Role of cytochrome P4502E1 activation in proximal tubular cell injury induced by hydrogen peroxide (original) (raw)
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Toxicology in Vitro, 2003
Deliberate exposure to solvents has been associated with kidney disorders. However, the mechanism by which solvents induce renal damage after acute exposure has not been studied. Proximal tubular cell (LLC-PK1) cytotoxicity after exposure for 48 h to either 5 mm of p-xylene (XY) or toluene (TL) was compared to control (C) by cell viability (MTS assay), LDH release, DNA fragmentation, and malondialdehyde (MDA) release. CYP2E1 activity with or without a free radical scavenger (catalase-CT), or the CYP2E1 inhibitor disulfiram (DSF), was examined. Both p-xylene and toluene significantly reduced cell viability (XY 53.9 8 AE1.6 vs TL 54.8AE 0.9 vs C 102.7 AE 2.1), increased CYP2E1 activity (mm/mg protein/min) (XY 3.6 AE 0.5 vs TL 3.7 AE0.7 vs C 1.3 AE0.4) and MDA release (mm/mg protein) (XY 29.1 AE3.9 vs TL 12.3AE 1.4 vs C 2.8 AE 0.3). LDH was increased (XY 59.9 AE 3.0 vs TL 27.6 AE 0.5 vs C 8.4 AE 1.2), but there was no significant change in DNA fragmentation (OD/mg protein) suggesting necrosis as the predominant mode of cell death. DSF significantly attenuated CYP2E1 activity (XY+DSF 1.4 AE0.9, TL+DSF 2.3AE 0.1), LDH release (XY+DSF 45.1 AE2.0, TL+DSF 13.0 AE 0.2) and MDA release (XY+DSF 4.3AE 0.5, TL+CT 6.2 AE 1.1). Moreover, CT attenuated LDH release (XY+CT 36.4AE 5.1, TL+DSF 15.6 AE0.5) and MDA release (XY+DSF 5.4 AE0.7, TL+DSF 6.6 AE 1.3) in XY and TL treated cells. This study confirms the pivotal role of CYP2E1 in solvent-induced oxidative stress and necrosis in proximal tubular cells after exposure to solvent at 5 mm for 48 h. #
Role of Calcium and Calcium-activated Proteases in CYP2E1-dependent Toxicity in HEPG2 Cells
Journal of Biological Chemistry, 2001
The objective of this work was to investigate whether CYP2E1-and oxidative stress-dependent toxicity in HepG2 cells is mediated by an increase of cytosolic Ca 2؉ and activation of Ca 2؉-modulated processes. HepG2 cells expressing CYP2E1 (E47 cells) or control cells not expressing CYP2E1 (C34 cells) were preloaded with arachidonic acid (AA, up to 10 M) and, after washing, incubated with iron-nitrilotriacetic acid (up to 100 M) for variable periods (up to 12 h). Toxicity was greater in E47 cells than in C34 cells at all times and combinations of iron/AA tested. Cytosolic calcium increased with incubation time in both cell lines, but the increase was higher in E47 cells than in C34 cells. The rise in calcium was an early event and preceded the developing toxicity. Toxicity in E47 cells and the increase in Ca 2؉ were inhibited by omission of Ca 2؉ from the extracellular medium, and toxicity was restored by reincorporation of Ca 2؉. An inhibitor of Ca 2؉ release from intracellular stores did not prevent the toxicity or the increase in Ca 2؉ , reflecting a role for the influx of extracellular Ca 2؉ in the toxicity. Reactive oxygen production was similar in media with or without calcium, indicating that calcium was not modulating CYP2E1-dependent oxidative stress. Toxicity, lipid peroxidation, and the increase of Ca 2؉ in E47 cells exposed to iron-AA were inhibited by ␣-tocopherol. E47 cells (but not C34 cells) exposed to iron-AA showed increased calpain activity in situ (40fold). The toxicity in E47 cells mirrorred calpain activation and was inhibited by calpeptin, suggesting that calpain activation plays a causal role in toxicity. These results suggest that CYP2E1-dependent toxicity in this model depends on the activation of lipid peroxidation, followed by an increased influx of extracellular Ca 2؉ and activation of Ca 2؉-dependent proteases.
Biochemical and Biophysical Research Communications, 2016
Supplemental oxygen, which is routinely administered to preterm infants with pulmonary insufficiency, contributes to bronchopulmonary dysplasia (BPD) in these infants. Hyperoxia also contributes to the development of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) in adults. The mechanisms of oxygen-mediated pulmonary toxicity are not completely understood. Recent studies have suggested an important role for cytochrome P450 (CYP)1A1/1A2 in the protection against hyperoxic lung injury. The role of CYP1B1 in oxygen-mediated pulmonary toxicity has not been studied. In this investigation, we tested the hypothesis that CYP1B1 plays a mechanistic role in oxygen toxicity in pulmonary cells in vitro. In human bronchial epithelial cell line BEAS-2B, hyperoxic treatment for 1-3 days led to decreased cell viability by about 50-80%. Hyperoxic cytotoxicity was accompanied by an increase in levels of reactive oxygen species (ROS) by up to 110%, and an increase of TUNEL-positive cells by up to 4.8-fold. Western blot analysis showed hyperoxia to significantly down-regulated CYP1B1 protein level. Also, there was a decrease of CYP1B1 mRNA by up to 38% and Cyp1b1 promoter activity by up to 65%. On the other hand, CYP1B1 siRNA appeared to rescue the cell viability under hyperoxia stress, and overexpression of CYP1B1 significantly attenuated hyperoxic cytotoxicity after 48 h of incubation. In immortalized lung endothelial cells derived from Cyp1b1-null and wild-type mice, hyperoxia increased caspase 3/7 activities in a time-dependent manner, but
Cisplatin toxicity reduced in human cultured renal tubular cells by oxygen pretreatment
Cisplatin is an effective and widely used chemotherapy agent and its side effects, particularly nephrotoxicity, limit its usage and related platinum-based drugs. Cisplatin nephrotoxicity is mainly due to extremely increase in reactive oxygen species (ROS) generation leading to kidney tubular cell death. Preconditioning with oxidative stress has been demonstrated to stimulate the cellular adaptation to subsequent severe oxidative stress. Short term oxygen pre-exposure as a mild oxidative stress may enhance some endogenous defense mechanisms, so its effect on Cisplatin induced cell death was investigated in present research. We studied the effects of hyperoxic environment pre-exposure on Cisplatin toxicity in an in-vitro model of cultured human embryonic tubular epithelial cells (AD293). Viability of AD293 cells, as evaluated by MTT-assay, was affected by Cisplatin in a time (1-4 h) dependent model. Biochemical markers of cell apoptosis were evaluated using immunoblotting. Pretreatment with nearly pure oxygen (!90%) for 2 h significantly reduced the level of cell damage. Activated caspase 3 and Bax/Bcl-2 ratio were significantly increased in Cisplatin-treated cells. Oxygen pretreatment inhibited caspase 3 activation and decreased Bax/Bcl-2 ratio. Oxygen pre-treatment itself not showed any cytotoxicity in exposure times up to 3 h. Our data indicate that hyperoxic preconditioning reduces Cisplatin toxicity in cultured human tubular epithelial cells. The exact mechanism of protection is unclear, though enhancement of some endogenous defense mechanisms and subsequently scavenging of free oxygen radicals may play an important role.
CYP3A induction aggravates endotoxemic liver injury via reactive oxygen species in male rats
Free Radical Biology and Medicine, 2004
We carried out this experiment to evaluate the relationship between isoforms of cytochrome P450 (P450) and liver injury in lipopolysaccharide (LPS)-induced endotoxemic rats. Male rats were intraperitoneally administered phenobarbital (PB), a P450 inducer, for 3 days, and 1 day later, they were intravenously given LPS. PB significantly increased P450 levels (200% of control levels) and the activities (300 -400% of control) of the specific isoforms (CYP), CYP3A2 and CYP2B1, in male rats. Plasma AST and ALT increased slightly more in PB-treated rats than in PBnontreated (control) rats with LPS treatment. Furthermore, either troleandomycin or ketoconazole, specific CYP3A inhibitors, significantly inhibited LPS-induced liver injury in control and PB-treated male rats. To evaluate the oxidative stress in LPS-treated rats, in situ superoxide radical detection using dihydroethidium (DHE), hydroxy-2-nonenal (HNE)modified proteins in liver microsomes and 8-hydroxydeoxyguanosine (8-OHdG) in liver nuclei were measured in control and PB-treated rats. DHE signal intensity, levels of HNE-modified proteins, and 8-OHdG increased significantly in PBtreated rats. LPS further increased DHE intensity, HNE-modified proteins, and 8-OHdG levels in normal and PB-treated groups. CYP3A inhibitors also inhibited the increases in these items. Our results indicate that the induction or preservation of CYP isoforms further promotes LPS-induced liver injury through mechanisms related to oxidative stress. In particular, CYP3A2 of P450 isoforms made an important contribution to this LPS-induced liver injury. D 2004 Elsevier Inc. All rights reserved.
Biochemistry, 1993
In vitro effects of cadmium 1on on vasoconstriction, particularly on vasoconstriction independent of extracellular Ca 2+, were investigated using isolated rat aorta Aorta incubation w~th CdCl 2 (0 01, 0 1 raM) significantly attenuated contractile responses to KC1 and phenylephrlne in the medium containing normal Ca 2+ (2 5 mM) The contractile response to phenylephrlne in the presence of calcium channel antagonists, nlfedlplne (1 /zM) or verapamll (1 #M), was markedly inhibited by CdC12 (0 1 mM) In the medium without Ca 2+, phenylephrlne (10 /xM) Induced a phasic contraction, which was markedly inhibited by CdCl 2 (0 1 raM) In the medium without Ca 2+, phorbol 12-myrlstate 13-acetate (1 /zM) and okadalc acid (10 /zM) caused tonic contractile responses, which were strongly attenuated by CdC1 z (0 1 raM) pretreatment Contractile response to sodium fluoride (5 ~ 15 raM) in the absence of extracellular Ca 2+ was strongly attenuated by CdCl 2 (0 1 mM) pretreatment These results suggest that cadmium ion depresses an extracellular Ca2+-independent component of agonlst-lnduced vasoconstriction by hindering an lntracellular contractile mechamsm(s)
The role of cytochrome P450 (CYP) enzymes in hyperoxic lung injury
Expert Opinion on Drug Metabolism & Toxicology, 2020
Introduction: Hyperoxic lung injury is a condition that can occur in patients in need of supplemental oxygen, such as premature infants with bronchopulmonary dysplasia or adults with acute respiratory distress syndrome. Cytochrome P450 (CYP) enzymes play critical roles in the metabolism of endogenous and exogenous compounds. Areas covered: Through their complex pathways, some subfamilies of these enzymes may contribute to or protect against hyperoxic lung injury. Oxidative stress from reactive oxygen species (ROS) production is most likely a major contributor of hyperoxic lung injury. CYP1A enzymes have been shown to protect against hyperoxic lung injury while CYP1B enzymes seem to contribute to it. CYP2J2 enzymes help protect against hyperoxic lung injury by triggering EET production, thereby, increasing antioxidant enzymes. The metabolism of arachidonic acid to ωterminal hydroxyeicosatetraenoic acid (20-HETEs) by CYP4A and CYP4F enzymes could impact hyperoxic lung injury via the vasodilating effects of 20-HETE. CYP2E1 and CYP2A enzymes may contribute to the oxidative stress in the lungs caused by ethanol-and nicotine-metabolism, respectively. Expert opinion: Overall, the CYP enzymes, depending upon the isoform, play a contributory or protective role in hyperoxic lung injury, and are, therefore, ideal candidates for developing drugs that can treat oxygen-mediated lung injury.
Extracellular Iron(II) Can Protect Cells from Hydrogen Peroxide
Archives of Biochemistry and Biophysics, 1996
We hypothesized that exposure of cells to H 2 O 2 plus It is well known that H 2 O 2 plus Fe 2/ will generate Fe 2/ would increase formation of cell-derived lipid peroxoxidants that initiate free radical reactions and that ides that would inactivate prostaglandin H synthase, rethese radicals may result in cell injury (1, 2). The prisulting in decreased prostaglandin synthesis. Therefore, mary oxidant generated by H 2 O 2 plus Fe 2/ is the hywe treated human endothelial cells with 0-100 mM H 2 O 2 droxyl radical (HO •) (3). followed immediately by addition of 0-200 mM Fe 2/. After oxidant exposure, cells were stimulated with 20 mM ara-Fe 2/ / H 2 O 2 r Fe 3/ / OH 0 / HO • (Fenton reaction) chidonic acid to induce prostaglandin I 2 (PGI 2) synthesis. The hydroxyl radical is highly reactive and is believed Adding 100 mM H 2 O 2 prior to arachidonic acid decreased to cause cytolysis by initiating peroxidation of cell lip-PGI 2 synthesis more than 80%. However, to our surprise, ids (4-6). the addition of Fe 2/ , in increasing amounts, progressively protected PGI 2 synthesis against the harmful effects of In addition to HO • , the reaction of H 2 O 2 with Fe 2/ H 2 O 2. A ratio of one part H 2 O 2 to two parts Fe 2/ offered will generate Fe 2/ /Fe 3/ ratios that may increase lipid almost complete protection, whereas Fe 3/ did not protect oxidation (7, 8). A 1:1 ratio of Fe 2/ to Fe 3/ has been PGI 2 synthesis from H 2 O 2. We found that 100 mM H 2 O 2 reported as ideal for maximizing lipid peroxidation (7was not cytolytic; however, 250 mM H 2 O 2 was cytolytic; 9). If a reaction mixture contains two parts Fe 2/ to one Fe 2/ protected against this cytotoxicity. In addition, expart H 2 O 2 , the products would be HO • , plus a 1:1 ratio tracellular Fe 2/ prevented the rise in intracellular calof Fe 2/ :Fe 3/. The resulting combination of HO • plus a cium caused by H 2 O 2 and extracellular Fe 2/ preserved 1:1 ratio of Fe 2/ :Fe 3/ might generate more lipid peroxintracellular glutathione in H 2 O 2-exposed cells. Electron ides than HO • alone. In addition, the Fe 2/ :Fe 3/ ratios paramagnetic resonance spin trapping demonstrated may generate the perferryl ion, Fe 2/ OO • S Fe 3/ O r0 2 , that extracellular Fe 2/ generated the hydroxyl free radiwhich may generate additional lipid peroxides (2, 10, cal, HO • , outside the cell. We speculate that extracellular 11). Therefore, it seems likely that a ratio of two parts Fe 2/ protects the intracellular space from H 2 O 2 by initiat-Fe 2/ to one part H 2 O 2 would increase cytolysis over ing the Fenton reaction outside the cell. This reductive either agent alone. cleavage of H 2 O 2 generates HO • in the extracellular Lipid peroxidation products may also injure specific space, where much of the HO • will react with noncellular cell functions without causing cytolysis. For example, components, thereby protecting the cell interior. ᭧ 1996 prostaglandin H synthase (PGHS) 2 is an enzyme that Academic Press, Inc.
Attenuation of Hyperoxic Lung Injury by the CYP1A Inducer -Naphthoflavone
Toxicological Sciences, 2005
Supplemental oxygen, frequently used in premature infants, has been implicated in the development of bronchopulmonary dysplasia (BPD). While the mechanisms of oxygen-induced lung injury are not known, reactive oxygen species (ROS) are most likely involved in the process. Here, we tested the hypothesis that upregulation of cytochrome P450 (CYP) 1A isoforms in lung and liver may lead to protection against hyperoxic lung injury. Adult male Sprague-Dawley rats were pretreated with the CYP1A inducer beta-naphthoflavone (b-NF) (80 mg/kg/day), once daily for 4 days, followed by exposure to hyperoxic environment (O 2 > 95%) or room air (normoxia) for 60 h. Pleural effusions were measured as estimates of lung injury. Activities of hepatic and pulmonary CYP1A1 were determined by measurement of ethoxyresorufin O-deethylation (EROD) activity. Northern hybridization and Western blot analysis of lung and liver were performed to assess mRNA and protein levels, respectively. Our results showed that b-NF-treated animals, which displayed the highest pulmonary and hepatic induction in EROD activity (10-fold and 8-fold increase over corn oil (CO) controls, respectively), offered the most protective effect against hyperoxic lung injury, p < 0.05. Northern and Western blot analysis correlated well with enzyme activities. Our results showed an inverse correlation between pulmonary and hepatic CYP1A expression and the extent of lung injury, which supports the hypothesis that CYP1A enzyme plays a protective role against oxygen-mediated tissue damage.