Animals

Animal experimentation was carried out following both Spanish and European legislations. Animals were housed in controlled conditions of temperature (22 °C) and humidity with dark/light cycles of 12 h, at the Instituto de Investigaciones Biomédicas Sols-Morreale (Madrid, Spain) animal facilities (PROEX 007/2019). Mice were fed with standard chow diet ad libitum and had free access to drinking water.

Male C57BL/6 J mice between 2 and 3 months of age were submitted to a model of APAP-induced ALF. Briefly, 30 mg of APAP (A7085 Merck Life Science, Darmstadt, Germany) was dissolved in 1.5 ml saline and warmed at 55 °C until totally dissolved. Overnight fasted mice were injected intraperitoneally with a single dose of 300 mg/kg APAP or saline (control group), and sacrificed 6 and 24 h after injection. Livers and serum samples were collected and conveniently stored for further analysis.

Histopathology assessment

Livers collected from mice were embedded in paraffin and 5 μm-thick liver tissue slices were made. Liver sections were stained with Hematoxylin and Eosin (H&E) and representative images were taken using an optical microscope Nikon Eclipse E400 (Nikon, Tokyo, Japan) equipped with a plan Apocromatic 4X, 10X and 20X objective (Nikon). Hepatic necrosis area was assessed in six different lobular areas, using ImageJ software (NIH, Bethesda, MD, USA).

Transaminase activity analysis

Blood samples were collected from animals after sacrifice and serum was used in a 1:4 dilution. ALT and AST activities were evaluated using colorimetric kits (41282, Spinreact, Girona, Spain, and MAK055, Merck Life Science, Darmstadt, Germany, respectively).

BMP6 detection by immunohistochemistry (IHQ)

Liver tissue slides were deparaffinized and rehydrated. Antigen retrieval was performed following HIER method by boiling the slides for 20 min in 10 mM sodium citrate pH 6. Sections were blocked prior to immunostaining with anti-BMP6 antibody 1:100 (ab155963, Abcam plc, Cambridge, UK) overnight. After incubation with the secondary antibody, DAB detection system (EnVision™ Flex Mini Kit, High pH (Link) Agilent, Santa Clara, CA, USA) was used for visualization according to the manufacturer’s instructions. For histological assessment, six representative images were taken per section using a Nikon Eclipse E400 optical microscope (Nikon) equipped with a plan Apocromatic 4X, 10X and 20X objective (Nikon). Intensity of BMP6 staining was quantified using ImageJ software (NIH) and reported as the average value in arbitrary units (a.u.).

Study population

This study included serum samples kindly provided by Professor James W. Dear from Centre for Cardiovascular Science, Queen’s Medical Research Institute, University of Edinburgh (Scotland, UK). Ethical approval for this study was provided by London—South East Research Ethics Committee (18/LO/0894) (ClinicalTrials.gov identifier: NCT03497104). Patients presenting to Royal Infirmary of Edinburgh, UK (RIE) following APAP overdose, who met the inclusion criteria, were asked to provide informed consent to participate in the prospective. Inclusion criteria: age 16 years and over, hospital attendance with APAP overdose alone or as part of a mixed overdose and patient is able to give informed consent. Exclusion criteria: patient detained under the Mental Health Act, inability to provide informed consent, unreliable history of overdose and prisoners.

Clinical characteristics of the study population are shown in Table 1: 18 patients with APAP overdose, from which 9 developed DILI, with ALT in serum values over 100 U/L (ALT = 4571 ± 3089 U/L), and 9 patients that did not develop DILI, reflected in normal serum levels of ALT (ALT = 16 ± 6.63 U/L). The serum samples from these patients were used to quantify the circulating concentration of BMP6. Serum samples analysis was blinded and performed in a random order.

Table 1 Characteristics of the study populationQuantitative analysis of serum BMP6 levels by ELISA

Serum BMP6 concentration was quantified by ELISA using the mouse BMP6 ELISA kit (CSB-E09279m) for mouse samples and the human BMP6 ELISA kit (CSB-E09277h) for human samples (Cusabio Technology LLC, Hubei, China), following the manufacturer´s instructions. Absorbance from samples was interpolated to a standard curve using a four-parameter logistic (4-PL) equation.

Cell culture and treatments

Immortalized neonatal mouse hepatocyte cell line (Pardo et al. 2015) and human hepatoma cell line Huh7 (ATCC, Manassas, VA, USA) were cultured in Dulbecco´s modified Eagle medium (DMEM, Cytiva, Marlborough, MA, USA) with high glucose supplemented with 10% heat-inactivated fetal bovine serum (FBS), HEPES and antibiotics (penicillin/streptomycin). Human leukemia monocytic-derived cell line THP1 (kindly provided by Dr. Elena Fernández-Ruiz, Hospital Universitario de la Princesa, Madrid, Spain) was cultured in suspension in RPMI (RPMI 1640, Gibco) supplemented with 10% heat-inactivated FBS and antibiotics (penicillin/streptomycin). All cells were maintained at 37 °C, 5% CO2 and relative humidity 95%.

To simulate APAP-induced damage, immortalized mouse hepatocytes and Huh7 cells were stimulated with APAP dissolved in ethanol at different concentrations (1 mM, 5 mM, 10 mM and 20 mM), or ethanol (control) for various time periods. For treatment with BMP6, THP1 cell line was serum-starved for 3 h, and then 1, 10 or 100 ng/ml of recombinant human BMP6 protein (507-BP/CF, R&D Systems, Inc. Minneapolis, USA) was added to the culture media. After treatment, culture media were collected and plates were washed with PBS and used for further analysis.

Transient silencing of BMP6 in Huh7 cells

After a confluence of 60% was achieved, Huh7 cells were transfected with small interfering RNA (siRNA) for BMP6 (siBMP6) or siRNA control (siControl, siC) (siGENOME SMARTpool Human BMP6 M021475-03–0005, siGENOME™ Control Pool Non-Targeting #1 D-001206-13-20, Dharmacon™ Inc., Lafayette, CO, USA) at a final concentration of 10 nM in serum-free DMEM without antibiotics for 36 h. Quantification of BMP6 gene silencing was evaluated by quantitative real-time PCR (RT-qPCR) (Table S1) and western blot analysis.

Cell survival analysis

Cell survival was measured with crystal violet staining. Plates were washed with PBS to remove unattached death cells and covered with 0.2% crystal violet diluted in 2% ethanol for 30 min. Then, staining excess was removed by washing with distilled water and plates were left to dry. Finally, colorant was dissolved in 1% sodium dodecyl sulfate (SDS) and optical density was measured with spectrophotometer Spectra MR (29,010, Dynex Technologies, Chantilly, VA, USA) at 560 nm. Cell viability was calculated from the absorbance measurement.

Cytotoxicity assay by lactate dehydrogenase (LDH)

Cellular toxicity was evaluated by measuring lactate dehydrogenase (LDH) release to the culture media due to cellular necrosis. Assay was performed using the Cytotoxicity Detection KitPLUS LDH (0474492600, Roche Diagnostics, Mannheim, Germany) following manufacturing indications. Percentage of cytotoxicity was calculated as percentage of cytotoxicity = ((Sample O.D.–Control O.D.) / (Positive control O.D–Control O.D.)) × 100.

DAPI staining

Immortalized mouse hepatocytes and Huh7 cells were grown on glass coverslips and treated with APAP as described previously in this section. After treatment, culture media were removed and cells were washed with PBS. Afterward, cells were permeabilized and fixed with methanol for 10 min. DAPI (4ʹ,6-diamidino-2-phenylindole) staining (Thermo Fisher Scientific Inc., Madrid, Spain) was added in 1:1000 proportion for 5 min after which excess was washed with PBS. Mounting medium used was Fluoromont G® (BioNova cientifica, Madrid, Spain). Representative images were taken using an optical microscope Nikon Eclipse E400 (Nikon) equipped with a plan Apocromatic 40X objective (Nikon). Presence of apoptotic nuclei was assessed with ImageJ software (NIH).

ROS quantification in vitro by fluorescence microplate reader

Cells were seeded in a 96 multi-well plate in a density of 2 × 104 cells/plate. The probe dihydroethidium (DHE, 2,140,299, Invitrogen by Thermo Fisher Scientific Inc.) was used at a concentration of 10 μM in DMEM FluoroBrite containing High Glucose and without L-Glutamine (Thermo Fisher Scientific Inc.) to measure superoxide. After 30 min with the probe, vehicle or APAP was added and ROS production was measured in a fluorescence microplate reader (CLARIOstarPlus BMG Labtech, Germany) for 2 h using 488/550–580 excitation/emission filter pairs. DAPI staining was used to normalize number of cells (358/455–465). Each condition was run in triplicate and antimycin A (10 μM) was used as a positive control.

Gene expression analysis

Total RNA was extracted from liver tissue and cell lysate using TRIzol reagent (Vitro, Sevilla, Spain). Obtained RNA purity and concentration were measured with Nanodrop (Termofisher Nanodrop 2000c) and cDNA was obtained by reverse transcription of RNA using ImProm-II™ Reverse transcription kit (Promega Inc., Madison, WI, USA) in a T100TM Thermal Cycler (BioRad Inc., Madrid, Spain). RT-qPCR was performed with SYBR Green method using StepOnePlusTM Real-Time PCR System sequence detector (Thermo Fisher Scientific, Inc.) and quantified with ΔΔCt method. Samples were run in duplicate and normalized with endogenous gene 36b4. Primer sequences used are listed in Table S1.

Western blot analysis

Protein content from culture media was precipitated with trichloroacetic acid (TCA; 0.85 g/ml), washed with acetone (cooled at −20 ºC) and boiled in Laemmli buffer. Total protein extracts from cell lysates were obtained using RIPA buffer (50 mM Tris–HCl, pH 7.4, 1% Triton X-100, 0.2% SDS, 1 mM EDTA, 1 mM PMSF and 5 μg/ml leupeptin) and boiled in Laemmli buffer. Protein samples were separated in 10% or 15% SDS-PAGE by electrophoresis following with transferring to Immunoblot nitrocellulose membrane. After blocking with 3% BSA, membranes were incubated overnight with primary antibodies at 4 °C: anti-BMP6 (1:1000) (ab155963, Abcam plc), anti-JNK (sc-7345, Santa Cruz Biotechnology Inc., Heidelberg, Germany), anti-cleaved caspase-3 (#9661), anti-phospho-JNK (#4668), anti-phospho-P38 MAPK (#9211) and anti-P38 (#9212) (Cell Signalling Technology, Danvers, MA, USA). Finally, membranes were incubated with the corresponding secondary antibody (Santa Cruz Biotechnology Inc.). Immunoreactive bands were visualized using the ECL Western blotting protocol (Bio-Rad). Densitometric analysis of the bands was performed using Image J software (NIH). Anti-Tubulin (sc-166729, Santa Cruz Biotechnology Inc.), anti-vinculin (sc-73614, Santa Cruz Biotechnology Inc.) and Ponceau staining were used as loading control.

Statistical analysis

Quantitative variables are expressed as measures of central tendency (mean) and dispersion (standard error of mean, SEM). Data between groups were compared with Student´s t test for variables following a normal distribution and Mann–Whitney U test for continuous variables following a non-parametric distribution. All statistical analyses were performed using GraphPad Prism 6.0 (GraphPad Software Inc., San Diego, CA, USA) and IBM SPSS Statistics 21.0 (SPSS Inc., IBM, Armonk, NY, USA) software, with a p-value of < 0.05 considered as statistically significant.