Marco Pelin1, Elena Genova2, Laura Fusco3, Monzer Marisat4, Ute Hofmann5, Diego Favretto6, Marianna Lucafò4, Andrea Taddio7, Stefano Martelossi6, Alessandro Ventura7, Gabriele Stocco8, Matthias Schwab9, Giuliana Decorti7. 1. Department of Life Sciences, University of Trieste, I-34127 Trieste, Italy. 2. PhD School in Reproduction and Developmental Sciences, University of Trieste, I-34127 Trieste, Italy. 3. PhD School in Chemistry, University of Trieste, I-34127 Trieste, Italy. 4. Department of Medical, Surgical and Health Sciences, University of Trieste, I-34127 Trieste, Italy. 5. Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, D-70376 Stuttgart, Germany; University of Tübingen, Tübingen, Germany. 6. Institute for Maternal and Child Health I.R.C.C.S. Burlo Garofolo, I-34137 Trieste, Italy. 7. Department of Medical, Surgical and Health Sciences, University of Trieste, I-34127 Trieste, Italy; Institute for Maternal and Child Health I.R.C.C.S. Burlo Garofolo, I-34137 Trieste, Italy. 8. Department of Life Sciences, University of Trieste, I-34127 Trieste, Italy. Electronic address: stoccog@units.it. 9. Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, D-70376 Stuttgart, Germany; University of Tübingen, Tübingen, Germany; Department of Clinical Pharmacology, University Hospital Tübingen, D-72076 Tübingen, Germany; Department of Pharmacy and Biochemistry, University of Tübingen, D-72076 Tübingen, Germany.
Abstract
AIM: To apply an innovative LC-MS/MS method to quantify thiopurine metabolites in human hepatocytes and to associate them to cytotoxicity. METHODS: Immortalized human hepatocytes (IHH cells) were treated for 48 and 96 h, with 1.4 × 10-4 M azathioprine and 1.1 × 10-3 M mercaptopurine, concentrations corresponding to the IC50 values calculated after 96 h exposure in previous cytotoxicity analysis. After treatments, cells were collected for LC-MS/MS analysis to quantify 11 thiopurine metabolites with different level of phosphorylation and viable cells were counted by trypan blue exclusion assay to determine thiopurines in vitro effect on cell growth and survival. Statistical significance was determined by analysis of variance (ANOVA). RESULTS: Azathioprine and mercaptopurine had a significant time-dependent cytotoxic effect (p-value ANOVA = 0.012), with a viable cell count compared to controls of 55.5% and 67.5% respectively after 48 h and 23.7% and 36.1% after 96 h; no significant difference could be observed between the two drugs. Quantification of thiopurine metabolites evidenced that the most abundant metabolite was TIMP, representing 57.1% and 40.3% of total metabolites after 48 and 96 h. Total thiopurine metabolites absolute concentrations decreased over time: total mean content decreased from 469.9 pmol/million cells to 83.6 pmol/million cells (p-value ANOVA = 0.0070). However, considering the relative amount of thiopurine metabolites, TGMP content significantly increased from 11.4% cells to 26.4% (p-value ANOVA = 0.017). A significant association between thiopurine effects and viable cell counts could be detected only for MeTIMP: lower MeTIMP concentrations were associated with lower cell survival (p-value ANOVA = 0.011). Moreover, the ratio between MeTIMP and TGMP metabolites directly correlated with cell survival (p-value ANOVA = 0.037). CONCLUSION: Detailed quantification of thiopurine metabolites in a human hepatocytes model provided useful insights on the association between thioguanine and methyl-thioinosine nucleotides with cell viability.
AIM: To apply an innovative LC-MS/MS method to quantify thiopurine metabolites in human hepatocytes and to associate them to cytotoxicity. METHODS: Immortalized human hepatocytes (IHH cells) were treated for 48 and 96 h, with 1.4 × 10-4 M azathioprine and 1.1 × 10-3 M mercaptopurine, concentrations corresponding to the IC50 values calculated after 96 h exposure in previous cytotoxicity analysis. After treatments, cells were collected for LC-MS/MS analysis to quantify 11 thiopurine metabolites with different level of phosphorylation and viable cells were counted by trypan blue exclusion assay to determine thiopurines in vitro effect on cell growth and survival. Statistical significance was determined by analysis of variance (ANOVA). RESULTS:Azathioprine and mercaptopurine had a significant time-dependent cytotoxic effect (p-value ANOVA = 0.012), with a viable cell count compared to controls of 55.5% and 67.5% respectively after 48 h and 23.7% and 36.1% after 96 h; no significant difference could be observed between the two drugs. Quantification of thiopurine metabolites evidenced that the most abundant metabolite was TIMP, representing 57.1% and 40.3% of total metabolites after 48 and 96 h. Total thiopurine metabolites absolute concentrations decreased over time: total mean content decreased from 469.9 pmol/million cells to 83.6 pmol/million cells (p-value ANOVA = 0.0070). However, considering the relative amount of thiopurine metabolites, TGMP content significantly increased from 11.4% cells to 26.4% (p-value ANOVA = 0.017). A significant association between thiopurine effects and viable cell counts could be detected only for MeTIMP: lower MeTIMP concentrations were associated with lower cell survival (p-value ANOVA = 0.011). Moreover, the ratio between MeTIMP and TGMP metabolites directly correlated with cell survival (p-value ANOVA = 0.037). CONCLUSION: Detailed quantification of thiopurine metabolites in a human hepatocytes model provided useful insights on the association between thioguanine and methyl-thioinosine nucleotides with cell viability.