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

•Female mice are resistant to acetaminophen-induced liver damage (AILD).•Male mice display mitochondrial dysfunction and pronounced AILD.•Peroxiredoxin-6 (Prdx6) is preferentially adducted by acetaminophen in male mice.•Gclm deficiency renders female mice sensitive to AILD, yet Prdx6 is not adducted.•Prdx6 adduction exacerbates oxidative stress caused by acetaminophen overdose.

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.

Graphical abstractAcetaminophen (APAP) is primarily metabolized and excreted as APAP–glucuronide (APAP–Gluc) and APAP–SO4 in hepatocytes. Under conditions of overdose, greater amounts of APAP are bioactivated, forming a reactive quinonimine (NAPQI). Glutathione (GSH) can reduce NAPQI, recycling to APAP, or conjugate NAPQI forming APAP–glutathione (APAP–SG), which is excreted from the hepatocyte. Depletion of GSH permits protein thiol arylation by NAPQI that may compromise protein function and contribute to hepatocellular death. Male and female C56BL/6 mice show similar formation of APAP adducts, yet male mice are more sensitive to APAP toxicity. In this study, we show that peroxiredoxin-6 (Prdx6), a 1-Cys peroxiredoxin that is dependent upon GSH for activity, is preferentially and sensitively adducted in male mice. Furthermore, this adduct is localized to liver fractions enriched for mitochondria. This, combined with greater intrahepatic accumulation of APAP–SG in male mice, points to parallel metabolic and molecular mechanisms of APAP induced liver damage and suggests a novel role for Prdx6 in mitochondrial function.Figure optionsDownload full-size imageDownload as PowerPoint slide