Mechanisms of chloroform-induced hepatotoxicity: Oxidative stress and mitochondrial permeability transition in freshly isolated mouse hepatocytes

Angela S. Burke; Kelly Redeker; Richard C. Kurten; Laura P. James; Jack A. Hinson

doi:10.1080/15287390701551399

Mechanisms of chloroform-induced hepatotoxicity: Oxidative stress and mitochondrial permeability transition in freshly isolated mouse hepatocytes

Angela S. Burke, Kelly Redeker, Richard C. Kurten, Laura P. James, Jack A. Hinson

Biology

Research output: Contribution to journal › Article › peer-review

Abstract

The role of mitochondrial permeability transition (MPT) and oxidative stress in chloroform toxicity was determined in freshly isolated female B6C3F1 mouse hepatocytes. Incubation of chloroform ( 12 mM) with hepatocytes resulted in cell death ( alanine aminotransferase release and propidium iodide fluorescence). Chloroform had volatilized from the incubation and glutathione was depleted by 1 h; however, toxicity was not significantly different between control and chloroform-incubated cells. Hepatocytes were washed and reincubated in fresh media at 1 h. Subsequent reincubation of chloroform-treated hepatocytes resulted in significant toxicity at 3-5 h. Inclusion of the MPT inhibitor cyclosporine A or the antioxidant N-acetylcysteine (NAC) in the reincubation media at 1 h prevented toxicity. Confocal microscopy studies with the dye calcein AM indicated MPT that was blocked by cyclosporine A or NAC. Fluorescence microscopy studies utilizing JC-1 indicated loss of mitochondrial membrane potential, which was also blocked by cyclosporine A or NAC. Dichlorofluorescein fluorescence increased during the reincubation phase, indicating increased oxidative stress, and the increase was blocked by cyclosporine A. Since oxidative stress may occur by peroxynitrite, its role in toxicity was examined. Either of the nitric oxide synthase inhibitors N-G-methyl-L-arginine (L-NMMA) and 7-nitroindazole (7-NI) at 1 h blocked toxicity. Western blot analysis of hepatocytes for 3-nitrotyrosine in proteins, a biomarker of peroxynitrite, indicated one major nitrated protein at 81 kD. Nitration of this protein was inhibited by cyclosporine A, L-NMMA, 7-NI, or NAC. The data indicate that chloroform-induced cell death occurs in two phases: a metabolic phase characterized by glutathione depletion, and an oxidative phase characterized by MPT and protein nitration.

Original language	English
Pages (from-to)	1936-1945
Number of pages	10
Journal	Journal of toxicology and environmental health-Part a-Current issues
Volume	70
Issue number	22
DOIs	https://doi.org/10.1080/15287390701551399
Publication status	Published - 2007

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10.1080/15287390701551399

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@article{154b7b9b658c4a30b3fefe30d352c6d8,

title = "Mechanisms of chloroform-induced hepatotoxicity: Oxidative stress and mitochondrial permeability transition in freshly isolated mouse hepatocytes",

abstract = "The role of mitochondrial permeability transition (MPT) and oxidative stress in chloroform toxicity was determined in freshly isolated female B6C3F1 mouse hepatocytes. Incubation of chloroform ( 12 mM) with hepatocytes resulted in cell death ( alanine aminotransferase release and propidium iodide fluorescence). Chloroform had volatilized from the incubation and glutathione was depleted by 1 h; however, toxicity was not significantly different between control and chloroform-incubated cells. Hepatocytes were washed and reincubated in fresh media at 1 h. Subsequent reincubation of chloroform-treated hepatocytes resulted in significant toxicity at 3-5 h. Inclusion of the MPT inhibitor cyclosporine A or the antioxidant N-acetylcysteine (NAC) in the reincubation media at 1 h prevented toxicity. Confocal microscopy studies with the dye calcein AM indicated MPT that was blocked by cyclosporine A or NAC. Fluorescence microscopy studies utilizing JC-1 indicated loss of mitochondrial membrane potential, which was also blocked by cyclosporine A or NAC. Dichlorofluorescein fluorescence increased during the reincubation phase, indicating increased oxidative stress, and the increase was blocked by cyclosporine A. Since oxidative stress may occur by peroxynitrite, its role in toxicity was examined. Either of the nitric oxide synthase inhibitors N-G-methyl-L-arginine (L-NMMA) and 7-nitroindazole (7-NI) at 1 h blocked toxicity. Western blot analysis of hepatocytes for 3-nitrotyrosine in proteins, a biomarker of peroxynitrite, indicated one major nitrated protein at 81 kD. Nitration of this protein was inhibited by cyclosporine A, L-NMMA, 7-NI, or NAC. The data indicate that chloroform-induced cell death occurs in two phases: a metabolic phase characterized by glutathione depletion, and an oxidative phase characterized by MPT and protein nitration.",

author = "Burke, {Angela S.} and Kelly Redeker and Kurten, {Richard C.} and James, {Laura P.} and Hinson, {Jack A.}",

year = "2007",

doi = "10.1080/15287390701551399",

language = "English",

volume = "70",

pages = "1936--1945",

journal = "Journal of toxicology and environmental health-Part a-Current issues",

issn = "1528-7394",

publisher = "Taylor & Francis",

number = "22",

}

Mechanisms of chloroform-induced hepatotoxicity: Oxidative stress and mitochondrial permeability transition in freshly isolated mouse hepatocytes. / Burke, Angela S.; Redeker, Kelly; Kurten, Richard C. et al.
In: Journal of toxicology and environmental health-Part a-Current issues, Vol. 70, No. 22, 2007, p. 1936-1945.

Research output: Contribution to journal › Article › peer-review

TY - JOUR

T1 - Mechanisms of chloroform-induced hepatotoxicity

T2 - Oxidative stress and mitochondrial permeability transition in freshly isolated mouse hepatocytes

AU - Burke, Angela S.

AU - Redeker, Kelly

AU - Kurten, Richard C.

AU - James, Laura P.

AU - Hinson, Jack A.

PY - 2007

Y1 - 2007

N2 - The role of mitochondrial permeability transition (MPT) and oxidative stress in chloroform toxicity was determined in freshly isolated female B6C3F1 mouse hepatocytes. Incubation of chloroform ( 12 mM) with hepatocytes resulted in cell death ( alanine aminotransferase release and propidium iodide fluorescence). Chloroform had volatilized from the incubation and glutathione was depleted by 1 h; however, toxicity was not significantly different between control and chloroform-incubated cells. Hepatocytes were washed and reincubated in fresh media at 1 h. Subsequent reincubation of chloroform-treated hepatocytes resulted in significant toxicity at 3-5 h. Inclusion of the MPT inhibitor cyclosporine A or the antioxidant N-acetylcysteine (NAC) in the reincubation media at 1 h prevented toxicity. Confocal microscopy studies with the dye calcein AM indicated MPT that was blocked by cyclosporine A or NAC. Fluorescence microscopy studies utilizing JC-1 indicated loss of mitochondrial membrane potential, which was also blocked by cyclosporine A or NAC. Dichlorofluorescein fluorescence increased during the reincubation phase, indicating increased oxidative stress, and the increase was blocked by cyclosporine A. Since oxidative stress may occur by peroxynitrite, its role in toxicity was examined. Either of the nitric oxide synthase inhibitors N-G-methyl-L-arginine (L-NMMA) and 7-nitroindazole (7-NI) at 1 h blocked toxicity. Western blot analysis of hepatocytes for 3-nitrotyrosine in proteins, a biomarker of peroxynitrite, indicated one major nitrated protein at 81 kD. Nitration of this protein was inhibited by cyclosporine A, L-NMMA, 7-NI, or NAC. The data indicate that chloroform-induced cell death occurs in two phases: a metabolic phase characterized by glutathione depletion, and an oxidative phase characterized by MPT and protein nitration.

AB - The role of mitochondrial permeability transition (MPT) and oxidative stress in chloroform toxicity was determined in freshly isolated female B6C3F1 mouse hepatocytes. Incubation of chloroform ( 12 mM) with hepatocytes resulted in cell death ( alanine aminotransferase release and propidium iodide fluorescence). Chloroform had volatilized from the incubation and glutathione was depleted by 1 h; however, toxicity was not significantly different between control and chloroform-incubated cells. Hepatocytes were washed and reincubated in fresh media at 1 h. Subsequent reincubation of chloroform-treated hepatocytes resulted in significant toxicity at 3-5 h. Inclusion of the MPT inhibitor cyclosporine A or the antioxidant N-acetylcysteine (NAC) in the reincubation media at 1 h prevented toxicity. Confocal microscopy studies with the dye calcein AM indicated MPT that was blocked by cyclosporine A or NAC. Fluorescence microscopy studies utilizing JC-1 indicated loss of mitochondrial membrane potential, which was also blocked by cyclosporine A or NAC. Dichlorofluorescein fluorescence increased during the reincubation phase, indicating increased oxidative stress, and the increase was blocked by cyclosporine A. Since oxidative stress may occur by peroxynitrite, its role in toxicity was examined. Either of the nitric oxide synthase inhibitors N-G-methyl-L-arginine (L-NMMA) and 7-nitroindazole (7-NI) at 1 h blocked toxicity. Western blot analysis of hepatocytes for 3-nitrotyrosine in proteins, a biomarker of peroxynitrite, indicated one major nitrated protein at 81 kD. Nitration of this protein was inhibited by cyclosporine A, L-NMMA, 7-NI, or NAC. The data indicate that chloroform-induced cell death occurs in two phases: a metabolic phase characterized by glutathione depletion, and an oxidative phase characterized by MPT and protein nitration.

U2 - 10.1080/15287390701551399

DO - 10.1080/15287390701551399

M3 - Article

SN - 1528-7394

VL - 70

SP - 1936

EP - 1945

JO - Journal of toxicology and environmental health-Part a-Current issues

JF - Journal of toxicology and environmental health-Part a-Current issues

IS - 22

ER -