Isolation and Characterization of the Protein Components of the Liver Microsomal O2-insensitive NADH-Benzamidoxime Reductase

Abstract

Drugs containing strong basic nitrogen functional groups can be N-oxygenated to genotoxic products. While the reduction of such products is of considerable toxicological significance, most in vitro studies have focused on oxygen-sensitive reductase systems. However, an oxygen-insensitive microsomal hydroxylamine reductase consisting of NADH, cytochrome b 5 , its reductase, and a third unidentified protein component has been known for some time (Kadlubar, F. F., and Ziegler, D. M. (1974) Arch. Biochem. Biophys. 162, 83-92). This report describes the isolation and identification of all of the components required for the reconstitution of an oxygen-insensitive liver microsomal system capable of catalyzing the efficient reduction of primary N-hydroxylated structures such as amidines, guanidines, amidinohydrazones, and similar functional groups. In addition to cytochrome b 5 and its reductase, the reconstituted system requires phosphatidylcholine and a P450 isoenzyme that has been purified to homogeneity from pig liver. The participation of cytochrome b 5 and NADH cytochrome b 5 reductase in cytochrome P450-dependent biotransformations has previously only been described for oxidative processes. The data presented suggest that this system may be an important catalyst in the reduction of genotoxic N-hydroxylated nitrogen components in liver. Their facile reduction by cellular NADH may be the reason why N-hydroxylated products can be missed by studies in vivo. Furthermore, the enzyme system is involved in the reduction of amidoximes and similar functional groups, which can be used as prodrug functionalities for amidines and related groups.The metabolism of nitrogen-containing functional groups has become a topic of considerable interest since the early discovery that N-hydroxylated intermediates are often responsible for the toxic and/or carcinogenic properties of aromatic amines, hydrazines, and amides (1). On the other hand, the more facile N-oxygenation of secondary and tertiary alkylamines to hydroxylamines and N-oxides was considered as a route for detoxication (2, 3), and it was generally assumed that the strongly basic nitrogen compounds were metabolically stable. However, we have demonstrated that even the protonated hydrophilic amidines (4 -6), as well as diamidines such as pentamidine and diminazene and also guanidines and amidinohydrazones, are capable of undergoing metabolic N-oxygenation by liver microsomal cytochrome P450 monooxygenases (7,8). The N-oxygenation of these functional groups produces more reactive metabolites, and the genotoxic properties of benzamidoxime are well known (9). During investigations of the metabolic fate of strongly basic N-hydroxylated xenobiotics, we observed that they were readily reduced both in vivo and in vitro by a microsomal system present in all mammalian species (rats, rabbits, pigs, and humans) tested to date (10 -13).Preliminary experiments indicated that this system (10) had many of the characteristics of the microsomal O 2 -insensitive hydroxylamine reductase descri...

Synthesis and Antiprotozoal Activity of N-Alkoxy Analogues of the Trypanocidal Lead Compound 4,4′-Bis(imidazolinylamino)diphenylamine with Improved Human Blood−Brain Barrier Permeability

“…In fact, the 2-aminoimidazolinyl moiety can be viewed as a cyclic guanidine 42 and the in vivo reduction of N-hydroxylated guanidines and amidines by microsomes and mitochondria of different organs from different species (e.g., kidney, liver, brain) has been established before. 12,43,44 This result is important because it is compound 16 that showed the highest in vitro BBB permeability in our assays. The respectively), may possibly be attributed to a poor bioactivation.…”

Amidoxime Reductase System Containing Cytochrome b5 Type B (CYB5B) and MOSC2 Is of Importance for Lipid Synthesis in Adipocyte Mitochondria

“…Furthermore, the reductase activity was dependent on the cofactor NADH and inhibited by potassium cyanide (KCN), whereas typical CYP inhibitors, such as carbon monoxide, were ineffective (6) indicating that the reductase is unlikely to be a cytochrome P450 as was suggested previously (10). Several studies have attempted to identify the third component, and the involvement of stearoyl-CoA desaturase (11) and cytochrome P450 isoenzyme CYP2D (10) in the microsomal fraction as well as a novel molybdenum-containing enzyme in the mitochondrial fraction (12) was suggested.…”

“…Furthermore, the reductase activity was dependent on the cofactor NADH and inhibited by potassium cyanide (KCN), whereas typical CYP inhibitors, such as carbon monoxide, were ineffective (6) indicating that the reductase is unlikely to be a cytochrome P450 as was suggested previously (10). Several studies have attempted to identify the third component, and the involvement of stearoyl-CoA desaturase (11) and cytochrome P450 isoenzyme CYP2D (10) in the microsomal fraction as well as a novel molybdenum-containing enzyme in the mitochondrial fraction (12) was suggested. In subsequent studies, two highly homologous molybdenum-containing proteins, namely molybdenum cofactor sulfurase C-terminal containing 1 and 2 (MOSC1 and MOSC2, respectively, referred to as mARC1 and mARC2), were shown to display amidoxime reductase activity in vitro when incubated in a reconstituted system with CYB5 and CYB5R3 (cytochrome b 5 reductase 3) (13,14).…”

Unusual Dehydroxylation of Antimicrobial Amidoxime Prodrugs by Cytochrome b5 and NADH Cytochrome b5 Reductase

“…Cytochrome b 5 , along with its reductase, is an electron transfer protein involved in reduction reactions with methemoglobin (Hultquist and Passon, 1971), oxidized ascorbate (Ito et al, 1981;Shirabe et al, 1995), fatty acid desaturases (Oshino et al, 1971), and some P450 enzymes (Hildebrandt and Estabrook, 1971). Controversy exists, however, over whether these two proteins can directly reduce xenobiotic amidoximes (Kurian et al, 2004) or whether a third P450 or other protein is required for reduction (Clement et al, 1997;Clement and Lopian, 2003;Andersson et al, 2005).…”

“…In support of the latter, recent metabolism results have demonstrated that CYP1A and CYP3A4 are involved in the oxidative O-demethylations (unpublished). Previous studies with aromatic amidoximes have shown that these functional groups are reduced to amidines by an NADdependent liver microsomal pathway (Clement et al, 1997;Trepanier and Miller, 2000), which includes cytochrome b 5 and NAD cytochrome b 5 reductase (Clement et al, 1997;Clement, 2002;Kurian et al, 2004;Andersson et al, 2005). Cytochrome b 5 , along with its reductase, is an electron transfer protein involved in reduction reactions with methemoglobin (Hultquist and Passon, 1971), oxidized ascorbate (Ito et al, 1981;Shirabe et al, 1995), fatty acid desaturases (Oshino et al, 1971), and some P450 enzymes (Hildebrandt and Estabrook, 1971).…”