Paracetamol/acetaminophen (N-acetyl-p-aminophenol, APAP) is a top selling analgesic used in more than 600 prescription and non-prescription pharmaceuticals. To study efficiently some of the potential undesirable effects associated with increasing APAP consumption (e.g., developmental disorders, drug-induced liver injury), there is a need to improve current APAP biomonitoring methods that are limited by APAP short half-life. Here, we demonstrate using high-resolution mass spectrometry (HRMS) in several human studies that APAP thiomethyl metabolite conjugates (S-methyl-3-thioacetaminophen sulfate and S-methyl-3-thioacetaminophen sulphoxide sulfate) are stable biomarkers with delayed excretion rates compared to conventional APAP metabolites, that could provide a more reliable history of APAP ingestion in epidemiological studies. We also show that these biomarkers could serve as relevant clinical markers to diagnose APAP acute intoxication in overdosed patients, when free APAP have nearly disappeared from blood. Using in vitro liver models (HepaRG and primary human hepatocytes), we then confirm that these thiomethyl metabolites are directly linked to the toxic N-acetyl-p-benzoquinone imine (NAPQI) elimination, and produced via an overlooked pathway called the thiomethyl shunt pathway. Further studies will be needed to determine whether the production of the reactive hepatotoxic NAPQI metabolites is currently underestimated in human. Nevertheless, these biomarkers could already serve to improve APAP human biomonitoring, and investigate, for instance, inter-individual variability in NAPQI production to study underlying causes involved in APAP-induced hepatotoxicity. Overall, our finding demonstrate the potential of exposomics-based HRMS approach to advance towards a better precision for human biomonitoring.

扑热息痛/对乙酰氨基酚（N-乙酰基-对氨基苯酚，APAP）是一种最畅销的镇痛药，用于 600 多种处方药和非处方药。为了有效地研究与增加 APAP 消耗相关的一些潜在不良影响（例如发育障碍、药物引起的肝损伤），需要改进目前受到 APAP 半衰期短限制的 APAP 生物监测方法。在这里，我们在几项人体研究中使用高分辨率质谱 (HRMS) 证明，APAP 硫甲基代谢物结合物（S-甲基-3-硫代乙酰氨基酚硫酸盐和 S-甲基-3-硫代乙酰氨基酚亚硫酸盐硫酸盐）是稳定的生物标志物，与与传统的 APAP 代谢物相比，这可以在流行病学研究中提供更可靠的 APAP 摄入历史。我们还表明，当游离 APAP 几乎从血液中消失时，这些生物标志物可以作为相关的临床标志物来诊断服药过量患者的 APAP 急性中毒。使用体外肝脏模型（HepaRG 和原代人肝细胞），我们确认这些硫甲基代谢物与有毒的 N-乙酰基对苯醌亚胺 (NAPQI) 消除直接相关，并通过称为硫甲基分流途径的被忽视的途径产生。需要进一步的研究来确定目前人类中反应性肝毒性 NAPQI 代谢物的产生是否被低估。尽管如此，这些生物标志物已经可以用于改善 APAP 人类生物监测，并研究 NAPQI 产生的个体间变异性，以研究 APAP 诱导的肝毒性的根本原因。总的来说，我们的发现证明了基于暴露组学的 HRMS 方法在提高人类生物监测精度方面的潜力。