Title

Acetominophen-induced hepatotoxicity: Alterations in protein phosphorylation, synthesis, and degradation

Date of Completion

January 1996

Keywords

Biology, Cell|Health Sciences, Toxicology

Degree

Ph.D.

Abstract

Cultured mouse hepatocytes maintained for up to 48 hr in medium supplemented with 15 mM nicotinamide to stabilize cytochrome P450 provided a valid model system for studying acetaminophen (APAP) hepatotoxicity. The lowered level of P450 was found to be advantageous in delaying the onset of toxicity and in temporally dissociating alterations in cellular metabolism that may contribute to the progression or reversal of cell injury. Alterations in protein synthesis, degradation, and phosphorylation were among the key metabolic processes examined as potential determinants of both adaptive and repair mechanisms in response to injury. All three processes were observed to decrease following exposure to APAP, however this is the first report to describe distinct changes in the synthesis and phosphorylation of specific proteins.^ The de novo synthesis of proteins migrating at 58 and 32 kDa (p58 and p32) decreased and increased respectively during APAP exposure. Recovery of p58 synthesis upon APAP removal correlated with the length of time that cultures were initially exposed to the drug whereas p32 synthesis remained elevated during the recovery period. These studies further defined a "window of reversibility" and suggested that recovery of the synthesis of p58 may ultimately prove to be a specific marker for predicting the reversibility of hepatocellular damage induced by APAP and related compounds.^ Exposure to APAP also selectively altered the phosphorylation state of several proteins: p22, p25a, p25b, p26, p28, p59. Evidence is also presented that these changes (1) are dependent on cytochrome P450 activation of APAP; (2) correlate with the progression of APAP toxicity; (3) are in part mimicked by menadione and several different inhibitors of protein phosphatase 1 and 2A; and (4) are paralleled by a decline in the activity of protein phosphatases. Given the pivotal role of reversible phosphorylations in the integration of metabolism, prevention of the dephosphorylation and/or persistent stimulation of specific kinase activity toward these proteins may contribute to or signal irreversible events leading to cell death. ^