Evaluation for Safety of Cultured Corneal Fibroblasts with Cotreatment of Alcohol and Mitomycin C

Tae-im Kim, Hungwon Tchah, Eun Hee Cho, Michael S. Kook

Research output: Contribution to journalArticle

23 Citations (Scopus)

Abstract

PURPOSE. To investigate the effects of alcohol and mitomycin C (MMC) on cultured corneal fibroblast of the rabbit to determine the safety of this compound for clinical use. METHOD. Corneal fibroblasts of New Zealand rabbits were cultured. Various concentrations (0%, 10%, 20%, 30%, 40%, and 60%) of ethanol were applied for 10, 20, 30, and 40 seconds to estimate dose- and time-dependent responses of cultured corneal fibroblasts. Cell viability was assessed using the MTT assay. Treated cells were additionally stained with Hoechst and annexin V for the identification of apoptosis. To investigate the coeffects of ethanol and MMC, dose and time dependency were evaluated after treatment with various concentrations of ethanol and MMC at different exposure times, and cell viability was established. To determine the latent effects of ethanol and MMC, cultured corneal fibroblasts were cotreated with various concentrations of ethanol and 0.02% MMC for various periods and washed out, and then one group was incubated for 24 hours and another group was not incubated. Cell viability was estimated, and Hoechst and annexin V staining were performed before and after incubation. To establish the pathway of cell death, caspase-3 activities were measured in cultured corneal fibroblasts treated with ethanol or MMC. RESULTS. Cell viability after ethanol treatment was dose and time dependent. After application of ethanol for 10 seconds, cell viability was significantly reduced with 20% ethanol (P = 0.001). At 20, 30, and 40 seconds of treatment with 10% ethanol, cell viability was significantly reduced (P < 0.01). Hoechst and annexin V staining revealed typical characteristics of apoptosis, such as bright fluorescent chromatin condensation, low fluorescence of nuclear fragmentation, and cell membrane shrinkage. Cell viability was more significantly reduced after cotreatment with alcohol and MMC, compared with treatment with alcohol alone. Moreover, cell viability was considerably decreased in the incubated group, compared with the nonincubated group. After 24 hours of incubation, cultured corneal fibroblasts cotreated with 10% ethanol and 0.02% MMC were Stained with Hoechst and annexin V. Results were similar to data obtained with ethanol-treated cells. However, after application of 20% alcohol and MMC, a significant number of cells were not viable and were detached from the well walls. Caspase-3 activity significantly increased after treatment with 30% ethanol only and 30% ethanol in conjunction with 0.02% MMC. CONCLUSIONS. Alcohol and MMC reduced cell viability in cultured corneal fibroblasts in a dose- and time-dependent manner and had synergistic effects. This is related to the caspase-3 pathway, especially with concentrations of ethanol over 30%. Cotreatment with these reagents may significantly damage cultured corneal fibroblasts.

Original languageEnglish
Pages (from-to)86-92
Number of pages7
JournalInvestigative Ophthalmology and Visual Science
Volume45
Issue number1
DOIs
Publication statusPublished - 2004 Jan 1

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Mitomycin
Ethanol
Fibroblasts
Alcohols
Safety
Cell Survival
Annexin A5
Caspase 3
Apoptosis
Staining and Labeling
Rabbits
Nuclear Envelope
Chromatin

All Science Journal Classification (ASJC) codes

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

Cite this

@article{cb78e4fef22849ecb2b6cf8cf8725f2c,
title = "Evaluation for Safety of Cultured Corneal Fibroblasts with Cotreatment of Alcohol and Mitomycin C",
abstract = "PURPOSE. To investigate the effects of alcohol and mitomycin C (MMC) on cultured corneal fibroblast of the rabbit to determine the safety of this compound for clinical use. METHOD. Corneal fibroblasts of New Zealand rabbits were cultured. Various concentrations (0{\%}, 10{\%}, 20{\%}, 30{\%}, 40{\%}, and 60{\%}) of ethanol were applied for 10, 20, 30, and 40 seconds to estimate dose- and time-dependent responses of cultured corneal fibroblasts. Cell viability was assessed using the MTT assay. Treated cells were additionally stained with Hoechst and annexin V for the identification of apoptosis. To investigate the coeffects of ethanol and MMC, dose and time dependency were evaluated after treatment with various concentrations of ethanol and MMC at different exposure times, and cell viability was established. To determine the latent effects of ethanol and MMC, cultured corneal fibroblasts were cotreated with various concentrations of ethanol and 0.02{\%} MMC for various periods and washed out, and then one group was incubated for 24 hours and another group was not incubated. Cell viability was estimated, and Hoechst and annexin V staining were performed before and after incubation. To establish the pathway of cell death, caspase-3 activities were measured in cultured corneal fibroblasts treated with ethanol or MMC. RESULTS. Cell viability after ethanol treatment was dose and time dependent. After application of ethanol for 10 seconds, cell viability was significantly reduced with 20{\%} ethanol (P = 0.001). At 20, 30, and 40 seconds of treatment with 10{\%} ethanol, cell viability was significantly reduced (P < 0.01). Hoechst and annexin V staining revealed typical characteristics of apoptosis, such as bright fluorescent chromatin condensation, low fluorescence of nuclear fragmentation, and cell membrane shrinkage. Cell viability was more significantly reduced after cotreatment with alcohol and MMC, compared with treatment with alcohol alone. Moreover, cell viability was considerably decreased in the incubated group, compared with the nonincubated group. After 24 hours of incubation, cultured corneal fibroblasts cotreated with 10{\%} ethanol and 0.02{\%} MMC were Stained with Hoechst and annexin V. Results were similar to data obtained with ethanol-treated cells. However, after application of 20{\%} alcohol and MMC, a significant number of cells were not viable and were detached from the well walls. Caspase-3 activity significantly increased after treatment with 30{\%} ethanol only and 30{\%} ethanol in conjunction with 0.02{\%} MMC. CONCLUSIONS. Alcohol and MMC reduced cell viability in cultured corneal fibroblasts in a dose- and time-dependent manner and had synergistic effects. This is related to the caspase-3 pathway, especially with concentrations of ethanol over 30{\%}. Cotreatment with these reagents may significantly damage cultured corneal fibroblasts.",
author = "Tae-im Kim and Hungwon Tchah and Cho, {Eun Hee} and Kook, {Michael S.}",
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Evaluation for Safety of Cultured Corneal Fibroblasts with Cotreatment of Alcohol and Mitomycin C. / Kim, Tae-im; Tchah, Hungwon; Cho, Eun Hee; Kook, Michael S.

In: Investigative Ophthalmology and Visual Science, Vol. 45, No. 1, 01.01.2004, p. 86-92.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Evaluation for Safety of Cultured Corneal Fibroblasts with Cotreatment of Alcohol and Mitomycin C

AU - Kim, Tae-im

AU - Tchah, Hungwon

AU - Cho, Eun Hee

AU - Kook, Michael S.

PY - 2004/1/1

Y1 - 2004/1/1

N2 - PURPOSE. To investigate the effects of alcohol and mitomycin C (MMC) on cultured corneal fibroblast of the rabbit to determine the safety of this compound for clinical use. METHOD. Corneal fibroblasts of New Zealand rabbits were cultured. Various concentrations (0%, 10%, 20%, 30%, 40%, and 60%) of ethanol were applied for 10, 20, 30, and 40 seconds to estimate dose- and time-dependent responses of cultured corneal fibroblasts. Cell viability was assessed using the MTT assay. Treated cells were additionally stained with Hoechst and annexin V for the identification of apoptosis. To investigate the coeffects of ethanol and MMC, dose and time dependency were evaluated after treatment with various concentrations of ethanol and MMC at different exposure times, and cell viability was established. To determine the latent effects of ethanol and MMC, cultured corneal fibroblasts were cotreated with various concentrations of ethanol and 0.02% MMC for various periods and washed out, and then one group was incubated for 24 hours and another group was not incubated. Cell viability was estimated, and Hoechst and annexin V staining were performed before and after incubation. To establish the pathway of cell death, caspase-3 activities were measured in cultured corneal fibroblasts treated with ethanol or MMC. RESULTS. Cell viability after ethanol treatment was dose and time dependent. After application of ethanol for 10 seconds, cell viability was significantly reduced with 20% ethanol (P = 0.001). At 20, 30, and 40 seconds of treatment with 10% ethanol, cell viability was significantly reduced (P < 0.01). Hoechst and annexin V staining revealed typical characteristics of apoptosis, such as bright fluorescent chromatin condensation, low fluorescence of nuclear fragmentation, and cell membrane shrinkage. Cell viability was more significantly reduced after cotreatment with alcohol and MMC, compared with treatment with alcohol alone. Moreover, cell viability was considerably decreased in the incubated group, compared with the nonincubated group. After 24 hours of incubation, cultured corneal fibroblasts cotreated with 10% ethanol and 0.02% MMC were Stained with Hoechst and annexin V. Results were similar to data obtained with ethanol-treated cells. However, after application of 20% alcohol and MMC, a significant number of cells were not viable and were detached from the well walls. Caspase-3 activity significantly increased after treatment with 30% ethanol only and 30% ethanol in conjunction with 0.02% MMC. CONCLUSIONS. Alcohol and MMC reduced cell viability in cultured corneal fibroblasts in a dose- and time-dependent manner and had synergistic effects. This is related to the caspase-3 pathway, especially with concentrations of ethanol over 30%. Cotreatment with these reagents may significantly damage cultured corneal fibroblasts.

AB - PURPOSE. To investigate the effects of alcohol and mitomycin C (MMC) on cultured corneal fibroblast of the rabbit to determine the safety of this compound for clinical use. METHOD. Corneal fibroblasts of New Zealand rabbits were cultured. Various concentrations (0%, 10%, 20%, 30%, 40%, and 60%) of ethanol were applied for 10, 20, 30, and 40 seconds to estimate dose- and time-dependent responses of cultured corneal fibroblasts. Cell viability was assessed using the MTT assay. Treated cells were additionally stained with Hoechst and annexin V for the identification of apoptosis. To investigate the coeffects of ethanol and MMC, dose and time dependency were evaluated after treatment with various concentrations of ethanol and MMC at different exposure times, and cell viability was established. To determine the latent effects of ethanol and MMC, cultured corneal fibroblasts were cotreated with various concentrations of ethanol and 0.02% MMC for various periods and washed out, and then one group was incubated for 24 hours and another group was not incubated. Cell viability was estimated, and Hoechst and annexin V staining were performed before and after incubation. To establish the pathway of cell death, caspase-3 activities were measured in cultured corneal fibroblasts treated with ethanol or MMC. RESULTS. Cell viability after ethanol treatment was dose and time dependent. After application of ethanol for 10 seconds, cell viability was significantly reduced with 20% ethanol (P = 0.001). At 20, 30, and 40 seconds of treatment with 10% ethanol, cell viability was significantly reduced (P < 0.01). Hoechst and annexin V staining revealed typical characteristics of apoptosis, such as bright fluorescent chromatin condensation, low fluorescence of nuclear fragmentation, and cell membrane shrinkage. Cell viability was more significantly reduced after cotreatment with alcohol and MMC, compared with treatment with alcohol alone. Moreover, cell viability was considerably decreased in the incubated group, compared with the nonincubated group. After 24 hours of incubation, cultured corneal fibroblasts cotreated with 10% ethanol and 0.02% MMC were Stained with Hoechst and annexin V. Results were similar to data obtained with ethanol-treated cells. However, after application of 20% alcohol and MMC, a significant number of cells were not viable and were detached from the well walls. Caspase-3 activity significantly increased after treatment with 30% ethanol only and 30% ethanol in conjunction with 0.02% MMC. CONCLUSIONS. Alcohol and MMC reduced cell viability in cultured corneal fibroblasts in a dose- and time-dependent manner and had synergistic effects. This is related to the caspase-3 pathway, especially with concentrations of ethanol over 30%. Cotreatment with these reagents may significantly damage cultured corneal fibroblasts.

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