Hepatitis C virus structural proteins can exacerbate or ameliorate acetaminophen-induced liver injury in mice

Abstract

Chronic hepatitis C virus (HCV) infection predisposes patients to develop liver failure after acetaminophen (APAP) overdose. Mechanisms involved in this were explored using transgenic mice expressing the HCV structural proteins core, E1 and E2. Treatment of C57BL/6J mice with 200 mg/kg body weight APAP resulted in significant liver injury at 6 h as indicated by elevated ALT levels, focal centrilobular necrosis and nuclear DNA fragmentation. HCV transgenic mice showed a variable response, with approximately half the animals showing exacerbation of all parameters of liver injury, while the other half was protected. HCV transgenic mice with higher liver injury had lower liver glutathione levels, elevated mitochondrial oxidative stress and enhanced release of apoptosis-inducing factor (AIF) from the mitochondria. This was accompanied by induction of a higher ER stress response and induction of autophagy. Transgenic animals showing protection against liver injury had a robust recovery of liver glutathione content at 6 h when compared to wild-type animals, accompanied by reduction in mitochondrial oxidative stress and AIF release. This was accompanied by an elevation in glutathione S-transferase mRNA levels and activity, which suggests that an efficient clearance of the reactive intermediate may contribute to the protection against APAP hepatotoxicity in these mice. These results demonstrate that while HCV infection could exacerbate APAP-induced liver injury due to induction and amplification of mitochondrial oxidant stress, it could also protect against injury by activation of APAP scavenging mechanisms.

Figure 1

(A) ALT levels in plasma from individual wild type (WT) and HCV transgenic mice 6 hours after APAP treatment (200mg/kg) (n=3 for Control and APAP WT and 12 for HCV core protein transgenic mice). (B) ALT values of individual HCV transgenic mice were plotted against their respective liver GSH content. (C) ALT data represented as mean ± SE for wild type and HCV transgenic mice showing protection (p) and exacerbation (e) of liver injury after APAP overdose. *P<0.05 (compared to WT Control; ^#P<0.05 (compared to WT APAP). (D) Mean of ALT values are plotted against mean ± SE of GSH values of the 4 different groups.

Figure 1

(A) ALT levels in plasma from individual wild type (WT) and HCV transgenic mice 6 hours after APAP treatment (200mg/kg) (n=3 for Control and APAP WT and 12 for HCV core protein transgenic mice). (B) ALT values of individual HCV transgenic mice were plotted against their respective liver GSH content. (C) ALT data represented as mean ± SE for wild type and HCV transgenic mice showing protection (p) and exacerbation (e) of liver injury after APAP overdose. *P<0.05 (compared to WT Control; ^#P<0.05 (compared to WT APAP). (D) Mean of ALT values are plotted against mean ± SE of GSH values of the 4 different groups.

Figure 2

Representative liver sections stained with H&E (A) and the TUNEL assay (B) in wild type controls and HCV transgenic mice 6 hours after treatment with APAP (200 mg/kg). HCV transgenic mice with exacerbated injury (HCV+E) or protection (HCV+P) are indicated.

Figure 3

(A) Cyp 2E1 protein levels in wild type and HCV transgenic mice 6 hours after APAP treatment (200 mg/kg). The lower histogram illustrates densitometric data from western blots. Values are mean ± SE of ≥3 mice per group. (B) Liver glutathione content in wild type and HCV transgenic mice 20 minutes after APAP treatment (200mg/kg). Values are mean ± SE of at least 3 mice per group. *P<0.05 (compared to WT Control; ^#P<0.05 (compared to WT APAP).

Figure 4

(A) Liver glutathione content in wild type and HCV transgenic mice 6 hours after APAP treatment (200 mg/kg). Values are mean ± SE of 3-5 mice per group. (B) Representative redox western blot for mitochondrial thioredoxin 2, 6 hours after APAP treatment, and its densitometric quantitation. Values are mean ± SE of 3-5 mice per group. (C) Western blot for apoptosis-inducing factor (AIF) in the cytosol 6 hours after APAP treatment in wild type control and HCV transgenic mice. *P<0.05 (compared to WT Control; ^#P<0.05 (compared to WT APAP).

Figure 5

(A) Quantitation of Bcl-x_L mRNA and a representative western blot (insert) of Bcl-x_L protein in wild type controls and HCV transgenic mice, 6 hours after APAP (200 mg/kg). Values are mean ± SE of ≥ 3 mice per group. (B) A representative western blot and densitometric quantitation of GADD153/CHOP protein in wild type and HCV transgenic mice 6 hours after APAP (200 mg/kg). Values are mean ± SE of ≥3 mice per group. *P<0.05 (compared to control), ^#P<0.05 (compared to APAP-WT)

Figure 6

(A) A representative western blot and densitometric quantitation of Grp78 protein in wild type and HCV transgenic mice 6 hours after APAP (200 mg/kg). Values are mean ± SE of ≥3 mice per group. *P<0.05 (compared to control). (B) Quantitation of LC3-I and LC3-II protein in liver from wild type and HCV transgenic mice 6 hours after APAP, with a representative western blot (insert). Values are mean ± SE of ≥3 mice per group. *P<0.05 (compared to control), ^#P<0.05 when compared to APAP-WT)

Figure 7

(A) Quantitation of metallothionein protein in liver from wild type and HCV transgenic mice 6 hours after APAP (200 mg/kg), with a representative western blot (insert). Values are mean ± SE of ≥3 mice per group. *P<0.05 (compared to control), ^#P<0.05 (compared to APAP-WT). (B) Quantitation of GST mu3 mRNA levels in liver from wild type and HCV transgenic mice 6 hours after APAP (200 mg/kg). Values are mean ± SE of at least 3 mice per group. (C) Glutathione S Transferase activity in liver homogenates from wild type and HCV transgenic mice 6 hours after APAP. Values are mean ± SE of ≥3 mice per group. *P<0.05 (compared to APAP control)