Kinetics of 6-Thioxanthine Metabolism by Allelic Variants of Xanthine Oxidase

Abstract

Our previous studies show that 10 xanthine oxidase (XO) variants (Arg149Cys, Pro555Ser, Arg607Gln, Thr623Ile, Ile703Val Asn909Lys, Thr910Lys, Pro1150Arg, His1221Arg, and Cys1318Tyr) exhibit altered activity toward the endogenous substrate xanthine. This study investigates whether these variants also exhibit altered kinetics for the exogenous substrate 6-thioxanthine (6-TX). To investigate the kinetics of wild-type XO and these variants, expression constructs were transfected into mammalian COS-7 cells. S-9 fractions containing the expressed proteins were used to determine their kinetic parameters, i.e., K(m), V(max), and intrinsic clearance (CL(int)), for the substrate 6-TX. Functional characterization of the 10 XO variants revealed that 4 of the variants (Arg149Cys, Asn909Lys, Thr910Lys, and Pro1150Arg) were inactive, 2 (Arg607Gln, and Cys1318Tyr) had reduced activity (CL(int), 55.5% and 64.7% less than that of wild-type XO, respectively). This study provides comprehensive data regarding how genetic variation in XO affects its activity toward 6-TX. We found that the in vitro activity of 8 of the XO variants toward 6-TX was functionally affected. These results suggeste that polymorphism in the gene encoding XO may increase the toxicity of thiopurine drugs such as 6-mercaputopurine.

Polymorphism of Genes Involved in Purine Metabolism (XDH, AOX1, MOCOS) in Kidney Transplant Recipients Receiving Azathioprine

“…21 Among the SNPs examined in this study, only rs17011368 (resulting in Ile703Val amino acid substitution, in complete linkage with rs17323225, Ile646Val in our population) has been investigated in relation to thiopurine metabolism in transfected human cell lines, showing about 30% reduction of V max , but the in vivo effect of those variants and common synonymous polymorphisms in XDH gene has been not studied up to date. 22 Smith et al 8 observed significantly reduced risk of ADRs in course of IBD therapy in individuals carrying the XDH rs4407290 silent polymorphism variant allele with particular underrepresentation of atypical side effects (headache, myalgia, rash, flulike symptoms, etc). Our results do not support conclusions of that study, possibly due to differences in study design, and also due to the fact that only serious ADRs of AZA were analyzed in our investigation.…”

Molybdenum in Human Health and Disease

“…The fact that xanthine accumulation is much higher than the one of hypoxanthine suggests an increased salvage of hypoxanthine, which was experimentally proven by feeding studies using radio-labeled purines [ 26 ]. Given the relatively broad substrate specifi city, XOR can hydroxylate a number of exogenous substrates such as thiopurines, which are chemotherapeutics used for the treatment of acute lymphoblastic leukemia and autoimmune diseases [ 27 ]. Therefore polymorphisms in the XDH gene may increase the toxicity of drugs such as 6-mercaptopurine.…”

Association of Mutations Identified in Xanthinuria with the Function and Inhibition Mechanism of Xanthine Oxidoreductase

“…Four gene mutations—Thr117Ser, Gly172Arg, Ile703Val, and Arg913Gln—have been reported to be involved in thiopurine intolerance [ 97 ]. Kudo et al [ 101 , 106 ] compared the oxidation of xanthine and 6-thioxanthine (6-TX) using XO mutants. The mutant of Arg149Cys showing type 1 xanthinuria was inactive for both xanthine and 6-TX ( Figure 7 A) [ 101 , 106 ].…”