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

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Drug Metab. Pharmacokinet. 25 (4): 361–366 (2010). Regular Article Kinetics of 6-Thioxanthine Metabolism by Allelic Variants of Xanthine Oxidase

Mutsumi KUDO1,TakamitsuSASAKI1, Masaaki ISHIKAWA1, Noriyasu HIRASAWA2 and Masahiro HIRATSUKA2,* 1Department of Clinical Pharmacotherapeutics, Tohoku Pharmaceutical University, Sendai, Japan 2Laboratory of Pharmacotherapy of Life-Style Related Diseases, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan

Full text of this paper is available at http://www.jstage.jst.go.jp/browse/dmpk

Summary: 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., Km, Vmax, and intrinsic clearance (CLint), 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 (CLint,55.5z and 64.7z 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.

Keywords: xanthine oxidase; 6-thioxanthine; genetic polymorphism; variant proteins; functional characterization

The thiopurine drug 6-mercaptopurine (6-MP) are cytotoxic polymorphisms in the TPMT gene are thought to be drugs that are currently used in the treatment of several dis- mainly responsible for the large variation in the toxicity eases, including childhood acute lymphoblastic leukemia.1) and therapeutic effects of 6-MP observed among individ- Figure 1 shows a schematic diagram of thiopurine uals.3) However, the 6-MP oxidation pathway has metabolism. 6-MP is metabolized in three pathways— received scant attention. catabolism to 6-thiouric acid (6-TUA) by the action of XO is distributed in many tissues, and its activity is xanthine oxidase (XO) and aldehyde oxidase (AO), strongest in the liver and intestine.4,5) Interindividual vari- methylation to methylmercaputopurine metabolites by ation in human liver XO activity exists, with approxi- the action of thiopurine S-methyltransferase (TPMT), and mately 20% of Caucasian subjects displaying relatively anabolism to 6-thioinosine-monophosphate by hypoxan- lower enzyme activity.6) Population phenotyping studies thine-guanine phosphoribosyltransferase (HGPRT). performed using caffeine indicate the existence of Anabolism to 6-thioinosine-monophosphate results in the marked interindividual and interethnic differences in XO formation of cytotoxic 6-thioguanine nucleotides (6- activity. Approximately 11% Japanese7) and 4% Spanish TGNs).2) Thiopurine methyltransferase appears to be lar- people8) are considered to exhibit poor XO metabolism. gely responsible for the metabolism of 6-MP. Genetic Further, studies have shown gender-dependent differ-

Received; March 24, 2010, Accepted; May 14, 2010 *To whom correspondence should be addressed: Masahiro HIRATSUKA,Ph.D.,Laboratory of Pharmacotherapy of Life-Style Related Diseases, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan, 6-3, Aoba, Aramaki, Aoba-ku, Sendai 980-8578, Japan. Tel & Fax. ＋81-22-717-7049, E-mail: mhira＠m.tohoku.ac.jp This work was supported by KAKENHI (20590154) from Japan Society for the Promotion of Science (JSPS), in part by a grant from the Smoking Research Foundation and a grant from Japan Research Foundation for Clinical Pharmacology.

361 362 Mutsumi KUDO, et al.

Fig. 1. Metabolic pathway of 6-mercaputopurine 6-MP, 6-mercaputopurine; 6-MMP, 6-methyl mercaputopurine; 6-TX, 6-thioxanthine; 6-TUA, 6-thiouric acid; 6-TIMP, 6-thioinosine monophosphate; 6-TIDP, 6-thioinosine-diphosphate; 6-TITP, 6-thioinosine-triphosphate; 6-TXMP, 6-thioxanthine monophosphate; 6-TGNs, 6- thioguanine nucleotides; TPMT, thiopurine S-methyltransferase; XO, xanthine oxidase; AO, aldehyde oxidase; HGPRT, hypoxanthine-guanine phosphoribosyltransferase; ITPase, inosine triphosphate pyrophosphatase; IMPDH, inosine monophosphate dehydrogenase; GMPS, guanosine monophosphate synthetase.

ences in the hepatic XO activity in humans, with male Table 1. Kinetic parameters for xanthine oxidation catalyzed subjects exhibiting stronger activity than female sub- by wild-type and variant XOs in previous study jects.6) XO deficiency, also known as classical xanthinur- K V V /K 9) Variant m max max m ia, is classified into 2 types-I and II. In the former type, (mM) (nmol/mg/min) (mL/mg/min)

XO activity is obliterated because of mutations in the Wild 1.68±0.38 40.4±7.4 26.9±9.9 XO-encoding gene on chromosome 2p22–23.10–14) The bioavailability of 6-MP has been estimated as ap- Arg149Cysa N.D. N.D. N.D. proximately 5–37% owing to the presence of large Pro555Ser 2.05±0.23 20.2±5.3** 9.8±1.9 amounts of XO in the liver.15) This bioavailability in- Arg607Gln 2.90±0.32** 33.3±3.2 11.5±1.1 creases when XO inhibitors such as methotrexate are ad- ministered concomitantly with 6-MP.16,17) Competitive in- Thr623Ile 3.68±0.37*** 38.5±0.3 10.5±1.0** hibition of XO with allopurinol pretreatment results in a Ile703Val 2.19±0.41 66.6±9.9*** 31.7±10.5 5-fold increase in the bioavailability of oral 6-MP.18) Con- Asn909Lys 2.51±0.27 12.4±1.8*** 8.6±3.8*** current use of allopurinol and 6-MP has been reported to cause severe myelotoxicity.19,20) Further, Wong et al. Thr910Lys N.D. N.D. N.D. report that the levels of active thiopurine metabolites are Pro1150Arg 1.67±0.64 27.1±0.8*** 18.0±6.7 undetectable when XO activity is strong.21) Thus, 6-MP- His1221Arg 1.46±0.07 60.6±11.1** 41.6±7.6** induced adverse effects may increase in poor XO metabolizers. Cys1318Tyr 2.04±0.32* 35.5±3.0* 17.6±1.6* Our previous studies show that 10 single nucleotide The kinetic parameters of wild type were average of seven wild types. polymorphisms (SNPs) with nonsynonymous substitu- aThis mutation is responsible for classical xanthinuria type I. *Pº0.05, **Pº0.01, and ***Pº0.005 compared to the wild type. ND, not tions and mutation functionally affect the activity of XO detectable. Values shown are means±SD of 3 independent experiments. toward the endogenous substrate xanthine (Table 1).22) It has been reported that the affinity of exogenous substrate 6-TX for XO is different from that of xanthine.23) To our knowledge, there is no study on the kinetic Thus possibly, XO genetic variant proteins might exhibit parameters for the oxidation of 6-TX by expressed XO substrate-dependent change in their kinetic parameters. variant proteins. This study investigates whether the 10 Kinetics of 6-Thioxanthine Metabolism by XO Variants 363

XO variants (Arg149Cys, Pro555Ser, Arg607Gln, triplicate. Statistical significance was determined by a Thr623Ile, Ile703Val, Asn909Lys, Thr910Lys, one-way analysis of variance (ANOVA) together with a Pro1150Arg, His1221Arg, and Cys1318Try) also exhibit post hoc Dunnett multiple comparisons test; differences altered kinetics for the exogenous substrate 6-TX. were considered to be statistically significant at p value º0.05. Materials and Methods Results Expression of XO variant proteins in COS-7 cells: Wild-type XO and 10 XO variants were expressed in The XO protein expression in COS-7 cells transfected COS-7 cells as previously described.22) XO protein ex- with recombinant XO cDNA was analyzed by immunob- pression in the COS-7 cells was then analyzed by western lotting. A 145-kDa band was successfully detected in the blotting. The S-9 fractions obtained from the transfected cells transfected with either wild-type XO or any of the cells, which correspond to 5.0 mgofprotein,weredena- variant proteins (Fig. 2). To determine the kinetic tured by heating the cells in a loading buffer containing parameters for the enzyme activity of wild-type XO and 2-mercaptoethanol and were then size-fractionated by XO variants, we studied the Michaelis-Menten kinetics of gel electrophoresis on a 7.5% polyacrylamide/0.1% sodi- 6-TX oxidation (Fig. 3). The calculated kinetic um dodecyl sulfate gel. The proteins were electrotran- parameters for 6-TX are shown in Table 2.Ourprevi- sferred onto polyvinylidene difluoride membranes (Im- ous study on xanthine oxidation revealed that the values mun-Blot; Bio-Rad, Hercules, California, USA), incubat- of Km, Vmax, and intrinsic clearance (CLint; Vmax/Km)for ed for 1.0 h with a polyclonal goat anti-human XO an- wild-type XO were 1.68 mM, 40.4 nmol･mg protein－1･ tibody (Santa Cruz, CA, USA) diluted in 5% milk in Tris- min－1, and 26.9 mL･mg protein－1･min－1, respectively. buffered saline (1:400), and finally incubated with the In the present study on 6-TX oxidation, wild-type XO ex- secondary antibody conjugated with horseradish peroxi- hibited a 32.8% decrease in its CLint value (8.49 mL･mg －1 －1 dase (Abcam, Cambridge, UK). The membrane was devel- protein ･min ), owing to a 2.9-fold increase in the Km oped using the Supersignal West Pico chemiluminescent value (4.97 mM), compared with wild-type of xanthine substrate (Pierce, Rockford, Illinois, USA) and scanned oxidation. using the Lumino imaging analyzer (FAS-1000; Toyobo, Among the 10 variants, 8 (Arg149Cys, Arg607Gln, Osaka, Japan). Thr623Ile, Ile703Val, Asn909Lys, Thr910Lys, Enzyme assay and kinetic analysis of XO activity Pro1150Arg, and Cys1318Try) exhibited significant in COS-7 cells: XO activity was measured using a differences in their kinetic parameters (Table 2). No oxi- 62.5-mL reaction mixture containing 50.0 mgoftheS-9 dation metabolite was detected in the S-9 fractions der- fraction protein and 0, 2.0, 3.0, 4.0, 6.0, 8.0, 16.0, and ived from the Arg149Cys, Asn909Lys, Thr910Lys, and 24.0 mM of 6-TX (BePharm Ltd., Shanghai, China) in 50 Pro1150Arg variants. Vmax values of the Arg607Gln and mM potassium phosphate (pH 8.1). After incubation at Cys1318Try variants were significantly lower than those 379C for 60 min, the reaction was terminated by adding of wild-type XO, by 45.0% and 57.9%, respectively 62.5 mL of 10% HCl. The reaction mixtures were subse- (Pº0.005). Further, these variants exhibited 55.5% quently centrifuged at 7,700×g for 5 min, and 50.0-mL (Arg607Gln) and 64.7% (Cys1318Try) decrease in their aliquots of the supernatants were subjected to reverse- CLint values, respectively. Vmax values of the Thr623Ile and phase high-performance liquid chromatography (HPLC). Ile703Val variants were significantly lower than those of HPLC analyses were performed using a Waters 2695 wild-type XO, by 65.1% and 69.4%, respectively separations module equipped with a SunFire C18 column (Pº0.01 and Pº0.05, respectively). However, their (4.6×150 mm; 5.0 mm; Waters Corporation, Milford, MA, USA). The eluent was monitored at 350 nm using a Waters 2996 Photodiode Array Detector. The mobile phase was 20 mM potassium phosphate (pH 2.8), the flow rate was 1.0 mL/min, and the column temperature was 409C. Under these conditions, the retention times of 6-TUA and 6-TX were 6.4 and 11.0 min, respectively. Statistical analysis: Kinetic data were applied to the Enzyme Kinetics Module in SigmaPlot 9.01 (Systat Fig. 2. Immunoblot analysis of XO proteins expressed in Software, Inc., Chicago, IL), a curve-fitting program COS-7 cells XO-immunoreactive proteins in COS-7 cells expressing wild-type based on nonlinear regression analysis, and the Km, Vmax, XO or XO variants are labeled as wild type, Arg149Cys, Pro555Ser, and intrinsic clearance (CLint, Vmax/Km) values were deter- Arg607Gln, Thr623Ile, Ile703Val, Asn909Lys, Thr910Lys, mined. All values were expressed as means±standard Pro1150Arg, His1221Arg, and Cys1318Try. The S-9 fraction (5.0 deviation (SD) of those obtained from 3 independent mg) of the proteins was loaded into each lane. Human liver cytosol- trnsfection experiments. Each assay was performed in ic fractions were used as the positive control. 364 Mutsumi KUDO, et al.

Fig. 3. The kinetics of 6-TX oxidation in S-9 fractions obtained from COS-7 cells expressing wild-type XO (white circles), Pro555Ser (black circles), Arg607Gln (white triangles), Thr623Ile (black triangles), Ile703Val (white squares), His1221Arg (black squares), and Cys1318Tyr (white diamonds) Each point represents the mean of 3 independent experiments.

Table 2. Kinetic parameters for 6-TX oxidation catalyzed by Table 3. Functional characterization of XO variants for xan- wild-type and variant XOs expressed in COS-7 cells thine and 6-TX

K V V /K Enzyme characterization change Variant m max max m (mM) (nmol/mg/min) (mL/mg/min) (vs. wild type) Variants Reference or SNP ID Wild 4.97±0.52 41.8±4.5 8.49±1.37 Xanthine 6-TX

Arg149Cys N.D. N.D. N.D. Arg149Cys Sakamoto et al. 2001 N.D. N.D.

Pro555Ser 5.29±1.54 39.8±4.2 8.24±3.69 Pro555Ser gs194465 Vmax: 53% —

Arg607Gln 4.28±1.37 18.8±5.9*** 4.71±1.96* Arg607Gln gs194477 CLint.: 69.0% CLint.: 55.5%

Thr623Ile 4.70±1.00 27.2±4.7** 6.02±1.90 Thr623Ile gs194490 CLint.: 63.0% Vmax: 65.1%

Ile703Val 5.75±1.90 29.0±6.4* 5.66±3.01 Ile703Val rs17011368 Vmax: 2.11-fold Vmax: 69.4%

Asn909Lys N.D. N.D. N.D. Asn909Lys rs566362 CLint.: 22% N.D. Thr910Lys N.D. N.D. N.D. Thr910Lys rs669884 N.D. N.D.

Pro1150Arg N.D. N.D. N.D. Pro1150Arg rs10381052 Vmax :66.0% N.D.

His1221Arg 5.29±1.07 42.3±5.8 8.09±0.97 His1221Arg Kudo et al. 2008 CLint.: 1.82-fold —

Cys1318Tyr 4.47±0.98 24.2±2.8*** 5.49±0.68* Cys1318Tyr rs2295474 CLint.: 43.0% CLint.: 64.7% *Pº0.05, **Pº0.01 and ***Pº0.005 compared to the wild type. ND, not detectable. Values shown are means±SD of 3 independent experiments. 2 (Arg607Gln and Cys1318Tyr) had reduced activity. It has been reported that substitutions of Arg149 and clearance was not significantly altered. The kinetic Thr910 lead to xanthinuria type I,12,14) and their xan- parameters of Pro555Ser and His1221Arg were similar thine-oxidizing activity was found to be below the detec- to those of wild-type XO. Comparisons between xanthine tion limits. Similarly, in our previous study, the and 6-TX oxidation on functional alterations of XO vari- Asn909Lys and Pro1150Arg variants exhibited reduced ants are shown in Table 3. activity when xanthine was used as substrate. These find- ings may be used to interpret clinically adverse effects as- Discussion sociated with the altered metabolism of 6-TX by the vari- In this study, we investigated whether 10 XO variants ants in human. (Arg149Cys, Pro555Ser, Arg607Gln, Thr623Ile, Il- Vmax and/or CLint values of 6-TX were significantly al- e703Val, Asn909Lys, Thr910Lys, Pro1150Arg, tered in the case of the Arg607Gln, Thr623Ile, Il- His1221Arg, and Cys1318Try) vary in their enzymatic e703Val, and Cys1318Tyr XO variants as compared to activity toward the exogenous substrate 6-TX. The wild-type XO. These results regarding alteration in the results revealed that 4 of the variants (Arg149Cys, kinetic parameters of 6-TX when Arg607Gln, Thr623Ile, Asn909Lys, Thr910Lys, and Pro1150Arg) were inactive, and Cys1318Tyr variants were used were similar to those Kinetics of 6-Thioxanthine Metabolism by XO Variants 365 obtained in a previous study on xanthine.22) In our previ- strate 6-TX. ous report,22) V value of xanthine was approximately 2- max References fold higher when the Ile703Val variant was used than when wild-type XO was used. In contrast, Vmax value of 1) Relling, M. V., Hancock, M. L., Boyett, J. M., Pui, C. H. and 6-TX decreased by 69.4% in the case of the Ile703Val Evans, W. E.: Prognostic importance of 6-mercaptopurine dose variant as compared to wild-type XO. Vmax value of xan- intensity in acute lymphoblastic leukemia. 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