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.