Cytotoxicity and Apoptosis Produced by Cytochrome P450 2E1 in Hep G2 Cells
Abstract
Two Hep G2 subclones overexpressing CYP2E1 were established with the use of transfection and limited dilution screening techniques. The Hep G2-CI2E1–43 and -47 (E47) cells (transduced Hep G2 subclones that overexpress CYP2E1) grew at a slower rate than parental Hep G2 cells or control subclones that do not express CYP2E1, but remained fully viable. When GSH synthesis was inhibited by treatment with buthionine sulfoximine, GSH levels rapidly declined in E47 cells but not control cells, which is most likely a reflection of CYP2E1-catalyzed formation of reactive oxygen species. Under these conditions of GSH depletion, cytotoxicity and apoptosis were found only with the E47 cells. Low levels of lipid peroxidation were found in the E47 cells, which became more pronounced after GSH depletion. The antioxidants vitamin E, vitamin C, or trolox prevented the lipid peroxidation as well as the cytotoxicity and apoptosis, as did transfection with plasmid containing antisense CYP2E1 or overexpression of Bcl-2. Levels of ATP were lower in E47 cells because of damage to mitochondrial complex I. When GSH was depleted, oxygen uptake was markedly decreased with all substrates in the E47 extracts. Vitamin E completely prevented the decrease in oxygen uptake. Under conditions of CYP2E1 overexpression, two modes of CYP2E1-dependent toxicity can be observed in Hep G2 cells: a slower growth rate when cellular GSH levels are maintained and a loss of cellular viability when cellular GSH levels are depleted. Elevated lipid peroxidation plays an important role in the CYP2E1-dependent toxicity and apoptosis. This direct toxicity of overexpressed CYP2E1 may reflect the ability of this enzyme to generate reactive oxygen species even in the absence of added metabolic substrate.
Footnotes
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Send reprint requests to: Dr. Arthur I. Cederbaum, Mount Sinai School of Medicine, Dept. of Biochemistry, Box 1020, One Gustave L. Levy Place, New York, NY 10029. E-mail:acederb{at}smtplink.mssm.edu
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This study was supported by Grants AA03312 and AA06610 from The National Institute on Alcohol Abuse and Alcoholism. These studies are in partial fulfillment of the requirement of the degree of Doctor of Philosophy from The City University of New York (Q.C.)
- Abbreviations:
- GSH
- reduced glutathione
- 4-HNE
- 4-hydroxy-2-nonenal
- 4-MP
- 4-methylpyrazole
- A14
- Hep G2-CIA-14, a Hep G2 subclone transduced with vector containing antisense bcl-2 cDNA
- B28
- Hep G2-CIBcl-2–28, a transduced Hep G2 subclone that overexpresses Bcl-2
- BSO
- buthionine sulfoximine
- C34
- Hep G2-CI-34, a Hep G2 subclone transduced with pCI-neo vector lacking any CYP2E1 cDNA insert
- C37
- Hep G2-CI-37, a Hep G2 subclone transduced with pCI-neo vector lacking any CYP2E1 cDNA insert
- DMSO
- dimethylsulfoxide
- E43
- Hep G2-CI2E1–43, a transduced Hep G2 subclone that overexpresses CYP2E1
- E47
- Hep G2-CI2E1–47, a transduced Hep G2 subclone that overexpresses CYP2E1
- HEPES
- 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
- LDH
- lactate dehydrogenase
- MDA
- malondialdehyde
- MEM
- minimum essential medium
- MTT
- 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
- PAGE
- polyacrylamide gel electrophoresis
- PBS
- phosphate-buffered saline
- PNP
- p-nitrophenol
- PUFA
- polyunsaturated fatty acid
- ROS
- reactive oxygen species
- SDS
- sodium dodecyl sulfate
- trolox
- 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid
- VitE
- vitamin E
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- Received October 24, 1997.
- Accepted December 16, 1997.
- The American Society for Pharmacology and Experimental Therapeutics
