Pelin Marco, Genova Elena, Fusco Laura, Marisat Monzer, Hofmann Ute, Favretto Diego, Lucafò Marianna, Taddio Andrea, Martelossi Stefano, Ventura Alessandro, Stocco Gabriele, Schwab Matthias, Decorti Giuliana
Department of Life Sciences, University of Trieste, I-34127 Trieste, Italy.
PhD School in Reproduction and Developmental Sciences, University of Trieste, I-34127 Trieste, Italy.
Chem Biol Interact. 2017 Sep 25;275:189-195. doi: 10.1016/j.cbi.2017.08.009. Epub 2017 Aug 12.
To apply an innovative LC-MS/MS method to quantify thiopurine metabolites in human hepatocytes and to associate them to cytotoxicity.
Immortalized human hepatocytes (IHH cells) were treated for 48 and 96 h, with 1.4 × 10 M azathioprine and 1.1 × 10 M mercaptopurine, concentrations corresponding to the IC 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).
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).
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.
应用一种创新的液相色谱 - 串联质谱法（LC-MS/MS）对人肝细胞中的硫嘌呤代谢物进行定量，并将其与细胞毒性相关联。
用1.4×10⁻⁶M硫唑嘌呤和1.1×10⁻⁶M巯嘌呤处理永生化人肝细胞（IHH细胞）48小时和96小时，这些浓度对应于先前细胞毒性分析中96小时暴露后计算出的IC值。处理后，收集细胞进行LC-MS/MS分析，以定量11种不同磷酸化水平的硫嘌呤代谢物，并通过台盼蓝排斥试验对活细胞进行计数，以确定硫嘌呤对细胞生长和存活的体外影响。通过方差分析（ANOVA）确定统计学显著性。
硫唑嘌呤和巯嘌呤具有显著的时间依赖性细胞毒性作用（方差分析p值 = 0.012），48小时后与对照组相比活细胞计数分别为55.5%和67.5%，96小时后分别为23.7%和36.1%；两种药物之间未观察到显著差异。硫嘌呤代谢物的定量表明，最丰富的代谢物是TIMP，在48小时和96小时后分别占总代谢物的57.1%和40.3%。硫嘌呤代谢物的总绝对浓度随时间下降：总平均含量从469.9 pmol/百万细胞降至83.6 pmol/百万细胞（方差分析p值 = 0.0070）。然而，考虑硫嘌呤代谢物的相对量，TGMP含量从11.4%显著增加至26.4%（方差分析p值 = 0.017）。仅对于MeTIMP可检测到硫嘌呤效应与活细胞计数之间存在显著关联：较低的MeTIMP浓度与较低的细胞存活率相关（方差分析p值 = 0.011）。此外，MeTIMP与TGMP代谢物之间的比率与细胞存活率直接相关（方差分析p值 = 0.037）。
在人肝细胞模型中对硫嘌呤代谢物进行详细定量，为硫鸟嘌呤和甲基硫代肌苷核苷酸与细胞活力之间的关联提供了有用的见解。
