Abstract

We previously demonstrated in in vitro and ex vivo models that physiological concentrations of unconjugated bilirubin (BR) prevent oxidative stress (OS)-induced hepatocanalicular dysfunction and cholestasis. Here, we aimed to ascertain, in the whole rat, whether a similar cholestatic OS injury can be counteracted by heme oxygenase-1 (HO-1) induction that consequently elevates endogenous BR levels. This was achieved through the administration of hemin, an inducer of HO-1, the rate-limiting step in BR generation. We found that BR peaked between 6 and 8 h after hemin administration. During this time period, HO-1 induction fully prevented the pro-oxidant tert-butylhydroperoxide (tBuOOH)-induced drop in bile flow, and in the biliary excretion of bile salts and glutathione, the two main driving forces of bile flow; this was associated with preservation of the membrane localization of their respective canalicular transporters, bile salt export pump (Bsep) and multidrug resistance-associated protein 2 (Mrp2), which are otherwise endocytosed by OS. HO-1 induction counteracted the oxidation of intracellular proteins and membrane lipids induced by tBuOOH, and fully prevented the increase in the oxidized-to-total glutathione (GSHt) ratio, a sensitive parameter of hepatocellular OS. Compensatory elevations of the activity of the antioxidant enzymes catalase (CAT) and superoxide dismutase (SOD) were also prevented. We conclude that in vivo HO-1 induction protects the liver from acute oxidative injury, thus preventing consequent cholestasis. This reveals an important role for the induction of HO-1 and the consequently elevated levels of BR in preserving biliary secretory function under OS conditions, thus representing a novel therapeutic tool to limit the cholestatic injury that bears an oxidative background.

Introduction

In aerobic organisms, the use of oxygen in the mitochondria as the last acceptor of the respiratory electron chain constitutes an evolutionary advantage. However, the main drawback is its high cytotoxic potentiality [1]. Cellular oxygen is used in 97–98% for water synthesis and the other 2–3% continuously generates reactive oxygen species (ROS) that can damage cellular structures. Oxidative stress (OS) arises from the imbalance between ROS generation during metabolic processes and the capacity of antioxidant systems to clear them out [2]. OS is a common feature in most hepatopathies, many of which concur with either mild or severe cholestasis. Obstructive cholestasis [3], sepsis-induced cholestasis [4], viral [5], toxic [6], and autoimmune hepatitis [7], alcoholic steatohepatitis [5], nonalcoholic fatty liver disease [8], and hepatic ischemia–reperfusion injury [9,10] are amongst those clinical entities bearing an oxidative background. Cumulative evidence indicates that ROS induce a number of functional changes either deleterious or adaptive in the capability of the hepatocytes to produce bile and to secrete exogenous and endogenous compounds. Functional changes involve impairment of biliary secretion through both, direct oxidative damage to the cellular structures involved in this process, and modification of intracellular redox-sensitive signal transduction pathways [11]. OS-induced impairment of bile flow generation has been demonstrated to occur soon after exposure to a number of pro-oxidant agents, including tert-butylhydroperoxide (tBuOOH) [12,13]. This compound is a synthetic analog of short-chain lipid hydroperoxides formed endogenously under OS conditions, which has been widely used as a model to study the effect of OS on biological systems [14–16]. Apart from inducing OS directly via reduction into both peroxyl- and alkoxyl-free radicals by both cytochrome P450 and the mitochondrial electron chain [17], tBuOOH promotes OS via ROS-induced formation of the mitochondrial permeability transition pore (MPTP) and further mitochondrial production of ROS [18]. Therefore, when administered acutely and studied in a short-term time scale, tBuOOH represents an ideal tool for the study of the effect of a ‘pure’ oxidant insult on liver secretory function and its prevention.

In previous work, our group demonstrated that endocytic internalization of canalicular transporters is a key factor in OS-induced cholestasis. We found in isolated rat hepatocyte couplets that tBuOOH induces endocytic internalization of the bile salt export pump (Bsep, Abcb11), a main canalicular carrier that transports into the bile canaliculus osmotically active monoanionic bile salts, the main driving forces involved in the generation of the so-called ‘bile salt-dependent bile flow’ [19]. The same holds true for the multidrug resistance associated protein 2 (Mrp2, Abcc2), as has been shown in isolated rat hepatocyte couplets [20,21], and perfused rat livers exposed to the pro-oxidant agent tBuOOH [20,21]; this transporter mediates the biliary excretion of glutathione, the main driving force of the so called ‘bile salt-independent bile flow’ [22].

Bilirubin (BR), one of the end products of heme catabolic pathway, has been historically considered not only a waste product but also a potentially toxic compound. However, this view began to change by the late 20th century, owing to the work of Stocker and colleagues [23,24] and today BR is regarded as a potent endogenous antioxidant agent with cytoprotective properties [25,26]. Antioxidant effects of BR in vitro comprise the scavenging of singlet oxygen [27] and superoxide anion [28], as well as peroxyl radicals [23], thus explaining its protective effects against membrane lipid peroxidation [29]. In addition, BR is a well-known scavenger of reactive nitrogen species [30]. In vivo, BR has been found to protect rodents against both OS-induced retinal degeneration [31] and diabetic nephropathy [32], and it also shows anti-genotoxic effects in humans and animal models [33]. BR has also been shown to bear an immunomodulatory function, as it prevents complement system-mediated hepatocyte lysis [34–36] and suppresses innate immune response after islet transplant [37]. In our previous work, we demonstrated that physiological concentrations of unconjugated BR prevent OS-induced hepatocellular damage, and that this cytoprotective effect is exerted through prevention of actin cytoskeleton disarrangement and the consequent endocytic internalization of canalicular transporters crucial to normal biliary secretory function [20]. These findings support the notion that unconjugated BR, an endogenously generated metabolite, may act as an efficient antioxidant agent, even at physiological concentrations.

Until nowadays, a myriad of studies has demonstrated the potential utility of exogenously administered BR in the treatment of several diseases, such as post-transplantation ischemia–reperfusion [38], cardiovascular [39], pulmonary [39], and metabolic diseases [40]. However, studies inquiring into the effect of endogenously elevated levels of BR occurring during pathological conditions of the liver are scarce. Moreover, the likely beneficial action of heme oxygenase-1 (HO-1) induction and consequently elevated BR levels as a therapeutic strategy under a ‘pure’ OS-induced cholestatic condition has never been addressed. The present work was aimed to extend our previous in vitro/ex vivo findings to the whole animal, an in vivo model much closer to the human pathophysiological context, and to use an experimental therapeutic approach safe and feasible enough to be translated into clinical use for the controlled elevation of the endogenous levels of BR, based upon induction of HO-1, the rate-limiting step in the production of BR from heme degradation. This NADPH-dependent enzyme opens the porphyrin ring via oxidation of the heme α-carbon bridge, with the release of Fe2+ and the formation of carbon monoxide (CO) and biliverdin; this latter compound is rapidly converted into BR by biliverdin reductase [41]. By using this approach, here we show for the first time that the induction of HO-1 at a level enough to significantly elevate endogenous BR levels is able to preserve the functional status of hepatocytes in terms of biliary secretory function under ‘pure’ OS conditions, acting as a crucial antioxidant defense to keep the secretory machinery operative after an oxidative challenge.

Materials and methods

Chemicals

Hemin, tBuOOH, thiobarbituric acid (TBA), 2,4-dinitrophenyhydrazine (DNPH), glutathione reductase from Saccharomyces cerevisiae, β-NAD 2′-phosphate reduced tetrasodium salt hydrate (β-NADPH), and 3α-hydroxysteroid dehydrogenase from Pseudomonas testosteroni were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). Rabbit anti-rat Bsep was from Kamiya Biomedical Co. (Seattle, WA, U.S.A.) and mouse anti-human MRP2 (M2III-6) was from Alexis Biochemicals (San Diego, CA, U.S.A.). Mouse anti-occludin, rabbit anti-occludin, Cy2–conjugated goat anti-mouse IgG, Cy2–conjugated donkey anti-rabbit IgG, and horseradish peroxidase (HRP)–conjugated goat anti-mouse IgG were from Thermo Fisher Scientific, Inc. (Waltham, MA, U.S.A.). Cy3–conjugated goat anti-rabbit IgG and Cy3–conjugated donkey anti-mouse IgG were obtained from Jackson ImmunoResearch Laboratory (West Grove, PA, U.S.A.). Mouse monoclonal antibody to HO-1 was from Abcam (Cambridge, U.K.). All the other chemicals were of the highest purity commercially available.

Animals and treatments

Animals were obtained from the Institute of Veterinary Sciences Litoral (ICiVet-Litoral, UNL - CONICET) and kept under identical conditions until use. All animals received humane care according to the criteria outlined in the ‘Guide for the Care and Use of Laboratory Animals’, written by the National Academy of Sciences and published by the National Institutes of Health (NIH publication 25-28, revised 1996) and the ARRIVE (Animal Research: Reporting In Vivo Experiments) guidelines (http://www.nc3rs.org/ARRIVE). All experimental protocols were approved by the Bioethical Committee for the Care and Use of Laboratory Animals of the Faculty of Biochemical and Pharmaceutical Sciences of the National University of Rosario (Resolution N° 489/2015). Studies were performed on male Wistar rats (90 days) weighing 300–350 g, maintained on a standard diet and water ad libitum, and housed in a temperature- and humidity-controlled room under a constant 12-h light, 12-h dark cycle. During the experiments, animal welfare was warranted by monitoring vital signs (heart and respiratory rates) and pain sensitivity (paw-withdrawal latency). At the end of the experiments, animals were killed by overdose of anesthesia, followed by exsanguination.

In studies aimed to assess the capability of hemin to induce HO-1, animals were randomly divided into two experimental groups: (i) Hemin group (n=10), which was administered hemin (20 mg/kg b.w., i.p., dissolved in DMSO plus saline); (ii) Control group (n=10), which received only the hemin vehicle.

In studies aimed to assess the capability of hemin to prevent tBuOOH-induced OS and hepatobiliary dysfunction, animals were randomly divided into four experimental groups: (i) ‘Hemin+tBuOOH’ group (n=12 for each experiment), which was administered hemin (20 mg/kg b.w., i.p., dissolved in DMSO plus saline), and 2 h later, tBuOOH (440 µmol/kg b.w., i.p., dissolved in saline); (ii) ‘Hemin’ group (n=12 for each experiment), which received hemin, followed 2 h later, by the i.p. administration of the tBuOOH vehicle; (iii) ‘tBuOOH’ group (n=12 for each experiment), which was administered the hemin vehicle, followed 2 h later, by tBuOOH administration; (iv) ‘Control’ group (n=12 for each experiment), which received only the hemin and tBuOOH vehicles (Figure 1).

Timeline of experimental procedures

Figure 1
Timeline of experimental procedures

In studies aimed to assess the capability of hemin to prevent tBuOOH-induced OS and hepatobiliary dysfunction, animals were randomly divided into four experimental groups: (i) ‘Hemin+tBuOOH’ group, which was administered hemin (20 mg/kg b.w., i.p., dissolved in DMSO plus saline) and tBuOOH (440 µmol/kg b.w., i.p., dissolved in saline); (ii) ‘Hemin’ group, which received hemin and tBuOOH vehicle; (iii) ‘tBuOOH’ group, which was administered hemin vehicle and tBuOOH; (iv) ‘Control’ group, which received hemin and tBuOOH vehicles. After surgical procedures, bile sampling started and continued for 2 h. At the end of the collection period, animals were killed and liver was removed and stored for further studies.

Figure 1
Timeline of experimental procedures

In studies aimed to assess the capability of hemin to prevent tBuOOH-induced OS and hepatobiliary dysfunction, animals were randomly divided into four experimental groups: (i) ‘Hemin+tBuOOH’ group, which was administered hemin (20 mg/kg b.w., i.p., dissolved in DMSO plus saline) and tBuOOH (440 µmol/kg b.w., i.p., dissolved in saline); (ii) ‘Hemin’ group, which received hemin and tBuOOH vehicle; (iii) ‘tBuOOH’ group, which was administered hemin vehicle and tBuOOH; (iv) ‘Control’ group, which received hemin and tBuOOH vehicles. After surgical procedures, bile sampling started and continued for 2 h. At the end of the collection period, animals were killed and liver was removed and stored for further studies.

All experiments started at 7 a.m. each day, with the corresponding injection according to the day protocol. Surgical procedures began 4 h after tBuOOH/vehicle injection. For this purpose, animals were anesthetized using ketamine/xylazine (100 mg/3 mg/kg i.p.) and bile duct was cannulated by means of a PE-10 catheter. Bile samples were collected every 10 min, for 2 h. At the end of the collection period, animals were killed, and liver was removed, washed, and stored according to the protocols of the different assays carried out afterward, i.e. part of the organ was frozen in isopentane, and stored at −70°C for immunofluorescence staining, while another portion was homogenized in 0.3 M sucrose plus protease inhibitors, for the remaining studies.

Evaluation of the impact of hemin treatment on HO-1 expression and endogenous BR levels

Hemin (ferriprotoporphyrin IX), a well-known inducer of HO-1 [42], was used throughout. Different doses of hemin were tested, as well as different time points after hemin injection at the selected dose. Biliary concentration of BR was used as a surrogate parameter to estimate BR endogenous levels. For this purpose, BR was assessed by spectrophotometric determination of absorbance at 462 nm of the bile samples collected; since the absorption coefficients of BR esters are similar to each other, the obtained absorbance value results from the sum of the absorbances of each of the esters [43].

Western blotting studies were carried out in hepatic homogenates, in order to confirm HO-1 induction by hemin. Briefly, liver tissue was homogenized in 0.3 M sucrose plus protease inhibitor cocktail (Sigma Chemical Co., St. Louis, MO, U.S.A.), and disrupted by sonication. Aliquots of equivalent protein content, as ascertained by the Lowry method [44], were separated by electrophoresis on an SDS/12% polyacrylamide gel and electrotransferred to PVDF membranes. These membranes were then incubated overnight with a mouse monoclonal antibody to HO-1 (1:1000), and HRP–conjugated goat anti-mouse IgG (1:3000, 1 h) was used as the secondary antibody. Blots were also probed for β-actin (Sigma–Aldrich Corp.) as a loading control. Protein bands were visualized using a chemiluminescence reagent (ECL™-GE Healthcare, Amersham, U.S.A.) and captured with Hyperfilm ECL™ (GE Healthcare, Amersham, U.S.A.). For quantitation, blots from at least four independent experiments were quantitated using ImageJ 1.51p software (NIH, U.S.A.).

Evaluation of the cellular redox status in OS-induced acute cholestasis

The magnitude of the oxidative hepatic impact induced by tBuOOH was evaluated as follows: (i) by measuring the generation of TBA-reactive substances (TBARS) in liver homogenates [45]; (ii) by measuring the concentration of protein carbonyls in liver homogenates by the method described by Levine and colleagues [46], which is based on the reaction of carbonyl groups with DNPH to form the corresponding hydrazone, which is spectrophotometrically detected at 370 nm; (iii) by determination of the ratio of oxidized glutathione (GSSG)-to-total glutathione (GSHt) in bile, for which bile samples were subjected to assessment of both GSSG and GSHt, by the recycling method of Tietze [47], as modified by Griffith [48]; (iv) by assessment of the activity of antioxidant enzymes catalase (CAT) and superoxide dismutase (SOD). The activity of CAT was assessed by recording the decomposition of H2O2 at 240 nm [49]. The activity of SOD was assessed by a commercial kit (Randox Ltd., Crumlin, U.K.), based on the utilization of xanthine and xanthine oxidase in order to generate superoxide anion, which then reacts with 5-phenyltetrazolium 2-(4-iodophenyl)-3-(4-nitrophenol)-chloride, forming a red complex, which is detectable at 420 nm.

On the other hand, presence of inflammatory cell infiltrate and levels of interleukin-6 were determined in liver slices and plasma samples respectively, of each studied group in order to discard any inflammatory changes occurring after tBuOOH treatment. Liver slices were stained with Hematoxylin–Eosin and interleukin-6 was assessed by means of a commercial ELISA kit provided by Thermo Fisher Scientific, Inc. (Waltham, MA, U.S.A.). Besides, in order to rule out steatosis as a component of our acute oxidative injury model, liver slices were stained with Sudan Black B and plasma samples were assayed for triglycerides and total cholesterol levels (CM250 Autoanalyzer, Wiener Lab, Rosario, Argentina).

Evaluation of biliary secretory function and canalicular transporter localization in OS-induced acute cholestasis

The impact of OS on normal secretory function was evaluated by studying the following parameters: (i) bile flow, gravimetrically assessed assuming a bile density of 1 g/ml; (ii) biliary excretion of bile salts, measured enzymatically by using 3α-hydroxysteroid dehydrogenase, as described by Talalay [50]; this parameter allows the estimation of the function of Bsep; (iii) biliary GSHt and BR concentrations, as described above, which estimate Mrp2 function; (iv) cellular localization of Bsep and Mrp2, by confocal immunofluorescent microscopy (LSM880, Carl Zeiss LLC, Thornwood, NY, U.S.A.). For Bsep and Mrp2 labeling, tissue sections were incubated overnight with the specific antibody to Bsep (1:100) or Mrp2 (1:100). Sections were then incubated either with Cy2–conjugated donkey anti-rabbit IgG (for the case of Bsep) or with Cy2–conjugated goat anti-mouse IgG (for the case of Mrp2). Immunolabeling of occludin was simultaneously processed (co-staining), so as to delimitate the canalicular region. For this purpose, mouse (for Bsep) or rabbit (for Mrp2) anti-occludin was used as primary antibody, followed by incubation with either Cy3–conjugated donkey anti-mouse IgG or Cy3–conjugated goat anti-rabbit IgG, respectively, for 1 h. Once stained, the specimens for Bsep and Mrp2 analysis were examined by confocal microscopy, and the images thus obtained were analyzed to quantitate the total intensity of fluorescence within the canalicular region, delimitated by occluding staining.

Statistical analysis

Results were expressed as mean ± S.D., with n representing the number of replicates of each experiment. The Student’s unpaired t test was used for comparison between two groups, as long as the requirements for parametric analysis were fulfilled; comparisons that did not meet these criteria were carried out using the Mann–Whitney’s rank sum test. When comparing more than two groups, the Kruskal–Wallis’ test (one-way ANOVA on ranks) was used; this was followed by the Dunn’s multiple-comparison, post hoc test for pairwise comparisons, if ANOVA reached any statistical significance amongst groups. Comparison of the variances of the densitometric profiles of Bsep and Mrp2 localization was performed using the Mann–Whitney U test. P-values <0.05 were considered to be statistically significant.

Results

Effect of hemin treatment on HO-1 expression and endogenous BR levels

Hemin was used as an inducer of HO-1, and biliary levels of BR increased consequently. Hemin doses of 5, 10, and 20 mg/kg b.w. were tested and the biliary BR concentration measured 6 h post-hemin injection was significantly elevated at the dose of 20 mg/kg b.w. (Figure 2A), which was then selected to perform a time-course study of hemin effect. Animals injected with 20 mg/kg b.w. hemin were subjected to surgery at 2, 4, 6, 8, 12, and 24 h after hemin treatment, and biliary concentration of BR was assessed; values peaked at 6–8 h post-hemin injection, reaching approximately three-fold increase in its basal biliary levels (Figure 2B). From these experiments, a hemin dose of 20 mg/kg b.w. and a time point of 6–8 h post-hemin injection were selected for further studies. Plasma levels of BR did not increase at any dose nor at any time point after hemin injection (data not shown); this may be due to the extremely efficient hepatic detoxification of BR in the rat, with maximal phase II metabolic rate and biliary transport, amongst many species tested [51]. Western blotting analyses of HO-1 protein expression corroborated the induction of HO-1 expression by hemin treatment (Figure 2C).

Effect of hemin treatment on HO-1 expression and endogenous BR levels

Figure 2
Effect of hemin treatment on HO-1 expression and endogenous BR levels

The (A) dose–response and (B) time-dependent effects of hemin on the endogenous levels of the BR produced by HO-1 were assessed by monitoring the increase in the biliary concentration of pigment as a surrogate parameter. A significant increase in BR biliary concentration measured 6 h post-hemin injection was obtained for 20 mg/kg b.w. hemin, and this dose was selected to carry out the time-course studies of hemin effect. For this purpose, animals were subjected to bile collection at different time points, and biliary concentration of BR was found to peak at 6–8 h post-hemin injection, reaching approximately three-fold increase in its basal biliary levels. Data are expressed as mean ± S.D. (*P<0.01 compared with t = 0 h, n=4 for each time point). (C) Western blotting analyses of HO-1 protein expression confirmed the hepatic HO-1 induction by hemin at the dose of 20 mg/kg b.w., 5 h after hemin administration. Data are expressed as mean ± S.D. (*P<0.05 compared with Control, n=4).

Figure 2
Effect of hemin treatment on HO-1 expression and endogenous BR levels

The (A) dose–response and (B) time-dependent effects of hemin on the endogenous levels of the BR produced by HO-1 were assessed by monitoring the increase in the biliary concentration of pigment as a surrogate parameter. A significant increase in BR biliary concentration measured 6 h post-hemin injection was obtained for 20 mg/kg b.w. hemin, and this dose was selected to carry out the time-course studies of hemin effect. For this purpose, animals were subjected to bile collection at different time points, and biliary concentration of BR was found to peak at 6–8 h post-hemin injection, reaching approximately three-fold increase in its basal biliary levels. Data are expressed as mean ± S.D. (*P<0.01 compared with t = 0 h, n=4 for each time point). (C) Western blotting analyses of HO-1 protein expression confirmed the hepatic HO-1 induction by hemin at the dose of 20 mg/kg b.w., 5 h after hemin administration. Data are expressed as mean ± S.D. (*P<0.05 compared with Control, n=4).

Effect of HO-1 induction on cellular redox status in acute oxidative cholestasis

Oxidative injury was evaluated through the ability of ROS to directly damage cellular macromolecules. For this purpose, hepatic lipid peroxidation and protein carbonylation were assessed. Animals treated with tBuOOH showed higher levels of TBARS and carbonylated proteins as compared with the Control group, while in animals pre-treated with hemin, similar values to Controls were obtained (Figure 3). GSSG/GSHt relation in bile, which is considered as a sensitive marker of hepatic OS [52], was also measured. We found a three-fold higher GSSG/GSHt ratio in animals subjected to the oxidative insult, while pre-treatment with hemin attenuated this rise (Figure 4). Altogether, these results clearly indicate a protective effect exerted by HO-1 induction against the oxidative injury induced by tBuOOH, likely due to increased levels of BR.

HO-1 induction prevents oxidative damage to lipids and proteins

Figure 3
HO-1 induction prevents oxidative damage to lipids and proteins

The effect of the oxidative insult on cellular molecules as lipids and proteins was studied in liver homogenates obtained from animals of the following groups: Control (vehicles only), Hemin (20 mg/kg b.w.), tBuOOH (440 μmol/kg b.w.), and Hemin (20 mg/kg b.w) + tBuOOH (440 μmol/kg b.w.). (A) Lipid peroxidation was assessed by the TBARS method. tBuOOH induced an increase in the generation of TBARS (expressed as malondialdehyde, MDA, as a product of lipid peroxidation) and hemin prevented this elevation. Data are expressed as mean ± S.D. aSignificantly different from Control; bSignificantly different from tBuOOH (P<0.05; n=5). (B) Protein carbonylation was determined colorimetrically using DNPH. Levels of protein carbonyls were significantly higher in the tBuOOH group as compared with Control group, while pre-treatment with hemin prevented this increase. Data are expressed as mean ± S.D. aSignificantly different from Control, bSignificantly different from tBuOOH (P<0.05; n=5).

Figure 3
HO-1 induction prevents oxidative damage to lipids and proteins

The effect of the oxidative insult on cellular molecules as lipids and proteins was studied in liver homogenates obtained from animals of the following groups: Control (vehicles only), Hemin (20 mg/kg b.w.), tBuOOH (440 μmol/kg b.w.), and Hemin (20 mg/kg b.w) + tBuOOH (440 μmol/kg b.w.). (A) Lipid peroxidation was assessed by the TBARS method. tBuOOH induced an increase in the generation of TBARS (expressed as malondialdehyde, MDA, as a product of lipid peroxidation) and hemin prevented this elevation. Data are expressed as mean ± S.D. aSignificantly different from Control; bSignificantly different from tBuOOH (P<0.05; n=5). (B) Protein carbonylation was determined colorimetrically using DNPH. Levels of protein carbonyls were significantly higher in the tBuOOH group as compared with Control group, while pre-treatment with hemin prevented this increase. Data are expressed as mean ± S.D. aSignificantly different from Control, bSignificantly different from tBuOOH (P<0.05; n=5).

HO-1 induction prevents the increase in GSSG/GSHt ratio in bile

Figure 4
HO-1 induction prevents the increase in GSSG/GSHt ratio in bile

GSSG and GSHt were assessed in bile samples collected from animals of the following groups: Control (vehicles only), Hemin (20 mg/kg b.w.); tBuOOH (440 μmol/kg b.w.), and Hemin (20 mg/kg b.w) + tBuOOH (440 μmol/kg b.w.). The relation between the two species of glutathione is a sensible marker of OS in bile. Animals treated with tBuOOH exhibited higher GSSG/GSHt ratio, and pre-treatment with hemin prevented such an increase, thus corroborating an antioxidant role for HO-1 induction. Data are expressed as mean ± S.D. aSignificantly different from Control, bSignificantly different from tBuOOH (P<0.05; n=4).

Figure 4
HO-1 induction prevents the increase in GSSG/GSHt ratio in bile

GSSG and GSHt were assessed in bile samples collected from animals of the following groups: Control (vehicles only), Hemin (20 mg/kg b.w.); tBuOOH (440 μmol/kg b.w.), and Hemin (20 mg/kg b.w) + tBuOOH (440 μmol/kg b.w.). The relation between the two species of glutathione is a sensible marker of OS in bile. Animals treated with tBuOOH exhibited higher GSSG/GSHt ratio, and pre-treatment with hemin prevented such an increase, thus corroborating an antioxidant role for HO-1 induction. Data are expressed as mean ± S.D. aSignificantly different from Control, bSignificantly different from tBuOOH (P<0.05; n=4).

In order to evaluate the status of antioxidant defenses, levels of CAT and SOD were determined in liver tissue homogenates. We found that hepatic levels of both antioxidant enzymes were increased by tBuOOH, and that this elevation was abolished by hemin pre-treatment. Superoxide anion radical metabolization by SOD converts it into hydrogen peroxide, which is then converted into water and oxygen by CAT. Therefore, our results show that HO-1 induction prevents the ROS-mediated triggering of the antioxidant defense machinery in the liver, an effect most likely due to the scavenging properties of BR, which prevents ROS from building up (Figure 5). Hemin treatment alone produced no significant variation in any of the parameters studied.

HO-1 induction prevents the triggering of the antioxidant response

Figure 5
HO-1 induction prevents the triggering of the antioxidant response

The activity of the antioxidant enzymes (A) CAT and (B) SOD were assessed in liver homogenates obtained from animals of the following groups: Control (vehicles only), Hemin (20 mg/kg b.w.), tBuOOH (440 μmol/kg b.w.), and Hemin (20 mg/kg b.w) + tBuOOH (440 μmol/kg b.w.). The activity of CAT was determined by recording the decomposition of H2O2 at 240 nm and that of SOD was assessed by means of the reaction of superoxide anion with 5-phenyltetrazolium 2-(4-iodophenyl)-3-(4-nitrophenol)-chloride, that renders a red complex detectable at 420 nm. Oxidative insult triggers the burst of antioxidant enzymes while pre-treatment with hemin prevented this phenomenon. Data are expressed as mean ± S.D. aSignificantly different from Control, bSignificantly different from tBuOOH (P<0.05; n=5).

Figure 5
HO-1 induction prevents the triggering of the antioxidant response

The activity of the antioxidant enzymes (A) CAT and (B) SOD were assessed in liver homogenates obtained from animals of the following groups: Control (vehicles only), Hemin (20 mg/kg b.w.), tBuOOH (440 μmol/kg b.w.), and Hemin (20 mg/kg b.w) + tBuOOH (440 μmol/kg b.w.). The activity of CAT was determined by recording the decomposition of H2O2 at 240 nm and that of SOD was assessed by means of the reaction of superoxide anion with 5-phenyltetrazolium 2-(4-iodophenyl)-3-(4-nitrophenol)-chloride, that renders a red complex detectable at 420 nm. Oxidative insult triggers the burst of antioxidant enzymes while pre-treatment with hemin prevented this phenomenon. Data are expressed as mean ± S.D. aSignificantly different from Control, bSignificantly different from tBuOOH (P<0.05; n=5).

At the same time, we corroborated that tBuOOH-induced injury is of strictly pro-oxidant nature, since no evidence of either inflammation (e.g. leukocytes infiltration and interleukin-6 elevations) or steatosis (e.g. hepatic lipid droplets and increased triglycerides and total cholesterol levels) was observed (Figure 6).

tBuOOH induces a ‘pure’ oxidative injury

Figure 6
tBuOOH induces a ‘pure’ oxidative injury

(A) Morphological analysis of Hematoxylin–Eosin stained specimens, (B) plasma levels of interleukin-6, (C) morphological analysis of Sudan Black B stained specimens, and (D) plasma levels of triglycerides and cholesterol corroborated that tBuOOH-induced injury is of a strictly pro-oxidant nature, since no evidence of inflammatory changes nor steatosis was observed in any of the groups studied: Control (vehicles only), Hemin (20 mg/kg b.w.); tBuOOH (440 μmol/kg b.w.), and Hemin (20 mg/kg b.w) + tBuOOH (440 μmol/kg b.w.); n=4.

Figure 6
tBuOOH induces a ‘pure’ oxidative injury

(A) Morphological analysis of Hematoxylin–Eosin stained specimens, (B) plasma levels of interleukin-6, (C) morphological analysis of Sudan Black B stained specimens, and (D) plasma levels of triglycerides and cholesterol corroborated that tBuOOH-induced injury is of a strictly pro-oxidant nature, since no evidence of inflammatory changes nor steatosis was observed in any of the groups studied: Control (vehicles only), Hemin (20 mg/kg b.w.); tBuOOH (440 μmol/kg b.w.), and Hemin (20 mg/kg b.w) + tBuOOH (440 μmol/kg b.w.); n=4.

Effect of HO-1 induction on biliary secretory function in acute oxidative cholestasis

Once evidenced the oxidative injury elicited by tBuOOH, we evaluated whether the main parameters of biliary secretory function were consequently altered. Bile flow was significantly decreased in animals treated with tBuOOH throughout the period of time studied, while pre-treatment with hemin completely prevented this drop (Figure 7). In order to investigate whether prevention exerted by pre-treatment with hemin on bile flow drop was due to its protective effect on the secretory capacity of key canalicular transporters, we evaluated the biliary excretion of Bsep and Mrp2 specific substrates. As expected, biliary excretion of bile salts decreased in animals treated with tBuOOH, and pre-treatment with hemin prevented this decrease, thus evidencing a protective effect of HO-1 induction on Bsep function (Figure 8A). A similar phenomenon was observed for Mrp2. Biliary excretion of the Mrp2 substrates, BR and GSHt, dropped after tBuOOH treatment, while hemin prevented such a decrease (Figure 8B,C). Taken together, these results show that HO-1 induction, most likely through elevation of endogenous BR levels, prevents the impairment in the function of the two main canalicular transporters involved in bile formation, thus preventing OS-induced acute cholestasis.

HO-1 induction prevents OS-induced bile flow impairment

Figure 7
HO-1 induction prevents OS-induced bile flow impairment

Bile flow was gravimetrically assessed in the following groups: Control (vehicles only), Hemin (20 mg/kg b.w.), tBuOOH (440 μmol/kg b.w.), and Hemin (20 mg/kg b.w) + tBuOOH (440 μmol/kg b.w.). Bile flow was significantly decreased in animals treated with tBuOOH throughout the period of time studied, while pre-treatment with hemin completely prevented this drop. Data are expressed as mean ± S.D. aSignificantly different from control; bSignificantly different from tBuOOH (P<0.01; n=6).

Figure 7
HO-1 induction prevents OS-induced bile flow impairment

Bile flow was gravimetrically assessed in the following groups: Control (vehicles only), Hemin (20 mg/kg b.w.), tBuOOH (440 μmol/kg b.w.), and Hemin (20 mg/kg b.w) + tBuOOH (440 μmol/kg b.w.). Bile flow was significantly decreased in animals treated with tBuOOH throughout the period of time studied, while pre-treatment with hemin completely prevented this drop. Data are expressed as mean ± S.D. aSignificantly different from control; bSignificantly different from tBuOOH (P<0.01; n=6).

HO-1 induction prevents the oxidative impairment in biliary secretory function

Figure 8
HO-1 induction prevents the oxidative impairment in biliary secretory function

Secretory activities of Bsep and Mrp2 were tested in animals subjected to the oxidative insult elicited by tBuOOH, and in those animals pre-treated with hemin, through determination of the excretion rate of specific substrates of each transporter into bile. Mean biliary excretion of (A) bile salts (Bsep substrates), (B) total BR, and (C) GSHt (Mrp2 substrates). In the three panels, the following groups are shown: Control (vehicles only), Hemin (20 mg/kg b.w.), tBuOOH (440 μmol/kg b.w.), and Hemin (20 mg/kg b.w) + tBuOOH (440 μmol/kg b.w.). Excretion rate of all three substrates decreased in animals treated with tBuOOH, while pre-treatment with hemin prevented these decreases in 40, 47, and 73%, respectively, thus evidencing an impairment in the function of both transporters due to the oxidative insult and a partial prevention by HO-1 induction. Data are expressed as mean ± S.D. aSignificantly different from control; bSignificantly different from tBuOOH (P<0.05; n=5).

Figure 8
HO-1 induction prevents the oxidative impairment in biliary secretory function

Secretory activities of Bsep and Mrp2 were tested in animals subjected to the oxidative insult elicited by tBuOOH, and in those animals pre-treated with hemin, through determination of the excretion rate of specific substrates of each transporter into bile. Mean biliary excretion of (A) bile salts (Bsep substrates), (B) total BR, and (C) GSHt (Mrp2 substrates). In the three panels, the following groups are shown: Control (vehicles only), Hemin (20 mg/kg b.w.), tBuOOH (440 μmol/kg b.w.), and Hemin (20 mg/kg b.w) + tBuOOH (440 μmol/kg b.w.). Excretion rate of all three substrates decreased in animals treated with tBuOOH, while pre-treatment with hemin prevented these decreases in 40, 47, and 73%, respectively, thus evidencing an impairment in the function of both transporters due to the oxidative insult and a partial prevention by HO-1 induction. Data are expressed as mean ± S.D. aSignificantly different from control; bSignificantly different from tBuOOH (P<0.05; n=5).

Since proper plasma membrane localization is crucial to Bsep and Mrp2 transport function, and OS alters their stability in membrane leading to endocytic internalization, we ascertained whether HO-1 induction with hemin can prevent this alteration, by fluorescent immunostaining of these transporters, followed by confocal microscopy. For this purpose, tissue sections were subjected to specific co-staining of Bsep or Mrp2 with occludin. In control livers, transporter-associated fluorescence was confined to the canalicular space, whereas tBuOOH induced redistribution of both transporters from the canalicular membrane into vesicles located either in cytosol or around the pericanalicular area. Pre-treatment with hemin significantly prevented these alterations, as can be seen both in confocal images (Figure 9) and through the quantitative analysis of the images obtained (Supplementary Figure S1). These results strongly suggest that the protective action of HO-1 induction, and consequently elevated levels of BR, on the oxidative impairment of biliary secretory function is also exerted at the level of canalicular localization of transporters that are crucial to the biliary secretion process.

Endocytic internalization of Bsep and Mrp2 induced by OS, and its prevention by HO-1 induction

Figure 9
Endocytic internalization of Bsep and Mrp2 induced by OS, and its prevention by HO-1 induction

The images were taken from samples of liver tissues obtained at the end of the experiments. Bsep and Mrp2 were immunostained with specific antibodies, and occludin was used to delimitate the canalicular area. (A) Co-staining of Bsep (green) and occludin (red). (B) Co-staining of Mrp2 (green) and occludin (red). In both sets of micrographs, the following groups are shown: Control (vehicles only), Hemin (20 mg/kg b.w.), tBuOOH (440 μmol/kg b.w.), and Hemin (20 mg/kg b.w) + tBuOOH (440 μmol/kg b.w.). Besides each representative confocal image, insets show (a) transporter and (b) occludin channels separately. In control livers, both Bsep and Mrp2 were mainly confined to the canalicular space, while in livers treated with tBuOOH, a significant relocalization of both transporters to the pericanalicular area was observed, thus suggesting endocytic internalization of these transporters. Hemin-induced HO-1 expression prevented this phenomenon, as evidenced by a control-like pattern of Bsep and Mrp2 distribution in livers pre-treated with hemin (for quantitative analysis, see Supplementary Figure S1).

Figure 9
Endocytic internalization of Bsep and Mrp2 induced by OS, and its prevention by HO-1 induction

The images were taken from samples of liver tissues obtained at the end of the experiments. Bsep and Mrp2 were immunostained with specific antibodies, and occludin was used to delimitate the canalicular area. (A) Co-staining of Bsep (green) and occludin (red). (B) Co-staining of Mrp2 (green) and occludin (red). In both sets of micrographs, the following groups are shown: Control (vehicles only), Hemin (20 mg/kg b.w.), tBuOOH (440 μmol/kg b.w.), and Hemin (20 mg/kg b.w) + tBuOOH (440 μmol/kg b.w.). Besides each representative confocal image, insets show (a) transporter and (b) occludin channels separately. In control livers, both Bsep and Mrp2 were mainly confined to the canalicular space, while in livers treated with tBuOOH, a significant relocalization of both transporters to the pericanalicular area was observed, thus suggesting endocytic internalization of these transporters. Hemin-induced HO-1 expression prevented this phenomenon, as evidenced by a control-like pattern of Bsep and Mrp2 distribution in livers pre-treated with hemin (for quantitative analysis, see Supplementary Figure S1).

Discussion

In the present study we show, for the first time, that induction of HO-1 by hemin protects the liver from a ‘pure’ oxidative cholestatic injury in vivo, most probably due to the consequently elevated levels of BR. Although it is a well-known antioxidant metabolite [53,54], BR has scarcely been regarded as a therapeutic strategy for hepatopathies. This may certainly be due to its also well-recognized deleterious effects when present at high concentrations. These effects have been described to mainly affect the nervous system [55,56]. Nevertheless, under controlled conditions, BR exhibits hepatoprotective properties from what to take benefit in the case of exogenously administered inducers of its synthesis.

In a previous work frorm our group, we found that physiological concentrations of unconjugated BR prevent the biliary secretory function to collapse under an OS insult in in vitro models, such as isolated rat hepatocytes, isolated rat hepatocyte couplets, and isolated and perfused rat liver [20]. In light of the potential use of this endogenous metabolite for the treatment of acute oxidative cholestasis, we tested the effect of HO-1 induction, a key step in BR formation, after in vivo exposure to mild doses of tBuOOH, a well-known model pro-oxidant agent. tBuOOH, administered i.p. to adult rats on a specific time point, and at a dose of 440 µmol/kg b.w., caused significant OS, as evidenced by the elevated tissular lipid and protein oxidation levels, the increased biliary GSSG/GSHt ratio, and the enhanced antioxidant compensatory response in liver tissue.

Here, we found that ROS generated by tBuOOH induce peroxidation of membrane lipids and carbonylation of cellular proteins. Lipid peroxidation is initiated when a hydrogen atom is extracted from an unsaturated fatty acid by a radical species, thus triggering a destructive chain reaction that generates lipid peroxides. Disruption of cellular membranes by these peroxides produces reactive metabolites like malondialdehyde and 4-hydroxy-2,3-transnonenal, causing cellular dysfunction [57]. On the other hand, carbonyl groups (aldehydes and ketones) are introduced into proteins by primary oxidative modifications, and the appearance of such carbonyl groups is taken as a likely evidence of oxidative modification [58]. Induction of HO-1 prevented such oxidative deleterious events most likely by the scavenging effect of BR on the radical species generated after tBuOOH injection. In this sense, many authors have demonstrated that BR is endowed with a strong antioxidant activity [25–30]. Amongst the most recent ones, Zelenka and colleagues [59] described that BR induces a dose-dependent decrease in cytoplasmic and mitochondrial levels of hydrogen peroxide in a human embryonic kidney cell line, and Jansen and colleagues [60] found that BR inhibits mitochondrial ROS formation, and that it is a potent scavenger of superoxide radical.

BR, at a concentration in the nM order, is able to protect against 10000-fold higher concentrations of H2O2 [61], since the antioxidant actions of BR may be amplified 10000 times or greater by the biliverdin reductase cycle [62]. Although biliverdin is the actual product of HO-1 activity, and BR is produced after a further reduction step catalyzed by biliverdin reductase, the possibility that biliverdin, rather than BR, is involved in the antioxidant effects of HO-1 induction, needs to be considered. There is agreement in that biliverdin bears some ROS-scavenging properties in vitro [60], but its antioxidant activity is 60-fold lower than BR, because, unlike BR, it lacks a γ-methene group [27]. In addition, biliverdin is immediately reduced to BR, so that its intracellular levels are expected to be low [61]. Furthermore, studies with full HO-1 activity but inhibition of biliverdin reductase, ensuring biliverdin but not BR formation, seems to support this concept, since the antioxidant effect afforded by HO-1 induction was fully inhibited [62,63]. In addition, inhibition of biliverdin reductase in vivo exacerbates OS in diseases with an oxidant background, such as diabetes [40] and Alzheimer disease [64]; this finding was reproduced in biliverdin reductase-depleted cells, where ROS-mediated cytotoxicity was exacerbated. Interestingly, the pro-oxidant effect of biliverdin reductase depletion was even higher than that recorded after glutathione depletion, suggesting that BR exerts an even higher antioxidant effect than glutathione in the cell [62]. Irrespective of their relative antioxidant potency, both compounds seem to have complementary antioxidant properties; unconjugated BR would protect preferentially membrane lipids and membrane-associated proteins due to its high hydrophobicity, whereas glutathione preferentially protects soluble proteins due to its hydrophilicity [25]. Our finding that HO-1 induction fully protected against tBuOOH-induced lipid peroxidation further points BR rather than biliverdin as the actual antioxidant compound, since unlike BR, biliverdin is highly water soluble [65].

Another HO-1 product that may bear, at least hypothetically, protective effects against tBuOOH-induced OS is CO. tBuOOH induces OS, in part, by inducing MPTP formation, and further mitochondrial production of ROS [18], and CO was shown to limit this process by inducing a mild-uncoupling state in isolated mitochondria by modulating heme-containing cytochrome oxidases [66]. However, tBuOOH only induced MPTP formation at a concentration of 500 µM [18], a level that was shown to virtually abolish canalicular secretory function in an isolated rat hepatocyte couplets model [67]; this is in contrast with our results here in the in vivo tBuOOH cholestatic model, where only a partial decrease in bile flow was recorded, suggesting that such a high tBuOOH level was not reached. Taken together, our own results and those available in the literature and summarized above rather point to endogenously produced BR as the major agent mediating HO-1 antioxidant effects.

Although abundant in in vitro experimental systems, compelling evidence in in vivo settings for an antioxidant cytoprotective role of BR is scarce. Boon and colleagues [53] demonstrated decreased circulating OS biomarker levels in Gunn rats, although they did not find alterations in cell-based OS markers, thus assuming that BR protective effects are confined to the vascular compartment. Contrarily, here we demonstrated a clear hepatocellular antioxidant protective effect of BR due to locally increased levels of this metabolite, while no elevations in systemic BR concentrations were found.

In the last years, evidence has accumulated that OS is cholestatic due to an impairment of hepatocellular biliary secretory machinery, mainly through direct oxidative damage to cellular structures by ROS [68]. However, as the liver constitutes an organ that is particularly susceptible to OS due to its high metabolic activity, it is equipped with special defense mechanisms to scavenge ROS, with glutathione and the antioxidant enzymes CAT and SOD amongst the main ones. For this purpose, the liver has redox-sensitive mechanisms that lead to the induction by ROS of these and other antioxidant enzymes, including HO-1 itself, as a compensatory response aimed to decrease ROS generated by the oxidative insult and re-establish the redox balance. This OS-triggered induction is mainly mediated by the redox-sensitive transcription factor Nuclear factor-erythroid related factor-2 (Nrf2) [69]. Under normal conditions, Nrf2 is bound in cytoplasm to its repressor, Kelch-like ECH-associated protein (Keap1), an OS sensor. Under OS conditions, Nrf2 is released and translocated to the nucleus, where it binds to the antioxidant response element (ARE) located at the promoter region of HO-1 and other antioxidant enzymes, such as SOD, CAT, glutathione S-transferase, and peroxidase, thus transcriptionally promoting their synthesis [70]. We studied this antioxidant adaptive response by evaluating the capability of tBuOOH to activate CAT and SOD, and the prevention of this effect by HO-1 induction. Both CAT and SOD activities were found to peak in animals treated with tBuOOH and pre-treatment with hemin prevented the activation of these antioxidant mechanisms of defense, likely due to BR scavenger action that provides a more reductive milieu, thus favoring a rapid and efficient protection against ROS produced by tBuOOH. Although tBuOOH is expected to induce HO-1 as a part of the antioxidant response induced by OS [41], it is apparent that the extent of this adaptive induction was not enough to counteract tBuOOH-oxidizing damage, and required the therapeutic addition of a potent inducer like hemin to complement this protection. Additionally, hemin is not only an inducer but also a substrate of HO-1, thus guaranteeing that its induction results in elevated levels of BR, the product of the reaction.

It has been well established that the oxidative challenge affects the hepatocyte secretory machinery by impairing both bile flow and the biliary excretion of endo- and xenobiotics [19]. Here, we demonstrated that the excretion of specific substrates of hepatocanalicular transporters crucial to bile formation, such as Bsep and Mrp2, are highly impaired due to the oxidative insult, thus producing a decrease in bile flow. Localization of Bsep and Mrp2 at the hepatocanalicular membrane is a key feature for the normal function of these transporters and OS has been demonstrated to induce their internalization to the pericanalicular area in in vitro studies [13]. Here, we reproduced the findings mentioned above in the whole animal context, thus enabling us to corroborate the delocalization phenomenon as the main cause of cholestasis. Taken together, our results suggest that the induction of HO-1 would counteract the outcome of cholestatic conditions that concur with OS.

Bile salt-independent bile flow mainly depends on the canalicular transport of glutathione, an Mrp2 substrate [13]. Glutathione biliary excretion rate was decreased by tBuOOH treatment, thus suggesting a functional alteration of this transporter. Our results strengthen the hypothesis that tBuOOH-associated OS would reduce Mrp2 functional capacity, thus resulting in decreased excretion of glutathione. Since conjugated BR is also a substrate of Mrp2 [71], this functional failure would also explain the decrease in the excretion of BR in animals exposed to tBuOOH. On the other hand, we confirmed that induction of HO-1 increased hepatic BR levels, since biliary excretion of BR was increased and that of glutathione was decreased in animals treated only with hemin. This indicates that elevated intrahepatic levels of BR competitively inhibited the transport of glutathione by Mrp2. For this reason, we observed a partial prevention of the decrease in the biliary excretion of glutathione and a complete prevention of the decrease in the biliary excretion of BR in animals pre-treated with hemin. Bile salts excretion rate was also decreased by OS and counteracted by hemin, thus evidencing a functional oxidative impairment of Bsep that could result from a competitive inhibition process exerted by GSSG, excessively generated under these conditions [12], together with the endocytic internalization of Bsep induced by tBuOOH [19–21], two phenomena that were prevented by HO-1 induction. Our results are partially in-line with those obtained by Donner and colleagues [72] in a model of cholestasis by hepatic ischemia–reperfusion, where cholestasis and internalization of Mrp2 and Bsep were partially prevented by HO-1 induction. However, unlike our ‘pure’ OS model based upon the acute administration of a direct pro-oxidizing agent like tBuOOH, hepatic ischemia–reperfusion has a complex pathogenesis, involving inflammatory and microcirculatory alterations, apart from oxidant ones, which may have significantly contributed to the cholestatic failure [73]. Importantly, they can be counteracted by other HO-1 products, as for example, CO, which has potent anti-ischemic and anti-inflammatory properties [74]. Nevertheless, there is considerable evidence implicating ROS as another cause of the hepatic injury, since ischemia results in a build-up of NADH that, on reperfusion, generates a burst of ROS from the mitochondrial electron transport chain, which can be protected by HO-1-derived BR in the same manner it afforded full protection in our pro-oxidant model [21].

In light of the crucial role of OS in hepatopathies, particularly in the cholestatic ones involving malabsorption of fat-soluble antioxidant vitamins [75], antioxidants are considered a valid therapeutic approach for their treatment. However, such antioxidant strategies are nowadays scarce and not completely free from deleterious side effects. This is particularly worrying in long-term treatments with lipophilic antioxidants, such as vitamins E and A, since it has been described that both increase the probability of developing different types of cancer [76], and increase mortality in those patients already suffering from them [77]. Taken together, our results allow us to postulate that an increase in the endogenous generation of BR by HO-1 induction could represent a much more beneficial and innocuous therapeutic strategy for chronic cholestatic diseases, such as primary sclerosing cholangitis or primary biliary cirrhosis, in order to minimize the deleterious effects associated with OS in such pathologies [78]. In this sense, a decrease in HO-1 levels has been recently described in human cholestatic diseases such as primary biliary cirrhosis [79] and primary sclerosing cholangitis [80]. In addition, intrahepatic bile salts accumulated in cholestasis, apart from being pro-oxidants themselves, also exacerbate OS by depleting hepatocytes of BR via induction of BR basolateral export pumps (e.g. Mrp3), and by exacerbating BR consumption via BR oxidation [81]. Therefore, inducing this enzyme would clearly result in a beneficial counteraction. Nevertheless, BR metabolism is somewhat different between rat and humans. For instance, unlike humans, rat lacks gallbladder [82], thus resulting in a more diluted bile, since the main function of the gallbladder is to concentrate the bile before it is released into the duodenum. Also, rat bears lower plasma levels of BR [83], as compared with humans, due to an extremely efficient detoxification system. These facts should be borne in mind at the time of designing an HO-1 induction protocol in humans, since the impact of hemin treatment and consequent HO-1 induction is expected to be quantitatively different in terms of BR production. Then, doses and timings should be adjusted in order to achieve supraphysiological, but not harmful, levels of BR, such as those obtained here for a hemin dose of 20 mg/kg b.w. and at a time-dependent response of 6–8 h post-treatment. All in all, our results strongly encourage the development of new strategies aiming at increasing the endogenous bioavailability of BR by means of the strictly controlled manipulation of HO-1 expression.

Clinical perspectives

  • Several studies have demonstrated the potential utility of exogenously administered BR in the treatment of various diseases. However, the likely beneficial action of HO-1 induction and consequently endogenously elevated BR as a therapeutic strategy under a ‘pure’ OS-induced cholestasis has never been addressed.

  • HO-1 induction and consequent elevation of BR fully prevented the OS-induced drop in bile flow and in the biliary excretion of bile salts and glutathione, two main driving forces of bile flow; this was associated with preservation of the localization of their canalicular transporters, otherwise endocytosed by OS. HO-1 induction counteracted the oxidation of proteins and lipids and the increase in the oxidized-to-GSHt ratio in bile, a sensitive marker of OS. Compensatory elevations of antioxidant enzymes were also prevented.

  • By extending our previous in vitro/ex vivo findings to the whole animal, an in vivo model much closer to the human pathophysiological context, the present study strongly encourages the development of new strategies aiming at the strictly controlled induction of HO-1 expression which renders elevated levels of the antioxidant metabolite BR.

Acknowledgements

We thank Dr Rodrigo Vena and Dr Verónica Livore for their valuable technical assistance in confocal imaging and protein carbonyls assay procedure, respectively.

Author contribution

P.L.M. and P.C. carried out all the experiments, equally contributing to the work. M.V.R. assisted in animal management and surgical procedures as well as in Western blotting technique. G.B.P. and A.I.M. performed all histological studies. D.E.A.F., S.M.M.A., and E.J.S.P. contributed to the interpretation of the results. C.L.B. wrote the manuscript with support from P.L.M., P.C., S.M.M.A., E.J.S.P., and M.G.R. C.L.B. designed and directed the project, aided by M.G.R. and E.J.S.P. All authors discussed the results and contributed to the final manuscript.

Competing interests

The authors declare that there are no competing interests associated with the manuscript.

Funding

This work was supported by the Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT) [grant numbers PICT 2015-1242, PICT 2016-1613]; and the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) [grant number PIP 2013-112 201201 00217 (to C.L.B. and M.G.R.)].

Abbreviations

     
  • BR

    bilirubin

  •  
  • Bsep

    bile salt export pump

  •  
  • CAT

    catalase

  •  
  • DNPH

    2,4-dinitrophenyhydrazine

  •  
  • GSHt

    total glutathione

  •  
  • GSSG

    oxidized glutathione

  •  
  • HO-1

    heme oxygenase-1

  •  
  • HRP

    horseradish peroxidase

  •  
  • MPTP

    mitochondrial permeability transition pore

  •  
  • Mrp2

    multidrug resistance-associated protein 2

  •  
  • Nrf2

    nuclear factor-erythroid related factor-2

  •  
  • OS

    oxidative stress

  •  
  • ROS

    reactive oxygen species

  •  
  • SOD

    superoxide dismutase

  •  
  • TBA

    thiobarbituric acid

  •  
  • TBARS

    TBA-reactive substance

  •  
  • tBuOOH

    tert-butylhydroperoxide

References

References
1.
Parreño
M.
,
Casanova
I.
,
Céspedes
M.V.
,
Vaqué
J.P.
,
Pavón
M.A.
,
Leon
J.
et al. .
(
2008
)
Bobel-24 and derivatives induce caspase-independent death in pancreatic cancer regardless of apoptotic resistance
.
Cancer Res.
68
,
6313
6323
[PubMed]
2.
Vincent
H.K.
,
Innes
K.E.
and
Vincent
K.R.
(
2007
)
Oxidative stress and potential interventions to reduce oxidative stress in overweight and obesity
.
Diabetes Obes. Metab.
9
,
813
839
[PubMed]
3.
Šmíd
V.
,
Petr
T.
,
Váňová
K.
,
Jašprová
J.
,
Šuk
J.
,
Vítek
L.
et al. .
(
2016
)
Changes in liver ganglioside metabolism in obstructive cholestasis - the role of oxidative stress
.
Folia Biol. (Praha)
62
,
148
159
[PubMed]
4.
Recknagel
P.
,
Gonnert
F.A.
,
Halilbasic
E.
,
Gajda
M.
,
Jbeily
N.
,
Lupp
A.
et al. .
(
2013
)
Mechanisms and functional consequences of liver failure substantially differ between endotoxaemia and faecal peritonitis in rats
.
Liver Int.
33
,
283
293
[PubMed]
5.
Gitto
S.
,
Vitale
G.
,
Villa
E.
and
Andreone
P.
(
2014
)
Update on alcohol and viral hepatitis
.
J. Clin. Transl. Hepatol.
2
,
228
233
6.
Malaguarnera
G.
,
Cataudella
E.
,
Giordano
M.
,
Nunnari
G.
,
Chisari
G.
and
Malaguarnera
M.
(
2012
)
Toxic hepatitis in occupational exposure to solvents
.
World J. Gastroenterol.
18
,
2756
2766
[PubMed]
7.
Kaffe
E.T.
,
Rigopoulou
E.I.
,
Koukoulis
G.K.
,
Dalekos
G.N.
and
Moulas
A.N.
(
2015
)
Oxidative stress and antioxidant status in patients with autoimmune liver diseases
.
Redox Rep.
20
,
33
41
[PubMed]
8.
Spahis
S.
,
Delvin
E.
,
Borys
J.M.
and
Levy
E.
(
2017
)
Oxidative stress as a critical factor in nonalcoholic fatty liver disease pathogenesis
.
Antioxid. Redox Signal.
26
,
519
541
[PubMed]
9.
Bellanti
F.
,
Mirabella
L.
,
Mitarotonda
D.
,
Blonda
M.
,
Tamborra
R.
,
Cinnella
G.
et al. .
(
2016
)
Propofol but not sevoflurane prevents mitochondrial dysfunction and oxidative stress by limiting HIF-1α activation in hepatic ischemia/reperfusion injury
.
Free Radic. Biol. Med.
96
,
323
333
10.
Tao
X.
,
Sun
X.
,
Xu
L.
,
Yin
L.
,
Han
X.
,
Qi
Y.
et al. .
(
2016
)
Total flavonoids from Rosa laevigata Michx fruit ameliorates hepatic ischemia/reperfusion injury through inhibition of oxidative stress and inflammation in rats
.
Nutrients
8
,
418
432
11.
Basiglio
C.L.
,
Toledo
F.D.
,
Sanchez Pozzi
E.J.
and
Roma
M.G.
(
2014
)
Radical oxygen species and bile secretion
. In
Systems Biology of Free Radicals and Antioxidants
(
Laher
I.
, ed.), pp.
1787
1808
,
Springer-Verlag
,
Berlin Heidelberg
12.
Ballatori
N.
I
and
Truong
A.T.
(
1989
)
Altered hepatic junctional permeability, bile acid excretion and glutathione efflux during oxidant challenge
.
J. Pharmacol. Exp. Ther.
251
,
1069
1075
[PubMed]
13.
Schmitt
M.
,
Kubitz
R.
,
Wettstein
M.
,
vom Dahl
S.
and
Häussinger
D.
(
2000
)
Retrieval of the mrp2 gene encoded conjugate export pump from the canalicular membrane contributes to cholestasis induced by tert-butyl hydroperoxide and chloro-dinitrobenzene
.
Biol. Chem.
381
,
487
495
[PubMed]
14.
Nieminen
A.L.
,
Saylor
A.K.
,
Tesfai
S.A.
,
Herman
B.
and
Lemasters
J.J.
(
1995
)
Contribution of the mitochondrial permeability transition to lethal injury after exposure of hepatocytes to t-butylhydroperoxide
.
Biochem. J.
307
,
99
106
[PubMed]
15.
Byrne
A.M.
,
Lemasters
J.J.
and
Nieminen
A.L.
(
1999
)
Contribution of increased mitochondrial free Ca2+ to the mitochondrial permeability transition induced by tert-butylhydroperoxide in rat hepatocytes
.
Hepatology
29
,
1523
1531
[PubMed]
16.
Imberti
R.
,
Nieminen
A.L.
,
Herman
B.
and
Lemasters
J.J.
(
1993
)
Mitochondrial and glycolytic dysfunction in lethal injury to hepatocytes by t-butylhydroperoxide: protection by fructose, cyclosporin A and trifluoperazine
.
J. Pharmacol. Exp. Ther.
265
,
392
400
[PubMed]
17.
Davies
M.J.
(
1989
)
Detection of peroxyl and alkoxyl radicals produced by reaction of hydroperoxides with rat liver microsomal fractions
.
Biochem. J.
257
,
603
606
[PubMed]
18.
Toledo
F.D.
,
Pérez
L.M.
,
Basiglio
C.L.
,
Ochoa
J.E.
,
Sanchez Pozzi
E.J.
and
Roma
M.G.
(
2014
)
The Ca2+-calmodulin-Ca2+/calmodulin dependent protein kinase II signaling pathway is involved in oxidative stress-induced mitochondrial permeability transition and apoptosis in isolated rat hepatocytes
.
Arch. Toxicol.
88
,
1695
1709
[PubMed]
19.
Pérez
L.M.
,
Milkiewicz
P.
,
Elias
E.
,
Coleman
R.
,
Sánchez Pozzi
E.J.
and
Roma
M.G.
(
2006
)
Oxidative stress induces internalization of the bile salt export pump, Bsep, and bile salt secretory failure in isolated rat hepatocyte couplets: A role for protein kinase C and prevention by protein kinase A
.
Toxicol. Sci.
91
,
150
158
20.
Basiglio
C.L.
,
Toledo
F.D.
,
Boaglio
A.C.
,
Arriaga
S.M.
,
Ochoa
J.E.
,
Sánchez Pozzi
E.J.
et al. .
(
2014
)
Physiological concentrations of unconjugated bilirubin prevent oxidative stress-induced hepatocanalicular dysfunction and cholestasis
.
Arch. Toxicol.
88
,
501
514
[PubMed]
21.
Toledo
F.D.
,
Basiglio
C.L.
,
Barosso
I.R.
,
Boaglio
A.C.
,
Zucchetti
A.E.
,
Sánchez Pozzi
E.J.
et al. .
(
2017
)
Mitogen-activated protein kinases are involved in hepatocanalicular dysfunction and cholestasis induced by oxidative stress
.
Arch. Toxicol.
91
,
2391
2403
[PubMed]
22.
Ballatori
N.
and
Truong
A.T.
(
1989
)
Relation between biliary glutathione excretion and bile acid-independent bile flow
.
Am. J. Physiol.
256
,
G22
G30
[PubMed]
23.
Stocker
R.
,
Yamamoto
Y.
,
McDonagh
A.F.
,
Glazer
A.N.
and
Ames
B.N.
(
1987
)
Bilirubin is an antioxidant of possible physiological importance
.
Science
235
,
1043
1046
[PubMed]
24.
Stocker
R.
,
Glazer
A.
and
Ames
B.
(
1987
)
Antioxidant activity of albumin-bound bilirubin
.
Proc. Natl Acad. Sci. U.S.A.
84
,
5918
5922
25.
Sedlak
T.W.
,
Saleh
M.
,
Higginson
D.S.
,
Paul
B.D.
,
Juluri
K.R.
and
Snyder
S.H.
(
2009
)
Bilirubin and glutathione have complementary antioxidant and cytoprotective roles
.
Proc. Natl Acad. Sci. U.S.A.
106
,
5171
5176
26.
Clark
J.E.
,
Foresti
R.
,
Green
C.J.
and
Motterlini
R.
(
2000
)
Dynamics of haem oxygenase-1 expression and bilirubin production in cellular protection against oxidative stress
.
Biochem. J.
348
,
615
619
[PubMed]
27.
Stevens
B.
and
Small
R.D.
Jr
(
1976
)
The photoperoxidation of unsaturated organic molecules–XV. O21Delta g quenching by bilirubin and biliverdin
.
Photochem. Photobiol.
23
,
33
36
[PubMed]
28.
Kaul
R.
,
Kaul
H.K.
,
Bajpai
P.C.
and
Murti
C.R.K.
(
1979
)
Evidence for the possible involvement of the superoxide radicals in the photodegradation of bilirubin
.
J. Biosci.
1
,
377
383
29.
Witting
P.K.
,
Westerlund
C.
and
Stocker
R.
(
1996
)
A rapid and simple screening test for potential inhibitors of tocopherol mediated peroxidation of LDL lipids
.
J. Lipid Res.
37
,
853
867
[PubMed]
30.
Kaur
H.
,
Hughes
M.N.
,
Green
C.J.
,
Naughton
P.
,
Foresti
R.
and
Motterlini
R.
(
2003
)
Interaction of bilirubin and biliverdin with reactive nitrogen species
.
FEBS Lett.
543
,
113
119
[PubMed]
31.
Oveson
B.C.
,
Iwase
T.
,
Hackett
S.F.
,
Lee
S.Y.
,
Usui
S.
,
Sedlak
T.W.
et al. .
(
2011
)
Constituents of bile, bilirubin and TUDCA, protect against oxidative stress-induced retinal degeneration
.
J. Neurochem.
116
,
144
153
[PubMed]
32.
Fujii
M.
,
Inoguchi
T.
,
Sasaki
S.
,
Maeda
Y.
,
Zheng
J.
,
Kobayashi
K.
et al. .
(
2010
)
Bilirubin and biliverdin protect rodents against diabetic nephropathy by downregulating NAD(P)H oxidase
.
Kidney Int.
78
,
905
919
[PubMed]
33.
Wallner
M.
,
Antl
N.
,
Rittmannsberger
B.
,
Schreidl
S.
,
Najafi
K.
,
Müllner
E.
et al. .
(
2013
)
Anti-genotoxic potential of bilirubin in vivo: damage to DNA in hyperbilirubinemic human and animal models
.
Cancer Prev. Res. (Phila)
6
,
1056
1063
[PubMed]
34.
Arriaga
S.M.
,
Basiglio
C.L.
,
Mottino
A.D.
and
Almará
A.M.
(
2009
)
Unconjugated bilirubin inhibits C1 esterase activity
.
Clin. Biochem.
42
,
919
921
[PubMed]
35.
Basiglio
C.L.
,
Arriaga
S.M.
,
Pelusa
F.
,
Almará
A.M.
,
Roma
M.G.
and
Mottino
A.D.
(
2007
)
Protective role of unconjugated bilirubin on complement‐mediated hepatocytolysis
.
Biochim. Biophys. Acta
1770
,
1003
1010
[PubMed]
36.
Basiglio
C.L.
,
Arriaga
S.M.
,
Pelusa
F.
,
Almará
A.M.
,
Kapitulnik
J.
and
Mottino
A.D.
(
2010
)
Complement activation and disease: protective effects of hyperbilirubinemia
.
Clin. Sci.
118
,
99
113
37.
Adin
C.A.
,
VanGundy
Z.C.
,
Papenfuss
T.L.
,
Xu
F.
,
Ghanem
M.
,
Lakey
J.
et al. .
(
2017
)
Physiologic doses of bilirubin contribute to tolerance of islet transplants by suppressing the innate immune response
.
Cell Transplant.
26
,
11
21
[PubMed]
38.
Ollinger
R.
,
Wang
H.
,
Yamashita
K.
,
Wegiel
B.
,
Thomas
M.
,
Margreiter
R.
et al. .
(
2007
)
Therapeutic applications of bilirubin and biliverdin in transplantation
.
Antioxid. Redox Signal.
9
,
2175
2185
[PubMed]
39.
Ryter
S.W.
,
Morse
D.
and
Choi
A.M.
(
2007
)
Carbon monoxide and bilirubin: potential therapies for pulmonary/vascular injury and disease
.
Am. J. Respir. Cell Mol. Biol.
36
,
175
182
[PubMed]
40.
Liu
J.
,
Wang
L.
,
Xu
Tian
,
Liu
L.
,
Wong
W.
,
Zhang
Y.
et al. .
(
2015
)
Unconjugated Bilirubin mediates heme oxygenase-1–induced vascular benefits in diabetic mice
.
Diabetes
64
,
1564
1575
[PubMed]
41.
Wegiel
B.
,
Nemeth
Z.
,
Correa-Costa
M.
,
Bulmer
A.C.
and
Otterbein
L.E.
(
2014
)
Heme oxygenase-1: a metabolic nike
.
Antioxid. Redox Signal.
20
,
1709
1722
[PubMed]
42.
Braggins
P.E.
,
Trakshel
G.M.
,
Kutty
R.K.
and
Maines
M.D.
(
1986
)
Characterization of two heme oxygenase isoforms in rat spleen: comparison with the hematin-induced and constitutive isoforms of the liver
.
Biochem. Biophys. Res. Commun.
141
,
528
533
[PubMed]
43.
Blanckaert
N.
(
1980
)
Analysis of bilirubin and bilirubin mono- and di-conjugates. Determination of their relative amounts in biological samples
.
Biochem. J.
185
,
115
128
[PubMed]
44.
Lowry
O.H.
,
Rosebrough
N.J.
,
Farr
L.L.
and
Randall
R.J.
(
1951
)
Protein measurement with the Folin phenol reagent
.
J. Biol. Chem.
193
,
265
275
[PubMed]
45.
Borgognone
M.
,
Pérez
L.M.
,
Basiglio
C.L.
,
Ochoa
J.E.
and
Roma
M.G.
(
2005
)
Signaling modulation of bile salt-induced necrosis in isolated rat hepatocytes
.
Toxicol. Sci.
83
,
114
125
[PubMed]
46.
Levine
R.L.
,
Williams
J.A.
,
Stadtman
E.R.
and
Shacter
E.
(
1994
)
Carbonyl assays for determination of oxidatively modified proteins
.
Methods Enzymol.
233
,
346
357
[PubMed]
47.
Tietze
F.
(
1969
)
Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: applications to mammalian blood and other tissues
.
Anal. Biochem.
27
,
502
522
[PubMed]
48.
Griffith
O.W.
(
1980
)
Determination of glutathione and glutathione disulfide using glutathione reductase and 2-vinylpyridine
.
Anal. Biochem.
106
,
207
212
[PubMed]
49.
Maehly
A.C.
and
Chance
B.
(
1954
)
The assay of catalases and peroxidases
.
Methods Biochem. Anal.
1
,
357
424
[PubMed]
50.
Talalay
P.
(
1960
)
Enzymatic analysis of steroid hormones
.
Methods Biochem. Anal.
8
,
119
143
[PubMed]
51.
Fevery
J.
,
Van de Vijver
M.
,
Michiels
R.
and
Heirwegh
K.P.
(
1977
)
Comparison in different species of biliary bilirubin-IX alpha conjugates with the activities of hepatic and renal bilirubin-IX alpha-uridine diphosphate glycosyltransferases
.
Biochem. J.
164
,
737
746
[PubMed]
52.
Lauterburg
B.H.
,
Smith
C.V.
,
Hughes
H.
and
Mitchell
J.R.
(
1984
)
Biliary excretion of glutathione and glutathione disulfide in the rat: regulation and response to oxidative stress
.
J. Clin. Invest.
73
,
124
133
[PubMed]
53.
Boon
A.C.
,
Lam
A.K.
,
Gopalan
V.
,
Benzia
I.F.
,
Briskey
D.
,
Coombes
J.S.
et al. .
(
2015
)
Endogenously elevated bilirubin modulates kidney function and protects from circulating oxidative stress in a rat model of adenine-induced kidney failure
.
Sci. Rep.
5
,
15482
[PubMed]
54.
O’Malley
S.S.
,
Gueorguieva
R.
,
Wu
R.
and
Jatlow
P.I.
(
2015
)
Acute alcohol consumption elevates serum bilirubin: an endogenous antioxidant
.
Drug Alcohol Depend.
149
,
87
92
[PubMed]
55.
Deganuto
M.
I
,
Cesaratto
L.
,
Bellarosa
C.
,
Calligaris
R.
,
Vilotti
S.
,
Renzone
G.
et al. .
(
2010
)
A proteomic approach to the bilirubin-induced toxicity in neuronal cells reveals a protective function of DJ-1 protein
.
Proteomics
10
,
1645
1657
[PubMed]
56.
Lakovic
K.
,
Ai
J.
,
D’Abbondanza
J.
,
Tariq
A.
,
Sabri
M.
,
Alarfaj
A.K.
et al. .
(
2014
)
Bilirubin and its oxidation products damage brain white matter
.
J. Cereb. Blood Flow Metab.
34
,
1837
1847
[PubMed]
57.
Benzie
I.F.
(
1996
)
Lipid peroxidation: a review of causes, consequences, measurement and dietary influences
.
Int. J. Food Sci. Nutr.
47
,
233
261
[PubMed]
58.
Levine
R.
(
2000
)
Methods in molecular biology
. In
Stress Response: Methods and Protocols
,
Keyse
S.M.
ed., pp.
15
24
,
Springer Science and Business Media
,
Luxemburg
59.
Zelenka
J.
,
Dvolák
A.
,
Alán
L.
,
Zadinová
M.
,
Haluzík
M.
and
Vítek
L.
(
2016
)
Hyperbilirubinemia protects against aging-associated inflammation and metabolic deterioration
.
Oxid. Med. Cell Longev.
,
2016
,
6190609
[PubMed]
60.
Jansen
T.
,
Hortmann
M.
,
Oelze
M.
,
Opitz
B.
,
Steven
S.
,
Schell
R.
et al. .
(
2010
)
Conversion of biliverdin to bilirubin by biliverdin reductase contributes to endothelial cell protection by heme oxygenase-1—evidence for direct and indirect antioxidant actions of bilirubin
.
J. Mol. Cell Cardiol.
49
,
186
195
[PubMed]
61.
Doré
S.
,
Takahashi
M.
,
Ferris
C.D.
,
Zakhary
R.
,
Hester
L.D.
,
Guastella
D.
et al. .
(
1999
)
Bilirubin, formed by activation of heme oxygenase-2, protects neurons against oxidative stress injury
.
Proc. Natl Acad. Sci. U.S.A.
96
,
2445
2450
[PubMed]
62.
Baranano
D.E.
,
Rao
M.
,
Ferris
C.D.
and
Snyder
S.H.
(
2002
)
Biliverdin reductase: a major physiologic cytoprotectant
.
Proc. Natl. Acad. Sci. U.S.A.
99
,
16093
16098
[PubMed]
63.
Chen
W.
,
Maghzal
G.J.
,
Ayer
A.
,
Suarna
C.
,
Dunn
L.L.
and
Stocker
R.
(
2018
)
Absence of the biliverdin reductase-a gene is associated with increased endogenous oxidative stress
.
Free Radic. Biol. Med.
115
,
156
165
[PubMed]
64.
Barone
E.
,
Di Domenico
F.
,
Mancuso
C.
and
Butterfield
D.A.
(
2014
)
The Janus face of the heme oxygenase/biliverdin reductase system in Alzheimer disease: it’s time for reconciliation
.
Neurobiol. Dis.
62
,
10
65.
McCandless
D.W.
(
2011
)
Biochemistry and physiology of bilirubin
. In
Kernicterus. Contemporary Clinical Neuroscience
, pp.
19
31
,
Humana Press
,
Totowa, NJ
66.
Lo Iacono
L.
,
Boczkowski
J.
,
Zini
R.
,
Salouage
I.
,
Berdeaux
A.
,
Motterlini
R.
et al. .
(
2011
)
A carbon monoxide-releasing molecule (CORM-3) uncouples mitochondrial respiration and modulates the production of reactive oxygen species
.
Free. Radic. Biol. Med.
50
,
1556
1564
67.
Ahmed-Choudhury
J.
,
Orsler
D.J.
and
Coleman
R.
(
1998
)
Hepatobiliary effects of tertiary-butylhydroperoxide (tBOOH) in isolated rat hepatocyte couplets
.
Toxicol. Appl. Pharmacol.
152
,
270
275
[PubMed]
68.
Roma
M.G.
and
Sanchez Pozzi
E.J.
(
2008
)
Oxidative stress: a radical way to stop making bile
.
Ann. Hepatol.
7
,
16
33
[PubMed]
69.
Espinosa-Diez
C.
,
Miguel
V.
,
Mennerich
D.
,
Kietzmann
T.
,
Sánchez-Pérez
P.
,
Cadenas
S.
et al. .
(
2015
)
Antioxidant responses and cellular adjustments to oxidative stress
.
Redox Biol.
6
,
183
197
[PubMed]
70.
Ndisang
J.F.
(
2017
)
Synergistic interaction between heme oxygenase (HO) and nuclear-factor E2-related factor-2 (Nrf2) against oxidative stress in cardiovascular related diseases
.
Curr. Pharm. Des.
23
,
1465
1470
[PubMed]
71.
Nies
A.T.
and
Keppler
D.
(
2007
)
The apical conjugate efflux pump ABCC2 (MRP2)
.
Pflugers Arch.
453
,
643
659
[PubMed]
72.
Donner
M.
,
Topp
S.A.
,
Cebula
P.
,
Krienen
A.
,
Gehrmann
T.
,
Sommerfeld
A.
et al. .
(
2012
)
HbG200-mediated preinduction of heme oxygenase-1 improves bile flow and ameliorates pericentral downregulation of Bsep and Mrp2 following experimental liver ischemia and reperfusion
.
Biol. Chem.
394
,
97
112
73.
Teoh
N.C.
and
Farrell
G.C.
(
2003
)
Hepatic ischemia reperfusion injury: pathogenic mechanisms and basis for hepatoprotection
.
J. Gastroenterol. Hepatol.
18
,
891
902
[PubMed]
74.
Motterlini
R.
(
2007
)
Carbon monoxide-releasing molecules (CO-RMs): vasodilatory, anti-ischaemic and anti-inflammatory activities
.
Biochem. Soc. Trans.
35
,
1142
1146
[PubMed]
75.
Floreani
A.
,
Baragiotta
A.
,
Martines
D.
,
Naccarato
R.
and
D’odorico
A.
(
2000
)
Plasma antioxidant levels in chronic cholestatic liver diseases
.
Aliment. Pharmacol. Ther.
14
,
353
358
[PubMed]
76.
Cuzick
J.
(
2017
)
Preventive therapy for cancer
.
Lancet Oncol.
18
,
e472
e482
[PubMed]
77.
Bjelakovic
G.
,
Nikolova
D.
,
Gluud
L.L.
,
Simonetti
R.G.
and
Gluud
C.
(
2015
)
Antioxidant supplements for prevention of mortality in healthy participants and patients with various diseases
.
Sao Paulo Med. J.
133
,
164
165
[PubMed]
78.
Abutwerat
A.
,
Pemberton
P.W.
,
Smith
A.
,
Burrows
P.C.
,
McMahon
R.F.
,
Jain
S.K.
et al. .
(
2003
)
Oxidant stress is a significant feature of primary biliary cirrhosis
.
Biochim. Biophys. Acta
1637
,
142
150
[PubMed]
79.
Wasik
U.
,
Milkiewicz
M.
,
Kempinska-Podhorodecka
A.
and
Milkiewicz
P.
(
2017
)
Protection against oxidative stress mediated by the Nrf2/Keap1 axis is impaired in primary biliary cholangitis
.
Sci. Rep.
7
,
44769
[PubMed]
80.
Tang
W.
,
Jiang
Y.F.
,
Ponnusamy
M.
and
Diallo
M.
(
2014
)
Role of Nrf2 in chronic liver disease
.
World J. Gastroenterol.
20
,
13079
13087
[PubMed]
81.
Muchova
L.
,
Vanova
K.
,
Zelenka
J.
,
Lenicek
M.
,
Petr
T.
,
Vejrazka
M.
et al. .
(
2011
)
Bile acids decrease intracellular bilirubin levels in the cholestatic liver: implications for bile acid-mediated oxidative stress
.
J. Cell. Mol. Med.
15
,
1156
1165
[PubMed]
82.
McMaster
P.D.
(
1922
)
Do species lacking a gallbladder possess its functional equivalent?
J. Exp. Med.
35
,
127
140
[PubMed]
83.
Guha
C.
,
Parashar
B.
,
Deb
N.J.
,
Garg
M.
,
Gorla
G.R.
,
Singh
A.
et al. .
(
2002
)
Normal hepatocytes correct serum bilirubin after repopulation of Gunn rat liver subjected to irradiation/partial resection
.
Hepatology
36
,
354
362
[PubMed]

Author notes

*

These authors contributed equally to this work.