It is well established that following a toxic dose of
acetaminophen (
APAP),
nitrotyrosine protein adducts (3-NT), a hallmark of
peroxynitrite production, were colocalized with necrotic hepatic centrilobular regions where
cytochrome P450 2E1 (
CYP2E1) is highly expressed, suggesting that 3-NT formation may be essential in
APAP-mediated toxicity. This study was aimed at investigating the relationship between
CYP2E1 and nitration (3-NT formation) followed by
ubiquitin-mediated degradation of
proteins in wild-type and Cyp2e1-null mice exposed to
APAP (200 and 400mg/kg) for 4 and 24h. Markedly increased centrilobular liver
necrosis and 3-NT formation were only observed in
APAP-exposed wild-type mice in a dose- and time-dependent manner, confirming an important role for
CYP2E1 in
APAP biotransformation and toxicity. However, the pattern of 3-NT
protein adducts, not accompanied by concurrent activation of
nitric oxide synthase (NOS), was similar to that of
protein ubiquitination. Immunoblot analysis further revealed that immunoprecipitated nitrated
proteins were ubiquitinated in
APAP-exposed wild-type mice, confirming the fact that nitrated
proteins are more susceptible than the native
proteins for
ubiquitin-dependent degradation, resulting in shorter half-lives. For instance, cytosolic
superoxide dismutase (SOD1) levels were clearly decreased and immunoprecipitated SOD1 was nitrated and ubiquitinated, likely leading to its accelerated degradation in
APAP-exposed wild-type mice. These data suggest that
CYP2E1 appears to play a key role in 3-NT formation, protein degradation, and liver damage, which is independent of NOS, and that decreased levels of many
proteins in the wild-type mice (compared with
Cyp2e1-null mice) likely contribute to
APAP-related toxicity.