Is nuclear factor erythroid 2-related factor 2 responsible for sex differences in susceptibility to acetaminophen-induced hepatotoxicity in mice?

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that positively regulates the expression and activity of cytoprotective genes during periods of oxidative stress. It has previously been shown that some Nrf2 genes are more highly expressed in livers of female than male mice. This could explain previously reported sex-related differences in susceptibility to acetaminophen (APAP) hepatotoxicity in mice, where females show greater resistance to APAP hepatotoxicity. Here, we examined, for the first time, differences in mRNA and protein expression for Nrf2 and a battery of Nrf2-dependent genes in naïve wild-type (WT) and overnight-fasted WT and Nrf2-null male and female mice following APAP treatment. Alanine aminotransferase (ALT) activity was measured as an indicator of hepatotoxicity. Hepatic mRNA and protein levels were measured by quantitative polymerase chain reaction and western blotting, respectively. Contrary to expectations, basal Nrf2 mRNA and protein expression were significantly lower in livers of naïve female than male mice. Although mRNA and/or protein expression of quinone oxidoreductase 1 and multidrug resistance-associated protein 4 was more pronounced in livers of female than male mice under some of the conditions examined, no higher global expression of Nrf2-dependent genes was detected in female mice. Furthermore, ALT activity was significantly elevated in overnight-fasted WT and Nrf2-null male mice following APAP treatment, but no increases in ALT were observed in either genotype of female mice. These results indicate that factors other than Nrf2 are responsible for the lower susceptibility of female mice to APAP hepatotoxicity.

Green tea extract provides extensive Nrf2-independent protection against lipid accumulation and NFκB pro- inflammatory responses during nonalcoholic steatohepatitis in mice fed a high-fat diet.

SCOPE: Green tea extract (GTE) reduces liver steatosis and inflammation during nonalcoholic steatohepatitis (NASH). We hypothesized GTE would mitigate NASH in a nuclear factor erythroid-2-related-factor-2 (Nrf2)-dependent manner in a high fat (HF) induced model. METHODS AND RESULTS: Nrf2-null and wild-type (WT) mice were fed an HF diet containing 0 or 2% GTE for eight weeks prior to assessing parameters of NASH. Compared to WT mice, Nrf2-null mice had increased serum alanine aminotransferase, hepatic triglyceride, expression of free fatty acid uptake and lipogenic genes, malondialdehyde and NFκB phosphorylation and expression of pro-inflammatory genes. In WT mice, GTE increased Nrf2 and NADPH:quinone oxidoreductase-1 mRNA, and lowered hepatic steatosis, lipid uptake and lipogenic gene expression, malondialdehyde, and NFκB-dependent inflammation. In Nrf2-null mice, GTE lowered NFκB phosphorylation and TNF-α and MCP1 mRNA to levels observed in WT mice fed GTE whereas hepatic triglyceride and lipogenic genes were lowered only to those of WT mice fed no GTE. Malondialdehyde was lowered in Nrf2-null mice fed GTE, but not to levels of WT mice, and without improving the hepatic antioxidants α-tocopherol, ascorbic acid and uric acid. CONCLUSION: Nrf2 deficiency exacerbates NASH whereas anti-inflammatory and hypolipidemic activities of GTE likely occur largely independent of Nrf2 signaling.

Tolerance to acetaminophen hepatotoxicity in the mouse model of autoprotection is associated with induction of flavin-containing monooxygenase-3 (FMO3) in hepatocytes.

Acetaminophen (APAP) pretreatment with a hepatotoxic dose (400 mg/kg) in mice results in resistance to a second, higher dose (600 mg/kg) of APAP (APAP autoprotection). Recent microarray work by our group showed a drastic induction of liver flavin containing monooxygenase-3 (Fmo3) mRNA expression in our mouse model of APAP autoprotection. The role of liver Fmo3, which detoxifies xenobiotics, in APAP autoprotection is unknown. The purpose of this study was to characterize the gene regulation and protein expression of liver Fmo3 during APAP hepatotoxicity. The functional consequences of Fmo3 induction were also investigated. Plasma and livers were collected from male C57BL/6J mice over a period of 72 h following a single dose of APAP (400 mg/kg) to measure Fmo3 mRNA and protein expression. Although Fmo3 mRNA levels increased significantly following APAP treatment, protein expression changed marginally. In contrast, both Fmo3 mRNA and protein expression were significantly higher in APAP autoprotected livers. Unlike male C57BL/6J mice, female mice have ∼80-times higher constitutive Fmo3 mRNA levels and are highly resistant to APAP hepatotoxicity. Coadministration of APAP with the FMO inhibitor methimazole rendered female mice susceptible to APAP hepatotoxicity, with no changes in susceptibility detected in male mice. Furthermore, a human hepatocyte cell line (HC-04) clone over-expressing human FMO3 showed enhanced resistance to APAP cytotoxicity. Taken together, these findings establish for the first time induction of Fmo3 protein expression and function by xenobiotic treatment. Our results also indicate that Fmo3 expression and function plays a role in protecting the liver from APAP-induced toxicity. Although the mechanism(s) of this protection remains to be elucidated, this work describes a novel protective function for this enzyme.