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P658. Effects of allopurinol on thiopurine metabolism and gene expression levels in HepG2 cells

S. Haglund1, A. Zimdahl2, S. Vikingsson2, S. Almer3, J. Söderman1, 1Ryhov County Hospital, Division of Medical Diagnostics, Jönköping, Sweden, 2Linköping University, Department of Medical and Health Sciences, Linköping, Sweden, 3Karolinska University Hospital, GastroCentrum, Stockholm, Sweden

Background

Combination therapy with thiopurines (TP) in reduced dose and the xanthine oxidase (XO) inhibitor allopurinol (AP) is safe and effective in patients with previous intolerance to TP. It reverses an unfavorable TP metabolite profile with high meTIMP and low 6TGN concentrations, thereby increasing the therapeutic 6TGN.

In this study we aimed to elucidate the effects of AP on the TP metabolism and gene expression levels in the liver cell line HepG2.

Methods

HepG2 cells (+/− transiently transfected to express XO) were incubated with 6-mercaptopurine (6MP) 6 µM, AP 130 µM or 6MP+AP. Genes previously identified in a microarray study of TP treated patients and genes with a potential relationship with TP metabolism [Haglund S et al. PLoS ONE 8(2): e56989 2013] were analyzed using quantitative real-time PCR. Metabolites (MMP base of meTIMP, 6TG base of 6TGN, 6MP base of TIMP and TX; stems from both TXMP and thioxanthine) were analyzed with IP-RP-HPLC. Experiments were run in triplicates. In Student's t-test a P < 0.05 was considered significant after Benjamini-Hochberg correction for multiple testing. Interactions between gene products identified here and prioritized genes present at susceptibility loci identified for IBD [Jostins L et al. Nature 2012 Nov 1; 491(7422): 119–124] were evaluated using the STRING database v 8.3.

Results

No differences in metabolite concentrations were observed between 6MP vs. 6MP+AP in HepG2 cells +/− XO. However, large variation in metabolite determinations over triplicates was observed.

In HepG2 cells not transfected to express XO 6MP resulted in an up-regulation of DPP4, ENTPD1, GMPR1 and SLX1A. No genes were regulated in these cells by AP alone. In a combined treatment (AP+6MP) compared with 6MP alone, these cells expressed reduced levels of DPP4, ENTPD1, FAM156A, GNB4 and SLC29A2, and increased levels of AOX1, MOCOS and PPAT.

Of these 10 regulated genes 5 interacted with 9 genes present at 9 IBD susceptibility loci. FAM156A, SLX1A1 and SLC29A2 were not present in any network. No genes were regulated by AP, 6MP or the combination treatment in HepG2 cells transfected to express XO.

Conclusion

The effect of AP on gene expression levels seems to require presence of 6MP. The interactions between genes identified here and candidate IBD susceptibility genes represented mainly biological processes associated with purine metabolism, T-cell activation, chemokine signaling and inflammation. Further exploration of these genes in vivo may lead to a better understanding of the mechanisms of AP in modulating the thiopurine metabolism. Possibly an effect of AP on metabolite concentrations in HepG2 cells (+/− XO) was obscured by the large variation in metabolite determinations and the small study number.