mARC1 Is the Main Contributor to Metabolic Reduction of N-Hydroxyurea

Abstract

N-Hydroxyurea has been known since the 1960s as an antiproliferative drug and is used both in oncology and for treatment of hematological disorders such as sickle cell anemia where very high daily doses are administered. It is assumed that the cellular effect of N-hydroxyurea is caused by inhibition of ribonucleotide reductase, while alternative mechanisms, e.g., generation of nitric oxide, have also been proposed. Despite its many therapeutic applications, the metabolism of hydroxyurea is largely unexplored. The major elimination pathway of N-hydroxyurea is the reduction to urea. Since the mitochondrial amidoxime reducing component (mARC) is known for its N-reductive activity, we investigated the reduction of NHU by this enzyme system. This study presents in vitro and in vivo evidence that this reductive biotransformation is specifically mediated by the mARC1. Inactivation by mARC1 is a possible explanation for the high doses of NHU required for treatment.

Figure 1

Elimination pathways of NHU-known enzymatic conversions are those by urease, peroxidase, catalase, and P450. In addition, a conversion to urea is known, which can be attributed to the mARC enzyme system.

Figure 2

Michaelis–Menten kinetics for the reduction of N-hydroxyurea by human mARC1 and mARC2. (A) Linear plot. (B) Logarithmic (Boltzmann-like) plot. Dashed vertical lines represent K_M values; dashed horizontal lines represent V_max values. (C) K_M and V_max values. Assay conditions are described in the Materials and Methods section. Standard deviations are calculated from n = 6. As mARC2 activities were not yet saturated at the highest NHU concentrations, we examined that the K_M and V_max values are only rough estimates based on extrapolation.

Figure 3

Quantification of Mtarc1 in GalNAc-siRNA-treated or genetically modified mice. (A) Hepatic Mtarc1 mRNA quantified by qPCR. (B) Ratio of hepatic mARC1 protein to vinculin quantified by immunoblotting. (C) Western blot bands for vinculin a.

Figure 4

Quantification of ¹³C,¹⁵N-labeled NHU and urea in murine plasma 15 min after bolus application. (A)¹³C,¹⁵N-NHU plasma concentration. (B) ¹³C,¹⁵N-urea plasma concentration. (C) Correlation between hepatic Mtarc1 mRNA abundance and plasma ¹³C,¹⁵N-NHU concentrations. (D) Correlation between the hepatic mARC1 protein abundance and ¹³C,¹⁵N-NHU plasma concentrations.