ROSs

The excessive accumulation of ROS is the main cause of oxidative stress, which can be caused by the DILI-causing drugs through different mechanisms [26]. Mitochondria are the main sites for ROS, which includes peroxides, hydrogen peroxide (H₂O₂), and hydroxyl groups, produced in the cell (Figure 1) [30, 31]. Under physiological conditions, most of the electrons donated to mitochondrial outer membrane (MOM) migrate downward to cytochrome c oxidase, where they react with O₂ and H⁺ to yield H₂O₂. However, some electrons may react directly with O₂ to form O₂^•–, and spontaneously disproportionate to O₂ and H₂O₂ with manganese superoxide dismutase (MnSOD) [32]. H₂O₂ is detoxified into H₂O₂ via mitochondrial glutathione (GSH) [33] peroxidase and peroxiredoxins, or active myeloperoxidase reacts with iron (Fe²⁺) to become the highly reactive hydroxyl radical (OH^•) [31, 34–36]. ROS is not only a signal transduction molecule under pathological conditions, but also important for maintaining physiological balance, depending on the intensity and duration of oxidative stress. Low-intensity ROS participates in immune regulation and signal transduction, maintains energy level, kills bacteria and parasites, and eliminates toxins, and high-level ROS promotes cell apoptosis or autophagy which cause damage to normal cells and tissues [37, 38].

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Figure 1. 

Oxidative stress mechanism caused by DILI. The main pro-oxidation mechanism is characterized by the production of OH^• from H₂O₂ and Fe²⁺ through the Fenton reaction. The main source of H₂O₂ is the peroxisome of long-branched fatty acids β-oxidation. OH^• can cause LPO of organelle membranes, damage mitochondrial metabolism through the production of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(mPTP) and mitochondrial DNA (mtDNA) mutation, increase the pro-apoptotic activity, and produce malondialdehyde (MDA). In addition, chronic Fe²⁺ overload may also activate nuclear factor-kappa B (NF-κB) enhancing the production of inducible nitric oxide synthase (iNOS), resulting in the increase of nitric oxide (NO), which leads to the reaction with the superoxide anion RNS. Figure was created with BioRender.com. ERK1/2: extracellular signal-regulated kinase 1/2; HO-1: heme oxygenase-1; JNK: c-Jun N-terminal kinase; SOD1: superoxide dismutase 1

The liver is a crucial organ for the production of ROSs, which are either converted from endogenous products during liver metabolism or by intake (e.g., APAP). APAP is a non-steroidal anti-inflammatory drug (NSAID) with definite hepatotoxicity in clinical application [12]. After rapid absorption, APAP is mainly metabolized through phase II reaction (sulfation or glucuronidation), converting into non-toxic compounds and excreting into urine. At present, the major reason of hepatotoxicity caused by APAP is its metabolite N-acetyl-p-benzoquinoneimine (NAPQI). A small amount of APAP is metabolized by cytochrome P450 isoenzyme 2E1 (Cyp2E1) to produce intermediates, which produce NAPQI. NAPQI is directly excreted into bile by interaction with GSH to play a detoxification role. After overdose, APAP overwhelms phase II reaction pathway. After NAPQI depletes GSH, excessive NAPQI interferes with the complex I/II of mitochondrial electron transfer chain (ETC), resulting in the leakage of electrons from ETC to O₂, thus forming O₂^•– [39, 40]. The interaction of NAPQI with target DNA and protein in mitochondria and the formation of protein adducts lead to oxidative stress, mitochondrial dysfunction [26, 41]. Furthermore, other drugs produce ROS through different mechanisms. Diethylaminoethoxyhexestrol, perhexiline, amiodarone, and tamoxifen selectively accumulate and reach high concentrations in the mitochondrial matrix, thereby blocking the transfer of electrons along the MOM, increasing production of ROS [42–44].

As mentioned earlier, cytochrome P450 (Cyp450) plays a major role in the detoxification process of APAP. The latest literature also shows that the liver injury caused by polymedication in coronavirus disease 2019 (COVID-19) patients is also considered to be concentrated in the catalytic cycle of Cyp450 with its isoforms and the production of ROS in the oxidative stress environment [45]. In some cases of idiosyncratic DILI caused by drugs, anti-Cyp450 was detected in the blood of patients, representing an association between these drugs and Cyp450 in line with the proposed involvement of the adaptive immune system [46]. Oxidative stress in idiosyncratic DILI includes mitochondrial stress and microsomal stress, which may be related to ROS produced by Cyp450 in these subcellular domains of hepatocytes, but the specific ROS type involved has not been determined [46].

RNS

Posttranslational modifications involving various active nitrogen species have a common precursor, NO that is the most well-known free radical acting as a signaling molecule. RNS includes nitrogen free radicals and non-free radicals, which are derived from NO and O₂^•– by iNOS and nicotinamide adenine dinucleotide phosphate (NADPH), respectively (Figure 1) [47, 48]. Under steady-state, NOS expression is regulated by signal pathways including mitogen-activated protein kinase (MAPK) and JNK/signal transducer of activators of transcription (STAT), which indicates that the inducible production of NO is strictly controlled [49]. In the pathological state, O₂^•− reacts with NO to increase ONOO^– via an up-regulation of NOS and endothelial NOS [50]. Upon APAP overdose, excessive NAPQI can also induce the formation of OH^• free radicals and react with NO to form ONOO^– [51]. Since O₂^•− hardly passes through the hepatocyte membrane, the production of ONOO^– only occurs in mitochondria. The ONOO^– can react with GSH to detoxify [52]. Consequently, GSH is exhausted due to these excessive reactions, resulting in the accumulation of ONOO^– [53]. High reactivity and powerful oxidant ONOO^– can cause nitration of protein tyrosine residues, resulting in mtDNA damage and membrane pore opening [53]. In addition ONOO^– reacts directly with carbon dioxide producing nitrosoperoxycarbonate (ONOOCO2^–), which decomposes into nitrogen dioxide (NO₂) and carbonate (^•CO₃^–) with a reactivity similar to that of OH^• radicals [54]. The production of ^•CO₃^– and OH^• radicals and the capacity of ONOO^– to react with metal centers leads to hepatocyte toxicity [49].

The JNK signaling pathway

The JNK is a subclass of the MAPK family, which was confirmed to play a vital role in APAP hepatotoxicity by mediating APAP-induced mitochondrial targeting and oxidative stress (Figure 1) [55, 56]. JNK has 10 subtypes from three genes, only JNK1 and JNK2 genes are widely expressed in the liver [57]. During JNK activation the first step occurs when the O₂^•− production from mitochondrial respiratory complex III spills into the cytosol and triggers a signaling cascade [58]. Then, NAPQI and subsequent mitochondrial-derived ROS activates upstream kinases, including protein kinase C (PKC), MAPK, such as mixed lineage kinase 3 (MLK3) and apoptosis signal-regulated kinase 1 (ASK1), eventually leading to JNK activation. MLK3 is activated by oxidative stress and regulates the early stage of JNK activation; however, ASK1 controls the late stage of JNK activation induced by APAP [13, 59, 60]. The activated JNK [phospho-c-Jun N-terminal kinase (p-JNK)] combines with its target SH3 domain-binding protein (Sab) on the mitochondrial membrane, leading to a self-sustaining activation circuit of ROS production and JNK activation, which eventually leads to the opening of mitochondrial membrane permeability transition pore (MPT) [61]. After the toxic stress of APAP, JNK phosphorylates Sab, which causes Sab to release type 6 protein tyrosine phosphatase (PTPN6) [62, 63]. The released PTPN6 is activated and transferred to the intima, where it dephosphorylates and inactivates Src in the membrane gap. With the deactivation of Src, the ETC chain is blocked and the generation of ROS is enhanced [13]. Switching on MPT induces the collapse of mitochondrial membrane potential, thus ceasing ATP synthesis and releasing the membrane proteins which initiate necrosis [64].

A great quantity of experiments demonstrated that hepatocytes can be protected from cell death by knocking down, knocking out, or inhibiting JNK/Sab to interfere with this pathway at any point [59–61, 65]. The key point is that docking site of Sab or inhibiting the binding between JNK and Sab can protect against APAP toxicity, indicating that the interaction of these two proteins is the key step of cell death [13, 66]. In Sab knockout mice, the mitochondrial translocation of p-JNK was totally inhibited regardless of various isoforms of JNK [66, 67]. Therefore, in animal studies, fomepizole can attenuate APAP-induced liver injury by inhibiting JNK during post-metabolic treatment, and its interference with Cyp2E1 metabolism [68, 69]. The latest research found that platanoides may reduce oxidative stress and nitrification stress by preventing continuous JNK activation, which can effectively prevent APAP-related hepatotoxicity [23]. Collectively, this evidence demonstrated that JNK plays an important role in activating oxidative stress and triggering DILI, which also provides targeted targets for clinical treatment.

LPO

Lipid peroxide acts as a vital role in the balance of oxidative stress between pro-oxidation and antioxidant activities, LPO may be involved in the mechanism of cell death during DILI [8]. Lipids present hydrophobicity in cells and serve as metabolic fuel, supporting membrane structure and selective cell membrane transport. When the oxidative metabolism in mitochondria is abnormal, the electronic leakage increases the uncoupling of oxidative phosphorylation, resulting in the production of oxygen free radicals which may induce LPO reactions (Figure 1) [70]. LPO is a free radical reaction process, which is triggered by the formation of OH^• radicals (Fe²⁺-dependent Fenton reaction) from H₂O₂ combined with the production of lipid radicals, leading to the destruction of polyunsaturated fatty acids (PUFAs) [71]. LPO triggered subsequently rapid destruction of membrane potential and ionic gradient, and promoted cell necrosis. This is also one of the mechanisms of DILI caused by anti-epileptic drugs [12]. Mitochondrial oxidative metabolism is blocked by drugs, resulting in abnormal metabolism of free fatty acids (FFAs), increased anaerobic fermentation, lactic acid accumulation, and generation of ROS. ROS oxidizes liposomes and causes LPO, induces mtDNA damage by changing mitochondrial membrane permeability, further promoting liver injury [72]. Furthermore, experimental models of APAP-induced hepatotoxicity showed that vitamin E pre-treatment can prevent massive LPO induce to liver damage [73]. However, this is a matter of debate since there are conflicting results and it is still uncertain whether vitamin E protects against liver injury caused by APAP [74–76]. Research showed that animals fed a regular diet with sufficient fat-soluble antioxidants to prevent a robust increase of LPO, therefore LPO was not the relevant injury mechanism of APAP-induced liver injury in quantity, in addition, LPO was the cause of the injury rather than the outcome [73, 77–80].

Moreover, there is a controversy whether Fe²⁺ as a promoter of LPO. Studies shown that protein nitration and LPO can lead to APAP hepatotoxicity after Fe²⁺ overload [81]. PUFAs react with ROS to generate lipid hydroperoxides (LOOHs), which react with Fe²⁺ via Fenton reaction to produce highly reactive lipid free radicals [80]. Lipid free radicals further react with other PUFAs propagating a chain reaction. If is an uncontrolled episode, it leads to the accumulation of lipid peroxides and induces Fe²⁺-mediated cell death [82]. In addition, Fe²⁺ has also been proved to catalyze the nitrification process of ONOO⁻ to proteins such as MnSOD, which is beneficial to oxidative stress and mitochondrial dysfunction by APAP-mediated cell death [83, 84]. Deferoxamine can inhibit ONOO^–-mediated protein nitrification, which demonstrates that Fe²⁺chelators have partial protective effect on APAP-induced liver injury [85]. As mentioned earlier, LPO was not sufficient in quantity to cause APAP-induced cell death. Therefore, under normal conditions, ferroptosis is not a cell death mode related to APAP hepatotoxicity [86], but may affect DILI through classical Fenton reaction and other mechanisms.

IRE1 signaling

As an essential ER stress sensor for UPR in animals and plants, IRE1 detects ER homeostasis and triggers UPR through cytoplasmic kinase domain and RNase domain under the stimulation [95–98]. Under ER stress, IRE1 RNase is activated by conformational changes, self-phosphorylation, and higher oligomerization (Figure 2) [96, 99, 100]. The primary function of IRE1-dependent spliced X-box binding protein 1 (sXBP1) signal transduction is to protect the liver from the stress caused by stimulation [101]. Upon activation of IRE1, multiple downstream signaling of UPR is initiated through unconventional splicing of the transcription factor XBP1 or post-transcriptional modification through controlled inositol-requiring enzyme 1 dependent decay (RIDD) of multiple substrates [97–99]. Splicing occurs through XBP1 mRNA to the translation of sXBP1 protein, which is a transcription factor that induces genes involved in chaperoning proteins and degrades misfolded proteins in ER, thus triggering inflammatory reaction and promoting cell apoptosis mediated by apoptosis ASK1 and JNK [99, 102].

With the main cause of DILI [7, 99, 103]—APAP-mediated hepatoxicity—a great deal of data indicated a role for ER stress. The mechanism of APAP hepatotoxicity is similar in humans and mice [104]. The sXBP1 or unspliced X-box binding protein 1 (uXBP1) in human HepaRG cells was consistent with the findings in mice [105]. Under ER stress, the IRE1α-XBP1 arm of the UPR response plays a critical role in APAP-induced liver injury via upregulating the activity of Cyp450 [106]. In XBP1-deleted mice, overexpression of IRE1 in liver led to reduced JNK activation, and protected against APAP via the inhibition of Cyp450 activity [105–107]. This was linked to the recovery of GSH, which prevented severe oxidative stress [105]. In recent years, increasing studies demonstrated that autophagy is activated to respond to APAP-induced liver injury [108].

Furthermore, IRE1α can also function via the RIDD pathway. IRE1α accelerates the degradation of mRNAs encoding mostly ER-targeted proteins, resulting in a decrease of protein entering the ER membrane during ER stress [109, 110]. Meanwhile, XBP1-deficient mice showed decreased damage caused by APAP due to enhancement of autophagy and activating RIDD [105, 106].

Once cell stress reaches its critical threshold, over-activation of the JNK signaling pathway and mitochondrial dysfunction are considered as major cellular events in APAP-induced liver injury [111, 112]. p-JNK translocates to the MOM and finally induces mitochondrial dysfunction which contributes to hepatocyte necrosis [80]. Mitochondrial injury is likely to positively regulate the sustained activation of JNK cell death pathways and the onset of ER stress, which may be an intertwined mechanism [113]. After APAP treatment, the intense JNK phosphorylation and upregulated JNK1 expression in JNK-deleted liver correlated with a sharp increase in serum transaminases [22]. Gunawan’s study [114] showed that disruption of JNK2 instead of JNK1 can suppress liver injury, indicating the importance of JNK2 in hepatotoxicity caused by APAP. Another paper indicated that extensive necrosis occurred in APAP-treated JNK hepatocyte-specific knockout mice, and further demonstrating that combined activity of JNK1 and JNK2 can prevent APAP-induced necrosis by controlling the generation of oxidative stress [114].

PERK signaling

PERK is a member of the eIF2α kinase family, which protects cells involved in various stimuli by biasing general protein synthesis, but initiating stress-related protein production [115]. In ER stress, after BiP/GRP78 dissociation, increasing expression of phosphorylated eIF2α/PERK signaling is activated by APAP hepatotoxicity (Figure 2) [94]. PERK temporarily inhibits the translation of general protein via elF2α phosphorylation. Notably, phosphorylated elF2α selectively enhanced mRNA translation including ATF4 transcription factor to mediate UPR [116–119]. During the ER stress regulation, ATF4 activates UPR target gene, which encodes proteins necessary for antioxidant reaction and amino acid synthesis and transport [120]. ATF4 is also a transcriptional activation CHOP, which acts as a transcriptional inhibitor downstream of PERK and IRE1. The main function of CHOP is to induce apoptosis by various kinds of mechanisms in ER stress [117], thereby mediating cell apoptosis via mitochondrial pathway or DR pathway that further exacerbate hepatotoxicity [1, 121]. Conversely, decreased expression of CHOP, BiP/GRP78, and JNK are related to protect against APAP-induced hepatotoxicity which are possibly mediated by increased proliferation [94, 105].

ATF signaling

Similar to IRE1 and PERK, ATF6 contains an ER luminal stress-sensing domain and a cytoplasmic enzymatic domain, which is one of ER transmembrane proteins. In response to a sublethal dose of APAP-mediated ER stress, the transcription factor ATF6 is activated (Figure 2) [122]. ATF6 dissociates from BiP/GRP78 to Golgi apparatus, where it is hydrolyzed into the nuclear form of ATF6 [123]. The cleavage transcription factor domain of ATF6 regulates UPR in the nucleus, including CHOP, XBP1, and protein chaperones such as BiP/GRP78 [120, 124, 125]. Additionally, ATF6 forms a heterodimer with IRE1-XBP1 pathway to activate the genes involved in ERAD [126].