The effect of acetaminophen on ubiquitin homeostasis in Saccharomyces cerevisiae

Abstract

Acetaminophen (APAP), although considered a safe drug, is one of the major causes of acute liver failure by overdose, and therapeutic chronic use can cause serious health problems. Although the reactive APAP metabolite N-acetyl-p-benzoquinoneimine (NAPQI) is clearly linked to liver toxicity, toxicity of APAP is also found without drug metabolism of APAP to NAPQI. To get more insight into mechanisms of APAP toxicity, a genome-wide screen in Saccharomyces cerevisiae for APAP-resistant deletion strains was performed. In this screen we identified genes related to the DNA damage response. Next, we investigated the link between genotype and APAP-induced toxicity or resistance by performing a more detailed screen with a library containing mutants of 1522 genes related to nuclear processes, like DNA repair and chromatin remodelling. We identified 233 strains that had an altered growth rate relative to wild type, of which 107 showed increased resistance to APAP and 126 showed increased sensitivity. Gene Ontology analysis identified ubiquitin homeostasis, regulation of transcription of RNA polymerase II genes, and the mitochondria-to-nucleus signalling pathway to be associated with APAP resistance, while histone exchange and modification, and vesicular transport were connected to APAP sensitivity. Indeed, we observed a link between ubiquitin levels and APAP resistance, whereby ubiquitin deficiency conferred resistance to APAP toxicity while ubiquitin overexpression resulted in sensitivity. The toxicity profile of various chemicals, APAP, and its positional isomer AMAP on a series of deletion strains with ubiquitin deficiency showed a unique resistance pattern for APAP. Furthermore, exposure to APAP increased the level of free ubiquitin and influenced the ubiquitination of proteins. Together, these results uncover a role for ubiquitin homeostasis in APAP-induced toxicity.

Acetaminophen reduces the protein levels of high affinity amino acid permeases and causes tryptophan depletion

“…In our previous research (Huseinovic et al 2017a , b ), we showed that APAP toxicity depends on cellular ubiquitin levels and that ubiquitin-deficient strains, such as ubi4∆ , doa1∆, doa4∆ , ubp6∆ and ∆ubp14, conferred resistance to APAP (Huseinovic et al 2017b ). Since it is known that the Tat2 expression level is stabilized in deletion strains doa4∆ , ubp6∆ and ubp14∆ under high pressure (Miura and Abe 2004 ), and based on our current finding that only Tat1 or Tat2 overexpression conferred resistance to APAP, we hypothesized that the Tat2 expression level is stabilized in the APAP resistant mutants during APAP treatment.…”

“…Previously, we used yeast Saccharomyces cerevisiae as a eukaryotic model organism to get more insight into NAPQI independent APAP toxicity, because yeast lacks the genes coding for drug-metabolizing P450 enzymes and is incapable of APAP metabolism and formation of NAPQI (Srikanth et al 2005 ). Our study revealed that APAP toxicity depends on the cellular concentration of ubiquitin: ubiquitin depletion confers resistance to APAP, whereas ubiquitin overexpression caused sensitivity (Huseinovic et al 2017b ). Based on the correlation between ubiquitin levels and APAP-induced toxicity, we also performed a deubiquitinase (DUB) gene deletion screen (Huseinovic et al 2017a ).…”

“…Interestingly, a study with human astrocytes showed that halothane, another volatile anesthetic similar in chemical structure to isoflurane, induced an increase in intracellular Glu (Miyazaki et al 1997 ). In our previous study, we showed that deletion of RTG genes RTG1, RTG2, RTG3 and MKS1, conferred resistance to APAP (Huseinovic et al 2017b ).…”

“…In our previous research, we made a link between ubiquitin levels and APAP toxicity in yeast (Huseinovic et al 2017b ) and showed, using a DUB deletion strain screen (Huseinovic et al 2017a ), that APAP had a comparable toxicity profile as tyrosine and an overlap with drugs causing internalization of high affinity AAP Tat2 (rapamycin and quinine) (Beck et al 1999 ; Khozoie et al 2009 ). In this study, we explored these observations further by investigating whether APAP can cause a nutrient starvation response in yeast and alter the intracellular concentration of amino acids.…”

Galangin Nanoparticles Protect Acetaminophen-Induced Liver Injury: A Biochemical and Histopathological Approach

“…With its analgesic and antifever properties, and its low risk at the recommended dose, acetaminophen is widely used as an over-the-counter drug [ 1 ]. Acetaminophen poisoning is one of the significant causes of liver damage, and the use of this drug in chronic form may cause serious health problems [ 2 , 3 ]. Acetaminophen is converted to non-toxic metabolites in phase II metabolism.…”