Bioactivation of diclofenac in human hepatocytes and the proposed human hepatic proteins modified by reactive metabolites
Creators: name: Inoue, Kazuko
name: Mizuo, Hitoshi
name: Ishida, Tomomi
name: Komori, Takafumi
name: Kazutomi Kusano
Publisher: name: Taylor & Francis
Year: 2020
Subjects: subject: Biophysics
subject: Biochemistry
subject: Genetics
subject: FOS: Biological sciences
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Physiology
subject: Pharmacology
subject: Biotechnology
subject: 39999 Chemical Sciences not elsewhere classified
subject: FOS: Chemical sciences
subject: FOS: Chemical sciences
subject: Ecology
subject: Cancer
subject: 110309 Infectious Diseases
subject: FOS: Health sciences
subject: FOS: Health sciences
subject: Plant Biology
subject: 60506 Virology

description: To reveal putative bioactivation pathways of diclofenac, <i>in vitro</i> human liver materials such as microsomal fractions and hepatocytes were used to confirm metabolic activation of diclofenac by <sup>35</sup>S-cysteine trapping assay and covalent binding assay. Candidate human liver proteins possibly targeted by <sup>14</sup>C-diclofenac via bioactivation were investigated using two-dimensional gel electrophoresis followed by detection of remaining radioactivity on the modified proteins with bio-imaging analyzer.In the <sup>35</sup>S-cysteine trapping assay, three and two adducts with <sup>35</sup>S-cysteine were observed in NADPH-fortified and UDPGA-fortified human liver microsomes, respectively. In the covalent binding assay using <sup>14</sup>C-diclofenac in human hepatocytes, the extent of covalent binding of diclofenac to human hepatic proteins increased time-dependently. Addition of glutathione attenuated the extent of covalent binding of <sup>14</sup>C-diclofenac to human liver microsomal proteins.Fifty-nine proteins from human hepatocytes were proposed as the candidate proteins targeted by reactive metabolites of diclofenac. Proteins modified by cytochrome P450-mediated reactive metabolites were identified by using a cytochrome P450 inhibitor, 1-aminobenzyltriazole and seven of the nine radioactive protein spots were removed by 1-aminobenzyltriazole treatment.In contrast, the remaining two radioactive protein spots, mainly containing human serum albumin and heat shock proteins, were not affected by the addition of 1-aminobenzyltriazole, which suggested the involvement of the acyl glucuronide of diclofenac, formed via uridine diphosphate-glucuronosyl transferases, in the covalent modifications induced by diclofenac. To reveal putative bioactivation pathways of diclofenac, <i>in vitro</i> human liver materials such as microsomal fractions and hepatocytes were used to confirm metabolic activation of diclofenac by <sup>35</sup>S-cysteine trapping assay and covalent binding assay. Candidate human liver proteins possibly targeted by <sup>14</sup>C-diclofenac via bioactivation were investigated using two-dimensional gel electrophoresis followed by detection of remaining radioactivity on the modified proteins with bio-imaging analyzer. In the <sup>35</sup>S-cysteine trapping assay, three and two adducts with <sup>35</sup>S-cysteine were observed in NADPH-fortified and UDPGA-fortified human liver microsomes, respectively. In the covalent binding assay using <sup>14</sup>C-diclofenac in human hepatocytes, the extent of covalent binding of diclofenac to human hepatic proteins increased time-dependently. Addition of glutathione attenuated the extent of covalent binding of <sup>14</sup>C-diclofenac to human liver microsomal proteins. Fifty-nine proteins from human hepatocytes were proposed as the candidate proteins targeted by reactive metabolites of diclofenac. Proteins modified by cytochrome P450-mediated reactive metabolites were identified by using a cytochrome P450 inhibitor, 1-aminobenzyltriazole and seven of the nine radioactive protein spots were removed by 1-aminobenzyltriazole treatment. In contrast, the remaining two radioactive protein spots, mainly containing human serum albumin and heat shock proteins, were not affected by the addition of 1-aminobenzyltriazole, which suggested the involvement of the acyl glucuronide of diclofenac, formed via uridine diphosphate-glucuronosyl transferases, in the covalent modifications induced by diclofenac.