Function-guided proximity mapping unveils electrophilic-metabolite sensing by proteins not present in their canonical locales.

Enzyme-assisted posttranslational modifications (PTMs) constitute a major means of signaling across different cellular compartments. However, how nonenzymatic PTMs-despite their direct relevance to covalent drug development-impinge on cross-compartment signaling remains inaccessible as current target-identification (target-ID) technologies offer limited spatiotemporal resolution, and proximity mapping tools are also not guided by specific, biologically-relevant, ligand chemotypes. Here we establish a quantitative and direct profiling platform (Localis-rex) that ranks responsivity of compartmentalized subproteomes to nonenzymatic PTMs. In a setup that contrasts nucleus- vs. cytoplasm-specific responsivity to reactive-metabolite modification (hydroxynonenylation), ∼40% of the top-enriched protein sensors investigated respond in compartments of nonprimary origin or where the canonical activity of the protein sensor is inoperative. CDK9-a primarily nuclear-localized kinase-was hydroxynonenylated only in the cytoplasm. Site-specific CDK9 hydroxynonenylation-which we identified in untreated cells-drives its nuclear translocation, downregulating RNA-polymerase-II activity, through a mechanism distinct from that of commonly used CDK9 inhibitors. Taken together, this work documents an unmet approach to quantitatively profile and decode localized and context-specific signaling/signal-propagation programs orchestrated by reactive covalent ligands.

Models of Idiosyncratic Drug-Induced Liver Injury.

Drug-induced liver injury (DILI) is a leading cause of attrition during the early and late stages of drug development and after a drug is marketed. DILI is generally classified as either intrinsic or idiosyncratic. Intrinsic DILI is dose dependent and predictable (e.g., acetaminophen toxicity). However, predicting the occurrence of idiosyncratic DILI, which has a very low incidence and is associated with severe liver damage, is difficult because of its complex nature and the poor understanding of its mechanism. Considering drug metabolism and pharmacokinetics, we established experimental animal models of DILI for 14 clinical drugs that cause idiosyncratic DILI in humans, which is characterized by the formation of reactive metabolites and the involvement of both innate and adaptive immunity. On the basis of the biomarker data obtained from the animal models, we developed a cell-based assay system that predicts the potential risks of drugs for inducing DILI. These findings increase our understanding of the mechanisms of DILI and may help predict and prevent idiosyncratic DILI due to certain drugs.

Primaquine-5,6-orthoquinone is directly hemolytic to older G6PD deficient RBCs in a humanized mouse model.

Primaquine and tafenoquine are the only approved drugs that can achieve a radical cure for Plasmodium vivax malaria but are contraindicated in patients who are deficient in glucose 6-phosphate dehydrogenase (G6PDd) due to risk of severe hemolysis from reactive oxygen species (ROS) generated by redox cycling of drug metabolites. 5-hydroxyprimaquine and its quinone-imine cause robust redox cycling in red blood cells (RBCs), but are so labile as to not be detected in blood or urine. Rather, the 5,8-quinoneimine is rapidly converted into primaquine-5,6-orthoquinone (5,6-POQ) that is then excreted in the urine. The extent to which 5,6-POQ contributes to hemolysis remains unclear, although some have suggested that it is a minor toxin that should be used predominantly as a surrogate to infer levels of 5-hydroxyprimaquine. In this report, we describe a novel humanized mouse model of the G6PD Mediterranean variant (hG6PDMed-) that recapitulates the human biology of RBC age dependent enzyme decay, as well as an isogenic matched control mouse with human non-deficient G6PD hG6PDND In vitro challenge of RBCs with 5,6-POQ causes increased generation of superoxide and methemoglobin. Infusion of treated RBCs shows that 5,6-POQ selectively causes in vivo clearance of older hG6PDMed- RBCs. These findings support the hypothesis that 5,6-POQ directly induces hemolysis and challenges the notion that 5,6-POQ is an inactive metabolic waste product. Indeed, given the extreme lability of 5-hydroxyprimaquine and the relative stability of 5,6-POQ, these data raise the possibility that 5,6-POQ is a major hemolytic primaquine metabolite in vivo. Significance Statement These findings demonstrate that 5,6-POQ, which has been suggested to be an inert waste product of active primaquine metabolites, directly induces ROS that lead specifically to removal of older G6PDd RBCs from circulation. As 5,6-POQ is relatively stable compared to other active primaquine metabolites, these data support the hypothesis that 5,6-POQ is a major toxin in primaquine induced hemolysis. In addition, a new model of G6PDd is used to show that young G6PDd RBCs are resistant to primaquine induced hemolysis.

Diversity-Orientated Synthesis and Biological Properties of Compounds Based on the N-Phenylquinoneimine Scaffold.

The N-phenylquinoneimine scaffold is a versatile synthetic platform that has gained significant attention in the field of drug discovery due to its structural diversity and capacity to interact with biologically relevant targets. This review explores established synthetic methodologies and highlights the significant biological activities exhibited by compounds derived from this scaffold, their implications for medicinal chemistry, and the development of novel therapeutics.

Metabolic activation of tyrosine kinase inhibitors: recent advance and further clinical practice.

At present, receptor tyrosine kinase signaling-related pathways have been successfully mediated to inhibit tumor proliferation and promote anti-angiogenesis effects for cancer therapy. Tyrosine kinase inhibitors (TKIs), a group of novel chemotherapeutic agents, have been applied to treat diverse malignant tumors effectively. However, the latent toxic and side effects of TKIs, such as hepatotoxicity and cardiotoxicity, limit their use in clinical practice. Metabolic activation has the potential to lead to toxic effects. Numerous TKIs have been demonstrated to be transformed into chemically reactive/potentially toxic metabolites following cytochrome P450-catalyzed activation, which causes severe adverse reactions, including hepatotoxicity, cardiotoxicity, skin toxicity, immune injury, mitochondria injury, and cytochrome P450 inactivation. However, the precise mechanisms of how these chemically reactive/potentially toxic species induce toxicity remain poorly understood. In addition, we present our viewpoints that regulating the production of reactive metabolites may decrease the toxicity of TKIs. Exploring this topic will improve understanding of metabolic activation and its underlying mechanisms, promoting the rational use of TKIs. This review summarizes the updated evidence concerning the reactive metabolites of TKIs and the associated toxicities. This paper provides novel insight into the safe use of TKIs and the prevention and treatment of multiple TKIs adverse effects in clinical practice.

Molecular mechanisms of hepatotoxicity induced by compounds occurring in Evodiae Fructus.

Evodiae Fructus (EF) is a common herbal medicine with thousands of years of medicinal history in China, which has been demonstrated with many promising pharmacological effects on cancer, cardiovascular diseases and Alzheimer's disease. However, there have been increasing reports of hepatotoxicity associated with EF consumption. Unfortunately, in a long term, many implicit constituents of EF as well as their toxic mechanisms remain poorly understood. Recently, metabolic activation of hepatotoxic compounds of EF to generate reactive metabolites (RMs) has been implicated. Herein, we capture metabolic reactions relevant to hepatotoxicity of these compounds. Initially, catalyzed by the hepatic cytochrome P450 enzymes (CYP450s), the hepatotoxic compounds of EF are oxidized to generate RMs. Subsequently, the highly electrophilic RMs could react with nucleophilic groups contained in biomolecules, such as hepatic proteins, enzymes, and nucleic acids to form conjugates and/or adducts, leading to a sequence of toxicological consequences. In addition, currently proposed biological pathogenesis, including oxidative stress, mitochondrial damage and dysfunction, endoplasmic reticulum (ER) stress, hepatic metabolism disorder, and cell apoptosis are represented. In short, this review updates the knowledge on the pathways of metabolic activation of seven hepatotoxic compounds of EF and provides considerable insights into the relevance of proposed molecular hepatotoxicity mechanisms from a biochemical standpoint, for the purpose of providing a theoretical guideline for the rational application of EF in clinics.

Chemical metabolite synthesis and profiling: Mimicking in vivo biotransformation reactions.

Biotransformation was previously viewed as merely the structural characterization of drug metabolites, and it was performed only when drug candidates entered clinical development. The synthesis of drug metabolites is crucial to the drug development process because it generates either pharmacologically active, inactive, or reactive molecules and hence their characterization and comprehensive pharmacological evaluation is necessary. The chemical metabolite synthesis is very challenging due to the complex structures of many drug molecules, presence of multiple stereocenters, poor reaction yields, and the formation of unwanted by-products. Drug metabolites and their chemical synthesis have immense significance in the drug discovery process. The chemical synthesis of metabolites facilitates on- or off-target pharmacological and toxicological evaluations at the easiest. In a broader view metabolite could be a target lead molecule for drug design, toxic reactive metabolites, pharmaceutical standards for bioanalytical methods, etc. Collectively these metabolite information dossiers will aid regulatory agencies such as the EMA and FDA in maintaining strict vigilance over drug manufacturers with regard to the safety of NCE's and their hidden metabolites. Herein, we are presenting a systematic compilation of chemical and biocatalytic strategies reported to date for pharmaceutical drug metabolite synthesis. This review report is very useful for the laboratory synthesis of new drug metabolites, and their preclinical biological evaluation could aid in the detection of early threats (alerts) in drug discovery, eliminate the toxicity profile, explore newer pharmacology, and delivering a promising and safe drug candidate to humankind.

The endoplasmic reticulum participated in drug metabolic toxicity.

Covalent binding of reactive metabolites formed by drug metabolic activation with biological macromolecules is considered to be an important mechanism of drug metabolic toxicity. Recent studies indicate that the endoplasmic reticulum (ER) could play an important role in drug toxicity by participating in the metabolic activation of drugs and could be a primarily attacked target by reactive metabolites. In this article, we summarize the generation and mechanism of reactive metabolites in ER stress and their associated cell death and inflammatory cascade, as well as the systematic modulation of unfolded protein response (UPR)-mediated adaptive pathways.

Covalent Protein Modification: An Unignorable Factor for Bisphenol A-Induced Hepatotoxicity.

Covalent modification of proteins by reactive pollutants/metabolites might trigger various toxicities resulting from the disruption of protein structures and/or functions, which is critical for understanding the mechanism of pollutants-induced toxicity. However, this mechanism has rarely been touched on due to the lack of a methodology. In this research, the protein modification of bisphenol A (BPA) in rats was characterized using a series of liquid chromatography-tandem mass spectrometry (LC-MS) approaches. BPA-modified cysteine (Cys1) was first released from proteins via enzymatic hydrolysis and identified using LC-MS. Moreover, the positive correlation between Cys1 and hepatotoxicity indicated the involvement of protein modification in BPA toxicity. Then, in vitro incubation of BPA with amino acids and protein confirmed that BPA could specifically modify cysteine residues of proteins after bioactivation and provided four additional modification patterns. Finally, 24 BPA-modified proteins were identified from the liver of BPA-exposed rats using proteomic analysis, and they were mainly enriched in oxidative stress-related pathways. The modification on superoxide dismutases, catalase, and glutathione S-transferases disrupted their enzymatic functions, leading to oxidative damage. These results revealed that the covalent protein modification is an unignorable factor for BPA hepatotoxicity. Moreover, the workflow can be applied to identify protein adducts of other emerging contaminants and possible risk.

Drug-drug interactions induced by Linderane based on mechanism-based inactivation of CYP2C9 and the molecular mechanisms.

Linderane (LDR) is a main furan-containing sesquiterpenoid of the common herbal medicine Lindera aggregata (Sims) Kosterm. Our early study indicated that LDR led to mechanism-based inactivation (MBI) of CYP2C9 in vitro, implying possible drug-drug interactions (DDIs) in clinic. In the present study, influence of LDR on the pharmacokinetics of the corresponding hydroxylated metabolites of CYP2C9 substrates in rats was investigated. Pharmacokinetic studies revealed that pretreatment with LDR at 20 mg/kg for 15 days inhibited the metabolism of both tolbutamide and warfarin catalyzed by CYP2C9. As for 4-hydroxytolbutamide, the Cmax was decreased, the t1/2z was prolonged, and the Vz/F was increased, all with significant difference. As for 7-hydroxywarfarin, the AUC0-t/AUC0-∞ and CLz/F were significantly decreased and increased, respectively. Furthermore, the underlying molecular mechanisms based on MBI of CYP2C9 by LDR were revealed. Two reactive metabolites of LDR, furanoepoxide and γ-ketoenal intermediates were identified in CYP2C9 recombinant enzyme incubation systems. Correspondingly, covalent modifications of lysine and cysteine residues of CYP2C9 protein were discovered in the CYP2C9 incubation system treated with LDR. The formation of protein adducts exhibited obvious time- and dose-dependence, which is consistent with the trend of enzyme inhibition caused by LDR in vitro. In addition to the apoprotein of CYP2C9, the heme content was significantly reduced after co-incubation with LDR. These data revealed that modification of both apoprotein and heme of CYP2C9 by reactive metabolites of LDR led to MBI of CYP2C9, therefore resulting in the inhibition of biotransformation of CYP2C9 substrates to their corresponding metabolites in vivo.

A cysteine trapping assay for risk assessment of reactive acyl CoA metabolites.

Some drugs with carboxylic acid moieties can potentially cause rare but severe hepatotoxicity. The reactive chemical species generated by drug metabolism are thought to be one reason for this event. Although the phase II conjugation metabolism of carboxylic acids generally renders a compound more polar and inactive, it is also responsible for the formation of reactive metabolites.This study aimed to provide a new approach towards the risk assessment of carboxylic acids in the aspect of reactive acyl CoA metabolites.Although acyl CoA metabolites have been concerned, it is difficult to detect them because of their instability. We investigated the trapping agents for acyl CoA metabolites. We found that cysteine is a good trapping agent and developed an assay method for the reactivity of acyl CoA metabolites. We evaluated 17 drugs with carboxylic acid moieties, all drugs concerned with hepatotoxicity displayed reactive potential. With consideration of the exposure of each parent drug, the correlation between drug labels and the calculated risk of carboxylic drugs was improved.These evaluations can be conducted without radiochemical reagents or the authentic standards of metabolites. We believe that the method will be beneficial for drug discovery.

Discovery and SAR studies of 3-amino-4-(phenylsulfonyl)tetrahydrothiophene 1,1-dioxides as non-electrophilic antioxidant response element (ARE) activators.

The transcription factor NRF2 controls resistance to oxidative insult and is thus a key therapeutic target for treating a number of disease states associated with oxidative stress and aging. We previously reported CBR-470-1, a bis-sulfone which activates NRF2 by increasing the levels of methylglyoxal, a metabolite that covalently modifies NRF2 repressor KEAP1. Here, we report the design, synthesis, and structure activity relationship of a series of bis-sulfones derived from this unexplored chemical template. We identify analogs with sub-micromolar potencies, 7f and 7g, as well as establish that efficacious NRF2 activation can be achieved by non-toxic analogs 7c, 7e, and 9, a key limitation with CBR-470-1. Further efforts to identify non-covalent NRF2 activators of this kind will likely provide new insight into revealing the role of central metabolism in cellular signaling.

Evaluation of covalent binding of flutamide and its risk assessment using 19F-NMR.

The formation of reactive metabolites (RMs) is a problem in drug development that sometimes results in severe hepatotoxicity. As detecting RMs themselves is difficult, a covalent binding assay using expensive radiolabelled tracers is usually performed for candidate selection. This study aimed to provide a practical approach toward the risk assessment of hepatotoxicity induced by covalent binding before candidate selection. We focused on flutamide because it contains a trifluoromethyl group that shows a strong singlet peak by 19F nuclear magnetic resonance (NMR) spectrometry. The covalent binding of flutamide was evaluated using quantitative NMR and its risk for hepatotoxicity was assessed by estimating the RM burden, an index that reflects the body burden associated with RM exposure by determining the extent of covalent binding, clinical dose and in vivo clearance. The extent of covalent binding and RM burden was 296 pmol/mg/h and 37.9 mg/day, respectively. Flutamide was categorised as high risk with an RM burden >10 mg/day consistent with its clinical hepatotoxicity. These results indicate that a combination of covalent binding assay using 19F-NMR and RM burden is useful for the risk assessment of RMs without using radiolabelled compounds.

Idiosyncratic Drug-Induced Liver Injury: Mechanistic and Clinical Challenges.

Idiosyncratic drug-induced liver injury (IDILI) remains a significant problem for patients and drug development. The idiosyncratic nature of IDILI makes mechanistic studies difficult, and little is known of its pathogenesis for certain. Circumstantial evidence suggests that most, but not all, IDILI is caused by reactive metabolites of drugs that are bioactivated by cytochromes P450 and other enzymes in the liver. Additionally, there is overwhelming evidence that most IDILI is mediated by the adaptive immune system; one example being the association of IDILI caused by specific drugs with specific human leukocyte antigen (HLA) haplotypes, and this may in part explain the idiosyncratic nature of these reactions. The T cell receptor repertoire likely also contributes to the idiosyncratic nature. Although most of the liver injury is likely mediated by the adaptive immune system, specifically cytotoxic CD8+ T cells, adaptive immune activation first requires an innate immune response to activate antigen presenting cells and produce cytokines required for T cell proliferation. This innate response is likely caused by either a reactive metabolite or some form of cell stress that is clinically silent but not idiosyncratic. If this is true it would make it possible to study the early steps in the immune response that in some patients can lead to IDILI. Other hypotheses have been proposed, such as mitochondrial injury, inhibition of the bile salt export pump, unfolded protein response, and oxidative stress although, in most cases, it is likely that they are also involved in the initiation of an immune response rather than representing a completely separate mechanism. Using the clinical manifestations of liver injury from a number of examples of IDILI-associated drugs, this review aims to summarize and illustrate these mechanistic hypotheses.

Mechanistic studies on the drug metabolism and toxicity originating from cytochromes P450.

Cytochromes P450 are oxidizing enzymes; a few families of cytochromes P450 are implicated in drug metabolism. These enzymatic reactions involve many processes including (i) prodrug to drug conversion, (ii) easy excretion of drug, (iii) generation of reactive metabolites, many of which cause toxicity. In this review, the fundamental biochemical mechanisms associated with the conversion of drugs into the useful or toxic metabolites have been discussed. The mechanisms can be established with the help of many experimental methods like mass spectral analysis, NMR and in vitro analysis etc. Computational methods provide detailed atomic level information, which is generally not available from experimental studies. Thus, the in silico efforts in elucidating the molecular mechanisms are complementary to the known experimental methods and are often clearer (especially in providing 3D information about the metabolites and their reactions). Quantum chemical methods and molecular docking become especially very useful. This review includes five case studies, which explain how the atomic level details were obtained to explore the reaction mechanisms of drug metabolism by cytochromes P450.

Synthesis and pharmacological evaluation of 11-(1,6-dimethyl-1,2,3,6-tetrahydropyridin-4-yl)-5H-dibenzo[b,e][1,4]diazepines with clozapine-like receptor occupancy at dopamine D1/D2 receptor.

Clozapine-like compound without agranulocytosis risk is need to cure the treatment resistant schizophrenia (TRS). We discovered (S)-3 as Clozapine-like dopamine D2/D1 receptor selectivity and improved reactive metabolites formation profile by the modification of piperazine moiety in Clozapine. The optimization of (S)-3 gave compound 5 to be best compound (approximately 10-fold stronger affinity for D2/D1 receptor and similar D2/D1 selectivity ratio with Clozapine). Clozapine-like D2/D1 receptor occupancy profile was proved by in vivo evaluation. In addition, the reactive metabolites derived agranulocytosis risk of compound 5 was considered to be lower than Clozapine. The pharmacology detail of compound 5 is being investigated to develop it for TRS treatment.

Detection and characterization of olmutinib reactive metabolites by LC-MS/MS: Elucidation of bioactivation pathways.

Olmutinib (Olita™) is an orally bioavailable third generation epidermal growth factor receptor tyrosine kinase inhibitor. Olmutinib was approved in South Korea in May 2016 for the treatment of patients suffering from locally advanced or metastatic epidermal growth factor receptor T790M mutation-positive non-small cell lung cancer. Reactive olmutinib intermediates may be responsible for the severe side effects associated with the treatment. However, literature review revealed no previous reports on the structural identification of reactive olmutinib metabolites. In this work, the formation of reactive olmutinib metabolites in rat liver microsomes was investigated. Methoxylamine, glutathione, and potassium cyanide were used as capturing agents for aldehyde, iminoquinones, and iminium intermediates, respectively. The stable complexes formed were identified using liquid chromatography-tandem mass spectrometry. The major phase I metabolic pathway observed in vitro was hydroxylation of the piperazine ring. Seven potential reactive intermediates were characterized, including three iminium ions, three iminoquinones, and one aldehyde. Based on the findings, various bioactivation pathways were postulated. Hence, identifying the reactive intermediates of olmutinib that may be the cause of severe side effects can provide new insights, leading to improved treatments for patients.

In vitro metabolites characterization of ponatinib in human liver microsomes using ultra-high performance liquid chromatography combined with Q-Exactive-Orbitrap tandem mass spectrometry.

Ponatinib is an oral drug for the treatment of chronic myeloid leukemia and acute lymphoblastic leukemia, which has been reported to increase the risk of hepatotoxicity. The aim of this study was to characterize the metabolites of ponatinib in human liver microsomes as well as its reactive metabolites. Ponatinib was incubated with human liver microsomes in the presence of NADPH and trapping agents (glutathione or potassium cyanide). The metabolites were characterized by liquid chromatography in combination with Q-Exactive-Orbitrap-MS. Under the current conditions, six metabolites were detected and structurally identified on the basis of their accurate masses, fragmentation patterns, and retention times. M3 (N-demethylation) was unambiguously identified by matching its retention time and fragment ions with those of its reference standard. N-demethylation and oxygenation were proved to be the predominant metabolic pathways of ponatinib. In addition, two reactive metabolites (cyano adducts) were detected in human liver microsomes in the presence of potassium cyanide and NADPH, suggesting that ponatinib underwent CYP450-mediated metabolic activation, which could be one of the causative mechanisms for its hepatotoxicity. The current study provides new information regarding the metabolic profiles of ponatinib and would be helpful in understanding the effectiveness and toxicity of ponatinib, especially the mechanism of hepatotoxicity.

Identification of novel glutathione conjugates of terbinafine in liver microsomes and hepatocytes across species.

1. Terbinafine (TBF), a common antifungal agent, has been associated with rare incidences of hepatotoxicity. It is hypothesized that bioactivation of TBF to reactive intermediates and subsequent binding to critical cellular proteins may contribute to this toxicity. In the present study, we have characterized the bioactivation pathways of TBF extensively in human, mouse, monkey, dog and rat liver microsomes and hepatocytes. 2. A total of twenty glutathione conjugates of TBF were identified in hepatocytes; thirteen of these conjugates were also detected in liver microsomes. To the best of our knowledge, only two of these conjugates have been reported previously. The conjugates were categorized into three groups based on their mechanism of formation: (a) alkene/alkyne oxidation followed by glutathione conjugation, with or without N-demethylation, (b) arene oxidation followed by glutathione conjugation, with or without N-demethylation, and (c) N-dealkylation followed by glutathione conjugation of the allylic aldehyde, alcohol and acid intermediates. 3. Differences were observed across species in the contributions of these pathways toward overall metabolic turnover. We conclude that, in addition to the glutathione conjugates known to form by Michael addition to the allylic aldehyde, there are other pathways involving the formation of arene oxides and alkene/alkyne epoxides that may be relevant to the discussion of TBF-mediated idiosyncratic drug reactions.

Cytotoxicity of safrole in HepaRG cells: studies on the role of CYP1A2-mediated ortho-quinone metabolic activation.

1. Safrole is a natural compound categorized as a group 2B carcinogen extracted from sassafras oil or certain other essential oils. The hepatotoxicity of safrole has always been highly concerned. So, the purpose of this study was to evaluate the role of cytochrome P450 (CYP450)-mediated reactive metabolites (RMs) formation and its induced cytotoxicity in HepaRG cells. 2. Safrole belongs to the methylenedioxyphenyl structure which could be activated to RMs. Two metabolites (M1, M2) and three new glutathione conjugates (M3-M5) of safrole ortho-oquinone RMs were found in HepaRG cells. Using human recombinant CYP450 enzymes and chemical inhibitor method, the metabolism of safrole RMs was predominantly carried out through the CYP1A2 with minor contributions by CYP2E1. 3. Induction of CYP1A2 by omeprazole (OME) enhanced safrole-induced cytotoxicity, compared with treatment with safrole alone, whereas inhibition of CYP1A2 by alpha-naphthoflavone (α-NAP) decreased the cytotoxicity. The cytotoxicity of cell induced by safrole was related to the amount of RMs formation. Besides, pretreatment with L-buthionine sulfoximine (BSO) to deplete intracellular GSH markedly enhanced safrole-induced cytotoxicity. OME induced the safrole-induced GSH exhaustion, and GSH depletion by safrole was not via oxidation of GSH and occurred prior to the increase in ROS. Furthermore, mitochondrial membrane potential (ΔΨm) could be aggravated by the inducer of CYP1A2 together with safrole. Collectively, these data suggest that the ortho-quinone RM may mediate safrole hepatotoxicity, and CYP1A2 was the core enzyme in ortho-quinone RMs-mediated safrole hepatotoxicity.