Archives of Biochemistry and Biophysics 555-556 (2014) 16–22

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Ammonia-induced energy disorders interfere with bilirubin metabolism in hepatocytes Qiongye Wang a,1, Yanfang Wang a,1, Zujiang Yu b, Duolu Li a, Bin Jia a, Jingjing Li a, Kelei Guan a, Yubing Zhou a, Yanling Chen b, Quancheng Kan a,⇑ a b

Department of Pharmacology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China Department of Infectious Diseases, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China

a r t i c l e

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Article history: Received 18 February 2014 and in revised form 5 May 2014 Available online 27 May 2014 Keywords: Hyperammonemia Mitochondria Energy Bilirubin metabolism

a b s t r a c t Hyperammonemia and jaundice are the most common clinical symptoms of hepatic failure. Decreasing the level of ammonia in the blood is often accompanied by a reduction in bilirubin in patients with hepatic failure. Previous studies have shown that hyperammonemia can cause bilirubin metabolism disorders, however it is unclear exactly how hyperammonemia interferes with bilirubin metabolism in hepatocytes. The purpose of the current study was to determine the mechanism or mechanisms by which hyperammonemia interferes with bilirubin metabolism in hepatocytes. Cell viability and apoptosis were analyzed in primary hepatocytes that had been exposed to ammonium chloride. Mitochondrial morphology and permeability were observed and analyzed, intermediates of the tricarboxylic acid (TCA) cycle were determined and changes in the expression of enzymes related to bilirubin metabolism were analyzed after ammonia exposure. Hyperammonemia inhibited cell growth, induced apoptosis, damaged the mitochondria and hindered the TCA cycle in hepatocytes. This led to a reduction in energy synthesis, eventually affecting the expression of enzymes related to bilirubin metabolism, which then caused further problems with bilirubin metabolism. These effects were significant, but could be reversed with the addition of adenosine triphosphate (ATP). This study demonstrates that ammonia can cause problems with bilirubin metabolism by interfering with energy synthesis. Ó 2014 Elsevier Inc. All rights reserved.

Introduction Hyperammonemia and jaundice are both symptoms of hepatic failure. Previous studies have demonstrated a relationship between hyperammonemia and bilirubin. One such study investigated eighty patients with acute liver failure and found that, among the many biochemical parameters examined, only bilirubin levels were found to correlate with ammonia levels [1]. However, drugs that reduce ammonia are not generally used in patients that have jaundice and hyperammonemia. Results from several clinical studies have shown significant reductions in bilirubin levels when ammonia-reducing drugs were used to treat icterohepatitis [2,3]. In order to validate these clinical observations, a study was done in which a hyperammonemic rat model was established via intragastric administration of an ammonium chloride solution and the

⇑ Corresponding author. Address: The First Affiliated Hospital of Zhengzhou University, No. 40, Daxue Road, Zhengzhou 450052, China. Fax: +86 37166970906. E-mail address: [email protected] (Q. Kan). 1 The authors contributed equally to the work. http://dx.doi.org/10.1016/j.abb.2014.05.019 0003-9861/Ó 2014 Elsevier Inc. All rights reserved.

relationship between ammonia and bilirubin was analyzed [4]. It was found that hyperammonemia seriously interfered with bilirubin metabolism, however the reason for is yet to be determined. Ammonia is regarded as the key precipitating factor in hepatic encephalopathy [5,6]. The possible mechanisms of ammonia neurotoxicity have been widely studied and include direct toxicity of ammonia, oxidative stress from ammonia exposure, possible interference with energy metabolism due to ammonia and changes in glutamine levels resulting from ammonia [7,8]. Energy disorders induced by hyperammonemia play a major role in hepatic encephalopathy. The multidrug resistance protein 2 (MRP2) is an ATPdependent conjugate export pump, which is very important for eliminating bilirubin glucuronosides from hepatocytes and transporting them into the bile [9,10]. We speculate that hyperammonemia-induced energy disorders may affect the expression of enzymes related to bilirubin metabolism, such as MRP2. A hepatocyte model of hyperammonemia was established using medium supplemented with ammonium chloride and the effects of ammonia on hepatic mitochondria and energy were examined. The effects of ammonia on enzymes related to bilirubin metabolism,

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such as heme oxygenase-1 (HO-1)2, UDP-glucuronosyltransferase 1 (UGT1A1) and MRP2, were also studied both before and after the addition of ATP. It appears that ammonia induces energy barriers, which interfere with bilirubin metabolism. Materials and methods Human primary hepatocyte culture Primary human hepatocytes were purchased from Lonza (Walkersville, MD, USA) and cultured following the manufacturer’s instructions. Briefly, hepatocytes were thawed and spread onto 24-well plates coated with type 1 collagen from the bovine dermis (Koken Co., Ltd., Tokyo, Japan), at a density of 150,000 cells/cm2, and hepatocyte culture medium (HCM) (Lonza) was added. The plates were then placed in a 37 °C incubator with 5% CO2. The HCM was composed of 500 ml of hepatocyte basal medium (Lonza) supplemented with ascorbic acid, bovine serum albumin, fatty acid free, hydrocortisone, human epidermal growth factor, and insulin. The collagen coating was prepared by dissolving 0.3 mg of type I collagen in 1 ml of hydrochloride solution (pH = 4.0). The solution was placed into each well and the plates were incubated at room temperature for 30 min. The solution was then removed and the wells were washed twice with phosphate buffered saline (PBS). Viability measurement Cell viability inhibition was evaluated by a colorimetric assay based on 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). 0.5 mg/ml MTT was added after wells were exposed to different concentrations of ammonia. After the wells were incubated with MTT for 4 h, the supernatants were removed, 150 lL Dimethyl sulfoxide (DMSO) was added to each well and the plates were shaken for 15 min in the dark. The absorption was measured using a microplate reader at a wavelength of 490 nm. Determination of apoptosis The DNA gap of 3-OH can be labeled by TdT-mediated dUTP nick end labeling (TUNEL) and can provide specific, accurate localization of apoptotic cells via fluorescence intensity. After exposure to ammonia, the cells were fixed in 4% paraformaldehyde, at room temperature for 30 min. Cells underwent TUNEL incubation (solution A:B = 1:50) for 2 h, at 37 °C, in the dark, followed by 4,6-diamidino-2-phenylindole (DAPI) staining. The cells were then examined and photographed using a fluorescence microscope (Nikon Corp., Japan). Quantification of the number of TUNEL-positive cells was done by counting the number of labeled cells in per section. The apoptotic index (AI) was expressed as a percentage of the TUNEL-positive cells. Analysis of mitochondrial morphology using transmission electron microscopy (TEM) Cells were harvested and fixed in 2.5% glutaraldehyde, embedded over night at 37 °C in propylene oxide and epoxy resin and cut into ultrathin sections (1–2 lm). Sections were double-stained with uranyl acetate and lead citrate and observed via electron microscopy at a magnification of 60,000 (H-7650, HITACHI Co., Ltd., Tokyo, Japan). 2 Abbreviations used: TCA, tricarboxylic acid; ATP, adenosine triphosphate; MRP2, multidrug resistance protein 2; HO-1, heme oxygenase-1; UGT1A1, UDP-glucuronosyltransferase 1; mPTP, mitochondrial permeability transition pore; GC–MS, gas chromatograph–mass spectrometer-computer.

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Observation of mitochondrial permeability Calcein fluorescence studies were used mitochondrial permeability transition pore (mPTP) measurements following the method of Petronilli et al., with slight modifications [11,12]. In brief, calcein/acetoxymethyl ester (Calcein/AM) freely enters the cell and becomes fluorescent upon de-esterification. Since mitochondrial membranes are impermeable to cobalt, co-loading of cells with cobalt chloride quenches the fluorescence everywhere in the cell, except the mitochondria. However, during induction of the mPTP, cobalt can enter the mitochondria and is able to quench the calcein fluorescence. The extent of such quenching represents a measure of mPTP. There are two modes of mPTP: transient and long-lasting. In the absence of any stimuli, cells show a gradual and spontaneous decrease in fluorescence, which is suggestive of transient pore opening under resting conditions. However, there is a rapid decline in mitochondrial fluorescence in cells in vitro after being treated with compounds known to open the mPTP [12]. Cultures were treated with 10 mM ammonia for 48 h and then washed three times with Hank’s balanced salt solution (HBSS; 144 mM NaCL, 10 mM HEPES, 2 mM CaCl2, 5 mM KCl, and 10 mM glucose, pH 7.4). Cells were then incubated at 37 °C, for 20 min, in fresh HBSS containing Calcein/AM (1 lM) and cobalt chloride (1 mM). Following cobalt treatment, cultures were washed with HBSS, and examined using a Nikon Diaphot inverted fluorescent microscope (excitation wavelength = 488 nm and emission wavelength = 505 nm). Images of fluorescence quenching were taken at 30 s-intervals for 15 min in cells treated with ammonia and in control cells. Fluorescent intensities were then analyzed using Image J software (Wayne Rasband, USA). The average pixel (fluorescent intensity) value in each image, containing an approximately equal number of cells, was obtained and expressed as the means of the total fluorescence intensity. This was derived from at least 6 random image fields in each group. Because the initial values of fluorescence intensities were different between the ammonia and control groups, the average fluorescence is reported as a percentage of the initial values. Analysis of TCA cycle intermediates using gas chromatograph–mass spectrometer-computer (GC–MS) The model cells were divided into 2 groups as follows: the ammonia treated group (n = 6) and the control group (n = 6). The treated group was exposed to 10 mM ammonia for 48 h, then extracted the cell fluid and derivatized metabolites. GC–MS analysis was performed using an Agilent 7890 gas chromatograph system, coupled with a Pegasus 4D time-of-flight mass spectrometer. The system utilized a DB-5MS capillary column coated with 5% diphenyl cross-linked with 95% dimethylpolysiloxane (30 m  250-lm inner diameter, 0.25-lm film thickness; J&W Scientific, Folsom, CA, USA). A 1-lL aliquot of the analyte was injected in splitless mode. Helium was used as the carrier gas. The front inlet purge flow rate was 3 mL min1 and the gas flow rate through the column was 1 mL min1. The initial temperature was set at 80 °C for 0.2 min and was subsequently raised to 180 °C at a rate of 10 °C min1. The temperature was then raised to 240 °C at a rate of 5 °C min1 and then to 290 °C at a rate of 20 °C min1, where it was maintained for 11 min. The injection, transfer line, and ion source temperatures were 280°, 270°, and 220 °C, respectively. The energy was 70 eV in the electron impact mode. The mass spectrometry data were acquired in full-scan mode, with an m/z range of 35–600, at a rate of 100 spectra per second, after a solvent delay of 492 s. Chroma TOF4.3X Software (LECO, St. Joseph, Michigan, USA) was used for auto acquisition of GC total ion chromatograms (TICs) and fragmentation patterns. Each com-

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pound had a unique fragmentation pattern composed of a series of split molecular ions. The mass–charge ratios and amount of the ions were compared to a standard mass chromatogram in the LECO-Fiehn Rtx5 mass spectra library in the Chroma TOF4.3X Software. For each peak, the software generated a list of similarities after comparing them with every substance that was in the LECO-Fiehn Rtx5 library [13]. Peaks that had more than 700 similarities were chosen for further research. Changes in index related to liver function The supernatant was extracted after cells were exposed to 0 and 10 mM ammonia for 12, 24, and 48 h. Alanine transaminases (ALT), aspartate transaminase (AST), glutathione (GSH) and cytochrome c oxidase (COX), as well as direct bilirubin (DBIL) were then measured using an ELISA kit (Suzhou Calvin biotechnology, Suzhou, China). The OD values were detected at 450 nm using a Thermo Multiskan MK3microplate reader (Thermo Fisher Scientific Inc., Waltham, MA, USA). Quantitative PCR (QT-PCR) for mRNA expression Total RNA was extracted at different time points, according to standard procedures. Samples with high-quality RNA were reverse-transcribed to cDNA. The GAPDH gene was selected as the internal reference gene. All primers were synthesized by TaKaRa (Dalian, China). hGAPDH: upstream primer 50 -ccagggctgcttttaactc-30 , downstream primer 50 -gctcccccctgcaaatga-30 ; HO-1: upstream primer 50 -ttgccagtgccaccaagttc-30 , downstream primer 50 -tcagcagctcctgca actcc-30 ; UGT1A1: upstream primer 50 -tggctgt tcccacttactgcac-30 , downstream primer 50 -agggtccgtcagcatgacatc30 ; MRP2, upstream primer 50 -cactgttggctttgttctgtcc-30 , downstream primer 50 -cagggtgcctcattttcca-30 . QT-PCR amplification was carried out according to the SYBRÒPremix Ex TapTMII kit instructions and tested with the ABI StepOnePlusTM Real-Time PCR System. Western blot for determination of proteasome content Cells were lysed in ice-cold RIPA buffer (P0013B, Beyotime, Shanghai, China). Lysates were adjusted so that the protein content was equal and were boiled in loading buffer. Lysates were then separated via electrophoresis (12% SDS–PAGE), transferred to a PVDF membrane and blocked with 5% skim milk in TBS 0.5% Tween 20. The membrane was incubated with a primary rabbit antiUGT1A1 IgG (NBP1-69448, NOVUS) or with rabbit anti-heme oxygenase-1(DA2045, BBI) and secondary antibody (goat anti-rabbit IRDyeÒ 680LT or goat anti-mouse IRDyeÒ 800CW; Li-CORÒ, Biosciences, Lincoln, Nebraska, USA) and were subsequently analyzed for immunoreactivity using the ODYSSEYÒ CLX Infrared Imaging System. Precision markers were used to assess the molecular weight. Statistical analysis The data were analyzed by ANOVA, followed by the Student–Newman–Keuls multiple-range test and are expressed as the mean ± SD. Statistical tests were performed with SAS 9.1 software and P < 0.05 was considered statistically significant.

by a dramatic time-dependent loss in cell viability (Fig. 1a). Exposure to 5 mM ammonia for 24 h was followed by a significant decline in cellular viability, however the cells recovered viability at 48 h. Microscopic analysis showed a loss in cell density that occurred in a concentration and time dependent manner (Fig. 1b). There were no obvious changes in cell morphology and growth density after treatment with 5 mM ammonia for 48 h. Decreased cell density and more dead cells were observed when at ammonia concentrations higher than 10 mM. There was an intracellular vacuolization phenomenon in cells that were treated with 10 and 20 mM; this phenomenon was more obvious in cells treated with 20 mM ammonia. Mitochondrial damage and apoptosis The classically recognized function of mitochondria is the synthesis of ATP, which is necessary for endergonic reactions [14]. The synthesis of ATP is affected by the integrity of the mitochondria. Injuries to the mitochondria were observed by TEM after exposure to 10 and 20 mM ammonia for 48 h (Fig. 1c). There was mitochondrial swelling and rounding, crista disorder, and vacuolar degeneration. Apoptotic cells with green nuclei were observed in both the ammonia-treated and control groups (Fig. 1d). The apoptosis indexes (AI) were 23% and 31% in 10 and 20 mM ammonia groups, respectively vs. 6% in the control group (P < 0.01). This indicated that apoptosis was significantly enhanced in ammonia-treated groups. Based on these results, both 10 and 20 mM ammonia can injure hepatocytes; however, the 20 mM ammonia concentration caused more cell damage. After exposure to 20 mM ammonia, the cell density was too low to continue the experiment, therefore a 10 mM ammonia was used for the following experiments and measurements. Effect of ammonia on mitochondrial permeability Cells were treated with 10 mM ammonia for 48 h and changes in mitochondrial permeability were examined using mitochondrial calcein fluorescence after quenching with cobalt. Cells in the absence of any stimuli (control group) showed a gradual and spontaneous decrease in fluorescence, which is suggestive of transient pore opening under resting conditions (Fig. 2a and b). Conversely, there was an abrupt decrease in mitochondrial fluorescence in cells treated with ammonia, which is suggestive of long-lasting pore opening with high ammonia levels (Fig. 2c and d). These results indicate that ammonia induces mPTP in hepatocytes. Reduction of TCA cycle intermediates TCA cycle intermediates were detected in hepatocytes, via GC– MS analysis, after 48 h exposure to 10 mM ammonia. The levels of citrate, alpha-ketoglutarate and malate were significantly lower in the ammonia-treated group compared with the control group. Glutamine, which is formed by the combination of ammonia and alpha-ketoglutarate, was significantly higher in the ammonia-treated group compared with the control group. The marked change in TCA cycle intermediates in hepatocytes treated with ammonia, compared to controls, suggests abnormalities within the TCA cycle (Fig. 3). This eventually led to a significant reduction in ATP release.

Results Changes in the index related to the liver function Effects of ammonia on the viability and morphology of hepatocytes A change in the pH value of less than ±0.025 was negligible. Exposure to 10 mM and 20 mM ammonia is clearly accompanied

After 12 h exposure to 10 mM ammonia, the level of GSH significantly decreased, but gradually recovered and was at the same level as the control group by 48 h (Fig. 4a). The decrease in GSH

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Fig. 1. Effects of ammonia on the viability and morphology of hepatocytes. (a) Hepatocytes were treated with ammonia at concentrations of 5, 10 and 20 mM for 12, 24, and 48 h. Cell viability was determined using an MTT assay. (b) Hepatocytes were treated with ammonia at concentrations of 0, 5, 10, and 20 mM for 48 h. Cell morphology was observed by microscopy (400). (c) Hepatocytes were treated with ammonia at concentrations of 0, 10, and 20 mM for 48 h. Morphologic features of mitochondria were analyzed using TEM (60000). Morphologic changes in the mitochondria in the high ammonia group: mitochondrial swelling and rounding, crista disorder, vacuolar degeneration. (d) Hepatocytes were treated with ammonia at concentrations of 0, 10, and 20 mM for 48 h. Apoptosis was determined using TUNEL assay (400). There were more apoptotic cells in the high ammonia group (green nuclei) than in the control group (blue nuclei). Values are expressed mean ± SD. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 2. Ammonia-induced mPTP as determined by calcein fluorescence (200). After hepatocytes were treated with ammonia at concentrations of 10 mM for 48 h, the cells then coloaded with Co2+ and calcein AM, and were imaged at 30-s intervals for 15 min. Baseline images were collected in the ammonia group and in the control group (a and c). Cells showed a rapid decline in the fluorescence in the ammonia group; this decline was almost complete after 10 min (b). The decrease in the fluorescence was negligible after 10 min in the control group (d). The plot shows the time course of fluorescence changes in the ammonia group and control group. Data are the mean of 6 random image fields expressed as the percentage of the initial values.

indicates an increase in intracellular sulfhydryl damage. The level of ALT in the ammonia group was significantly higher at 24 h compared to the control group, but was markedly decreased by 48 h (Fig. 4b). The level of AST in the ammonia-treated group was not

significantly different compared with the control group at 24 h, but was significantly higher at 48 h (Fig. 4c). Changes in the levels of ALT and AST are correlated with changes in the degree of liver damage. The level of COX in the ammonia-treated group was

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Fig. 3. Schematic representation summarizing changes in TCA cycle intermediates. The green color represents a decrease and the red color represents an increase. The y-axis of the box plot indicates relative concentration (peak area/internal standard peak area); Values are expressed mean ± SD, ⁄P < 0.05, ⁄⁄P < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 4. Changes in the index related to liver function. Values are expressed mean ± SD; ⁄P < 0.05, ⁄⁄P < 0.0001. ALT: glutamate pyruvate transaminase; AST: aspartate aminotransferase; GSH: glutathione; COX: cytochrome c oxidase.

significantly higher at both 24 h and 48 h compared with the control group (Fig. 4d). The increased level of COX suggests impaired oxidation of mitochondrial respiratory function. Effect of ammonia the DBIL levels and on enzymes related to bilirubin metabolism After exposure to 10 mM ammonia, the change of mRNA and protein of enzymes related to bilirubin metabolism and the DBIL

levels were detected at 6 h intervals for 72 h, the most significant change in mRNA expression was observed at 24 h, and the most significant change in protein expression was 12 h later than that in mRNA, and the most significant change in DBIL level was 6 h later than that in protein, so the expression of mRNA at 24 h, protein at 48 h and the DBIL level at 54 h showed up as a representative (Fig. 5). The level of UGT1A1 mRNA expression was higher in the group treated with 10 mM ammonia for 24 h compared with the control group and the level of MRP2 mRNA expression had significantly decreased compared with the control group. However, there was no difference in HO-1 mRNA expression between the 2 groups (Fig. 5a). The addition of ATP increased the level of UGT1A1 mRNA expression and significantly increased the level of MRP2 mRNA expression (Fig. 5c and d). The results of the western blots are consistent with those from the QT-PCR (Fig. 5e and f). The energy barrier caused by the ammonia reduced the level of MRP2. This blocked the extracellular transport of DBIL and resulted in a decreased level of DBIL in the cell supernatants (Fig. 5b). Expression of MRP2 significantly increased after the addition of ATP, resulting in an acceleration of bilirubin extracellular transport, an elevation in the level of extracellular DBIL. These results indicate that ammonia-induced energy disorders hinder the excretion of bilirubin by interfering with MRP2 in hepatocytes.

Discussion In the current study, we found that ammonia interfered with bilirubin metabolism via disturbance of energy metabolism in hepatocytes. Ammonia, a breakdown product of amino acid catabolism, can be a potent neurotoxin if allowed to accumulate [15]. The neurotoxicity of ammonia is commonly studied as a potential cause of encephalopathy. However, there are few studies that have examined the effect of ammonia on the liver and bilirubin

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Fig. 5. Effect of ammonia on the levels of DBIL and the bilirubin metabolism related enzyme. Cells were divided into four groups: control group (0 mM ammonia); 10 mM ammonia group;+ATP group (0.3 mM ATP was added 30 min before the addition of 10 mM ammonia).+GSH group (1.5 mM GSH was added 30 min before the addition of 10 mM ammonia). (a) QT-PCR analysis of the mRNA expression of enzymes related to bilirubin metabolism in the ammonia and control groups (n = 3 in each group); (b) The level of extracellular DBIL at 54 h in the four groups (n = 6 in each group). (c) and (d) The mRNA expression of UGT1A1 and MRP2 at 24 h in the four groups (n = 3 in each group); (e) and (f) Western blots of UGT1A1 and MRP2 at 48 h in the four groups (n = 3 in each group). Data are shown as mean ± SD. ⁄P < 0.05, ⁄⁄P < 0.01.

metabolism. Recent studies, including results obtained from our laboratory [4], have demonstrated that hyperammonemia has a direct adverse effect on hepatocytes and is therefore both a cause and an effect of hepatic failure. Furthermore, hyperammonemia can cause disorders in bilirubin metabolism in hepatocytes. In the current study, the role that hyperammonemia plays in bilirubin metabolism disorders was investigated. Ammonia is not toxic to all cells. Hassel et al. [16] demonstrated that different cell types have different growth inhibitions when exposed to the same concentrations of ammonia, suggesting that the effect of ammonia is cell specific. In the current study, there was a marked change in the viability and morphology of hepatocytes as the ammonia concentration and time of exposure increased, indicating that hepatocytes are sensitive to ammonia (Fig. 1a and b). Hepatocyte mitochondrial morphology began to show abnormalities as the concentration of ammonia increased (swelling and rounding, crista disorder, vacuolar degeneration) (Fig. 1c) and apoptosis occurred after the mitochondria had been damaged (Fig. 1d). Ammonia exposure also induced a long-lasting opening of mPTP (Fig. 2). The mPTP is a protein pore that is formed in the inner membrane of the mitochondria under certain pathological conditions and is indicative of an increase in mitochondrial permeability [17,18]. Induction of long-lasting mPTP openings can lead to mitochondrial swelling and cell death through apoptosis or necrosis depending on the particular biological setting [19–21]. In the current study, the ammonia-induced, prolonged opening of the hepatocyte mPTP may have been a factor in mitochondrial dysfunction and apoptosis. These results are consistent with reports of ammonia neurotoxicity in astrocytes, in which induction of mPTP has been described in cultured astrocytes exposed to ammonia [22,23].

The mPTP is associated with movement of metabolites across the inner mitochondrial membrane, swelling of the mitochondrial matrix, defective oxidative phosphorylation and ATP production [24]. In the current study, the TCA cycle intermediates, citrate, alpha-ketoglutarate and malate, were significantly lower in the ammonia-treated compared with the control group (Fig. 3). The decrease in alpha-ketoglutarate observed in hepatocytes treated with ammonia could be because ammonia chemically combines with alpha-ketoglutarate to form glutamine. This is consistent with the results of a study done by Rao and Norenberg [25]. It is apparent that any reaction that tends to remove alpha-ketoglutarate will also diminish the rate of formation of the succeeding members in the TCA cycle. Interestingly, the level of glutamine increased in hepatocytes after exposure to ammonia. This is in line with the results of other studies examining the effects of ammonia on astrocytes [26,27]. According to the hypothesis of ‘‘Trojan horse’’ [26], it is the glutamine-derived ammonia within the mitochondria that interferes with mitochondrial function, giving rise to excessive production of free radicals and the induction of mPTP. This may be one of reasons that the mitochondria are also damaged in hepatocytes. This indicates that the ammonia caused an energy disorder in the hepatocytes, which eventually led to a decrease in the production of ATP. The levels of index related to the liver function were markedly changed after exposure to ammonia (Fig. 4). Within 24 h of ammonia treatment, we observed that ALT increased with increasing time, but there is no obvious change in AST. ALT is completely extramitochondrial, however AST is both intra- and extramitochondrial. Therefore only ALT was released during the first 24 h since the mitochondria were still intact. AST levels had increased by 48 h, due to mitochondrial damage and the level of ALT had declined by this point due to impairment of the hepatic synthesis

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of ALT. This indicates that the degree of hepatocyte injury aggravated with increasing exposure times. The above results support the hypothesis that dysfunction of mitochondria and disorders in hepatocyte energy metabolism may be caused by ammonia. HO-1 catalyzes the rate-limiting step in the generation of indirect bilirubin [28]. And indirect bilirubin is extensively glucuronidated by UDP-glucuronosyltransferase 1 (UGT1A1) [30,31]. Eventually, the glucuronide metabolite, conjugated bilirubin, is excreted via MRP2 [31,32]. MRP2 is the ATP-dependent conjugate export pump [33,34] and plays a decisive role in the elimination of bilirubin glucuronosides from hepatocytes into bile [35]. Accordingly, the absence of functional MRP2 from the canalicular membrane leads to conjugated hyperbilirubinemia [36–38], as observed in a hereditary disorder described by Dubin and Johnson [39]. These results suggest that up-regulation of MRP2 will improve hyperbilirubinemia in patients with Dubin–Johnson syndrome. In this study, we found an increase in the expression of UGT1A1, which may be associated with the accumulation of intracellular DBIL itself can activate its metabolic enzyme (Fig. 5a). We also found a significant decrease in the expression of MRP2, along with a corresponding a reduction in extracellular DBIL (Fig. 5b and d), in ammonia-treated cells. This is likely due to the fact that there low level of MRP2 cannot effectively excrete DBIL from hepatocytes. However, these effects were reversed when ATP was added to the ammonia-treated cells, suggesting that adding ATP can repair metabolic disorders of bilirubin caused by ammonia. Reduction of ATP plays a very important role in bilirubin metabolism disorders. Dysfunctions in bilirubin metabolism may be explained by disorders in energy metabolism caused by ammonia. Conclusions In this study, it was found that hyperammonemia can damage the mitochondria and TCA cycle and can decrease the expression of MRP2 in hepatocytes, however this can be reversed with the addition of ATP. Hyperammonemia appears to cause an energy disorder, which then interferes with bilirubin metabolism. This study may provide insight into the possible molecular mechanism of jaundice induced by hyperammonemia and targets for the treatment or prevention of bilirubin metabolic disorders.

References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14]

[15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [30] [31] [32] [33] [34] [35] [36]

Acknowledgment We thank manuscript.

professor

[37]

Dongliang

Yang

for

revising

the

[38] [39]

V. Bhatia, R. Singh, S.K. Acharya, Gut 55 (2006) 98–104. Y. Wang, X. Zhang, X. Wang, J. Clin. Intern. Med. 4 (2007) 014. H.-k. Yin, Q.-x. Li, D. Luo, Chin. J. Clin. Hepatol. 4 (2007) 013. B. Jia, Z.J. Yu, Z.F. Duan, X.Q. Lu, J.J. Li, X.R. Liu, R. Sun, X.J. Gao, Y.F. Wang, J.Y. Yan, Q.C. Kan, Liver Int. 34 (2013) 748–758. P. Ferenci, A. Lockwood, K. Mullen, R. Tarter, K. Weissenborn, A.T. Blei, Hepatology 35 (2002) 716–721. E.A. Jones, K.D. Mullen, Clin. Liver Dis. 16 (2012) 7–26. D.L. Shawcross, S.S. Shabbir, N.J. Taylor, R.D. Hughes, Hepatology 51 (2010) 1062–1069. I. Coltart, T.H. Tranah, D.L. Shawcross, Arch. Biochem. Biophys. 534 (2013) 71– 87. A.T. Nies, D. Keppler, Pflugers Arch. 453 (2007) 643–659. G. Jedlitschky, I. Leier, U. Buchholz, J. Hummel-Eisenbeiss, B. Burchell, D. Keppler, Biochem. J. 327 (1997) 305–310. K.V.R. Rao, M.D. Norenberg, J. Biol. Chem. 279 (2004) 32333–32338. V. Petronilli, G. Miotto, M. Canton, M. Brini, R. Colonna, P. Bernardi, F. Di Lisa, Biophys. J. 76 (1999) 725–734. P. Bernardi, M. Forte, Novartis Found. Symp. 287 (2007) 157. V. Calabrese, R. Lodi, C. Tonon, V. D’Agata, M. Sapienza, G. Scapagnini, A. Mangiameli, G. Pennisi, A. Stella, D.A. Butterfield, J. Neurol. Sci. 233 (2005) 145–162. V. Felipo, R.F. Butterworth, Prog. Neurobiol. 67 (2002) 259–279. T. Hassell, S. Gleave, M. Butler, Appl. Biochem. Biotechnol. 30 (1991) 29–41. P. Bernardi, M. Forte, Novartis Found. Symp. 287 (2007) 157–164. discussion 164–159. P. Bernardi, Front. Physiol. 4 (2013) 95. K.W. Kinnally, P.M. Peixoto, S.-Y. Ryu, L.M. Dejean, Biochim. et Biophys. Acta (BBA) – Mol. Cell Res. 2011 (1813) 616–622. A. Nicotra, S. Parvez, Neurotoxicol. Teratol. 24 (2002) 599–605. J.S. Kim, L. He, J.J. Lemasters, Biochem. Biophys. Res. Commun. 304 (2003) 463– 470. P.V. Reddy, K.V. Rama Rao, M.D. Norenberg, J. Neurosci. Res. 87 (2009) 2677– 2685. K. Rama Rao, M. Chen, J. Simard, M. Norenberg, J. Neurosci. Res. 74 (2003) 891– 897. M. Zoratti, I. Szabo, U. De Marchi, Biochim. Biophys. Acta 1706 (2005) 40–52. K.V. Rao, M.D. Norenberg, Metab. Brain Dis. 16 (2001) 67–78. J. Albrecht, M.D. Norenberg, Hepatology 44 (2006) 788–794. A. Jayakumar, K. Rao, C.R. Murthy, M. Norenberg, Neurochem. Int. 48 (2006) 623–628. G.H. Oakes, J.R. Bend, J. Biochem. Mol. Toxicol. 19 (2005) 244–255. A. Rowland, J.O. Miners, P.I. Mackenzie, Int. J. Biochem. Cell Biol. 45 (2013) 1121–1132. D. Keppler, Drug Metab. Dispos. 42 (2014) 561–565. dmd.113.055772. K.-C. Sung, J. Shin, Y.-H. Lim, S.H. Wild, C.D. Byrne, Am. J. Cardiol. 112 (2013) 1873–1879. I. Leier, J. Hummel-Eisenbeiss, Y. Cui, D. Keppler, Kidney Int. 57 (2000) 1636– 1642. Y. Cui, J. König, U. Buchholz, H. Spring, I. Leier, D. Keppler, Mol. Pharmacol. 55 (1999) 929–937. G.D. Kruh, M.G. Belinsky, J.M. Gallo, K. Lee, Cancer Metastasis Rev. 26 (2007) 5– 14. T. Kawabe, Z.-S. Chen, M. Wada, T. Uchiumi, M. Ono, S.-i. Akiyama, M. Kuwano, FEBS Lett. 456 (1999) 327–331. J. Kartenbeck, U. Leuschner, R. Mayer, D. Keppler, Hepatology 23 (1996) 1061– 1066. I. Dubin, F.B. Johnson, Medicine 33 (1954) 155–198. J.E. Skandalakis, R.H. Johnson, E.O. Rand, J. Med. Assoc. Ga. 47 (1958) 392–393.