Altered responsiveness to extracellular ATP enhances acetaminophen hepatotoxicity
- Sylvia S Amaral†1,
- André G Oliveira†1,
- Pedro E Marques1,
- Jayane L D Quintão1,
- Daniele A Pires1,
- Rodrigo R Resende2,
- Bruna R Sousa2,
- Juliana G Melgaço3,
- Marcelo A Pinto3,
- Remo C Russo4,
- Ariane k C Gomes4,
- Lidia M Andrade4,
- Rafael F Zanin5,
- Rafaela V S Pereira1,
- Cristina Bonorino5,
- Frederico M Soriani6,
- Cristiano X Lima7,
- Denise C Cara1,
- Mauro M Teixeira2,
- Maria F Leite†4, 8 and
- Gustavo B Menezes†1, 9Email author
© Amaral et al.; licensee BioMed Central Ltd. 2013
Received: 22 October 2012
Accepted: 26 January 2013
Published: 5 February 2013
Adenosine triphosphate (ATP) is secreted from hepatocytes under physiological conditions and plays an important role in liver biology through the activation of P2 receptors. Conversely, higher extracellular ATP concentrations, as observed during necrosis, trigger inflammatory responses that contribute to the progression of liver injury. Impaired calcium (Ca2+) homeostasis is a hallmark of acetaminophen (APAP)-induced hepatotoxicity, and since ATP induces mobilization of the intracellular Ca2+ stocks, we evaluated if the release of ATP during APAP-induced necrosis could directly contribute to hepatocyte death.
APAP overdose resulted in liver necrosis, massive neutrophil infiltration and large non-perfused areas, as well as remote lung inflammation. In the liver, these effects were significantly abrogated after ATP metabolism by apyrase or P2X receptors blockage, but none of the treatments prevented remote lung inflammation, suggesting a confined local contribution of purinergic signaling into liver environment. In vitro, APAP administration to primary mouse hepatocytes and also HepG2 cells caused cell death in a dose-dependent manner. Interestingly, exposure of HepG2 cells to APAP elicited significant release of ATP to the supernatant in levels that were high enough to promote direct cytotoxicity to healthy primary hepatocytes or HepG2 cells. In agreement to our in vivo results, apyrase treatment or blockage of P2 receptors reduced APAP cytotoxicity. Likewise, ATP exposure caused significant higher intracellular Ca2+ signal in APAP-treated primary hepatocytes, which was reproduced in HepG2 cells. Quantitative real time PCR showed that APAP-challenged HepG2 cells expressed higher levels of several purinergic receptors, which may explain the hypersensitivity to extracellular ATP. This phenotype was confirmed in humans analyzing liver biopsies from patients diagnosed with acute hepatic failure.
We suggest that under pathological conditions, ATP may act not only an immune system activator, but also as a paracrine direct cytotoxic DAMP through the dysregulation of Ca2+ homeostasis.
KeywordsLiver injury Sterile inflammation Acetaminophen Remote injury Cell death Immune system Purinergic signaling Inflammation
The abusive use of medications is a major health issue and accounts every year to huge hospitalization costs and several deaths. Analgesics are one of the first alternatives to treat fever and pain, and acetaminophen is the most frequent drug found in these formulations. It is not surprising that the cases of acetaminophen overdose are still frequent in the clinics, and since the liver is a central organ in acetaminophen detoxification, hepatocytes are directly damaged during overdose. Despite the liver ability to regenerate after injury, a massive cell death may trigger an inflammatory response that accounts to additional injury. One of the strategies to restrict organ injury is to control liver inflammation, avoiding organ failure. Here we show that ATP, a key molecule in cell bioenergetics, is also involved in liver inflammation. When cells die, they release ATP to the extracellular environment and this may directly cause additional cell hepatocyte death. These effects may be explained by ATP ability to directly cause intracellular ionic dysregulation in acetaminophen-treated cells. Mice that received an overdose of acetaminophen had significantly less liver damage when extracellular ATP actions were inhibited. Also, human-derived cells cultivated in vitro were also protected from these toxic effects when the same blockage strategy was employed. Finally, we established that liver samples from patients suffering from acute hepatitis expressed more receptors to ATP, which suggests that a similar amplifying effect happened during their disease. In this sense, we provided evidence that managing liver response to extracellular ATP released from dead cells may hold future opportunities to avoid liver failure, transplantations and death.
Drug-induced liver injury (DILI) is an adverse drug reaction that causes acute hepatocyte death. There are several different grades of DILI, which range from an asymptomatic lesion (detectable only by serum transaminases analysis) to severe cases that require liver transplantation . However, 20% to 50% of eligible patients die before a transplant becomes available as a result of hepatic encephalopathy and multiple organ failure , indicating that novel therapies aimed to control the progression of liver damage are extremely necessary. The most common cause of DILI is the overdose of acetaminophen (APAP), a popular antipyretic and analgesic drug. Following APAP administration, its reactive metabolite NAPQI (N-acetyl-p-benzoquinone imine) accumulates within hepatocytes, causing cell death mainly by oncotic necrosis .
When cells die under such stressing situations, their intracellular contents are spilled to the interstitium and trigger inflammation by directly causing damage to adjacent cells or activating resident cells to release pro-inflammatory mediators. In the latter case, these molecules are called damage-associated molecular patterns (DAMPs) . In general, immune cells express receptors to almost all molecules that originally inhabit the intracellular compartment [5, 6], but not all DAMPs are exclusively associated with immune responses. For example, cells can secrete ATP to modulate intracellular functions, including cytosolic calcium (Ca2+) concentration and energetic balance [7–9]. However, extracellular ATP concentration significantly increases during necrosis, which in turn activates inflammasome assembling via P2X7 receptor, leading to release of IL-1β [5, 10]. The inflammatory response triggered by necrosis-derived ATP was recently described as an important factor to liver injury progression, and activation of P2X7 receptor is required for manifestations of APAP-induced injury .
Impaired intracellular Ca2+ management is also observed during APAP-induced hepatotoxicity, and it is closely related to the onset of cell death . Moreover, intracellular Ca2+ accumulation, particularly into the nucleus, causes DNA fragmentation by endonucleases, accelerating the progression of APAP-dependent cellular necrosis , indicating that molecules with ability to increase Ca2+ signaling may cause catastrophic consequences to APAP-challenged cells. ATP induces Ca2+ mobilization from intracellular stocks  and also by opening Ca2+ permeable channels in the membrane via P2 receptors . Taking into account the increased extracellular ATP concentration found during necrosis, we hypothesized that excessive interstitial ATP might contribute to liver injury progression not only via immune system stimulation, but also by worsening intracellular Ca2+ imbalance observed during APAP administration, acting as a direct cytotoxic DAMP.
Acetaminophen-induced liver damage, but not remote lung inflammation, is dependent on extracellular ATP signaling
Additional file 1: Video 1: Three-dimensional Z-stack rendering from liver confocal intravital microscopy – Control mouse. Lysm-eGFP mice were used to visualize neutrophil infiltration and sinusoidal perfusion. Sinusoids were stained by i.v. injection of PE-coupled anti-PECAM-1. Z-stacks were made (depth: 40 μm) and TIFF-acquired images were mounted by using Volocity software (NIH, USA). Mice were imaged by sequential laser scans (2.71 seconds). (MOV 3 MB)
Additional file 2: Video 2: Three-dimensional Z-stack rendering from liver confocal intravital microscopy – Acetaminophen-treated mouse. Lysm-eGFP mice were treated with acetaminophen (APAP; 500 mg/Kg; 24 h) and prepared to visualization of neutrophil infiltration and liver sinusoidal perfusion. Sinusoids were stained by i.v. injection of PE-coupled anti-PECAM-1. Z-stacks were made (depth: 40 μm) and TIFF-acquired images were mounted by using Volocity software (NIH, USA). Mice were imaged by sequential laser scans (2.71 seconds). (MOV 3 MB)
Additional file 3: Video 3: Three-dimensional Z-stack rendering from liver confocal intravital microscopy – Apyrase treated acetaminophen-challenged mouse. Lysm-eGFP mice were treated with acetaminophen (APAP; 500 mg/Kg; 24 h) and apyrase (25 U/mice, 2 hours after APAP) and prepared to visualization of neutrophil infiltration and liver sinusoidal perfusion. Sinusoids were stained by i.v. injection of PE-coupled anti-PECAM-1. Z-stacks were made (depth: 40 μm) and TIFF-acquired images were mounted by using Volocity software (NIH, USA). Mice were imaged by sequential laser scans (2.71 seconds). (MOV 3 MB)
HepG2 cells release ATP following acetaminophen incubation
To prove that stressed/necrotic hepatocytes could be a relevant source of extracellular ATP, we incubated HepG2 cells with APAP and measured ATP and ADP concentrations in the supernatant by high-performance liquid chromatography (HPLC). Supernatant from untreated cells had undetectable amounts of both ATP and ADP. However, following APAP incubation, extracellular ATP concentration rapidly increased, while ADP was detected in later timepoints (Figure 3F). Interestingly, cleavage of extracellular ATP reverted APAP cytotoxicity (Figure 3G), which was reproduced by unspecific blockage of P2 receptors by suramin (0.01-0.1 mM; 24 h) (Figure 3H). Selective participation of different P2 receptors subfamilies was also investigated. Specific P2X receptors antagonism by PPADS (0.1-100 μM; 24 h) or TNP-ATP (0.1-100 μM) also partially prevented APAP cytotoxicity, as well as selective blockage of P2Y receptors by reactive blue-2 (3-30 μM) (Figure 3H). While selective P2X7 blockage (by oxi-ATP) prevented APAP hepatotoxicity in vivo, no detectable effects were observed in vitro using a large dose range (10-100 μM; 24 h) (Additional file 5: Figure S2). These data suggest that not a specific subtype, but several ATP and ADP receptors may be involved in extracellular purinergic signaling during necrosis, and their combined stimulation may enhance cell death independently of the immune system activation.
In this context, we hypothesized that ATP might be directly harmful in concentrations that are biologically relevant. In fact, in the same titers found in medium recovered from APAP-challenged cells, both ATP and ADP were directly cytotoxic to “naïve” HepG2 cells (10 μM, Figure 3I, Additional file 4: Figure S1). ATP-mediated toxicity was detected in early timepoints (6 h), persisting until the end of incubation period (24 h, Figure 3I), which was also confirmed by ethidium bromide/acridine orange staining (Figure 3J; control in comparison to APAP). Subsequently, we examined whether the effects observed following ATP incubation were derived only from its metabolism to ADP. For this, we used a non-hydrolysable ATP (ATP-γ-S) in the same dose range and confirmed that this stable analogue was equally able to deflagrate cell death (Figure 3I).
Increased intracellular Ca2+ availability underlies the mechanisms by which ATP/P2 receptors activation contribute to APAP-mediated cell death
Additional file 6: Video 4: Detection of intracellular Ca2+ signals with confocal microscopy - APAP treated HepG2 cells. Nuclear and cytosolic Ca2+ were monitored in individual cells by using time-lapse confocal microscopy. HepG2 cells were cultured on glass coverslips in a density of 3×105 cells/well in 6 wells plates and kept in a Hepes-buffered solution during experiments. Cells were incubated with APAP (10 mM) and after 24 hours 4 μM cell permeant Fluo4-AM (fluo-4 acetoxymethyl ester; Molecular Probes) was added to the culture. ATP (10 μm) was used to trigger intracellular calcium signal. (MOV 6 MB)
Additional file 7: Video 5: Detection of intracellular Ca2+ signals with confocal microscopy - Control HepG2 cells. Nuclear and cytosolic Ca2+ were monitored in individual cells by using time-lapse confocal microscopy. HepG2 cells were cultured on glass coverslips in a density of 3×105 cells/well in 6 wells plates and kept in a Hepes-buffered solution during experiments. Cells were incubated with 4 μM cell permeant Fluo4-AM (fluo-4 acetoxymethyl ester; Molecular Probes) was added to the culture. ATP (10 μm) was used to trigger intracellular calcium signal. (MOV 5 MB)
To confirm that unbalanced calcium signal was involved in APAP cytotoxicity, we treated HepG2 cells with an intracellular Ca2+ scavenger (BAPTA-AM; 1 nM) throughout APAP challenge. Incubation with BAPTA-AM reduced APAP-mediated cell death in 50% (Figure 4H), suggesting that increased intracellular calcium availability contributed to APAP-mediated cell death.
Primary mouse hepatocytes also developed hyper-responsiveness to extracellular ATP following APAP exposure, which is prevented by P2 receptor antagonism
Additional file 8: Video 6: Detection of intracellular Ca2+ signals with confocal microscopy – Control primary hepatocytes. Nuclear and cytosolic Ca2+ were monitored in individual cells by using time-lapse confocal microscopy. Primary mouse hepatocytes were cultured on glass coverslips in a density of 3×105 cells/well in 6 wells plates and kept in a Hepes-buffered solution during experiments. Cells were incubated with APAP (20 mM) and after 6 hours 4 μM cell permeant Fluo4-AM (fluo-4 acetoxymethyl ester; Molecular Probes) was added to the culture. ATP (10 μm) was used to trigger intracellular calcium signal. (MP4 401 KB)
Additional file 10: Video 8: Detection of intracellular Ca2+ signals with confocal microscopy – APAP-treated primary hepatocytes displayed repeated calcium signal following ATP administration. Nuclear and cytosolic Ca2+ were monitored in individual cells by using time-lapse confocal microscopy. Primary mouse hepatocytes were cultured on glass coverslips in a density of 3×105 cells/well in 6 wells plates and kept in a Hepes-buffered solution during experiments. Cells were incubated with APAP (20 mM) and after 6 hours 4 μM cell permeant Fluo4-AM (fluo-4 acetoxymethyl ester; Molecular Probes) was added to the culture. ATP (10 μm) was used to trigger intracellular calcium signal. (MP4 1 MB)
Additional file 11: Video 9: Detection of intracellular Ca2+ signals with confocal microscopy – responsiveness to exogenous ATP. Nuclear and cytosolic Ca2+ were monitored in individual cells by using time-lapse confocal microscopy. Primary mouse hepatocytes were cultured on glass coverslips in a density of 3×105 cells/well in 6 wells plates and kept in a Hepes-buffered solution during experiments. Cells were incubated with APAP (20 mM) and suramin (0.1 mM) and after 6 hours 4 μM cell permeant Fluo4-AM (fluo-4 acetoxymethyl ester; Molecular Probes) was added to the culture. ATP (10 μm) was used to trigger intracellular calcium signal. (MP4 347 KB)
Increased expression of several purinergic receptors may explain the hyper-responsiveness to ATP during necrosis
To validate our findings in humans, we investigated the profile of purinergic receptors expression in patients diagnosed with drug-induced acute hepatitis. In agreement, several P2 receptors were also upregulated in acute hepatitis patients in comparison to healthy donors (Figure 6E), suggesting that altered cell responsiveness to extracellular purines may be also relevant in the context of liver injury progression. In addition, enhanced purinergic receptor expression (P2X2, P2X7 and P2X1; Figure 6F) was correlated with higher grades of liver injury (assessed by serum ALT levels) in several acute hepatitis patients (Pearson’s correlation; r).
Extracellular ATP is a well-characterized damage-associated molecular pattern (DAMP), which activates the NLRP3 inflammasome via P2X7 receptors, inducing production of inflammatory cytokines, including interleukin-1β [5, 11, 19, 20]. The majority of in vitro studies commonly use elevated concentrations of ATP (ranging from 1 to 10 mM) and short incubation regimes to induce cell activation, particularly leukocytes [21–23]. However, these conditions are unlikely to represent any in vivo environment found under either physiological or pathological circumstances . For instance, extracellular ATP concentration increases during inflammatory responses, but it reaches maximum concentrations in the range of hundred micromolars . So far, the damage exerted by increased extracellular concentrations of ATP has been solely attributed to indirect, inflammatory effects over immune cells [11, 26]. In this study, we showed that ATP and ADP have direct cytotoxic effects in hepatic cells and may have profound influence in the pathogenesis of acute liver failure. We provided evidence that ATP signaling during necrosis might have a double-faceted action by i) enhancing inflammatory response via IL-1β release and ii) directly causing hepatotoxicity due to a hyper-responsiveness behavior to ATP and increased intracellular Ca2+ availability.
In this study, we used a murine model of APAP poisoning to show that activation of several P2 receptors is detrimental during liver injury progression. Confocal intravital microscopy revealed remarkable changes in the liver environment during APAP overdose that were dependent on extracellular ATP signaling. Also, harmful effects of P2 receptor agonists seems to be restricted to the liver, since blockage of ATP/ADP sensing was not effective in preventing remote lung injury. In fact, despite the hepatoprotection and the reduced levels of circulating cytokines promoted by apyrase treatment, DAMPs released probably from remote injury were sufficient to support lung inflammation . In line with this interpretation, recent work demonstrated that increased serum levels of ATP found during hepatectomy were only transient (~ 5 minutes) and rapidly returned to baseline values . These data indicate that while extracellular ATP and ADP play a key role in local liver injury, they are not mediators of remote inflammatory responses.
Also, we performed a series of in vitro experiments to determine if hepatocytes could constitute a relevant source of extracellular ATP. Subsequently to APAP incubation, higher concentrations of ATP and ADP were found in the culture medium recovered from stressed/necrotic HepG2 cells in comparison to controls. Taking into account that these titers were sufficient to directly induce HepG2 death, we postulated that ATP released from a suffering or necrotic cell binds to P2 receptors in a challenged, neighboring cell and increases its intracellular Ca2+ concentration in sufficiently high levels to accelerate cell damage or even death. It is possible that off-target factors, including alterations in probe hydrolysis or intracellular pH variations could contribute to Fluo-4AM fluorescence variations observed in the current study. However, these factors possibly had a minor impact in our results, as we were able to significantly abrogate ATP-mediated increase in intracellular calcium signal with P2R antagonist treatment. Therefore, our data suggest that the main pathway involved in altered cell responsiveness in our model is related to P2R signaling. In fact, paracrine communication between hepatocytes mediated by ATP was previously reported , and blockage of cell-cell interaction via gap junctions may hold opportunities to restrict liver damage during APAP overdose . Also, recent data have confirmed that extracellular ATP may enhance APAP-mediated liver damage in vivo via activation of P2X7 and P2Y2 [30, 31]. Thus, we suggest that necrotic/suffering hepatocytes may efficiently supply extracellular ATP to fuel both immune system activation and direct hepatotoxicity. Likewise, in vitro effects were not restricted to single receptor activation (e.g. P2X7), but rather mediated by different P2 receptors subfamilies, suggesting that larger spectrum inhibition might be necessary to promote appreciable cytoprotection.
ATP induces intracellular signals by mobilizing Ca2+ from cytosolic and nuclear stocks  or allowing its influx across the membrane . Interestingly, in situations of deprivation of mitochondrial ATP, as seen throughout APAP challenge, cells may upregulate P2 receptors to increase intracellular Ca2+ and stimulate ATP synthesis within mitochondria as a tentative to escape from an irreversible damage [7–9]. Accordingly, both primary mouse hepatocytes and HepG2 cells developed a hyper-responsive behavior to exogenous ATP following APAP administration, which may be correlated with increased expression of different purinergic receptors. Therefore, we propose that such change in P2R levels observed in both APAP-treated cells and in liver biopsies from acute hepatitis patients may be a strategy to restrict cell suffering through P2 receptors-mediated intracellular Ca2+ increase. However, APAP challenge may disturb important intracellular mechanisms that manage Ca2+ compartmentalization, since challenged cells displayed elevated intracellular Ca2+ signal in our in vitro model. In this context, cytoprotection promoted by intracellular calcium sequestration (using BAPTA-AM) provided interesting insights for future investigations focused on elucidate the most important calcium sources that extracellular ATP recruits to boost APAP-triggered hepatotoxicity. It is worth taking into consideration that although we observed P2 receptor upregulation in HepG2 cells following APAP challenge, it is conceivable that purinergic signaling in other cell types (i.e. liver resident cells and infiltrating leukocytes) is also involved in the development of acute liver failure caused by APAP administration. Furthermore, although we confirmed APAP cytotoxicity by two different methodologies, alterations in cell metabolic pathways following APAP overdose (i.e. mitochondrial activity and cell proliferation rate) may also contribute to the mechanisms involved in APAP cytotoxicity.
C57BL/6 mice were from Centro de Bioterismo in UFMG (Brazil). Lysm-eGFP mice were donated by Dr. Paul Kubes (University of Calgary, Canada). All procedures were approved by Animal Care and Use Committee in UFMG (CEBIO n°051/2011). The investigation conformed to the standards of Guide for the Care and Use of Laboratory Animals (National Institutes of Health publication 85–23, 1996 revision).
Model of APAP-induced liver injury
Mice were fasted for 15 hours before oral APAP administration (500 mg/kg; Sigma, USA) or vehicle (warm sterile saline). After different time-points, mice were anesthetized and euthanized for blood (serum), liver, BAL (bronchial-alveolar lavage) and lung harvesting. ALT determination was performed using a kinetic test (Bioclin, Brazil) and cytokines and chemokines were quantified by ELISA kits (R&D systems, USA) both in serum and tissues. Fragments of liver and lung were fixed in formalin and sectioned for histology (H&E). Histological score was assessed by an experienced pathologist, in which 0: no lesion present; 1/2: individual necrotic cells seen at the first cell layer adjacent to the central vein, and hyaline degeneration present; 1: necrotic cells extending two or three cell layers from the central veins; 2: necrotic cells extending three to six cell layers from the central veins, but limited in peripheral distribution; 3: the same as 2, but with necrosis extending from one central vein to another; 4: more severe than 3, with extensive centrilobular necrosis throughout the section. A final score was given for each liver section . Serum mitDNA (cytochrome C primer) was estimated by Real-Time PCR as previously described . Neutrophil infiltration into the liver was estimated by the myeloperoxidase activity assay (MPO) . In a separated set of experiments, the liver was imaged using confocal intravital microscopy as described previously . Three-dimensional video reconstructions were made using confocal Z-stacks (40 μm; 1 μm step) and mounted using Volocity software (Perkin-Elmer, USA). BAL was collected for leukocyte counting . All experimental groups included N ≥ 5.
In vivo drugs and treatments
Mice received APAP (500 mg/Kg; Sigma) by oral gavage diluted in warm saline. This dose of APAP is almost completely metabolized within 1.5-2 hours . Therefore, all pharmacological treatments were performed two hours after APAP gavage to avoid interference in APAP bioactivation and off-target effects. Suramin (5 mg/Kg; i.v.), TNP-ATP (1 mg/Kg; i.v.), oxidized-ATP (oxi-ATP; 9 mg/Kg; i.v.), reactive blue-2 (10-100 mg/Kg; i.p.), apyrase (25 U/mice; i.v.) and theophylline (20 mg/Kg) were dissolved in sterile saline following supplier instructions (Sigma, USA).
In vitro HepG2 assays
HepG2 (American Type Culture Collection) cells were maintained at 37°C under an atmosphere of 5% CO2 in complete RPMI1640 medium containing 10% FBS and cultured in 105 cells/well in 96 wells plates. After 24 hours of incubation the supernatant was replaced by medium without FBS containing the treatments . APAP (5-20 mM), suramin (0.01-0.1 mM), TNP-ATP (0.1-100 μM), PPADS (0.1-100 μM), oxi-ATP (10-1000 μM), apyrase grade IV (10 U/mL), reactive blue-2 (3-30 μM, a generous gift from Dr. Tomoyuki Saino, Iwate Medical University, Japan), ATP (1-100 μM), ATP-γ-S (10 μM), ADP (1-10 μM), adenosine (0.1-10 μM) and BAPTA-AM (1 nM) were dissolved in DMSO or water following supplier instructions (Sigma, USA), and added into culture medium to incubation throughout the experiments. Working doses were chosen based on dose-response curves previously established in our group (Additional file 5: Figure S2). Cell viability was assessed by MTT metabolism assay (Sigma, USA) or by ethidium bromide/acridine orange viability assay . Purinergic receptors and ectonucleotidases expression was quantified by Real-Time PCR as described previously [40, 41]. Experiments were repeated at least three times, using 12 replicates per group.
Primary mouse hepatocytes (PMH) isolation and culture
PMH were isolated as previously described [42, 43]. Briefly, the portal vein was cannulated, and a solution collagenase B (4 mg/ml; Roche; from C. histolyticum) was perfused during 8 minutes (5 ml/min). Following initial digestion, liver was separated from the diaphragm and stomach, and carefully minced in a plastic Petri dish in Williams’s medium and the resultant cells were double filtered in serial nylon mesh filters. The cell suspension was washed twice with William’s medium and ressuspended in William’s medium containing 50 U of penicillin and 50 mg of streptomycin. This protocol yielded a high rate of viable hepatocytes (>95% as assessed by commercial fast hematological staining kit and Trypan blue exclusion). Cells were then seeded on collagen I-treated glass coverslips (3 × 105 cells/well) and cultured using 6-wells plates to further MTT and calcium signal assays (as described previously for HepG2 cells).
Analysis of extracellular nucleotides by HPLC
Cells were incubated in presence or absence of APAP and supernant was recovered in different timepoints. Aliquots of 40 μL were applied to a reverse phase HPLC system using a C18 Shimadzu column (Shimadzu, Japan) with absorbance measured at 260 nm. The mobile phase was 60 mM KH2PO4, 5 mM tetrabutylammonium chloride, pH 5.9, in 15% methanol. Retention times were assessed using standard samples of ATP and its metabolites.
Detection of intracellular Ca2+ signals with confocal microscopy
Nuclear and cytosolic Ca2+ were monitored in individual cells by using time-lapse confocal microscopy, as described . Cells were incubated with 4 μM cell permeant fluo-4-AM (fluo-4 acetoxymethyl ester; Molecular Probes) and all fluorescence analyses were performed offline using ImageJ software (NIH) as previously described .
Clinical data of patients enrolled in the study
serum bilirubin (mg/dl)
Acute Hepatitis (n = 15)
Healthy donors (n = 5)
Statistical analyses were performed using one-way ANOVA (Dunnett or Bonferroni post-test) or Student’s t test. P values less than 0.05 were considered statistically significant. Pearson’s correlation was calculated by Excel 2011 for Mac (Microsoft). All data are presented as mean ± SEM.
Damage-associated molecular patterns
Hematoxilin and eosin
Thiazolyl blue tetrazolium bromide
High performance liquid chromatography
Lysozyme M promoter for enhanced green fluorescent protein
Platelet-endothelial cell adhesion molecule-1
Quantitative polymerase chain reaction.
Authors would like to Dr. André Bafica, Dr. Aristóbolo Silva, Dr. Greg Kitten and Gilson Nogueira for providing reagents and technical assistance.
CNPq, CAPES, FAPEMIG, PRONEX and HHMI.
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