Rabbit P450 2E1 Expressed in CHO-K1 Cells Has a Short Half-Life

Barmada, Suzanne; Kienle, Eric; Koop, Dennis R.

doi:10.1006/bbrc.1995.1085

Cited by 25 publications

(8 citation statements)

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“…The cell culture medium was removed and levels of 6-hydroxychlorzoxazone were determined by HPLC as previously described. 19 Fluorescence Microscopy. The numbers of apoptotic and necrotic cells were quantified by fluorescence microscopy (Nikon Diaphot-TMD, Tokyo, Japan) as described by Duke et al 20 In short, acridine orange (250 g/mL) and ethidium bromide (250 g/mL) were added to the medium of cultured cells, and the cells were visualized under fluorescence microscopy at a magnification of ϫ200.…”

Section: Methodsmentioning

confidence: 99%

CYP2E1 overexpression alters hepatocyte death from menadione and fatty acids by activation of ERK1/2 signaling

et al. 2004

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Chronic oxidative stress induced by overexpression of the cytochrome P450 isoform 2E1 (CYP2E1) has been implicated in hepatocyte injury and death. However, the mechanism by which CYP2E1 overexpression may promote cell death is unknown. Acute oxidative stress activates mitogen-activated protein kinases (MAPK), suggesting that chronic oxidant generation by CYP2E1 may regulate cellular responses through these signaling pathways. The effect of CYP2E1 overexpression on MAPK activation and their function in altering death responses of CYP2E1-overexpressing hepatocytes were investigated. Chronic CYP2E1 overexpression led to increased extracellular signal-regulated kinase 1/2 (ERK1/2) activation constitutively and in response to oxidant stress from the superoxide generator menadione. CYP2E1-overexpressing cells were resistant to menadione toxicity through an ERK1/2-dependent mechanism. Similar to menadione, the polyunsaturated fatty acid (PUFA) arachidonic acid (AA) induced an increased activation of ERK1/2 in hepatocytes that overexpressed CYP2E1. However, CYP2E1-overexpressing cells were sensitized to necrotic death from AA and the PUFA ␥-linolenic acid, but not from saturated or monounsaturated fatty acids. Death from PUFA resulted from oxidative stress and was blocked by inhibition of ERK1/2, but not p38 MAPK or activator protein-1 signaling. CYP2E1 expression induced ERK1/2 activation through increased epidermal growth factor receptor (EGFR)/c-Raf signaling. Inhibition of EGFR signaling reversed CYP2E1-induced resistance to menadione and sensitization to AA toxicity. In conclusion, chronic CYP2E1 overexpression leads to sustained ERK1/2 activation mediated by EGFR/c-Raf signaling. This adaptive response in hepatocytes exposed to chronic oxidative stress confers differential effects on cellular survival, protecting against menadione-induced apoptosis, but sensitizing to necrotic death from PUFA. (HEPATOLOGY 2004;39:444 -455.)

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Section: Methodsmentioning

confidence: 99%

CYP2E1 overexpression alters hepatocyte death from menadione and fatty acids by activation of ERK1/2 signaling

et al. 2004

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“…Established methods for assessing CYP2E1 protein stabilization were performed according to published protocols (Barmada et al, 1995;Huan and Koop, 1999). In brief, Chinese hamster ovary cells (CHO-K1) that constitutively express rabbit CYP2E1 protein (Barmada et al, 1995) and human fibroblast cells (GM0637) that express rat CYP2E1 (Lin et al, 1998) were treated with various concentrations of nicotine (5-0.00005 M). CYP2E1 catalytic activity was assessed by the 6-hydroxylation of chlorzoxazone.…”

Section: Methodsmentioning

confidence: 99%

“…CYP2E1 catalytic activity was assessed by the 6-hydroxylation of chlorzoxazone. After overnight treatment with or without nicotine, cells were incubated with 200 M chlorzoxazone for 6 h. Media were then extracted and 6-OH-chlorzoxazone was determined by highperformance liquid chromatography (Barmada et al, 1995). Cells were allowed to lyse passively, insoluble debris was removed by centrifugation, and the supernatants were frozen until use for immunoblot analysis.…”

Section: Methodsmentioning

confidence: 99%

Rat Hepatic CYP2E1 Is Induced by Very Low Nicotine Doses: An Investigation of Induction, Time Course, Dose Response, and Mechanism

Micu

Miksys

Sellers

et al. 2003

J Pharmacol Exp Ther

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CYP2E1 is an ethanol-and drug-metabolizing enzyme that can also activate procarcinogens and hepatotoxicants and generate reactive oxygen species; it has been implicated in the pathogenesis of liver diseases and cancer. Cigarette smoke increases CYP2E1 activity in rodents and in humans and we have shown that nicotine (0.1-1.0 mg/kg s.c. ϫ 7 days) increases CYP2E1 protein and activity in the rat liver. In the current study, we have shown that the induction peaks at 4 h postnicotine (1 mg/kg s.c. ϫ 7 days) treatment and recovers within 24 h. No induction was observed after a single injection, and 18 days of treatment did not increase the levels beyond that found at 7 days. We found that CYP2E1 is induced by very low doses of chronic (ϫ 7 days) nicotine with an ED 50 value of 0.01 mg/kg s.c.; 0.01 mg/kg in a rat model results in peak cotinine levels (nicotine metabolite) similar to those found in people exposed to environmental tobacco smoke (passive smokers; 2-7 ng/ml). Previously, we have shown no change in CYP2E1 mRNA, and our current mechanistic study indicates that nicotine does not regulate CYP2E1 expression by protein stabilization. We postulated that a nicotine metabolite could be causing the induction but found that cotinine (1 mg/kg ϫ 7 days) did not increase CYP2E1. Our findings indicate that nicotine increases CYP2E1 at very low doses and may enhance CYP2E1-related toxicity in smokers, passive smokers, and people treated with nicotine (e.g., smokers, patients with Alzheimer's disease, ulcerative colitis or Parkinson's disease).

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“…The production of 6-hydroxy-chlorzoxazone was used to monitor the catalytic activity of CYP2E1 by high pressure liquid chromatography using a modification of the procedure described previously. 29 It should be noted that other P450 isozymes could catalyze the oxidation of chlorzoxazone, including CYP1A1. 30 However, the rate of CYP1A1 for chlorzoxazone is approximately 10-fold lower at V max compared with CYP2E1.…”

Section: Animals and Treatmentsmentioning

confidence: 99%

Cytochrome P450 CYP2E1, but not nicotinamide adenine dinucleotide phosphate oxidase, is required for ethanol-induced oxidative DNA damage in rodent liver

et al. 2005

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The occurrence of malignant tumors of the upper gastrointestinal tract and liver is, based largely on epidemiological evidence, causally related to the consumption of ethanol. It is widely recognized that oxidants play a key role in alcohol-induced liver injury; however, it is unclear how oxidants may be involved in DNA damage. We asked whether nicotinamide adenine dinucleotide phosphate oxidase, cytochrome P450 CYP2E1, or both are responsible for the production of DNA damage. The rodent Tsukamoto-French model of intragastric ethanol infusion was used. Wistar rats, Cyp2e1-, p47 phox -null, and hCyp2e1 transgenic mice were used. The abundance of oxidative DNA adducts, mutagenic apurinic/apyrimidinic sites, and expression of base excision DNA repair genes was determined. In rats and wild-type mice, ethanol treatment for 4 weeks led to an increase in oxidative DNA damage and induction of expression of the base excision DNA repair genes that are known to remove oxidative DNA lesions. No increase in either of the endpoints was observed in ethanol-treated Cyp2e1-null mice, whereas the magnitude of response in p47 phox -null mice and transgenic hCyp2e1 was identical to that in wild types. The increase in expression of DNA repair genes was completely abolished by treatment with the P450 inhibitor 1-aminobenzotriazole. In conclusion, the data support the hypothesis that oxidative stress to DNA is induced in liver by ethanol. Furthermore, although it was shown that nicotinamide adenine dinucleotide phosphate oxidase-derived oxidants are critical for the development of ethanol-induced liver injury, CYP2E1 is required for the induction of oxidative stress to DNA, and thus may play a key role in ethanol-associated hepatocarcinogenesis. (HEPATOLOGY 2005;41: 336-344.)

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Rabbit P450 2E1 Expressed in CHO-K1 Cells Has a Short Half-Life

Cited by 25 publications

References 0 publications

CYP2E1 overexpression alters hepatocyte death from menadione and fatty acids by activation of ERK1/2 signaling

CYP2E1 overexpression alters hepatocyte death from menadione and fatty acids by activation of ERK1/2 signaling

Rat Hepatic CYP2E1 Is Induced by Very Low Nicotine Doses: An Investigation of Induction, Time Course, Dose Response, and Mechanism

Cytochrome P450 CYP2E1, but not nicotinamide adenine dinucleotide phosphate oxidase, is required for ethanol-induced oxidative DNA damage in rodent liver

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