Acute ethanol causes hepatic mitochondrial depolarization in mice: role of ethanol metabolism.

Hepatic mitochondrial polarization, permeability transition (MPT), and reduce pyridine nucleotides, and steatosis in mice were monitored by intravital confocal/multiphoton microscopy of the fluorescence of rhodamine 123 (Rh123), calcein, NAD(P)H, and BODIPY493/503, respectively, after gavage with ethanol (1–6 g/kg).

Materials and Methods

Animals and chemicals

Sources for animals and reagents are listed in Table 1 . 10.1371/journal.pone.0091308.t001 Table 1 Sources for Animals and Chemicals. Items Sources Animals: C57BL/6 mice Jackson Laboratory, Bar Harbor, Maine Cyp2E1 -/- mice Dr. Frank Gonzales, National Cancer Institute Deer mice Peromyscus Genetic Stock Center, Columbia, SC Reagents: Actin antibody ICN, Costa Mesa, CA Alcohol analytical kit BioVision, San Francisco, CA Alda-1 Dr. Daria Mochly-Rosen, Stanford University ALT analytical kit Pointe Scientific, Uncoln Park, MI Aminobenzotriazole Sigma-Aldrich, St. Louis, MO BODIPY493/503 Invitrogen, Carlsbad, CA Bromosulfophthalein Sigma-Aldrich, St. Louis, MO Calcein-AM Biotium Inc., Hayward, CA Chemiluminescence kit Pierce Biotec., Rockford, IL Disulfiram Sigma-Aldrich, St. Louis, MO Enliten ATP Assay System Promega Corp., Madison, WI MAA antibody Dr. Todd Wyatt, Univ. of Nebraska Medical Center 4-HNE antibody Alpha Diagnostics, Inc., San Antonio, TX Polyethoxylated castor oil Sigma-Aldrich, St. Louis, MO Propidium iodide Sigma-Aldrich, St. Louis, MO Rhodamine 123 Sigma-Aldrich, St. Louis, MO TMRM Invitrogen, Carlsbad, CA Triglyceride analytical kit Enzymatic Standbio, Boerne, TX Alda-1, N -(1,3-benzodioxol-5-ylmethyl)-2,6-dichlorobenzamide; ALT, alanine aminotransferase; calcein-AM, calcein acetoxymethyl ester, 4-HNE, 4-hydroxynonenal adducts; MAA, malondialdehyde-acetaldehyde adducts; TMRM, tetramethylrhodamine methylester.

Animals and ethanol treatment Male

C57BL/6 mice (8–9 weeks), Cyp2E1–null mice, and alcohol dehydrogenase (ADH)-positive and ADH-negative deer mice had access to chow diet ad libitum before a single gavage with alcohol (1–6 g/kg) or vehicle (saline). Some wild-type mice were pretreated with a cytochrome P450 inhibitor, aminobenzotriazole (ABT, 100 mg/kg, i.g. ), an aldehyde dehydrogenase (ALDH) inhibitor, disulfiram (DSF, 200 mg/kg, i.p. ), or an ALDH activator, Alda-1 [18] ( N -(1,3-benzodioxol-5-ylmethyl)-2,6-dichlorobenzamide, 50 mg/kg, i.p. ) 30 min prior to ethanol or with cyclosporin A (CsA, 10 mg/kg, i.g. ) at 1 h before ethanol. Vehicles for ABT, DSF, Alda-1 and CsA were saline, DMSO, DMSO and 8.3% polyethoxylated castor oil with 8.3% ethanol, respectively.

Clinical chemistry and histology

Blood was collected 1–6 h after ethanol treatment. Alcohol and alanine aminotransferase (ALT) were measured using commercial analytical kits ( Table 1 ) according to the manufacturers' protocols. Apoptosis was assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) [19] . Some liver tissue was frozen-sectioned and stained with Oil-Red-O staining to detect steatosis [20] . Paraffin sections after paraformaldehyde fixation were stained with periodic acid Schiff (PAS) to assess glycogen.

Measurement of hepatic triglycerides

Liver tissue (200 mg) was homogenized in normal saline and extracted with 2∶1 chloroform/methanol. After centrifugation at 1700 rpm for 5 min, the chloroform phase was separated, dried in a speed vacuum centrifuge and resuspended in 1 ml of chloroform. Triglyceride was measured using an analytical kit ( Table 1 ) [20] .

Hepatic ATP measurement

To assess the effects of ethanol on hepatic ATP, mice were treated with saline or ethanol (6 g/kg, i.g .), and livers were harvested 6 h later by freeze-clamping using aluminum tongs chilled in liquid nitrogen followed by storage at −80°C. ATP in trichloroacetic acid extracts was detected by luciferin-luciferase assay using an Enliten ATP Assay System ( Table 1 ) [21] , [22] .

Immunoblotting

Acetaldehyde is highly reactive, volatile and thus difficult to measure accurately. However, acetaldehyde rapidly reacts with other aldehydes/proteins to form adducts. Such adducts are potentially toxic to mitochondria and linked to ethanol hepatotoxicity [23] . Accordingly, malondialdehyde-acetaldehyde adducts (MAA), a hybrid acetaldehyde adduct [23] – [25] , and 4-hydroxynonenal (4-HNE) adducts, an indicator of lipid peroxidation, were detected by immunoblotting, as described [20] . Immunoblotting was performed with primary antibodies specific for MAA, 4-HNE, and actin ( Table 1 ) at 1∶1000, 1∶1000, and 1∶3000 dilutions, respectively, over night at 4°C. Horseradish peroxidase-conjugated secondary antibodies were applied, and detection was by chemiluminescence [20] . Assay of hypoxia inducible factor (HIF)-1a mRNA by quantitative real-time PCR Livers were harvested at 1 h and 6 h after ethanol treatment. HIF-1α mRNA was detected by quantitative real-time PCR, as described [26] . HIF-1α mRNA in liver was determined using a forward primer of 5′-GAAATGGCCCAGTGAGAAAA-3′ and a reverse primer of 5′-CTTCCACGTTGCTGACTTGA-3′ . The abundance of mRNAs was normalized against hypoxanthine phospho-ribosyl-transferase (HPRT) using the ΔΔ Ct method.

Intravital confocal and multiphoton microscopy

Intravital confocal and multiphoton microscopy was performed at 1 h to 7 days after saline or ethanol treatment, as described [16] , [26] . Mice had access to chow diet and water ad libitum until imaging. Rhodamine 123 (Rh123) and tetramethylrhodamine methylester (TMRM), cationic fluorophores that are taken up by polarized mitochondria in response to the negative mitochondrial membrane potential, were used to monitor mitochondrial polarization after ethanol treatment. Propidium iodide (PI) and 4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza- s -indacene (BODIPY493/503) were used to label nuclei of non-viable cells and fat droplets, respectively. Endogenous mitochondrial NAD(P)H was detected by blue autofluorescence. Onset of inner membrane permeabilization characteristic of the mitochondrial permeability transition (MPT) was detected after portal infusion of calcein acetoxymethyl ester (calcein-AM), which is cleaved by intracellular esterases to release calcein free acid into the cytosol, as described [27] . Under normal conditions calcein fluoresces in the cytosol, revealing mitochondria as dark voids due to the impermeability of mitochondrial membranes to calcein. These dark voids disappear after onset of the MPT as calcein enters the mitochondrial matrix space through MPT pores [16] , [26] , [27] [28] [22] . Under pentobarbital anesthesia (80 mg/kg, i.p .), a tracheotomy was performed, and an intravenous catheter (20 gauge) was inserted into the trachea, secured with a 5-0 silk suture, and connected to a small animal ventilator. The carotid artery was then cannulated with polyethylene (PE10) tubing. Rh123 (2 µmol/mouse), TMRM (1 µmol/mouse), PI (0.04 µmol/mouse), and/or BODIPY493/503 (25 µg/mouse) were infused singly or in combination in 0.4 mL of normal saline via the PE10 tubing over 10 min. Calcein-AM (1 mg/mouse) was injected slowly into the rectal vein. Bromosulfophthalein (6.6 µmol/mouse), an anion channel inhibitor, was injected into the rectal vein 5 min before calcein-AM to prevent biliary excretion of calcein. Imaging was started within 10 min after fluorophore infusion. Direct infusion of the fluorophores into the blood stream eliminated the potential influence of ethanol on absorption rates for the fluorophores and allowed rapid distribution of fluorophores into organs and cells. After fluorophore loading, the abdomen was opened transversely to expose the liver. Laparotomized mice were then placed prone on the stage of a Zeiss LSM 510 NLO laser scanning confocal/multiphoton microscope. The liver was gently positioned over a coverslip and imaged with 25x and 63x water-immersion objective lenses. Settings for simultaneous imaging of Rh123 (green) plus PI (red), Rh123 (green) plus NAD(P)H autofluorescence (blue), TMRM (red) plus BODIPY493/503 (green), TMRM (red) alone or calcein (green) alone are listed in Table 2 . During image acquisition, the respirator was turned off during ∼8 sec image scans to eliminate breathing movement artifacts. In preliminary studies, we observed that the preparations were stable for at least 1 h, but for the experiments presented imaging was completed within 30 min of fluorophore loading. 10.1371/journal.pone.0091308.t002 Table 2 Intravital Confocal/Multiphoton Microscopy Settings. Fluorophores Imaging Mode Excitation Emission Filters Color Rh123+PI Two-photon 800 nm 500–550 & 650–710 nm Green; red Rh123+NAD(P)H Two-photon 720 nm 500–550 & 435–485 nm Green; blue Calcein Two-photon 720 nm 500–550 nm Green TMRM± BODIPY493/503 Single-photon 543&488 nm 565–615 & 500–530 nm Red; green Calcein-AM (1 mg/mouse); Rh123, rhodamine 123 (2 µmol/mouse); PI, propidium iodide (0.04 µmol/mouse); TMRM, tetramethylrhodamine methylester (0.8 µmol/mouse); BODIPY493/503 (25 µg/mouse). To quantify labeling, 10 or more images were collected randomly from the liver of each mouse. Hepatocytes in these fields (∼250 cells) were scored in a blinded fashion for bright punctate Rh123 or TMRM fluorescence representing cells with polarized mitochondria versus a dimmer diffuse cytosolic fluorescence representing cells with depolarized mitochondria. For individual hepatocytes, ethanol caused mitochondrial depolarization almost always in an all-or-none manner. Thus, the distinction between punctate and diffuse staining was generally unambiguous. Rarely, mitochondria were not all depolarized within a single hepatocyte, and we scored such cells based on whether the majority of mitochondria had lost punctate labeling. Nonviable PI positive cells, indicated by red nuclear fluorescence, were also counted. In some mice, simultaneous imaging of BODIPY493/503-labeled fat droplets (green) and mitochondrial polarization by TMRM (red) was performed at 2 h after ethanol treatment (6 g/kg, i.g. ). The number of fat droplets in hepatocytes with and without mitochondrial depolarization was assessed in a blinded manner in 10 random fields per liver. Reduced pyridine nucleotides (NADH plus NADPH) fluoresce blue, and previous studies showed that nearly all NAD(P)H autofluorescence arises from mitochondria, whereas cytosolic NAD(P)H fluorescence is highly quenched [29] , [30] . Mitochondrial NADPH/NADP is in dynamic equilibrium with mitochondrial NADH/NAD via the electrogenic mitochondrial transhydrogenase. Blue autofluorescence in liver represents mitochondrial NAD(P)H regardless of whether autofluorescence is measured from whole liver by whole organ fluorometry, from sublobular locations with microlight guides, or from single cells/mitochondria by confocal/multiphoton microscopy [29] – [31] . Reduced pyridine nucleotides are relatively fragile and readily undergo photooxidation and photodamage. To minimize such changes, we used a low laser power setting that yielded images with lower signal-to-noise characteristics than for our other imaging. Measurement of serum Rh123 At 6 h after gavage with ethanol (6 g/kg) or an equal volume of saline, Rh123 was infused into the carotid artery under anesthesia as described above, and blood was collected from the vena cava immediately and after 10 and 30 min. Serum was obtained by centrifugation at 12,000 rpm for 10 min and stored in the dark at −80°C. After thawing, serum was diluted 10-fold in saline, and fluorescence was measured using excitation and emission wavelengths of 511 and 534 nm, respectively, with a Spectra Max M2 plate reader (Molecular Devices, Sunnyvale, CA) in comparison to Rh123 standards. Hepatic ischemia/reperfusion in mice The effect of cyclosporin A (CsA) on the MPT was tested in a hepatic ischemia/reperfusion (I/R) model as a positive control for its effectiveness in vivo since CsA is a classical MPT inhibitor. Our previous studies demonstrated that hepatic I/R causes the MPT onset in vivo , leading to mitochondrial depolarization [16] , [32] . At 1 h after vehicle- and CsA (10 mg/kg, i.g.) treatment, hepatic ischemia was induced by clamping the artery and portal vein to the upper three lobes of the liver ( i.e. , about 70% of total liver). The ischemic liver was reperfused by opening the vascular clamp 1 h later as described previously [16] . Mitochondrial polarization status was detected by intravital multiphoton microscopy of Rh123 at 2 h after reperfusion.

Statistical analysis

All groups were compared using ANOVA plus Student-Newman-Keuls' post-hoc test or Student's t-test, as appropriate. Values are means ± SEM. Differences were considered significant at p