2). At a lower dose, in which both groups of mice survive,

ALT levels were significantly higher in CD1d−/− mice, compared to WT mice, at 24 and 48 hours post-APAP challenge (230 mg/kg; Fig. 1D). The mechanism of AILI involves APAP biotransformation into NAPQI, which depletes GSH in the liver. Upon GSH depletion, NAPQI binds to hepatocellular proteins, forming APAP protein Anti-infection Compound Library solubility dmso adducts.16 To assess whether differential amounts of APAP protein adducts are formed in WT and CD1d−/− mice after APAP challenge, female WT and CD1d−/− mice were treated for 2 hours with APAP (350 mg/kg). Levels of hepatic protein adducts were significantly increased in CD1d−/− mice, compared to WT mice (Fig. 2A,C). Studies have shown that adduct formation in the mitochondria is essential in APAP toxicity, because this leads to induction of mitochondrial ROS formation and mitochondrial permeability transition.17 Therefore, mitochondria were isolated after 2-hour APAP challenge to measure APAP protein adducts. Levels of mitochondrial protein adducts were significantly higher in CD1d−/− than WT mice (Fig. 2B,D). Mitochondrial ROS induction has been

demonstrated after APAP challenge.17 In agreement with these findings, we observed a significant GPCR & G Protein inhibitor increase in mitochondrial superoxide after 1-hour APAP challenge in WT mice. Importantly, CD1d−/− mice exhibited significantly higher superoxide levels in mitochondria, compared to WT mice (Fig. 3A). Interestingly, we also observed a significant increase in superoxide levels after 16-hour starvation of CD1d−/− mice, but not in WT mice (Fig. this website 3A). Associated with the increase in mitochondrial ROS, there was a significant decrease in mitochondrial membrane potential (MMP) in CD1d−/− mice, compared to WT mice, after starvation as well as 1 and 2 hours after APAP challenge (Fig. 3B). These data indicate that CD1d−/− mice are uniquely

susceptible to mitochondrial oxidative stress and dysfunction after starvation and APAP challenge. Covalent binding of NAPQI by GSH represents an important defense mechanism against APAP toxicity. To assess whether GSH levels were innately different between WT and CD1d−/− mice, liver GSH levels in naïve mice were measured, and data showed similar levels in WT and CD1d−/− mice. Starvation of mice for 16 hours caused a similar reduction in GSH levels (approximately 50%) in WT and CD1d−/− mice (Fig. 4). After APAP challenge, GSH levels in WT and CD1d−/− mice decreased to the lowest level at 2 hours and began to rebound at 8 and 19 hours. GSH levels were lower in CD1d−/− than WT mice at 8 hours post-APAP challenge, perhaps the result of enhanced hepatotoxicity in these mice. CYP2E1 is the major metabolizing enzyme in the biotransformation of APAP into NAPQI.3 Therefore, we compared expression levels of CYP2E1 in WT and CD1d−/− mice. No difference in CYP2E1 protein levels between naïve WT and CD1d−/− mice was observed.