Lysosomal Cholesterol Accumulation Sensitizes To Acetaminophen Hepatotoxicity by Impairing Mitophagy

The role of lysosomes in acetaminophen (APAP) hepatotoxicity is poorly understood. Here, we investigated the impact of genetic and drug-induced lysosomal cholesterol (LC) accumulation in APAP hepatotoxicity. Acid sphingomyelinase (ASMase)−/− mice exhibit LC accumulation and higher mortality after APAP overdose compared to ASMase+/+ littermates. ASMase−/− hepatocytes display lower threshold for APAP-induced cell death and defective fusion of mitochondria-containing autophagosomes with lysosomes, which decreased mitochondrial quality control. LC accumulation in ASMase+/+ hepatocytes caused by U18666A reproduces the susceptibility of ASMase−/− hepatocytes to APAP and the impairment in the formation of mitochondria-containing autolysosomes. LC extraction by 25-hydroxycholesterol increased APAP-mediated mitophagy and protected ASMase−/− mice and hepatocytes against APAP hepatotoxicity, effects that were reversed by chloroquine to disrupt autophagy. The regulation of LC by U18666A or 25-hydroxycholesterol did not affect total cellular sphingomyelin content or its lysosomal distribution. Of relevance, amitriptyline-induced ASMase inhibition in human hepatocytes caused LC accumulation, impaired mitophagy and increased susceptibility to APAP. Similar results were observed upon glucocerebrosidase inhibition by conduritol β-epoxide, a cellular model of Gaucher disease. These findings indicate that LC accumulation determines susceptibility to APAP hepatotoxicity by modulating mitophagy, and imply that genetic or drug-mediated ASMase disruption sensitizes to APAP-induced liver injury.


Biochemical measurements
To determine liver injury, serum ALT and AST were determined by standard procedures of the Clinical Chemistry laboratory of Hospital Clinic de Barcelona. GSH levels in homogenates, mitochondria or cytosols were determined by the recycling method.

Hematoxylin and Eosin and TUNEL staining
For hematoxylin and eosin staining, fresh liver samples were collected and fixed in formalin for 48h. Fixed livers were embedded in paraffin and placed in blocks and 7 μm slices were cut on a microtome. For TUNEL staining, paraffin-embedded liver tissue was cut into slices of 4 μm in thickness and fixed to glass slides. The TUNEL assay was performed by incubation of the slices with 2 μg/ml proteinase K (Sigma-Aldrich) in 10 mM Tris-HCl buffer containing 5 mM EDTA at a pH of 7.4. Sections were then labeled following the instructions of a commercial Kit (In situ Cell Death Detection Kit; Roche Diagnostics), stained with 3,3′-diaminobenzidine (Roche Diagnostics), and counterstained with hematoxylin and eosin.

Immunohistochemical Staining
Paraffin molds containing liver sections were cut into 5-μm sections and mounted on HistoGrip-coated slides. The sections were deparaffinized in xylene and dehydrated in a graded alcohol series. When necessary, endogenous peroxidase (3% H 2 O 2 ) and endogenous avidin and biotin were blocked. Slides were incubated with primary antibody (PCNA, Santa Cruz or Ki67, AbCam) overnight in a wet chamber at 4°C.
After a rinse with 1× PBS, the slides were incubated with a biotinylated antibody for 45 minutes in a wet chamber and developed with an ABC Kit with peroxidase substrate Baulies et al,3 (diaminobenzidine) and peroxidase buffer. After the slides were rinsed with tap water, they were counterstained with hematoxylin and mounted with Aquatex.

Mitochondria preparation
Livers were excised and homogenized in medium A (225 mM Mannitol; 75 mM Sucrose; 0,1 mM EGTA; 1 mg/ml FFA-BSA and 10 mM Hepes-KOH, pH 7.4). The homogenate was centrifuged at 700 g, 15 min at 4 ˚C, the pellet removed, and the supernatant centrifuged at 10.000 g, 15 min at 4˚C. After this centrifugation, the supernatant was discarded and the pellet containing the mitochondrial pellet was resuspended in buffer B (395 mM Sucrose; 0,1 mM EGTA and 10 mM Hepes-KOH, pH 7.4). The resuspended pellet was added to the top of a Percoll gradient (3 ml of 60% Percoll (v/v) and 7 ml of 26% Percoll (v/v) with buffer B) and was ultracentrifuged at 15.000 rpm, 35 min at 4˚C. Mitochondria was collected from the middle of the two Percoll layers and transferred to another tube where was resuspended with buffer B and centrifuged at 10.000 g, 15 min at 4˚C, to remove excess Percoll. Supernatant was discarded and the pellet containing mitochondria was resuspended with 350uL of Buffer A.

Immunofluorescence
Freshly isolated primary hepatocytes were plated on collagen-coated coverslips on 12 well plates at a 125000 cells per well. After the different treatments, cells were fixed with 4% paraformaldehyde (PFA) at room temperature and washed twice with PBS before proceeding with next steps. Then, cells were blocked and permeabilized for 30 min at room temperature with blocking buffer (PBS-0.5% glycine; 1% FFA-BSA; 0.025% saponinin) and incubated with Lamp2 (AbCam) primary antibody overnight.
Cells were washed twice and then incubated with the secondary antibody (anti-rat IgG Baulies et al,4 antibody conjugated with Alexa Fluor 488; Molecular Probes) 1h at room temperature.

Western blotting
To analyze liver samples by western blotting, 100mg of liver were homogenized in pestle homogenizer with 1ml of homobuffer supplemented with an antiprotease cocktail (Complete, Roche). Liver homogenates were diluted 4 times in RIPA lysis buffer supplemented with antiproteases and antiphosphatases (PhosSTOP, Roche). Samples were incubated 10 min at 4ºC, vortexed and spin down 5 min at 10.000 rpm. All supernatants were collected and quantified for protein concentration using Bradford reagent (Bio-Rad). From 20 to 80μg of liver lysates was run in polyacrylamide gels. To analyze the mitochondrial fraction, isolated mitochondria resuspended in buffer A were diluted 4 times in RIPA lysis buffer. For the analysis of cultured cells by western blotting, media was removed and cells were washed twice with PBS and scrapped with RIPA lysis buffer supplemented with antiprotease and antiphophatase cocktail at 4ºC. Samples were incubated 10 min at 4ºC, vortexed and spin down 5 min at 10.000 rpm. 5% BSA solution in TBST was used to block the nitrocellulose blotting membranes (Amersham). Membranes were incubated with primary antibodies against P-JNK (Cell Signaling), JNK (Cell Signaling), CYP2E1 (AbCam), C-32 (Invitrogen), PINK1 (AbCam), Parkin (AbCam), VDAC (Millipore), Hsp60 (AbCam), TOM20 (Invitrogen) or β-actin (Sigma). NAPQI protein adducts antiserum was provided by Drs. Hinson and James from the University of Arkansas. Membranes were incubated for 45min with HRP-conjugated secondary antibodies and developed with Pierce ECL Western Blotting Substrate (Thermo Scientific). Images were collected with the image-capturing instrument, LAS4000 (GE Healthcare) and analyzed with Image J free software.

RNA and DNA isolation
Total RNA or DNA from mouse and primary mouse hepatocytes were isolated with TRIzol® Reagent (Invitrogen). After homogenizing the sample with TRIzol®, chloroform was added, and the homogenate was allowed to separate into a clear upper aqueous layer (containing RNA), an interphase, and a red lower organic layer (containing the DNA and proteins). RNA was precipitated from the aqueous layer with isopropanol. Isopropanol was removed and the RNA pellet was air-dryed for 10min.
RNA pellet was resuspended in RNase-free water and incubated at least 30min at 4ºC to allow complete resuspension. RNA was quantified and purity was checked measuring the sample absorbance at 260nm and 280nm with the NanoDrop spectrophotometer (260/280 ratio between 1.7-2.0). To isolate DNA, any remaining aqueous phase from the red organic layer was removed carefully to ensure the quality of the isolated DNA.
Absolute ethanol was added per 1 mL TRIzol® used for the initial homogenization and tubes were inverted for mixing and incubated at room temperature during 3 minutes.
Samples were centrifuged at 2000 x g for 5 minutes and the supernatant was removed to proceed with the washes. 1 mL of sodium citrate/ ethanol solution (0,1M sodium citrate in 10% ethanol, pH 8,5) was added to the pellet per 1 mL of TRIzol® used for the initial homogenization. Samples were incubated 30 min at room temperature and the wash out step was repeated twice. 75% ethanol was added in the last purification step and after a 20 min incubation, pellets were air dried and resuspended with buffer AE (10 mM Tris-HCl, 0,5 mM EDTA, pH 9). DNA was quantified and purity was checked Baulies et al,6 measuring the sample absorbance at 260nm and 280nm with the NanoDrop spectrophotometer (260/280 ratio between 1.6-1.8).      Eight-week-old mice C57BL6 wild type (ASMase +/+ ) and age-matched ASMase knockout mice (ASMase -/-) were subcutaneously injected with a 30 mg/kg dose of 25-HC during 5 days. The last day, mice were intraperitoneally injected with either APAP (300mg/Kg body weight) or equal volume of saline as vehicle control. (a,c) Representative images of H&E staining of ASMase +/+ liver sections harvested 6 hours after the APAP injection. (b,d) Serum ALT levels measured 6 hours after APAP injection. Data are expressed as mean  SEM; n=5-9 mice per group. *p0,05 vs vehicle-treated group; #p0,05 vs APAP-treated group. Scale bar represents 100 μm.