Lysosomal instability and cathepsin B release during acetaminophen hepatotoxicity

Abstract

Acetaminophen (APAP) overdose is currently the most frequent cause of drug-induced liver failure in the United States. Recently, it was shown that lysosomal iron translocates to mitochondria where it contributes to the collapse of the mitochondrial membrane potential. Therefore, the purpose of this study was to investigate whether cathepsin B, a lysosomal protease, is involved in APAP-induced hepatotoxicity. Cathepsin B activity was measured in subcellular liver fractions of C57Bl/6 mice 3 hr after 300 mg/kg APAP treatment. There was a significant increase in cytoplasmic cathepsin activity, concurrent with a decrease in microsomal activity, indicative of lysosomal cathepsin B release. To investigate the effect of cathepsin B on hepatotoxicity, the cathepsin inhibitor AC-LVK-CHO was given 1 hr prior to 300 mg/kg APAP treatment along with vehicle control. There was no difference between groups in serum alanine aminotransferase (ALT) values, or by histological evaluation of necrosis, although cathepsin B activity was inhibited by 70-80% compared with controls. These findings were confirmed with a different inhibitor (z-FA-fmk) in vivo and in vitro. Hepatocytes were exposed to 5 mM acetaminophen. Lysotracker staining confirmed lysosomal instability and cathepsin B release, but there was no reduction in cell death after treatment with cathepsin B inhibitors. Finally, cathepsin B release was measured in clinical samples from patients with APAP-induced liver injury. Low levels of cathepsin B were released into plasma from overdose patients. APAP overdose causes lysosomal instability and release of cathepsin B into the cytosol but does not contribute to liver injury under these conditions.

Figure 1

Plasma alanine aminotransferase (ALT) activity (A), intracellular cathepsin B activity (B) and plasma cathepsin B activity (C) were measured in untreated control animals and 3 hr after 300 mg/kg acetaminophen (APAP). Cathepsin B activities (100%) were 440±48 RFU/mg protein/min (microsomal fraction), 138±11 RFU/mg/min (cytosol) and 1.4±1.0 RFU/mg/min (plasma). Data represent means ± SE of n = 4 animals per group. *P<0.05 (compared to controls, C)

Figure 2

Hepatic cathepsin B activities (A) and plasma alanine aminotransferase (ALT) activities (B) were measured after 12-hr treatment with 300 mg/kg APAP. Mice were pre-treated for 1 hr with saline (vehicle) or with the cathepsin B inhibitor AC-LVK-CHO (5 mg/kg). All data represent means ± SE of n=4 animals per group. *P<0.05 (compared to controls). (C) Representative images of H&E and TUNEL staining in each group. All images: 50x

Figure 3

(A) Total glutathione (GSH+GSSG) and (B) glutathione disulfide (GSSG) were measured 30 min. or 12 hr after treatment with 300 mg/kg APAP or in untreated controls. The GSSG-to-GSH ratio (C) for each animal was calculated. Mice were pre-treated for 1 hr with saline (vehicle) or with the cathepsin B inhibitor AC-LVK-CHO (5 mg/kg). Data represent means ± SE of n = 4 animals per group. *P<0.05 (compared to controls, Ctrl).

Figure 4

Hepatic cathepsin B activities (A) and plasma alanine aminotransferase (ALT) activities (A) were measured after 12-hr treatment with 600mg/kg APAP. Mice were treated with saline, Saline/DMSO vehicle or 10 mg/kg z-FA-FMK, 2 hr after APAP administration. *P<0.05 (compared to control). ^#P<0.05 (compared to saline or DMSO). (C) Representative images of H&E and TUNEL staining in each group. All images: 50x

Figure 5

Quantification of TUNEL staining (A) of galactosamine/endotoxin (Gal/ET) treated mice injected with either vehicle or 10mg/kg z-FA-FMK. Activities of hepatic caspase-3 (B), plasma ALT, (C) and hepatic cathepsin B (D) were also measured at 6 hr after Gal/ET treatment. Cathepsin B activities were 321±64 RFU/mg protein/min (vehicle) and 119±21 RFU/mg/min (z-FA-FMK). Representative images of TUNEL-stained slides are shown (E) (x100). TUNEL stain was only considered positive for specifically stained nuclei.

Figure 6

(A) Lysotracker fluorescence in control hepatocytes and in cells 6 hr after treatment with 5 mM acetaminophen (APAP). Primary mouse hepatocytes were isolated and treated with APAP; cells were loaded with Lysotracker after 6 hr and subjected to live cell imaging. (B) Primary mouse hepatocytes were either untreated or exposed to 5 mM APAP for 9 hr. Some cells were pre-treated for 30 min. with DMSO/cell culture medium (0.1% DMSO per well) or 50 μM of the cathepsin B inhibitors z-FA-FMK or CA-074Me. Cell viability was assessed by LDH release. Data represent means ± SE of n = 4 independent experiments. *P<0.05 (compared to untreated controls, C)