The mode of cisplatin-induced cell death in CYP2E1-overexpressing HepG2 cells: modulation by ERK, ROS, glutathione, and thioredoxin

In a previous study, E47 HepG2 cells that overexpress human CYP2E1 were shown to be more sensitive to cisplatin than C34 cells that do not express CYP2E1. In this study, we found that this sensitivity was due to an earlier activation of ERK in the E47 cells compared to the C34 cells. Glutathione depletion by L-buthionine sulfoximine (BSO) enhanced cisplatin cytotoxicity via increasing production of reactive oxygen species (ROS) and activation of ERK. In contrast, elevation of glutathione by glutathione ethyl ester (GSHE) decreased cisplatin/BSO cytotoxicity by decreasing ROS production and ERK activation. Inhibition of ERK activation by U0126 protected against cisplatin/BSO cytotoxicity via inhibiting ROS production but not restoring intracellular glutathione content. Examination of the mode of cell death showed that U0126 inhibited cisplatin-induced necrosis but not apoptosis. Cisplatin-induced apoptosis was caspases-dependent; BSO switched cisplatin-induced apoptosis to necrosis via decreasing activity of caspases, and GSHE switched cisplatin/BSO-induced necrosis back to apoptosis through maintaining activity of caspases. Similar to GSHE, U0126 partially switched cisplatin/BSO induced necrosis to apoptosis via restoring activity of caspases. Cisplatin lowered levels of thioredoxin, especially in the presence of BSO. Although U0126 failed in restoring intracellular glutathione levels, it restored thioredoxin levels, which maintain the activity of the caspases. These results suggest that thioredoxin can replace glutathione to promote the active thiol redox state necessary for caspase activity, and thus glutathione and thioredoxin regulate the mode of cisplatin toxicity in E47 cells via redox regulation of caspase activity.     

Serum deprivation-induced reactive oxygen species production is mediated by Romo1

Serum deprivation-triggered increases in reactive oxygen species (ROS) are known to induce apoptotic cell death. However, the mechanism by which serum deprivation causes ROS production is not known. Since mitochondria are the main source of ROS and since mitochondrial ROS modulator 1 (Romo1) is involved in ROS production, we sought to determine if serum deprivation triggered ROS production through Romo1. To examine the relationship between Romo1 and the serum deprivation-triggered increase in ROS, we transfected Romo1 siRNA into various cell lines and looked for inhibition of mitochondrial ROS generation. Romo1 knockdown by Romo1 siRNA blocked the mitochondrial ROS production caused by serum deprivation, which originates in the mitochondrial electron transport chain. We also found that Romo1 knockdown inhibited serum deprivation-induced apoptosis. These findings suggest that Romo1-derived ROS play an important role in apoptotic cell death triggered by withdrawal of cell survival factors.     

Lycopene attenuates alcoholic apoptosis in HepG2 cells expressing CYP2E1

To test the hypothesis that ethanol-induced hepatic apoptosis is secondary to the oxidative stress generated by cytochrome P4502E1 (CYP2E1), we assessed the effects of the carotenoid lycopene, a potent antioxidant extracted from tomatoes, on oxidative stress and apoptosis in HepG2 cells overexpressing CYP2E1 (2E1 cells). These were exposed for 5 days to 100mM ethanol and 10 microM lycopene or an equal volume of placebo (vehicle). Ethanol significantly increased apoptosis measured by flow cytometry and by TUNEL assay. This was accompanied by an ethanol-induced oxidative stress: hydrogen peroxide production was significantly increased and mitochondrial GSH was strikingly decreased. Both were restored by lycopene, with a significant decrease in apoptosis. The placebo had no protective effect. In conclusion, Lycopene opposes the ethanol-induced oxidative stress and apoptosis in 2E1 cells. The parallelism between these effects suggests a causal link. Furthermore, these beneficial effects and the innocuity of lycopene now justify an in vivo trial.     

CYP2E1 enhances ethanol-induced lipid accumulation but impairs autophagy in HepG2 E47 cells

The regulation and function of autophagy and lipid metabolism have recently been reported to be reciprocally related. Macroautophagy mediates the breakdown of lipids stored in lipid droplets. An inhibition of autophagy leads to the development of a fatty liver. We evaluated the ability of CYP2E1 to modulate the effects of ethanol on lipid accumulation and autophagy in vitro. The E47 HepG2 cell which expresses CYP2E1 was treated with ethanol at 50, 100 and 150 mM for 4 or 5 days. Ethanol-induced lipid accumulation and an increase of triglycerides (TG) in E47 cells to a greater extent than in control C34 cells which do not express CYP2E1. In contrast, autophagy (LC3 II/LC3 I ratio) was significantly induced by ethanol in C34 cells to a greater extent than in E47 cells. P62 was significantly increased in E47 cells after ethanol treatment. Thus, there is a reciprocal relationship between the effects of ethanol on lipid accumulation and autophagy in the CYP2E1-expressing cells. Inhibition of autophagy by 3-methyladenine (3MA), increased lipid accumulation and TG levels in C34 cells which display elevated autophagy, but enhanced lipid accumulation and TG level to a lesser extent in E47 cells which displayed lower autophagy. Ethanol induced CYP2E1 activity and oxidative stress in E47 cells compared with C34 cells. These experiments suggest that the expression of CYP2E1 may impair autophagy formation which contributes to lipid accumulation in the liver. We hypothesize that CYP2E1-induced oxidative stress promotes the accumulation of lipid droplets by ethanol and this may be responsible for the suppression of autophagy in the liver.     

Hypoxia-induced cell death of HepG2 cells involves a necrotic cell death mediated by calpain

To elucidate mechanism of cell death in response to hypoxia, we attempted to compare hypoxia-induced cell death of HepG2 cells with cisplatin-induced cell death, which has been well characterized as a typical apoptosis. Cell death induced by hypoxia turned out to be different from cisplatin-mediated apoptosis in cell viability and cleavage patterns of caspases. Hypoxia-induced cell death was not associated with the activation of p53 while cisplatin-induced apoptosis is p53 dependent. In order to explain these differences, we tested involvement of μ-calpain and m-calpain in hypoxia-induced cell death. Calpains, especially μ-calpain, were initially cleaved by hypoxia, but not by cisplatin. Interestingly, the treatment of a calpain inhibitor restored PARP cleavage that was absent during hypoxia, indicating the recovery of activated caspase-3. The inhibition of calpains prevented proteolysis induced by hypoxia. In addition, hypoxia resulted in a necrosis-like morphology while cisplatin induced an apoptotic morphology. The calpain inhibitor prevented necrotic morphology induced by hypoxia and converted partially to apoptotic morphology with nuclear segmentation. Our result suggests that calpains are involved in hypoxia-induced cell death that is likely to be necrotic in nature and the inhibition of calpain switches hypoxia-induced cell death to apoptotic cell death without affecting cell viability.     

Ca2+-dependent and independent mitochondrial damage in HepG2 cells that overexpress CYP2E1

The effect of detergents on electron and proton transfer in bovine cytochrome c oxidase was studied using steady-state and transient-state methods. Cytochrome c oxidase in lauryl maltoside has high maximal turnover (TN(max)=400 s(-1)), whereas activity is low (TN(max)=10 s(-1)) in Triton X-100. Single turnover studies of intramolecular electron transfer show similar rates in either detergent. Transient proton uptake experiments show the oxidase in lauryl maltoside consumes 1.8+/-0.3 H(+)/aa(3) during either partial reduction of the oxidase or reaction of fully reduced enzyme with O(2). However, the oxidase in Triton X-100 consumes 2.6+/-0.4 H(+)/aa(3) during partial reduction and 1.0+/-0.2 H(+)/aa(3) in the O(2) reaction. Absorption spectra recorded during turnover show that the enzyme undergoes activation in lauryl maltoside, but does not activate in Triton X-100. We propose that cytochrome c oxidase in different detergents allows access to different sites of protonation, which in turn influences steady-state activity.     

Overexpression of CYP2E1 in mitochondria sensitizes HepG2 cells to the toxicity caused by depletion of glutathione

Induction of CYP2E1 by ethanol is one mechanism by which ethanol causes oxidative stress and alcohol liver disease. Although CYP2E1 is predominantly found in the endoplasmic reticulum, it is also located in rat hepatic mitochondria. In the current study, chronic alcohol consumption induced rat hepatic mitochondrial CYP2E1. To study the role of mitochondrial targeted CYP2E1 in generating oxidative stress and causing damage to mitochondria, HepG2 lines overexpressing CYP2E1 in mitochondria (mE10 and mE27 cells) were established by transfecting a plasmid containing human CYP2E1 cDNA lacking the hydrophobic endoplasmic reticulum targeting signal sequence into HepG2 cells followed by G418 selection. A 40-kDa catalytically active NH2-terminally truncated form of CYP2E1 (mtCYP2E1) was detected in the mitochondrial compartment in these cells by Western blot analysis. Cell death caused by depletion of GSH by buthionine sulfoximine (BSO) was increased in mE10 and mE27 cells as compared with cells transfected with empty vector (pCI-neo). Antioxidants were able to abolish the loss of cell viability. Increased levels of reactive oxygen species and mitochondrial 3-nitrotyrosine and 4-hydroxynonenal protein adducts and decreased mitochondrial aconitase activity and mitochondrial membrane potential were observed in mE10 and mE27 cells treated with BSO. The mitochondrial membrane stabilizer, cyclosporine A, was also able to protect these cells from BSO toxicity. These results revealed that CYP2E1 in the mitochondrial compartment could induce oxidative stress in the mitochondria, damage mitochondria membrane potential, and cause a loss of cell viability. The accumulation of CYP2E1 in hepatic mitochondria induced by ethanol consumption might play an important role in alcohol liver disease.     

Green tea polyphenol epigallocatechin-3-gallate protects HepG2 cells against CYP2E1-dependent toxicity

Chronic ethanol consumption causes oxidative damage in the liver, and induction of cytochrome P450 2E1 (CYP2E1) is one pathway involved in oxidative stress produced by ethanol. The hepatic accumulation of iron and polyunsaturated fatty acids significantly contributes to ethanol hepatotoxicity in the intragastric infusion model of ethanol treatment. The objective of this study was to analyze the effect of the green tea flavanol epigallocatechin-3-gallate (EGCG), which has been shown to prevent alcohol-induced liver damage, on CYP2E1-mediated toxicity in HepG2 cells overexpressing CYP2E1 (E47 cells). Treatment of E47 cells with arachidonic acid plus iron (AA + Fe) was previously reported to produce synergistic toxicity in E47 cells by a mechanism dependent on CYP2E1 activity and involving oxidative stress and lipid peroxidation. EGCG protected E47 cells against toxicity and loss of viability induced by AA+Fe; EGCG had no effect on CYP2E1 activity. Prevention of this toxicity was associated with a reduction in oxidative damage as reflected by decreased generation of reactive oxygen species, a decrease in lipid peroxidation, and maintenance of intracellular glutathione in cells challenged by AA+Fe in the presence of EGCG. AA+Fe treatment caused a decline in the mitochondrial membrane potential, which was also blocked by EGCG. In conclusion, EGCG exerts a protective action on CYP2E1-dependent oxidative stress and toxicity that may contribute to preventing alcohol-induced liver injury, and may be useful in preventing toxicity by various hepatotoxins activated by CYP2E1 to reactive intermediates.     

Role of phospholipase A2 activation and calcium in CYP2E1-dependent toxicity in HepG2 cells

Previous studies suggested a role for calcium in CYP2E1-dependent toxicity. The possible role of phospholipase A2 (PLA2) activation in this toxicity was investigated. HepG2 cells that overexpress CYP2E1 (E47 cells) exposed to arachidonic acid (AA) +Fe-NTA showed higher toxicity than control HepG2 cells not expressing CYP2E1 (C34 cells). This toxicity was inhibited by the PLA2 inhibitors aristolochic acid, quinacrine, and PTK. PLA2 activity assessed by release of preloaded [3H]AA after treatment with AA+Fe was higher in the CYP2E1 expressing HepG2 cells. This [3H]AA release was inhibited by PLA2 inhibitors, alpha-tocopherol, and by depleting Ca2+ from the cells (intracellular + extracellular sources), but not by removal of extracellular calcium alone. Toxicity was preceded by an increase in intracellular calcium caused by influx from the extracellular space, and this was prevented by PLA2 inhibitors. PLA2 inhibitors also blocked mitochondrial damage in the CYP2E1-expressing HepG2 cells exposed to AA+Fe. Ca2+ depletion and removal of extracellular calcium inhibited toxicity at early time periods, although a delayed toxicity was evident at later times in Ca2+-free medium. This later toxicity was also inhibited by PLA2 inhibitors. Analogous to PLA2 activity, Ca2+ depletion but not removal of extracellular calcium alone prevented the activation of calpain activity by AA+Fe. These results suggest that release of stored calcium by AA+Fe, induced by lipid peroxidation, can initially activate calpain and PLA2 activity, that PLA2 activation is critical for a subsequent increased influx of extracellular Ca2+, and that the combination of increased PLA2 and calpain activity, increased calcium and oxidative stress cause mitochondrial damage, that ultimately produces the rapid toxicity of AA+Fe in CYP2E1-expressing HepG2 cells.     

Adenovirus mediated overexpression of CYP2E1 increases sensitivity of HepG2 cells to acetaminophen induced cytotoxicity

To study the biochemical and toxicological properties of cytochrome P450 2E1 (CYP2E1), an adenovirus containing human CYP2E1 cDNA (Ad-CYP2E1) was constructed and was shown to successfully mediate the overexpression of CYP2E1 in HepG2 cells. Acetaminophen (APAP) toxicity to HepG2 cells infected with Ad-CYP2E1 was characterized as a preliminary proof of principle experiment to validate the functionality of the CYP2E1 adenovirus. Compared with cells infected with Ad-LacZ, HepG2 cells infected with Ad-CYP2E1 were more sensitive to APAP induced necrosis and apoptosis when the cells were depleted of intracellular reduced glutathione (GSH). The APAP cytotoxicity was dependent on both the concentration of APAP and the multiplicity of infection of the Ad-CYP2E1 virus. Apoptosis induced by APAP in HepG2 cells overexpressing CYP2E1 was caspase dependent and could be inhibited by the pan-caspase inhibitor Z-VAD-fmk. After treatment with APAP, mitochondrial membrane potential was dramatically decreased in the CYP2E1-expressing cells. APAP protein adducts were elevated in HepG2 cells infected with Ad-CYP2E1 compared with that in cells infected with Ad-LacZ; two bands around 90 KD were found only in the CYP2E1-expressing cells. These results demonstrate that adenovirus-mediated overexpression of human CYP2E1 activates APAP to reactive metabolites which damage mitochondria, form protein adducts, and result in toxicity to HepG2 cells. The Ad-CYP2E1 may be useful for studies designed to investigate the role of CYP2E1 in APAP and alcoholic liver injury and to further characterize the actions and effects of CYP2E1.     

Distinct roles of CysLT1 and CysLT2 receptors in oxygen glucose deprivation-induced PC12 cell death

Cysteinyl leukotrienes are involved in ischemic brain injury, and their receptors (CysLT(1) and CysLT(2)) have been cloned. To clarify which subtype mediates the ischemic neuronal injury, we performed permanent transfection to increase CysLT(1) and CysLT(2) receptor expressions in PC12 cells. Oxygen glucose deprivation (OGD)-induced cell death was detected by Hoechst 33258 and propidium iodide fluorescent staining as well as by flow cytometry. OGD induced late phase apoptosis mainly and necrosis minimally. Over-expression of CysLT(1) receptor decreased and over-expression of CysLT(2) receptor increased OGD-induced cell death. An agonist LTD(4) (10(-7)M) also induced apoptosis, especially in CysLT(2) receptor over-expressing cells. A selective CysLT(1) receptor antagonist montelukast did not affect OGD-induced apoptosis; while non-selective CysLT receptor antagonist Bay u9773 inhibited OGD-induced apoptosis, especially in CysLT(2) receptor over-expressing cells. Thus, CysLT(1) and CysLT(2) receptors play distinct roles in OGD-induced PC12 cell death; CysLT(1) attenuates while CysLT(2) facilitates the cell death.     

Nitric oxide-dependent CYP2B degradation is potentiated by a cytokine-regulated pathway and utilizes the immunoproteasome subunit LMP2

CYP2B proteins in rat hepatocytes undergo NO-dependent proteolytic degradation, but the mechanisms and the reasons for the specificity towards only certain P450 (cytochrome P450) enzymes are yet unknown. In the present study we found that down-regulation of CYP2B proteins by the NO donor NOC-18 is accelerated by pretreatment of the hepatocytes with IL-1 (interleukin-1β) in the presence of an NO synthase inhibitor, suggesting that an NO-independent action of IL-1 contributes to the lability of CYP2B proteins. The immunoproteasome subunit LMP2 (large multifunctional peptidase 2) was significantly expressed in hepatocytes under basal conditions, and IL-1 induced LMP2 within 6-12 h of treatment. CYP2B protein degradation in response to IL-1 was attenuated by the selective LMP2 inhibitor UK-101, but not by the LMP7 inhibitor IPSI. The results show that LMP2 contributes to the NO-dependent degradation of CYP2B proteins, and suggest that induction of LMP2 may be involved in the potentiation of this degradation by IL-1.     

CYP2E1 and oxidative liver injury by alcohol

Ethanol-induced oxidative stress seems to play a major role in mechanisms by which ethanol causes liver injury. Many pathways have been suggested to contribute to the ability of ethanol to induce a state of oxidative stress. One central pathway seems to be the induction of cytochrome P450 2E1 (CYP2E1) by ethanol. CYP2E1 metabolizes and activates many toxicological substrates, including ethanol, to more reactive, toxic products. Levels of CYP2E1 are elevated under a variety of physiological and pathophysiological conditions and after acute and chronic alcohol treatment. CYP2E1 is also an effective generator of reactive oxygen species such as the superoxide anion radical and hydrogen peroxide and, in the presence of iron catalysts, produces powerful oxidants such as the hydroxyl radical. This review article summarizes some of the biochemical and toxicological properties of CYP2E1 and briefly describes the use of cell lines developed to constitutively express CYP2E1 and CYP2E1 knockout mice in assessing the actions of CYP2E1. Possible therapeutic implications for treatment of alcoholic liver injury by inhibition of CYP2E1 or CYP2E1-dependent oxidative stress will be discussed, followed by some future directions which may help us to understand the actions of CYP2E1 and its role in alcoholic liver injury.