Student Research: Isaac Mohar
PhD, , 2008
Faculty Advisor: Terrance J. Kavanagh
Gender and Glutamate Cysteine Ligase Modifier Subunit Expression as Determinants of Acetaminophen-induced Liver Damage in Mice
Glutathione (GSH) is an endogenous tripeptide that protects cells against oxidative injury and is an important co-factor in drug and xenobiotic metabolism. The often rate-limiting step in de novo GSH synthesis is the formation of ϒ-glutaylcysteine, catalyzed by glutamate cysteine ligase (GCL). GCL consists of catalytic (Gclc) and modifier subunits (Gclm). GSH is known to protect against acetaminophen (APAP)-induced liver damage (AILD). The hypothesized mechanism involves GSH-conjugation to a reactive matabolite of APAP, N-acetyl-p-benzoquinoneimine (NAPQI). Without adequate GSH, NAPQI adducts to proteins, which is associated with mitochondrial dysfunction and hepatocellular necrosis. This study examines the role of GSH in gender-mediated AILD using male and female mice with variable GSH synthesis. Mice with severely impaired GSH synthesis (Gclm null) displayed 20% of normal hepatic GSH. At a dose of 300 mg/kg APAP, little to no AILD was observed in female Gclm wild-type (WT) or heterozygous (HZ) mice to 24 hours. In contrast, female Gclm null and all male mice displayed significant AILD within 6 hours. GSH was rapidly depleted in all mice but recovered to control levels between 6 and 12h. ATP depletion was observed in male mice 1 to 2 hours after treatment, suggesting mitochondrial dysfunction. Liver APAP-protein adducts and APAP metabolites, quantified by HPLC-electrochemical detection (ECD), demonstrated greater adducts in Gclm null mice, which peaked 2 hours after treatment. Relatice to the opposite gender, female WT and HZ mice showed greater APAP-cysteine metabolite, while all male mice displayed higher levels of an unknown metabolite. Immunoblot visualization of APAP-protein adducts identified a 55-kDa cytosolic protein and 27-kDa mitochondrial protein to be more heavily adducted in male mice. Within gender, Gcllm null mice displayed a similar adduction pettern but enhanced banding. These results support the prevailing hypothesis that NAPQI adduction of key proteins induces mitochondrial dysfunction and leads to hepatocellular necrosis under overdose conditions. Impaired GSH synthesis enhances adduction, thereby predisposing otherwise resistant female mice to AILD and increasing the severity of liver damage to male mice.