Acetaminophen-induced inhibition of mitochondrial dehydrogenases and changes in mitochondrial NAD/NADH

The covalent binding of acetaminophen (APAP) to mitochondrial proteins has been postulated to alter the function of the organelle and contribute to the development of hepatotoxicity upon APAP overdose. There were 5 mitochondrial APAP-bound proteins with approximate molecular weights of 75, 60, 54, 44, and 35 kDa on western blots of SDS-PAGE probed with an anti-APAP antibody. On 2D gels of mitochondrial fractions, the 54 kDa band resolved into 3 proteins with pls of 6.4, 6.6 and 7.1. The pl 7.1 protein was the most prominent, and protein sequencing identified it as mitochondrial aldehyde dehydrogenase (ALDH), a protein which oxidizes aldehydes to acids using NAD. ALDH was inhibited 40% 4 hr after a toxic dose of APAP, and the time course of inhibition correlated with covalent binding to the 54 kDa protein suggesting that covalent binding causes this inhibition. Since the endogenous substrates of ALDH are not known, effects of ALDH inhibition on mitochondrial NAD/NADH were studied. NAD/NADH decreased by 40% at 4 hr but increased to 1.2x control by 8 hr after APAP. Use of an ALDH inhibitor, cyanamide, did not support a major role for aldehyde dehydrogenase in the APAP-induced NAD/NADH changes. Total ketone bodies were increased between 1 and 4 hr after APAP which suggests $\beta$-oxidation of fatty acids may play a role in the decreased NAD/NADH seen during that time. This was also supported by a time study with a subtoxic dose of APAP in which total ketone bodies increased and NAD/NADH decreased only at 8 hr after APAP. Attempts to normalize NAD/NADH with $\beta$-hydroxybutyrate and acetoacetate in preparation for protection experiments were unsuccessful, but led to the finding that $\beta$-hydroxybutyrate dehydrogenase was inhibited 35% 4 hr after APAP. Because the functioning of this enzyme in vivo is critical to the determination of NAD/NADH in these studies, detectable changes in NAD/NADH were probably not representative of areas of the liver where this enzyme has been inhibited by APAP. These data suggest that early changes in mitochondrial NAD/NADH resulted from changes in metabolism in an attempt by the liver to overcome the toxic insult while late changes were due to the liver being overwhelmed by APAP.