Puromycin aminonucleoside metabolism by glomeruli and glomerular epithelial cells in vitro

Abstract

Two puromycin aminonucleoside (PAN) excretion products were purified by HPLC from urine of PAN-treated rats and characterized by nuclear magnetic resonance as N6-dimethyl-3'amino-3'deoxyadenosine (DA-Ado) and N6-methyl-3'amino-3'deoxyadenosine (MA-Ado), respectively, the former corresponding to unmodified PAN. DA-Ado was not a substrate for adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP) or xanthine oxidase (XO), while MA-Ado was consecutively converted into hypoxanthine by a mixture of ADA and PNP. A different rate of transformation of DA-Ado and MA-Ado into hypoxanthine by isolated glomeruli was observed and was higher for the monomethylated analogue by a factor of 3 (79% vs. 21%); this was ascribed to the rate-limiting level of a demethylase activity acting on DA-Ado. Furthermore, DA-Ado was not transformed by glomerular epithelial cells in culture, while a little amount of MA-Ado was converted into hypoxanthine after six hours of incubation. In spite of this different metabolic behavior, the same order of cytotoxicity on glomerular epithelial cells in culture was observed for MA-Ado, DA-Ado and commercial PAN. All these molecules induced a dose response inhibition of [3H]thymidine incorporation into DNA after exposure for two hours and a marked alteration of cell viability which was not inhibited by free radical scavengers and deferoxamine. This study provides the first evidence for a glomerular metabolism of PAN and its urinary metabolite MA-Ado involving their transformation via the purine cycle enzymes. The rate of glomerular transformation of PAN in isolated glomeruli and the lack of its metabolism in cultured epithelial cells implies the presence of demethylase enzymes within the glomerulus and their absence in cultured epithelial cells. However, the similar cytotoxic effect of PAN and MA-Ado on cultured epithelial cells supports the concept that a mechanism which does not involve the purine cycle is responsible for PAN glomerular toxicity in vitro.