Regulation of cytochrome P450 2e1 expression by ethanol: role of oxidative stress-mediated pkc/jnk/sp1 pathway

As previously shown in U937 monocytic cells,we examined whether ethanol also induces ROS in SVGA astrocytes at 100 mℳ ethanol (near physiological concentration in binge drinkers) at 12–36 h. Single treatment of 100 mℳ ethanol induced ROS production by >20% at 24 and 36 h (). Further, to examine whether CYP2E1 is responsible for the generation of ROS, we knocked down CYP2E1 expression through transfection using 10 nℳ predesigned CYP2E1 siRNA and 10 nℳ scrambled siRNA as control. In all, 10 nℳ CYP2E1 siRNA effectively diminished CYP2E1 protein expression (, right panel), which significantly reduced ethanol-induced formation of ROS at 24 h (, left side). Although not significant, CYP2E1 siRNA alone slightly increased ROS level compared with scramble siRNA. These results suggested the role of CYP2E1 in ethanol-induced ROS production in SVGA astrocytes. ¹⁵ Figure 1a Figure 1b Figure 1b

As caspase-3 cleavage is a marker of early apoptosis, we examined caspase-3 cleavage activity at 100 mℳ ethanol treatment for 24 h in SVGA astrocytes. The results showed that ethanol increased caspase-3 cleavage activity by more than twofold compared with control. In addition, knocking down CYP2E1 expression through CYP2E1 siRNA almost completely abolished ethanol-induced caspase-3 cleavage (). Furthermore, diallyl sulfide (DAS), a selective chemical inhibitor of CYP2E1, which is also a food additive and has protective effect on immune cells,abolished ethanol-induced apoptosis (). In addition, 100ℳ vitamin C, as well as vitamin E, blocked the effect of ethanol on induction of caspase-3 cleavage activity (), suggesting that ethanol-induced apoptosis is mediated through ROS production. Vitamin C alone also showed decreased caspase-3 cleavage activity compared with control, and it appeared to be more effective than vitamin E. However, other anti-oxidants tested, N-acetyl cysteine (NAC) and butylated hydroxyltoluene (BHT), did not reduce ethanol-induced apoptosis, rather they further induced caspase-3 cleavage activity in combination with ethanol (). Therefore, we used vitamin C as an antioxidant in subsequent experiments. Figure 1c Figure 1d Figures 1e and f Supplementary Figure S1 ¹⁶ μ

To further confirm the effect of ethanol, as well as the role of CYP2E1 and oxidative stress on apoptosis, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed in SVGA astrocytes. The results showed that 24 h ethanol treatment at 100 mℳ significantly increased formation of DNA fragments (). Although DAS alone showed some DNA fragmentation, both DAS and vitamin C effectively reduced ethanol-induced DNA fragmentation in SVGA astrocytes (). Finally, we tested whether DAS and antioxidant rescue ethanol-induced cell death using MTT assay. Ethanol showed a time- (12–48 h) and dose-dependent (100–200 mℳ) effect on the cell death of SVGA astrocytes (). Further, 100 mℳ ethanol showed 27% cell death, which was rescued by DAS and vitamin C (). Similar to TUNEL assay, DAS alone caused ∼15% cell death compared with control. Although DAS has a protective effect,it is also known to cause toxicity at high concentration and when used for longer time.Therefore, we performed a subsequent experiment using CYP2E1 siRNA to assess the specificity of DAS. Similar to the increase in oxidative stress by CYP2E1 siRNA alone (), it also caused significant cell death (), suggesting that a basal level of CYP2E1 is required for cell survival. In fact, a physiological role of CYP2E1 is documented in dopamine metabolism and nuclear factor-E2-related factor 2 induction in brain cells.However, as expected, CYP2E1 siRNA abolished ethanol-induced cell death (). Overall, our results clearly suggested the role of CYP2E1 and ROS in ethanol-induced apoptosis and cell death in SVGA astrocytes. Figure 1g Figure 1g Supplementary Figures S2A and B Figure 1h Figure 1b Supplementary Figure S2C Supplementary Figure S2C ^{16 17} ,, ^{18 19 20}

The basal levels of mRNA expression of CYP enzymes were earlier detected in SVGA astrocytes.Compared with the two most abundant CYP enzymes, CYP2A6 (56%) and CYP1A1 (43%), CYP2E1 showed relatively low mRNA expression (3%). However, its relative level in SVGA astrocytes compared with other CYPs is similar to that in the liver.Further, as previously shown in U937 monocytic cells,we investigated whether ethanol induces CYP2E1 in SVGA astrocytes. Initial results showed that 50 mℳ ethanol is optimum to induce CYP2E1 for up to 24 h (data not shown). The ethanol concentration at ≥100 mℳ caused significant cell death in SVGA astrocytes (). Therefore, we used 100 mℳ ethanol for oxidative stress, apoptosis, and cell death experiments at 24 h (), while we used 50 mℳ ethanol for examining the induction of CYP2E1 in SVGA astrocytes (). Kinetic profile of CYP2E1 expression showed that 50 mℳ ethanol resulted in significant upregulation of CYP2E1 mRNA at 3 h (>150%) and 6 h (<150%) compared with control (). Ethanol also showed 150% increased expression of CYP2E1 protein at 6 h, compared with control (). Both mRNA and protein expression levels of CYP2E1 decreased to the level of control at ≥12 h. ^{21 22 15} Supplementary Figures S2A and B Figure 1 Figure 2 Figure 2a Figure 2b

To examine whether CYP2E1 induction is associated with ethanol metabolism-mediated ROS production, we measured ROS production at early time points up to 4 h in the absence and presence of 50 mℳ ethanol in SVGA astrocytes (). The data showed that ROS production was increased at 2 h (>30%) by ethanol treatment. This result is consistent with the other observations, in which nicotine treatments also generated ROS at early time points (30 min–2 h) in SVGA astrocytes.To complement the finding in, we used 100 mℳ ethanol at 1 and 2 h, which showed higher increase in ROS () than the ROS generated at 50 mℳ ethanol (). As expected, CYP2E1 selective inhibitor, DAS, significantly decreased ethanol-induced oxidative stress at 2 h (), suggesting the role of CYP2E1 in the production of ROS by ethanol metabolism. Furthermore, to determine whether CYP2E1-mediated ethanol metabolism and subsequent ROS production are responsible for CYP2E1 induction, SVGA astrocytes were pretreated with 100ℳ DAS and vitamin C followed by ethanol treatment for 6 h. DAS significantly reduced ethanol-mediated CYP2E1 induction at both mRNA and protein levels (). Similarly, 100 mℳ vitamin C also abolished ethanol-mediated induction of CYP2E1 mRNA as well as protein (). DAS and vitamin C alone did not alter CYP2E1 expression significantly. In order to confirm that CYP2E1 is the primary enzyme responsible for ethanol metabolism in SVGA astrocytes, we measured ADH mRNA in astrocytes. However, the level of ADH in SVGA astrocytes was undetectable. These results suggested that ethanol-induced CYP2E1 expression is mediated through CYP2E1-mediated ethanol metabolism and subsequent production of ROS. Figure 2c Figure 1a Supplementary Figure S3 Figures 2c and d Figure 2d Figures 2e and f Figures 2g and h ²¹ μ

To determine the underlying mechanism responsible for ethanol-mediated CYP2E1 induction, SVGA astrocytes were pretreated with staurosporine, an inhibitor of protein kinase C (PKC), as well as inhibitors of c-Jun N-terminal kinase (JNK) inhibitor (SP600125) and mitogen-activated protein kinase kinase (MEK) inhibitor (U0126). Staurosporine abrogated ethanol-mediated induction of CYP2E1 mRNA and protein (). In addition, while JNK inhibitor abolished ethanol-mediated CYP2E1 induction (), the MEK inhibitor showed no effect (). Furthermore, as PKCis the major subtype of PKC family that mediates JNK activation,we tested whether selective inhibitor of PKC(PKCpseudo-substrate inhibitor (PPSI)), as well as PKCsiRNA, abrogates ethanol-mediated CYP2E1 expression in SVGA astrocytes. As expected, 10ℳ PPSI significantly reduced ethanol-induced CYP2E1 mRNA expression () and 10 nℳ PKCsiRNA completely blocked ethanol-induced CYP2E1 mRNA expression (). Overall, these results suggest that the expression of CYP2E1 is regulated by the activation of the PKC/JNK pathway. Figures 3a and b Figures 3c and d Supplementary Figure S4A Figure 3e Figure 3f ζ ζ ζ ζ μ ζ ²³

To further examine the transcription factor that is involved in ethanol-mediated CYP2E1 induction, 10M pomalidomide, a selective inhibitor of CCAAT/enhancer-binding protein-(C/EBP-), and 200 nM mithrimycin A, a selective inhibitor of specificity protein 1 (SP1), were used in SVGA astrocytes, followed by treatment with 50 mM ethanol. Although mithrimycin A alone slightly downregulated CYP2E1 mRNA expression, it completely abolished ethanol-mediated induction of CYP2E1 (). By contrast, although pomalidomide alone also reduced CYP2E1 expression, it did not alter ethanol-mediated induction of CYP2E1 (), in spite of the fact that pomalidomide reduced C/EBP-protein expression (). Thus, our results suggest that SP1 is responsible for the regulation of CYP2E1. μ β β β Figure 4a Figure 4b Figure 4c

As shown in SVGA astrocytes (), we examined the role of CYP2E1 and vitamin C on apoptosis in U937 monocytes using annexin V assay under different conditions with respect to treatment times and ethanol concentrations. Ethanol showed a minor increase in apoptosis, which to some extent, was rescued by DAS, vitamin C, and vitamin E (). However, the changes in these results were not conclusive. Furthermore, we performed cell death assay using 200 mℳ ethanol at 48 h (optimal conditions), which showed >15% cell death (). As expected 100ℳ DAS as well as 100ℳ antioxidants (vitamins C and E) both rescued cell death induced by 100 mℳ ethanol (). Unlike SVGA astrocytes (), DAS did not cause significant cell death in U937 monocytes (). However, similar to SVGA astrocytes, vitamin C was relatively more effective than vitamin E in U937 monocytes. Figures 1c–g Supplementary Figure S5 Figure 5 Figure 5 Figure 1 Figure 5 μ μ

As in SVGA astrocytes, we investigated the mechanism by which CYP2E1 is regulated by ethanol in U937 monocytes. The results showed that DAS and vitamin C inhibited ethanol-induced CYP2E1 mRNA expression (). Treatment of U937 cells with either DAS or vitamin C also slightly increased CYP2E1 expression. Similarly, PKC inhibitor (staurosporine), JNK inhibitor (SP600125), and SP1 inhibitor (Mithramycin A), completely abolished ethanol-induced CYP2E1 mRNA expression (). These inhibitors did not show any effect on the basal levels of CYP2E1 expression. Similar to SVGA astrocytes, MEK inhibitor (U0126) () and C/EBP-inhibitor (pomalidomide) (), these inhibitors did not alter induction of CYP2E1 mRNA expression by ethanol in U937 monocytes (). Thus, the expression of CYP2E1 is also regulated by oxidative stress-mediated activation of PKC/JNK/SP1 pathway in U937 monocytes. Figures 6a and b Figures 7c–e Supplementary Figure S4B Figure 4b Figure 6f β

The U937 monocytic cell line was obtained from ATCC (Manassas, VA, USA). The SVGA astrocyte cell line was generously provided by Dr. Avindra Nath, NIH/NIDA. DAS, vitamin C, vitamin E, staurosporine, U0126, SP600125, pomalidomide, and protease inhibitor cocktail, NAC and BHT were bought from Sigma-Aldrich, St. Louse, MO, USA. Roswell Park Memorial Institute (RPMI) 1640 and Dulbecco's Modified Eagle Medium (DMEM) media were purchased from Mediatech Inc., Manassas, VA, USA. Qiagen RNeasy kit was obtained from Qiagen, Valencia, CA, USA. Gene expression kit and primer probes were obtained from Applied Biosystems (Carlsbad, CA, USA). MTT proliferation assay and mithramycin A were from R&D systems, Inc. (Minneapolis, MN, USA). TUNEL apoptosis and Annexin V/PE apoptosis kits were from Genscript Inc. (Piscataway, NJ, USA) and BD Biosciences (San Jose, CA, USA), respectively. BCA protein assay kit was purchased from Thermo Scientific (Rockford, IL, USA). Dichlorofluoroscein diacetate (DCFDA) was purchased from Invitrogen (Grand Island, NY, USA). Radioimmunoprecipitation assay buffer and protease inhibitor cocktail were bought from Boston Bioproducts (Ashland, MA, USA). Primary and secondary antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Scramble, predesigned CYP2E1, and PKCsiRNA, as well as lipofectamine, were purchased from Life Technologies (Grand Island, NY, USA). PPSI was obtained from Santa Cruz Biotechnology, Inc. Caspase-3 colorimetric assay kit was from Clontech Laboratories, Inc. (Mountain View, CA, USA). ζ

U937 cells were grown in RPMI 1640 media with 1% gentamicin at 37 °C in a humidified incubator containing 5% CO. SVGA cells were grown in DMEM, containing 1% gentamicin. Ethanol treatment of monocytes was performed as previously described,and samples treated at an ethanol concentration of 100 mℳ for 12 h were selected for inhibitor studies. Ethanol treatment of astrocytes was performed 12 h after seeding cells in six-well plates. Desiccator-like containers containing 150 ml of 100 mℳ ethanol were pre-incubated for 1 h for ethanol saturation. Then, 50 mℳ ethanol was added to each well, and the plates within the ethanol-saturated containers were incubated in the incubator. Both ethanol concentrations (10–50 mℳ) and time course (1–24 h) were optimized for ethanol-mediated CYP2E1 induction. A dose of 50 mℳ ethanol at 3 h was found to be optimal for further experiments in the case of astrocytes. Treatments with vitamin C, vitamin E, NAC, BHT, staurosporine, U0126, SP600125, pomalidomide, and mithrimycin were initiated 1 h before ethanol treatment. However, cells treated with DAS were pre-treated for 15 min, before ethanol treatment according to the previous protocol.We used controls for each time point with or without these agents. 2 ^{52 53}

The production of ROS was measured by flow cytometry using DCFDA as previously described.Briefly, the astrocytes were treated with alcohol, either with or without inhibitors, using serum-free medium at different times in a six-well plate followed by addition of 10ℳ DCFDA. Cells were then harvested and dissolved in 1 ml PBS to measure the DCF emission at 525±20 nm by flow cytometry and mean fluorescence intensity was measured and analyzed. ²⁴ μ

Caspase 3 cleavage activity was measured according to the manufacturer's protocol. Briefly, 2 × 10cells were collected and resuspended in 50l cell lysate buffer for 10 min on ice and then centrifuged at maximum speed for 10 min at 4 °C. Further, 50l reaction reagent, including 1% DTT, was then added to each supernatant and mixed properly. After adding 5l caspase 3 substrate individually, samples were kept in the water bath at 37 °C for 1 h. Samples were then measured using microplate reader at 405 nm. 6 μ μ μ

A TUNEL apoptosis test was applied in SVGA astrocytes, which were adherent, to measure cellular apoptosis induced by treatments. Cells in each well were cultured on a cover slip. After termination of treatments, cells were fixed in fresh 4% formaldehyde for 30 min and then incubated with 70% ethanol for 30 min to increase membrane permeability. After incubation with permeabilization solution on ice for 2 min, cells were labeled through incubation with labeling solution (containing 2% FITC-12-dUTP) for 1 h in the dark. After washing, cells were mounted on a slide and fluorescence was detected using a confocal microscope with an emission wavelength of 515 nm.

Annexin V detection was performed in U937 monocytes to measure the effect of ethanol and inhibitors on apoptosis and cell death. Briefly, media was removed and cells from each well were suspended in binding solution at a final concentration of 1 × 10cells per ml, 100l of which was transferred into a 5-ml tube. In all, 5l PE, as indicator of early apoptosis, and 5l of 7-AAD were added to the 100l cell solution, followed by 15 min incubation at room temperature in dark. After incubation, 400l binding solution was added to each tube and fluorescence was detected using flow cytometer (BD Biosciences). Mean fluorescence intensity was measured and analyzed. 6 μ μ μ μ μ

Cell viability test was performed on six-well plates, using MTT assay. Media was removed from each well to terminate cell treatments. After washing twice with PBS, cells from each well were incubated for 4 h with a mixture of 200l 0.5 mg/ml MTT solution in PBS and 300l fresh media. This media was then replaced by 500l of a mixture of 400l DMSO and 100l Sorenson's glycine buffer. Absorbance was obtained at an emission wavelength of 570 nm in micro plate reader. The total number of cells remaining alive in each well was calculated using a standard curve. μ μ μ μ μ

Total RNA was extracted using Qiagen RNeasy kit based on the manufacturer's protocols. For each reaction, RNA (100 ng) from the samples was reverse-transcribed into cDNA using High-capacity cDNA Reverse Transcription Kit. qRT-PCR was performed using cDNA generated from the reverse transcription of RNA according to the manufacturer's instructions (TaqMan Gene Expression Kit, Applied Biosystems). PCR reactions were performed on the iCycler iQ system. (Bio-Rad Laboratories, Hercules, CA, USA). Relative gene expression was calculated using GAPDH as an endogenous control.

Total cell lysates were prepared using radioimmunoprecipitation assay buffer, containing 1 × protease inhibitor cocktail. The protein concentrations were measured using BCA protein assay kit. Western blotting was performed essentially as described.Briefly, 20g of total proteins were run on SDS-PAGE and then transferred to polyvinylidene fluoride membranes. Transferred blots were blocked in 5% nonfat dry milk followed by overnight incubation with primary antibody (1 : 1000) and 2-h incubation with an appropriate secondary antibody (1 : 1500). Proteins were detected by LuminataTM crescendo western HRP substrate (Millipore corporation, Billerica, MA, USA), using the Alpha Innotech FluorChem HD2 gel documentation system (Alpha Innotech, San Leandro, CA, USA). The densitometry data were analyzed using AlphaEase FC StandAlone software (version 6.0.0.14; Alpha Innotech).-Tubulin or GAPDH served as internal loading control to normalize the expression of proteins. ²⁴ μ β

SVGA astrocytes were transfected with predesigned human CYP2E1 siRNA or scramble siRNA (10 nℳ) in six-well plates for 24 h with lipofectamine transfection reagent in serum-free and antibiotic-free media. Transfection media were then discarded, and SVGA astrocytes were incubated overnight with complete media (containing 10% FBS and 1% gentamicin), followed by 24 h ethanol treatment at 100 mℳ.

Statistical analysis for qRT-PCR, western blotting, ROS measurement, caspase-3 cleavage activity, and MTT assay was performed to determine mean±SD. One-way ANOVA was applied to determinevalues. Avalue of ≤0.05 was considered significant. P P