Elsevier

Biochemical Pharmacology

Volume 58, Issue 3, 1 August 1999, Pages 439-445
Biochemical Pharmacology

Molecular and Cellular Pharmacology
Microsomal formation of nitric oxide and cyanamides from non-physiological N-hydroxyguanidines: N-hydroxydebrisoquine as a model substrate

https://doi.org/10.1016/S0006-2952(99)00102-1Get rights and content

Abstract

The microsomal oxidative transformation of a non-physiological N-hydroxyguanidine was demonstrated for the first time for N-hydroxydebrisoquine as a model substrate (Clement et al., Biochem Pharmacol 46: 2249–2267, 1993). The objective of the present work was to further compare this reaction with the analogous oxidation of arginine via N-hydroxyarginine to citrulline and nitric oxide. The oxidation of N-hydroxydebrisoquine by liver microsomes from rats pretreated with dexamethasone not only produced nitric oxide and the urea, but also the cyanamide derivative as the main metabolite. The low stability of the cyanamide derivative, which easily hydrolyzed to the urea derivative, was noted. The formation of all compounds required cosubstrate and the enzyme source. Experiments with catalase, superoxide dismutase, and H2O2 showed that the O2 formed from the enzyme and the substrate apparently participated in the reaction. While the N-hydroxylation of the guanidine involves the usual monooxygenase activity of cytochrome P-450 (Clement et al., Biochem Pharmacol 46: 2249–2267, 1993), the resultant N-hydroxyguanidine decoupled the monooxygenase. Nitric oxide was detected by the oxyhemoglobin assay. To examine the influence of enzymatically formed nitric oxide on the formation of the metabolites, the N-hydroxydebrisoquine was incubated with SIN-1 as nitric oxide donor under aerobic conditions. It was again possible to detect the cyanamide and urea derivatives, with the latter as main metabolite. It was concluded that the microsomal transformation of N-hydroxydebrisoquine produces a cyanamide and nitric oxide which reacts with N-hydroxydebrisoquine to form the urea derivative. The purely chemical reaction of the unsubstituted N-hydroxyguanidine with nitric oxide gave similar results (Fukuto et al., Biochem Pharmacol 43: 607–613, 1992). In conclusion, similarities (formation of a urea derivative) and differences (formation of a cyanamide derivative) between the physiological oxidation of N-hydroxy-l-arginine by nitric oxide synthases and non-physiological N-hydroxyguanidines by cytochrome P-450 were observed. Furthermore, non-physiological N-hydroxyguanidines can be regarded as nitric oxide donors.

Section snippets

Chemicals and reagents

Catalase, SOD, and human hemoglobin were purchased from Sigma Chemical Co. Oxyhemoglobin was prepared from hemoglobin as described previously [6] and SIN-1 was supplied by Alexis Co. NADPH (tetrasodium salt) as well as all other chemicals and solvents were from Merck unless otherwise stated.

Synthesis

The cyanamide and urea derivatives of debrisoquine were obtained by reaction of tetrahydroisochinoline and bromocyanogen or potassium cyanate, respectively, as described earlier [7]. N-Hydroxydebrisoquine

Qualitative and quantitative analysis

The incubation of N-hydroxydebrisoquine with microsomes and NADPH resulted not only in the urea derivative [14], but also the cyanamide derivative (Fig. 1). The evidence for its formation was obtained unambiguously by HPLC. Addition of the reference substrate to the incubation mixture gave rise to an increase in the area of the metabolic peak, even when the eluent was varied (data not shown). A representative HPLC chromatogram recorded after the incubation of N-hydroxydebrisoquine with

Metabolism of N-hydroxydebrisoquine

The microsomal incubation of N-hydroxydebrisoquine produced not only the urea but also the cyanamide derivative as the main metabolites. The formation of a cyanamide was unexpected since during previous studies, also with amidoximes, similar metabolites were not identified [5]. Very recently, oxidation of various N-hydroxyguanidines and amidoximes by rat liver microsomes was found to lead to the corresponding cyanamides and nitriles, respectively∗. Moreover, investigations on the purely

Acknowledgements

The financial support of the “Fonds der Chemischen Industrie” is gratefully acknowledged.

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