To the Editor: Mohammadi and co-workers published a recent paper on the beneficial effect of the plant flavonoid chrysin in a model of acetaminophen (APAP) overdose in rats.(1) The authors treated rats for 14 days with various doses of chrysin and then challenged the animals with 3 doses of 1 500 mg/kg APAP every 2 h. Subsequently, the authors measured a number of parameters related to liver injury, antioxidant defense, and inflammation and concluded that chrysin was hepatoprotective due to strengthen of antioxidant defenses and due to its anti-inflammatory effect.(1) There are a number of serious concerns regarding the model used, the experimental design and the mechanistic conclusions drawn from these experiments. First, the rat is a poor model for APAP hepatotoxicity. The limited injury in rats even after very high overdoses does not reflect the human pathophysiology.(2) Although there is drug metabolism and reactive metabolite formation in rats as indicated by hepatic glutathione depletion and protein adducts formation, there seems to be very limited mitochondrial oxidant stress, JNK activation, and nuclear DNA fragmentation,(2) which are all hallmarks of APAP-induced liver injury in mice and humans.(3) Importantly, extensive centrilobular liver cell necrosis as shown by histology and indicated by >100-fold increases in plasma ALT activities are characteristic for APAP hepatotoxicity after an overdose in mice(2) and in humans.(4) In contrast, the injury in rats is modest even after severe overdoses.(2) These results were confirmed by the authors who showed a minimal, nonsignificant increase in plasma ALT activities and no cell necrosis.(1) Based on these data, it is clear that the rat model does not reflect the degree of injury and the mechanisms of cell death as observed in humans or mice. Hence, it is not the appropriate model to assess the potential hepatoprotective effect of any natural products. The second fundamental problem is the mechanistic conclusions that were proposed by the authors. Chrysin is a flavonoid and these compounds are well-established inhibitors of multiple cytochrome P450 enzymes. Specifically, a recent paper directly showed that chrysin prevents the metabolic activation of APAP in vivo and in vitro by inhibiting cytochrome P450 2E1, 1A2, and 3A4.(5) Thus, chrysin blocks the most upstream event in the toxicity, the formation of the reactive metabolite NAPQI, and as such will completely prevent any downstream events of the toxicity including the secondary sterile inflammatory response, which is based on the degree of injury (Figure 1).(3) Thus, a P450 inhibitor does not allow any conclusions whether the compound has additional effects such as acting as antioxidant or inhibiting inflammatory mediator formation. This raises serious questions regarding the validity of the mechanistic conclusions of the authors about the effect of chrysin. Figure 1. General mechanisms of acetaminophen (APAP) hepatotoxicity and protection by chrysin. The signaling mechanisms of toxicity start with the metabolism of APAP to the reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI) by cytochrome P450 enzymes. NAPQI can be detoxified by glutathione (GSH). However, after an overdose of APAP, GSH is depleted and excess NAPQI binds to mitochondrial proteins, triggers a limited oxidant stress, which is amplified by c-jun N-terminal kinase (JNK) activation. The enhanced oxidant stress causes the mitochondrial membrane permeability transition pore opening with the collapse of the membrane potential and swelling of the matrix, which ruptures the outer membrane and releases intermembrane proteins such as endonuclease G and apoptosis inducing factor (AIF). Both proteins move to the nucleus and cause DNA fragmentation. Extensive mitochondrial damage and nuclear degradation results in cell necrosis with release of damage associated molecular patterns (DAMPs), which trigger a sterile inflammatory response that mainly assists in the removal of cellular debris to prepare for regeneration. Chrysin protects against APAP hepatotoxicity by inhibiting cytochrome P450 enzymes, the most upstream event in the toxicity. Importantly, all other downstream toxic events are also blocked due to inhibition of reactive metabolite formation. The third issue is the experimental design. Most investigations into the protective effect of chemicals using the APAP model, emphasize the clinical importance of APAP-induced hepatotoxicity and acute liver failure. However, although frequently used in studies testing natural products, a pretreatment period of 14 days before APAP exposure is clinically irrelevant. It makes no sense to take an antidote in anticipation of being exposed to an overdose of APAP. The clinically relevant scenario is that the patient is exposed intentionally or unintentionally to an APAP overdose and then seeks medical attention either before or after the onset of liver injury. Thus, antidotes need to be able to interfere with the injury mechanisms and promote recovery. A P450 inhibitor may be partially effective when the overdose of APAP is not yet completely metabolized assuming the compound is rapidly available. Ideally, a good antidote has additional protective mechanisms beyond just inhibiting P450 enzymes as is known for the only clinically approved antidote N-acetylcysteine(6) or a more recently suggested compound 4-methylpyrazole.(7,8) More studies using appropriate models and a clinically relevant experimental design are necessary to fully evaluate the protective mechanisms of chrysin before it can be concluded that chrysin may be a viable candidate as a new antidote against APAP hepatotoxicity. This article references 8 other publications.

中文翻译：

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Mohammadi和同事（2019年）就有关“胰蛋白酶对预防对乙酰氨基酚引起的大鼠肝毒性的作用”致编辑的信。

致编辑：Mohammadi和他的同事发表了一篇关于植物类黄酮菊花精对大鼠对乙酰氨基酚（APAP）过量使用的有益作用的最新论文。（1）作者用不同剂量的菊花精对大鼠进行了14天的治疗，然后每2小时用3剂1500 mg / kg APAP攻击动物。随后，作者测量了许多与肝损伤，抗氧化剂防御和炎症相关的参数，并得出结论认为，由于抗氧化剂防御作用增强和抗炎作用，菊花素对肝脏具有保护作用。（1）有许多严重的问题关于使用的模型，实验设计以及从这些实验中得出的机械结论。首先，大鼠是APAP肝毒性的不良模型。血浆ALT活性无明显增加，且无细胞坏死。（1）基于这些数据，很明显，大鼠模型不能反映人或小鼠的损伤程度和细胞死亡机制。因此，它不是评估任何天然产物潜在的肝保护作用的合适模型。第二个基本问题是作者提出的力学结论。菊花蛋白是类黄酮，这些化合物是多种细胞色素P450酶的公认抑制剂。具体而言，最近的一篇论文直接表明，菊花蛋白可以阻止APAP的代谢活化 它不是评估任何天然产品潜在的肝保护作用的合适模型。第二个基本问题是作者提出的力学结论。菊花蛋白是类黄酮，这些化合物是多种细胞色素P450酶的公认抑制剂。具体而言，最近的一篇论文直接表明，菊花蛋白可以阻止APAP的代谢活化 它不是评估任何天然产品潜在的肝保护作用的合适模型。第二个基本问题是作者提出的力学结论。菊花蛋白是类黄酮，这些化合物是多种细胞色素P450酶的公认抑制剂。具体而言，最近的一篇论文直接表明，菊花蛋白可以阻止APAP的代谢活化体内和体外通过抑制细胞色素P450 2E1、1A2和3A4。（5）因此，菊花素阻断了毒性中最上游的事件，形成了反应性代谢产物NAPQI，因此将完全阻止毒性的任何下游事件，包括继发性无菌炎症反应取决于损伤的程度（图1）。（3）因此，P450抑制剂无法就该化合物是否具有其他作用（例如抗氧化剂或抑制炎症介质的形成）做出任何结论。这就提出了关于作者关于胰蛋白酶作用的机械结论的有效性的严重问题。图1.对乙酰氨基酚（APAP）肝毒性和菊花蛋白保护的一般机制。毒性的信号传导机制始于APAP代谢成反应性代谢物Ñ乙酰基p-苯醌亚胺（NAPQI）通过细胞色素P450酶。NAPQI可以被谷胱甘肽（GSH）解毒。但是，过量服用APAP后，GSH耗尽，过量的NAPQI与线粒体蛋白结合，触发有限的氧化应激，该氧化应激会通过c-jun N末端激酶（JNK）激活而放大。增强的氧化应激导致线粒体膜通透性转变孔打开，膜电位下降，基质膨胀，从而破裂外膜并释放出膜蛋白，例如核酸内切酶G和凋亡诱导因子（AIF）。两种蛋白质都移动到细胞核并引起DNA片段化。广泛的线粒体损伤和核降解导致细胞坏死并释放损伤相关分子模式（DAMP），会触发无菌的炎症反应，主要帮助清除细胞碎片，为再生做准备。菊花蛋白通过抑制细胞色素P450酶（毒性中最上游的事件）来防止APAP的肝毒性。重要的是，由于抑制了反应性代谢产物的形成，所有其他下游毒性事件也被阻断。第三个问题是实验设计。使用APAP模型对化学制品的保护作用进行的大多数研究都强调了APAP诱导的肝毒性和急性肝衰竭的临床重要性。但是，尽管经常在测试天然产物的研究中使用，但在暴露于APAP之前的14天的预处理期与临床无关。在预期会过量服用APAP时服用解毒剂是没有意义的。临床相关情况是患者有意或无意地暴露于过量的APAP，然后在肝损伤发作之前或之后寻求医疗救助。因此，解毒剂需要能够干扰损伤机制并促进恢复。如果过量服用APAP并不能快速代谢，则该化合物可能部分有效。理想地，一种好的解毒剂除了抑制P450酶外，还具有其他保护机制，这是唯一获得临床批准的解毒剂 解毒剂必须能够干扰损伤机制并促进恢复。如果过量服用APAP并不能快速代谢，则该化合物可能部分有效。理想地，一种好的解毒剂除了抑制P450酶外，还具有其他保护机制，这是唯一获得临床批准的解毒剂 解毒剂必须能够干扰损伤机制并促进恢复。如果过量服用APAP并不能快速代谢，则该化合物可能部分有效。理想地，一种好的解毒剂除了抑制P450酶外，还具有其他保护机制，这是唯一获得临床批准的解毒剂N-乙酰半胱氨酸（6）或较新近提出的化合物4-甲基吡唑（7,8）必须进一步进行适当模型和临床相关实验设计的研究，才能全面评估Chrysin的保护机制，然后才能得出结论Chrysin可能可以作为抗APAP肝毒性的新解药的可行候选药物。本文引用了其他8个出版物。