelial cells from aorta and brain are more resistant to free radical injury than PAE. The presence or absence of key media components (iron, pyruvate, cysteine, histidine) likely influences the extent of OFR injury.
先前的研究在培养的猪主动脉内皮(PAE)和大鼠冠状动脉内皮中使用H(2)O(2)或黄嘌呤/黄嘌呤氧化酶(X/XO)建立了氧源性自由基(OFR)损伤模型。0.1 mM的H(2)O(2)对两种细胞的存活率均为50%。为了确定H(2)O(2)或X/XO浓度是否对其他来源的内皮具有相同的损伤作用，我们建立了牛主动脉内皮(BAE)和牛脑微血管内皮(BBME)的OFR损伤模型。在评估细胞损伤前24小时，在培养基中加入不同浓度的H(2)O(2)(0.01至6 mM)或X/XO(10微米/0.1至0.3 U/mL)。采用台盼蓝排除试验(%存活率)和乳酸脱氢酶释放量测定来评估损伤。当细胞在Dulbecco改良Eagle培养基(DMEM)中生长时，产生50%活力所需的H(2)O(2)浓度在BAE和BBME中为0.6 mM，在PAE中为1 mM。同样，BAE和BBME对X/XO伤害的敏感性低于PAE。与DMEM相比，两种细胞在Medium 199 (M199)中生长时对H(2)O(2)或X/XO损伤更敏感。最显著的差异是PAE，在0.12和1.05 mM H(2)O(2)下，M199和DMEM的存活率分别为50%。结果表明，牛主动脉和脑内皮细胞对自由基损伤的抵抗能力强于PAE。关键介质成分(铁、丙酮酸、半胱氨酸、组氨酸)的存在或缺失可能影响OFR损伤的程度。
{"title":"Marked variation in free radical injury between bovine and porcine endothelial cells cultured in different media.","authors":"J. I. Ram, L. Hiebert","doi":"10.1089/109793301753407966","DOIUrl":"https://doi.org/10.1089/109793301753407966","url":null,"abstract":"Previous studies produced models of oxygen-derived free radical (OFR) injury, using H(2)O(2) or xanthine/xanthine oxidase (X/XO), in cultured porcine aortic endothelium (PAE) and rat coronary endothelium. H(2)O(2) at 0.1 mM resulted in 50% viability in both cell types. To determine if comparable H(2)O(2) or X/XO concentrations have the same injurious effect on endothelium from other sources, models of OFR injury were developed for bovine aortic endothelium (BAE) and bovine brain microvessel endothelium (BBME). Varying concentrations of H(2)O(2) (0.01 to 6 mM) or X/XO (10 microM/0.1 to 0.3 U/mL) were added to medium 24 h prior to evaluating cell damage. Injury was assessed using the Trypan blue exclusion test (% viability) and by measuring the release of lactate dehydrogenase into medium. H(2)O(2) concentrations required to produce 50% viability were >6 mM in BAE and BBME versus 1 mM in PAE when cells were grown in Dulbecco's modified Eagle's medium (DMEM). Similarly, BAE and BBME were less sensitive than PAE to damage by X/XO. Cells from both species were more sensitive to H(2)O(2) or X/XO injury when grown in Medium 199 (M199) versus DMEM. The most profound difference was observed with PAE where 50% viability was obtained with 0.12 versus 1.05 mM H(2)O(2) in M199 versus DMEM. These results indicate that bovine endothelial cells from aorta and brain are more resistant to free radical injury than PAE. The presence or absence of key media components (iron, pyruvate, cysteine, histidine) likely influences the extent of OFR injury.","PeriodicalId":80284,"journal":{"name":"In vitro & molecular toxicology","volume":"14 3 1","pages":"209-17"},"PeriodicalIF":0.0,"publicationDate":"2001-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1089/109793301753407966","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"60636804","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2001-01-01DOI: 10.1089/109793301753407975
M. McAleer, R. Tuan
Oxidative stress induces cellular apoptosis. Many agents producing intracellular oxidative stress, including H(2)O(2) and steroid hormones, have also been found to induce metallothionein (MT) expression. Recently, MT has been recognized as potentially having antioxidant activity. This action may be essential for survival of terminally differentiated cells subject to oxidative stress, such as syncytiotrophoblasts, placental cells producing pregnancy hormones and forming the maternal-fetal barrier. We previously demonstrated an inverse relationship between basal MT expression and apoptotic incidence in the trophoblastic cell line, JEG-3. Using JEG-3 cells transfected with MT in sense or antisense orientation, we have examined here the effect of altered basal MT levels on trophoblastic function and apoptosis following treatment with H(2)O(2) or diethylstilbestrol (DES). Induction of MT mRNA was observed in control and transfected JEG-3 cells following exposure to severe oxidative stress. Changes in the localization of MT protein, however, were apparent after a low oxidative stress challenge. Exposure to H(2)O(2) resulted in a dose-dependent decrease in human chorionic gonadotropin secretion in all JEG-3 cultures regardless of basal MT expression, whereas no change was detected following DES treatment. With respect to apoptosis, a significant protective effect was observed proportional to the basal MT level. These results suggest that although MT does not ameliorate oxidative stress-induced perturbation of some trophoblastic functions, its expression is critical for protection of these cells from severe oxidative stress-induced apoptosis. MT thus appears to act as an anti-apoptotic antioxidant in trophoblastic cells.
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