Browsing by Author "Merrill, Gary F."

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Carbonyl Reductase 1 Plays a Significant Role in Converting Doxorubicin to Cardiotoxic Doxorubicinol in Mouse Liver, but the Majority of the Doxorubicinol-Forming Activity Remains Unidentified
(2020-03) Breysse, Daniel H.; Boone, Ryan M.; Long, Cameron M.; Merrill, Miranda E.; Schaupp, Christopher M.; White, Colin C.; Kavanagh, Terrance J.; Schmidt, Edward E.; Merrill, Gary F.
Doxorubicin is a widely used cancer therapeutic, but its effectiveness is limited by cardiotoxic side effects. Evidence suggests cardiotoxicity is due not to doxorubicin, but rather its metabolite, doxorubicinol. Identification of the enzymes responsible for doxorubicinol formation is important in developing strategies to prevent cardiotoxicity. In this study, the contributions of three murine candidate enzymes to doxorubicinol formation were evaluated: carbonyl reductase (Cbr) 1, Cbr3, and thioredoxin reductase 1 (Tr1). Analyses with purified proteins revealed that all three enzymes catalyzed doxorubicin-dependent NADPH oxidation, but only Cbr1 and Cbr3 catalyzed doxorubicinol formation. Doxorubicin-dependent NADPH oxidation by Tr1 was likely due to redox cycling. Subcellular fractionation results showed that doxorubicin-dependent redox cycling activity was primarily microsomal, whereas doxorubicinol-forming activity was exclusively cytosolic, as were all three enzymes. An immunoclearing approach was used to assess the contributions of the three enzymes to doxorubicinol formation in the complex milieu of the cytosol. Immunoclearing Cbr1 eliminated 25% of the total doxorubicinol-forming activity in cytosol, but immunoclearing Cbr3 had no effect, even in Tr1 null livers that overexpressed Cbr3. The immunoclearing results constituted strong evidence that Cbr1 contributed to doxorubicinol formation in mouse liver but that enzymes other than Cbr1 also played a role, a conclusion supported by ammonium sulfate fractionation results, which showed that doxorubicinol-forming activity was found in fractions that contained little Cbr1. In conclusion, the results show that Cbr1 accounts for 25% of the doxorubicinol-forming activity in mouse liver cytosol but that the majority of the doxorubicinol-forming activity remains unidentified. SIGNIFICANCE STATEMENT: Earlier studies suggested carbonyl reductase (Cbr) 1 plays a dominant role in converting chemotherapeutic doxorubicin to cardiotoxic doxorubicinol, but a new immunoclearing approach described herein shows that Cbr1 accounts for only 25% of the doxorubicinol-forming activity in mouse liver cytosol, that two other candidate enzymes-Cbr3 and thioredoxin reductase 1-play no role, and that the majority of the activity remains unidentified. Thus, targeting Cbr1 is necessary but not sufficient to eliminate doxorubicinol-associated cardiotoxicity; identification of the additional doxorubicinol-forming activity is an important next challenge.
Cytoprotective Nrf2 pathway is induced in chronically txnrd 1-deficient hepatocytes
(2009-07) Suvorova, Elena S.; Lucas, Olivier; Weisend, Carla M.; Rollins, MaryClare F.; Merrill, Gary F.; Capecchi, Mario R.; Schmidt, Edward E.
"Background Metabolically active cells require robust mechanisms to combat oxidative stress. The cytoplasmic thioredoxin reductase/thioredoxin (Txnrd1/Txn1) system maintains reduced protein dithiols and provides electrons to some cellular reductases, including peroxiredoxins. Principal Findings Here we generated mice in which the txnrd1 gene, encoding Txnrd1, was specifically disrupted in all parenchymal hepatocytes. Txnrd1-deficient livers exhibited a transcriptome response in which 56 mRNAs were induced and 12 were repressed. Based on the global hybridization profile, this represented only 0.3% of the liver transcriptome. Since most liver mRNAs were unaffected, compensatory responses were evidently effective. Nuclear pre-mRNA levels indicated the response was transcriptional. Twenty-one of the induced genes contained known antioxidant response elements (AREs), which are binding sites for the oxidative and chemical stress-induced transcription factor Nrf2. Txnrd1-deficient livers showed increased accumulation of nuclear Nrf2 protein and chromatin immunoprecipitation on the endogenous nqo1 and aox1 promoters in fibroblasts indicated that Txnrd1 ablation triggered in vivo assembly of Nrf2 on each. Conclusions Chronic deletion of Txnrd1 results in induction of the Nrf2 pathway, which contributes to an effective compensatory response."
Hepatocytes lacking thioredoxin reductase 1 have normal replicative potential during development and regeneration
(2010-07) Rollins, MaryClare F.; van der Heide, Dana M.; Weisend, Carla M.; Kundert, Jean A.; Comstock, Kristin M.; Suvorova, Elena S.; Capecchi, Mario R.; Merrill, Gary F.; Schmidt, Edward E.
Cells require ribonucleotide reductase (RNR) activity for DNA replication. In bacteria, electrons can flow from NADPH to RNR by either a thioredoxin-reductase- or a glutathione-reductase-dependent route. Yeast and plants artificially lacking thioredoxin reductases exhibit a slow-growth phenotype, suggesting glutathione-reductase-dependent routes are poor at supporting DNA replication in these organisms. We have studied proliferation of thioredoxin-reductase-1 (Txnrd1)-deficient hepatocytes in mice. During development and regeneration, normal mice and mice having Txnrd1-deficient hepatocytes exhibited similar liver growth rates. Proportions of hepatocytes that immunostained for PCNA, phosphohistone H3 or incorporated BrdU were also similar, indicating livers of either genotype had similar levels of proliferative, S and M phase hepatocytes, respectively. Replication was blocked by hydroxyurea, confirming that RNR activity was required by Txnrd1-deficient hepatocytes. Regenerative thymidine incorporation was similar in normal and Txnrd1-deficient livers, further indicating that DNA synthesis was unaffected. Using genetic chimeras in which a fluorescently marked subset of hepatocytes was Txnrd1-deficient while others were not, we found that the multigenerational contributions of both hepatocyte types to development and to liver regeneration were indistinguishable. We conclude that, in mouse hepatocytes, a Txnrd1-independent route for the supply of electrons to RNR can fully support DNA replication and normal proliferative growth.