Related Experiment Videos
Covalent binding of acetaminophen to N-10-formyltetrahydrofolate dehydrogenase in mice
N R Pumford1, N C Halmes, B M Martin
1Division of Toxicology, University of Arkansas for Medical Sciences, Little Rock, USA.
Abstract:
The analgesic acetaminophen is frequently used as a model chemical to study hepatotoxicity; however, the critical mechanisms by which it produces toxicity within the cell are unknown. It has been postulated that covalent binding of a toxic metabolite to crucial proteins may inhibit vital cellular functions and may be responsible for, or contribute to, the hepatotoxicity. To further understand the importance of covalent binding in the toxicity, a major cytosolic acetaminophen-protein adduct of 100 kDa has been purified by a combination of anion exchange chromatography and preparative electrophoresis. N-Terminal and internal amino acid sequences of peptides from the purified 100-kDa acetaminophen-protein adduct were found to be homologous with the deduced amino amino acid sequence from the cDNA of N-10-formyltetrahydrofolate dehydrogenase. Antiserum specific for N-10-formyltetrahydrofolate dehydrogenase and acetaminophen react in a Western blot with the purified 100-kDa acetaminophen-protein adduct. Administration of a toxic dose of acetaminophen (400 mg/kg) to mice resulted in a 25% decrease in cytosolic N-10-formyltetrahydrofolate dehydrogenase activity at 2 hr. The covalent binding of acetaminophen to proteins such as N-10-formyltetrahydrofolate dehydrogenase and the subsequent decreases in their enzyme activity may play a role in acetaminophen hepatotoxicity.
Insights
Acetaminophen overdose can harm the liver. This study identifies N-10-formyltetrahydrofolate dehydrogenase as a protein that binds acetaminophen, potentially causing liver damage.
Area of Science:
- Biochemistry
- Toxicology
- Molecular Biology
Background:
- Acetaminophen (APAP) is a common pain reliever, but its mechanism of liver toxicity is not fully understood.
- A proposed mechanism involves the covalent binding of toxic APAP metabolites to cellular proteins, impairing vital functions.
- Identifying specific protein targets is crucial for understanding APAP-induced hepatotoxicity.
Purpose of the Study:
- To investigate the role of protein adduct formation in acetaminophen hepatotoxicity.
- To identify the specific cellular protein(s) that form covalent adducts with acetaminophen metabolites.
- To determine if binding to identified proteins affects their enzymatic activity.
Main Methods:
- Purification of a major 100 kDa cytosolic acetaminophen-protein adduct using anion exchange chromatography and preparative electrophoresis.
- Peptide sequencing (N-terminal and internal) of the purified adduct.
- Homology analysis of peptide sequences against known protein databases.
- Western blot analysis using specific antiserum for N-10-formyltetrahydrofolate dehydrogenase and acetaminophen.
- Measurement of cytosolic N-10-formyltetrahydrofolate dehydrogenase activity in mice after acetaminophen administration.
Main Results:
- A 100 kDa acetaminophen-protein adduct was successfully purified.
- Peptide sequences from the adduct showed homology to N-10-formyltetrahydrofolate dehydrogenase.
- Western blot confirmed the presence of acetaminophen and N-10-formyltetrahydrofolate dehydrogenase in the purified adduct.
- Mice treated with a toxic acetaminophen dose exhibited a 25% reduction in cytosolic N-10-formyltetrahydrofolate dehydrogenase activity within 2 hours.
Conclusions:
- Acetaminophen covalently binds to N-10-formyltetrahydrofolate dehydrogenase, forming a 100 kDa adduct.
- This covalent binding leads to a significant decrease in the enzyme's activity.
- The interaction between acetaminophen and N-10-formyltetrahydrofolate dehydrogenase may contribute to acetaminophen-induced liver toxicity.