Effect of ferroptosis inhibitors in a murine model of acetaminophen-induced liver injury

Abstract

Liver injury caused by acetaminophen (APAP) overdose is the leading cause of acute liver failure in western countries. The mode of APAP-induced cell death has been controversially discussed with ferroptosis emerging as a more recent hypothesis. Ferroptosis is characterized by ferrous iron-catalyzed lipid peroxidation (LPO) causing cell death, which can be prevented by the lipophilic antioxidants ferrostatin-1 and UAMC-3203. To assess the efficacy of these ferroptosis inhibitors, we used two murine models of APAP hepatotoxicity, APAP overdose alone or in combination with FeSO₄ in fasted male C57BL/6J mice. APAP triggered severe liver injury in the absence of LPO measured as hepatic malondialdehyde (MDA) levels. In contrast, ferrous iron co-treatment aggravated APAP-induced liver injury and caused extensive LPO. Standard doses of ferrostatin-1 did not affect MDA levels or the injury in both models. In contrast, UAMC-3203 partially protected in both models and reduced LPO in the presence of ferrous iron. However, UAMC-3203 attenuated the translocation of phospho-JNK through downregulation of the mitochondrial anchor protein Sab resulting in reduced mitochondrial dysfunction and liver injury. Thus, APAP toxicity does not involve ferroptosis under normal conditions. The lack of effects of ferroptosis inhibitors in the pathophysiology indicates that ferroptosis signaling pathways are not relevant therapeutic targets.

Keywords: UAMC‐3203; acetaminophen hepatotoxicity; ferroptosis; ferrostatin‐1; lipid peroxidation.

Figure 1:. Structures of the ferroptosis inhibitor Ferrostatin-1 and UAMC-3203.

Figure 2:. Ferrostatin-1 does not reduce APAP induced liver injury in mice at 6 h after APAP.

Overnight fasted mice were treated with 10 mg/kg ferrostatin-1 or vehicle (50% PEG-400 + 5% Tween-80) 1 h before 200 mg/kg APAP. (A) Plasma alanine amino transferase (ALT) activities, (B) necrotic area quantification, (C) representative H&E images (200X, scale bar=100 μm) (Marked areas are enlarged in adjacent panels) (D). Hepatic malondialdehyde (MDA) concentration and (E) relative hepatic mRNA expression of Ptgs2 normalized to 18s mRNA at 6 h after APAP overdose. Bars represent mean ± SEM for n=5 mice. *p<0.05 compared to untreated controls.

Figure 3:. Ferrostatin-1 does not mitigate injury or expression of ferroptosis markers in an iron-induced lipid peroxidation model.

Overnight fasted mice were treated with 10 mg/kg ferrostatin-1 or vehicle (50% PEG-400 + 5% Tween-80) 1 h before co-treatment of APAP 300 mg/kg and 0.15 mmol/kg FeSO₄. (A) Plasma alanine amino transferase (ALT) activities, (B) necrotic area quantification, (C) representative H&E images (100X, scale bar=100 μm) (Marked areas are enlarged in adjacent panels) (D). Hepatic malondialdehyde (MDA) concentration and (E) relative hepatic mRNA expression of Ptgs2 normalized to 18s mRNA at 6 h after APAP and FeSO4. Bars represent mean ± SEM for n=4 mice. #p<0.05 compared to untreated controls, *p<0.05 compared to APAP + Vehicle.

Figure 4:. UAMC-3203 reduces APAP-induced liver injury 6 h after APAP.

Overnight fasted mice were treated with 9.5 mg/kg UAMC-3203 or vehicle (2% DMSO) 1 h before 500 mg/kg APAP. (A) Plasma alanine amino transferase (ALT) activities, (B) necrotic area quantification, (C) representative H&E images (100X, scale bar=100 μm) (Marked areas are enlarged in adjacent panels) (D). Hepatic malondialdehyde (MDA) concentration and (E) relative hepatic mRNA expression of Ptgs2 normalized to 18s mRNA at 6 h after APAP overdose. Bars represent mean ± SEM for n=5 mice. #p<0.05 compared to APAP, *p<0.05 compared to APAP + Vehicle.

Figure 5:. Effect of UAMC-3203 on early events of APAP toxicity.

Overnight fasted mice were treated with 9.5 mg/kg UAMC-3203 or vehicle (2% DMSO) 1 h before 500 mg/kg APAP. (A) Hepatic APAP-Cys adduct levels and (B) Total hepatic glutathione content at 2 h after APAP (C) Immunoblot showing cytosolic and mitochondrial pJNK levels at 6 h after APAP overdose. The shown blots were quantified, i.e., the bars represent means ± SEM of the samples for n=3 or 4 mice. *p<0.05 compared to untreated controls, #p<0.05 compared to APAP.

Figure 6:. Effect of UAMC-3203 on mitochondrial Sab levels at 2 and 6 h after APAP.

Overnight fasted mice were treated with 9.5 mg/kg UAMC-3203 or vehicle (2% DMSO) 1 h before 500 mg/kg APAP. Western blot and quantification showing (A) mitochondrial Sab expression at 6 h after APAP (B) mitochondrial pJNK levels and (C) mitochondrial Sab expression at 2 h after APAP. The shown blots were quantified, i.e., the bars represent means ± SEM of the samples for n=3 or 4 mice. *p<0.05 compared to APAP, #p<0.05 compared to APAP + Vehicle.

Figure 7:. UAMC-3203 mitigates injury and ferroptosis markers in an iron-induced lipid peroxidation model.

Overnight fasted mice were treated with 9.5 mg/kg UAMC-3203 or vehicle (2% DSMO) 1 h before co-treatment of APAP 300 mg/kg and 0.15 mmol/kg FeSO₄. (A) Plasma alanine amino transferase (ALT) activities, (B) necrotic area quantification, (C) representative H&E images (100X, scale bar=100 μm) (Marked areas are enlarged in adjacent panels) (D). Hepatic malondialdehyde (MDA) levels and (E) relative hepatic mRNA expression of Ptgs2 normalized to 18s mRNA at 6 hours after APAP and FeSO4. Bars represent mean ± SEM for n=4 mice. &p<0.05 compared to untreated controls, *p<0.05 compared to APAP + Vehicle, #p<0.05 compared to APAP + Fe + Vehicle.

Figure 8:. UAMC-3203 inhibits mitochondrial permeability transition and mitochondrial Sab expression in an iron-induced lipid peroxidation model.

Overnight fasted mice were treated with 9.5 mg/kg UAMC-3203 or vehicle (2% DMSO) 1 h before 300 mg/kg APAP and 0.15 mmol/kg FeSO₄. Western blot and quantification showing (A) cytosolic and mitochondrial pJNK levels (B) mitochondrial Sab (C) cytosolic AIF and cytochrome c levels at 6 h after APAP and FeSO₄. The shown blots were quantified, i.e., the bars represent means ± SEM of the samples for n=3 or 4 mice. *p<0.05 compared to APAP + Vehicle, #p<0.05 compared to APAP + Fe + Vehicle.