Acetaminophen-induced liver damage in mice is associated with gender-specific adduction of peroxiredoxin-6

Abstract

The mechanism by which acetaminophen (APAP) causes liver damage evokes many aspects drug metabolism, oxidative chemistry, and genetic-predisposition. In this study, we leverage the relative resistance of female C57BL/6 mice to APAP-induced liver damage (AILD) compared to male C57BL/6 mice in order to identify the cause(s) of sensitivity. Furthermore, we use mice that are either heterozygous (HZ) or null (KO) for glutamate cysteine ligase modifier subunit (Gclm), in order to titrate the toxicity relative to wild-type (WT) mice. Gclm is important for efficient de novo synthesis of glutathione (GSH). APAP (300 mg/kg, ip) or saline was administered and mice were collected at 0, 0.5, 1, 2, 6, 12, and 24 h. Male mice showed marked elevation in serum alanine aminotransferase by 6 h. In contrast, female WT and HZ mice showed minimal toxicity at all time points. Female KO mice, however, showed AILD comparable to male mice. Genotype-matched male and female mice showed comparable APAP-protein adducts, with Gclm KO mice sustaining significantly greater adducts. ATP was depleted in mice showing toxicity, suggesting impaired mitochondria function. Indeed, peroxiredoxin-6, a GSH-dependent peroxiredoxin, was preferentially adducted by APAP in mitochondria of male mice but rarely adducted in female mice. These results support parallel mechanisms of toxicity where APAP adduction of peroxiredoxin-6 and sustained GSH depletion results in the collapse of mitochondria function and hepatocyte death. We conclude that adduction of peroxiredoxin-6 sensitizes male C57BL/6 mice to toxicity by acetaminophen.

Keywords: Acetaminophen; Gclm; Gender; Glutathione; Mitochondria; Peroxiredoxin-6.

Graphical abstract

Fig. 1

APAP overdose induces liver damage in male and Gclm KO mice. Mice were treated with saline or 300 mg/kg APAP. At the indicated times, liver damage was assessed by (A) measurement of serum alanine aminotransferase activity (ALT) and (B) histopathological scoring of H&E stained paraffin-embedded liver tissue (C). (A) Mean of serum ALT (+SE) at 0, 2, 6, 12, and 24 h following treatment shows sensitivity of male and Gclm KO mice to AILD (n≥4 for each point). (B) Histopathological scoring supports ALT data (n≥3, except male Gclm KO at 2 h where n=2). (C) H&E stained liver illustrates centrilobular hepatotoxcity in male and Gclm KO but minimal toxicity in female WT liver sections 6 h following treatment (bar 200 µm). Pairwise comparisons of log-transformed ALT and untransformed damage score using t-test assuming equal variance; ^#p<0.05, ^##p<0.01, ^###p<0.001, difference by Gclm genotype relative to WT within gender and time; ^⁎p<0.05, ^⁎⁎p<0.01, difference by gender within Gclm genotype and time point. Note: APAP treatment was lethal to two male Gclm KO mice, for which ALT values are not reported.

Fig. 2

Rapid, transient GSH depletion occurs in male and female mice. Total GSH was measured in cytosol (A, B) and mitochondria (C) fractions. APAP overdose induced rapid but transient depletion of hepatic cytosolic and mitochondrial GSH in all mice (significance not indicated). Cytosolic depletion was greater in male Gclm WT and HZ mice with slower recovery compared to female mice. Gclm KO mice showed significantly less GSH in both cytosol and mitochondria fractions. Points and bars represent mean with SE on bar graphs. ^#p<0.05, ^##p<0.01, ^###p<0.001 difference by Gclm genotype; ^⁎p<0.05, ^⁎⁎p<0.01, ^⁎⁎⁎p<0.001 difference by gender.

Fig. 3

APAP overdose impairs mitochondria function in male mice. Cytosolic ATP (A) measured at 0, 1, and 2 h illustrates significant loss of ATP in male mice (^⁎p<0.05, ^⁎⁎p<0.01, relative to t=0). Mitochondrial membrane potential (ΔΨ) (B), quantified by JC-1 uptake, measured in fresh liver homogenate isolated from female and male Gclm HZ mice 2 h following treatment shows lower basal and less inducible (+PMN) ΔΨ in male mice (n=3 each). (C) Area under the curve (AUC) relative to APAP-treated male. Mean (+/−SE) ^⁎p<0.05, difference by gender; ^##p<0.01, difference by substrate. Follow-up analysis in male C57BL/6J mice analyzed 6 h following 300 mg/kg APAP reproduces these effects (D–G) (n=5, each group). ^⁎p<0.05, relative to saline by paired t-test, except for ATP analyzed by nonparametric Wilcoxon rank-sum.

Fig. 4

Peroxiredoxin-6 adducted in male mitochondria. Cytosolic (A) and mitochondrial (B) APAP-protein adducts were isolated from female and male Gclm WT, HZ, and KO mice treated at 1 h following treatment. All mice show significant protein adduction in cytosolic and mitochondria fractions. Gclm KO mice generally show greater adducts. A protein of ~26-kDa was dichotomously adducted in mitochondria isolated from male mice. Mass spectral analysis of the 26-kDa band identified a few candidate proteins (Table 1). (C) Serial Western blot analysis of mitochondria isolated 2 h following treatment demonstrated that peroxiredoxin-6 (Prdx6) (bottom panel of C, blue-shaded open arrowhead) is the mitochondrial protein that is preferentially adducted in male mice. Cytosolic (25 µg) and mitochondrial (25 µg) proteins were resolved on 12% polyacrylamide gels under denaturing and reducing and probed as indicated. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

Prdx6 protein is extremely sensitive to adduction. Dose-response in male Gclm HZ mice illustrated the sensitivity of mitochondrial Prdx6 to adduction by APAP (A, B). The degree of Prdx6 adduction is diminished at 6 h following treatment with 150 mg/kg APAP (A), which does not induce sustained GSH depletion or elevated ALT (B). Furthermore, N-acetyl-cysteine (NAC) administration at indicated times following 300 mg/kg APAP reduces Prdx6 adduction, total protein arylation (C) and mitigates toxicity (D). Mean (SE); for dose response n≥4, for NAC rescue n≥3 except for A+NAC (0 h) where n=2. Pairwise comparisons of log-transformed ALT and untransformed GSH using t-test; ^⁎p<0.05, ^⁎⁎p<0.01, ^⁎⁎⁎p<0.001, ^⁎⁎⁎⁎p<0.0001 relative to saline control.

Fig. 6

Gender-specific metabolism of APAP. Previously frozen liver was analyzed for metabolites of APAP by HPLC-ECD: (A) APAP+APAP–SO₄, (B) APAP–glutathione (APAP–SG), (C) APAP–glucuronide (APAP–Gluc), (D) APAP–cysteine (APAP–Cys), (E) APAP–(OH)–SG, and (F) APAP–Cys adducts. Overall, male and female mice metabolize APAP distinctly, with differences in most major metabolites. No difference in absolute APAP–Cys adducts was observed between genders. Geometric mean values (pmol/mg liver) are indicated (n≥4). ANOVA performed on log-transformed data was used to determine differences within a metabolite, followed by pairwise comparison using t-test assuming equal variance. ^⁎p<0.05, ^⁎⁎p<0.01, ^⁎⁎⁎p<0.001, difference by gender within Gclm genotype and time.

Fig. 7

Rapid formation of APAP–Cys metabolite associated with protection against AILD. Comparison of the AUC from t=0.5 h to 2 h of APAP metabolites shows a relative bias of female WT and HZ mice to convert APAP to APAP–Cys. Although a lesser metabolite, appreciable formation of APAP–(OH)–SG is only occurs in male WT and HZ mice. This data also illustrates roughly gender equivalent net ADME despite differences in metabolite formation. Stacked bars represent the sum of geometric mean integrated from t=0.5 h to t=2 h.