Metabolism-dependent inhibition of CYP2E1 by isoniazid, a mediator of idiosyncratic liver injury.

Liver injury is a well-known adverse effect of the anti-tuberculosis drug isoniazid (INH); however, animal models that accurately replicate this effect as seen in humans have not been constructed, and the mechanism of its pathogenesis remains unclear. Recently, an immune-mediated mechanism have been proposed based on clinical studies, suggesting the involvement of cytochrome P450-mediated formation of reactive metabolites and covalent adducts in severe cases. In the present study, we investigated the role of CYP2E1 in this mechanism. Liver microsomes from humans, rats, and mice were preincubated with INH and NADPH; thereafter, residual CYP2E1 activity was measured. The inhibition of CYP2E1 by INH was potentiated by preincubation, indicating time-dependent inhibition. There were no major species-based differences in inhibition among humans, rats, and mice. Further to our findings on the inhibition kinetics, resistance of the inhibition to glutathione and catalase indicated that the reactive metabolites of INH covalently bonded to CYP2E1 in a suicidal manner. A similar time-dependent inhibition was also observed for the known metabolites acetylhydrazine and hydrazine; however, the conditions that inhibited the hydrolysis or activated the acetylation of INH did not affect inhibition by INH, suggesting that the reactive metabolites contributing to the inhibition were generated via alternative pathways. This indicates that CYP2E1 alone generates reactive INH metabolites and that haptenized CYP2E1 may be involved in immune-mediated liver injury.

Iminium ion metabolites are formed from nintedanib by human CYP3A4.

Nintedanib is used to treat idiopathic pulmonary fibrosis, systemic sclerosis, interstitial lung disease, and progressive fibrotic interstitial lung disease. It is primarily cleared via hepatic metabolism, hydrolysis, and glucuronidation. In addition, formation of the iminium ion, a possible reactive metabolite, was predicted based on the chemical structure of nintedanib. To obtain a hint which may help to clarify the cause of nintedanib-induced liver injury, we investigated whether iminium ions were formed in the human liver. To detect unstable iminium ions using liquid chromatography-tandem mass spectrometry (LC-MS/MS), potassium cyanide was added to the reaction mixture as a trapping agent. Human liver and intestinal microsomes were incubated with nintedanib in the presence of NADPH to form two iminium ion metabolites on the piperazine ring. Their formation is strongly inhibited by ketoconazole, a potent cytochrome P450 (CYP) 3A4 inhibitor. Among the recombinant P450s, only CYP3A4 formed cyanide adducts. The role of CYP3A4 was supported by the positive correlation between CYP3A4 protein abundance, as determined by LC-MS-based proteomics, and the formation of cyanide adducts in 25 individual human liver microsomes. In conclusion, we have demonstrated that iminium ion metabolites are formed from nintedanib by CYP3A4 as potential reactive metabolites.

Development of a Novel In Silico Classification Model to Assess Reactive Metabolite Formation in the Cysteine Trapping Assay and Investigation of Important Substructures.

Predicting whether a compound can cause drug-induced liver injury (DILI) is difficult due to the complexity of drug mechanism. The cysteine trapping assay is a method for detecting reactive metabolites that bind to microsomes covalently. However, it is cumbersome to use 35S isotope-labeled cysteine for this assay. Therefore, we constructed an in silico classification model for predicting a positive/negative outcome in the cysteine trapping assay. We collected 475 compounds (436 in-house compounds and 39 publicly available drugs) based on experimental data performed in this study, and the composition of the results showed 248 positives and 227 negatives. Using a Message Passing Neural Network (MPNN) and Random Forest (RF) with extended connectivity fingerprint (ECFP) 4, we built machine learning models to predict the covalent binding risk of compounds. In the time-split dataset, AUC-ROC of MPNN and RF were 0.625 and 0.559 in the hold-out test, restrictively. This result suggests that the MPNN model has a higher predictivity than RF in the time-split dataset. Hence, we conclude that the in silico MPNN classification model for the cysteine trapping assay has a better predictive power. Furthermore, most of the substructures that contributed positively to the cysteine trapping assay were consistent with previous results.

Role of microsomal metabolism in bromfenac-induced cytotoxicity.

This study delves into the intricate mechanisms underlying drug-induced liver injury (DILI) with a specific focus on bromfenac, the withdrawn nonsteroidal anti-inflammatory drug. DILI is a pervasive concern in drug development, prompting market withdrawals and posing significant challenges to healthcare. Despite the withdrawal of bromfenac due to DILI, the exact role of its microsomal metabolism in inducing hepatotoxicity remains unclear. Herein, employing HepG2 cells with human liver microsomes and UDP-glucuronic acid (UDPGA), our investigation revealed a substantial increase in bromfenac-induced cytotoxicity in the presence of UDPGA, pointing to the significance of UDP-glucuronosyltransferase (UGT)-dependent metabolism in augmenting toxicity. Notably, among the recombinant UGTs examined, UGT2B7 emerged as a pivotal enzyme in the metabolic activation of bromfenac. Metabolite identification studies disclosed the formation of reactive intermediates, with bromfenac indolinone (lactam) identified as a potential mediator of hepatotoxic effects. Moreover, in cytotoxicity experiments, the toxicity of bromfenac lactam exhibited a 34-fold increase, relative to bromfenac. The toxicity of bromfenac lactam was mitigated by nicotinamide adenine dinucleotide phosphate-dependent metabolism. This finding underscores the role of UGT-dependent metabolism in generating reactive metabolites that contribute to the observed hepatotoxicity associated with bromfenac. Understanding these metabolic pathways and the involvement of specific enzymes, such as UGT2B7, provides crucial insights into the mechanisms of bromfenac-induced liver injury. In conclusion, this research sheds light on the metabolic intricacies leading to cytotoxicity induced by bromfenac, especially emphasizing the role of UGT-dependent metabolism and the formation of reactive intermediates like bromfenac lactam. These findings offer insight into the mechanistic basis of DILI and emphasize the importance of understanding metabolism-mediated toxicity.

Metabolic bioactivation of antidepressants: advance and underlying hepatotoxicity.

Many drugs that serve as first-line medications for the treatment of depression are associated with severe side effects, including liver injury. Of the 34 antidepressants discussed in this review, four have been withdrawn from the market due to severe hepatotoxicity, and others carry boxed warnings for idiosyncratic liver toxicity. The clinical and economic implications of antidepressant-induced liver injury are substantial, but the underlying mechanisms remain elusive. Drug-induced liver injury may involve the host immune system, the parent drug, or its metabolites, and reactive drug metabolites are one of the most commonly referenced risk factors. Although the precise mechanism by which toxicity is induced may be difficult to determine, identifying reactive metabolites that cause toxicity can offer valuable insights for decreasing the bioactivation potential of candidates during the drug discovery process. A comprehensive understanding of drug metabolic pathways can mitigate adverse drug-drug interactions that may be caused by elevated formation of reactive metabolites. This review provides a comprehensive overview of the current state of knowledge on antidepressant bioactivation, the metabolizing enzymes responsible for the formation of reactive metabolites, and their potential implication in hepatotoxicity. This information can be a valuable resource for medicinal chemists, toxicologists, and clinicians engaged in the fields of antidepressant development, toxicity, and depression treatment.

Reactive metabolite of trovafloxacin activates inflammasomes: Implications for trovafloxacin-induced liver injury.

Trovafloxacin is a quinolone antibiotic drug with broad-spectrum activity, which was withdrawn from a global market relatively soon after approval because of serious liver injury. The characteristics of trovafloxacin-induced liver injury are consistent with an idiosyncratic reaction; however, the details of the mechanism have not been elucidated. We examined whether trovafloxacin induces the release of damage-associated molecular patterns (DAMPs) that activate inflammasomes. We also tested ciprofloxacin, levofloxacin, gatifloxacin, and grepafloxacin for their ability to activate inflammasomes. Drug bioactivation was performed with human hepatocarcinoma functional liver cell-4 (FLC-4) cells, and THP-1 cells (human monocyte cell line) were used for the detection of inflammasome activation. The supernatant from the incubation of trovafloxacin with FLC-4 cells for 7 days increased caspase-1 activity and production of IL-1ß by THP-1 cells. In the supernatant of FLC-4 cells that had been incubated with trovafloxacin, heat shock protein (HSP) 40 was significantly increased. Addition of a cytochrome P450 inhibitor to the FLC-4 cells prevented the release of HSP40 from the FLC-4 cells and inflammasome activation in THP-1 cells by the FLC-4 supernatant. These results suggest that reactive metabolites of trovafloxacin can cause the release of DAMPs from hepatocytes that can activate inflammasomes. Inflammasome activation may be an important step in the activation of the immune system by trovafloxacin, which, in some patients, can cause immune-related liver injury.

Development of a fluorescent-labeled trapping reagent to evaluate the risk posed by acyl-CoA conjugates.

Although acyl-CoA conjugates are known to have higher reactivity than acyl glucuronides, few studies have been conducted to evaluate the risk of the conjugates. In the present study, we aimed to develop a trapping assay for acyl-CoA conjugates using trapping reagents we have developed previously. It was revealed that Cys-Dan, which has both a thiol and an amino group, was the most effective in forming stable adducts containing an amide bond after intramolecular acyl migration. Additionally, we also developed a hepatocyte-based trapping assay in the present study to overcome the shortcomings of liver microsomes. Although liver microsomes are commonly used as enzyme sources in trapping assays, they lack some of the enzymes required for drug metabolism and detoxification systems. In human hepatocytes, our three trapping reagents, CysGlu-Dan, Dap-Dan and Cys-Dan, captured CYP-dependent reactive metabolites, reactive acyl glucuronides, and reactive acyl-CoA conjugates, respectively. The work suggests that the trapping assay with the reagents in hepatocytes is useful to evaluate the risk of reactive metabolites in drug discovery.

S-lactoyl modification of KEAP1 by a reactive glycolytic metabolite activates NRF2 signaling.

KEAP1 (Kelch-like ECH-associated protein), a cytoplasmic repressor of the oxidative stress responsive transcription factor Nuclear factor erythroid 2-related factor 2 (NRF2), senses the presence of electrophilic agents by modification of its sensor cysteine residues. In addition to xenobiotics, several reactive metabolites have been shown to covalently modify key cysteines on KEAP1, although the full repertoire of these molecules and their respective modifications remain undefined. Here, we report the discovery of sAKZ692, a small molecule identified by high-throughput screening that stimulates NRF2 transcriptional activity in cells by inhibiting the glycolytic enzyme pyruvate kinase. sAKZ692 treatment promotes the buildup of glyceraldehyde 3-phosphate, a metabolite which leads to S-lactate modification of cysteine sensor residues of KEAP1, resulting in NRF2-dependent transcription. This work identifies a posttranslational modification of cysteine derived from a reactive central carbon metabolite and helps further define the complex relationship between metabolism and the oxidative stress-sensing machinery of the cell.

Mechanism of non-steroidal anti-androgen-induced liver injury: Reactive metabolites of flutamide and bicalutamide activate inflammasomes.

Flutamide is a non-steroidal anti-androgen agent, which is mainly used for the treatment of prostate cancer. Flutamide is known to cause severe adverse events, which includes idiosyncratic liver injury. However, details of the mechanism of these adverse reactions have not been elucidated. We investigated whether flutamide induces the release of damage-associated molecular patterns (DAMPs) that activate inflammasomes. We also tested bicalutamide, enzalutamide, apalutamide, and darolutamide for their ability to activate inflammasomes in differentiated THP-1 cells. The supernatant from the incubation of flutamide and bicalutamide with human hepatocarcinoma functional liver cell-4 (FLC-4) cells increased caspase-1 activity and production of IL-1ß by differentiated THP-1 cells. In the supernatant of FLC-4 cells with flutamide and bicalutamide, the heat shock protein (HSP) 40 or 60 was significantly increased. Addition of a carboxylesterase or a CYP inhibitor to the FLC-4 cells prevented release of HSPs from the FLC-4 cells. These results suggested that the reactive metabolites of flutamide and bicalutamide can cause the release of DAMPs from hepatocytes and activate inflammasomes. Inflammasome activation may be an important step in the activation of the immune system by flutamide or bicalutamide, which in some patients, can cause immune-related adverse events.

Role of Oxidative Stress and Reactive Metabolites in Cytotoxicity & Mitotoxicity of Clozapine, Diclofenac and Nifedipine in CHO-K1 Cells In Vitro.

AIM: To provide in vitro data on toxicity mechanisms of clozapine, diclofenac and nifedipine. BACKGROUND: CHO-K1 cells were used as in vitro model to explore mechanisms of cytotoxicity of the test drugs. OBJECTIVE: Cytotoxic mechanisms of clozapine (CLZ), diclofenac (DIC) and nifedipine (NIF) were studied in CHO-K1 cells in vitro. All three drugs induce adverse reactions in some patients with partially unknown mechanisms. METHOD: Following the determination of time- and dose-dependency of cytotoxicity by the MTT test, cytoplasmic membrane integrity was explored by the LDH leakage test. Both end-points were further examined in the presence of soft and hard nucleophilic agents, glutathione (GSH) and potassium cyanide (KCN), respectively, and either individual or general cytochrome P450 (CYP) inhibitors, whether CYP-catalysed formation of electrophilic metabolites play a role in the observed cytotoxicity and membrane damage. The generation of reactive metabolites during the incubations was also explored. Formation of malondialdehyde (MDA) and oxidation of dihydrofluorescein (DCFH) were monitored whether peroxidative membrane damage and oxidative stress take place in cytotoxicity. Incubations were also conducted in the presence of chelating agents of EDTA or DTPA to explore any possible role of metals in cytotoxicity by facilitating electron transfer in redox reactions. Finally, mitochondrial membrane oxidative degradation and permeability transition pore (mPTP) induction by the drugs were tested as markers of mitochondrial damage. RESULTS: The presence of an individual or combined nucleophilic agents significantly diminished CLZ- and NIF-induced cytotoxicities, while the presence of both agents paradoxically increased DIC-induced cytotoxicity by a factor of three with the reason remaining unknown. The presence of GSH significantly increased DIC-induced membrane damage too. Prevention of membrane damage by the hard nucleophile KCN suggests the generation of a hard electrophile upon DIC and GSH interaction. The presence of CYP2C9 inhibitor sulfaphenazol significantly diminished DIC-induced cytotoxicity, probably by preventing the formation of 4-hydroxylated metabolite of DIC, which further converts to an electrophilic reactive intermediate. Among the chelating agents, EDTA caused a marginal decrease in CLZ-induced cytotoxicity, while DIC-induced cytotoxicity was amplified by a factor of five. Both reactive and stable metabolites of CLZ could be detected in the incubation medium of CLZ with CHO-K1 cells, which are known to have low metabolic capacity. All three drugs caused a significant increase in cytoplasmic oxidative stress by means of DCFH oxidation, which was confirmed by increased MDA from cytoplasmic as well as mitochondrial membranes. The addition of GSH paradoxically and significantly increased DIC-induced MDA formation, in parallel with the increase in membrane damage when DIC and GSH combined. CONCLUSION: Our results suggested that the soft electrophilic nitrenium ion of CLZ is not responsible for the observed in vitro toxicities, and this may originate from a relatively low amount of the metabolite due to the low metabolic capacity of CHO-K1. A hard electrophilic intermediate may contribute to cellular membrane damage incubated with DIC, while a soft electrophilic intermediate seems to exacerbate cell death by a mechanism other than membrane damage. A significant decrease in cytotoxicity of NIF by GSH and KCN suggested that both soft and hard electrophiles contribute to NIF-induced cytotoxicity. All three drugs induced peroxidative cytoplasmic membrane damage, while only DIC and NIF induced peroxidative mitochondrial membrane damage, which suggested mitochondrial processes may contribute to adverse effects of these drugs in vivo.

Sulfation of 12-hydroxy-nevirapine by human SULTs and the effects of genetic polymorphisms of SULT1A1 and SULT2A1.

Nevirapine (NVP) is an effective drug for the treatment of HIV infections, but its use is limited by a high incidence of severe skin rash and liver injury. 12-Hydroxynevirapine (12-OH-NVP) is the major metabolite of nevirapine. There is strong evidence that the sulfate of 12-OH-NVP is responsible for the skin rash. While several cytosolic sulfotransferases (SULTs) have been shown to be capable of sulfating 12-OH-NVP, the exact mechanism of sulfation in vivo is unclear. The current study aimed to clarify human SULT(s) and human organs that are capable of sulfating 12-OH-NVP and investigate the metabolic sulfation of 12-OH-NVP using cultured HepG2 human hepatoma cells. Enzymatic assays revealed that of the thirteen human SULTs, SULT1A1 and SULT2A1 displayed strong 12-OH-NVP-sulfating activity. 1-Phenyl-1-hexanol (PHHX), which applied topically prevents the skin rash in rats, inhibited 12-OH-NVP sulfation by SULT1A1 and SULT2A1, implying the involvement of these two enzymes in the sulfation of 12-OH-NVP in vivo. Among five human organ cytosols analyzed, liver cytosol displayed the strongest 12-OH-NVP-sulfating activity, while a low but significant activity was detected with skin cytosol. Cultured HepG2 cells were shown to be capable of sulfating 12-OH-NVP. The effects of genetic polymorphisms of SULT1A1 and SULT2A1 genes on the sulfation of 12-OH-NVP by SULT1A1 and SULT2A1 allozymes were investigated. Two SULT1A1 allozymes, Arg37Asp and Met223Val, showed no detectable 12-OH-NVP-sulfating activity, while a SULT2A1 allozyme, Met57Thr, displayed significantly higher 12-OH-NVP-sulfating activity compared with the wild-type enzyme. Collectively, these results contribute to a better understanding of the involvement of sulfation in NVP-induced skin rash and provide clues to the possible role of SULT genetic polymorphisms in the risk of this adverse reaction.

Feature MS fragments-based method for identification of toxic furanoids in biological samples.

Numerous furan-containing compounds have been reported to be toxic. The toxicity may be attributed to the metabolic activation of the furan ring to cis-enediones. Identification of unknown furans that undergo bioactivation is challenging. Here, we present a novel approach that enables non-targeted profiling of bioactivation of unknown furanoids both in vitro and in vivo. Cyclic pyrrole-glutathione conjugate was the predominant product of cis-enediones with glutathione. The shared glutathione substructure of conjugates was capable of generating four constant and signature fragments under collision-induced dissociation (CID) in the mass spectrometer, including neutral loss fragments 103.0269 Da and 146.0691 Da and product ions at m/z 130.0499 and 177.0328. The unique structure and high abundance of conjugates in combination with the consistency and specificity of CID fragmentation brought extraordinarily high selectivity and reliability for the four fragments as a fingerprint of bioactivated furanoids. The bioactivated furanoids can be identified by screening the four fragments in high-resolution MS/MS datasets using the neutral loss filtering and diagnostic fragmentation filtering of data post-acquisition software MZmine. The simultaneous formation of four individual signal points in the filtering channel with the same precursor ion and retention time was assigned to be furanoids. The method has been rigorously validated. In the pooled urine samples from nine model furanoids-treated mice, nine cis-enediones from the parent furanoids and two from furanoid metabolites were accurately detected and identified. The method showed great performance in non-targeted profiling bioactivated furanoids and their metabolites in urine samples of herbal extract-treated mice.

Assessment of metabolic activation of felbamate in chimeric mice with humanized liver in combination with in vitro metabolic assays.

Felbamate (FBM) is an antiepileptic drug that has minimal toxicity in preclinical toxicological species but has a serious idiosyncratic drug toxicity (IDT) in humans. The formation of reactive metabolites is common among most drugs associated with IDT, and 2-phenylpropenal (2-PP) is believed to be the cause of IDT by FBM. It is important to consider the species difference in susceptibility to IDT between experimental animals and humans. In the present study, we used an in vitro and in vivo model system to reveal species difference in IDT of FBM. Human cytochrome P450 (CYP) and carboxylesterase (CES) expressing microsomes were used to clarify the isozymes involved in the metabolism of FBM. The remaining amount of FBM was significantly reduced in incubation with microsomes expressing human CYP2C8, 2C9, 2E1, and CES1c isozymes. Chimeric mice with humanized liver are expected to predict IDT in humans. Therefore, metabolite profiles in chimeric mice with humanized liver were investigated after administration of FBM. Metabolites after glutathione (GSH) conjugation of 2-phenylpropenal (2-PP), which is the reactive metabolite responsible for FBM-induced IDT, were detected in chimeric mice plasma and liver homogenate. Mass spectrometry imaging (MSI) visualizes distribution of FBM and endogenous GSH, and GSH levels in human hepatocyte were decreased after administration of FBM. In this study, we identified CYP and CES isozymes involved in the metabolism of FBM and confirmed reactive metabolite formation and subsequent decrease in GSH using humanized animal model. These results would provide useful information for the susceptibility to IDT between experimental animals and humans.

Evidence for adduction of biologic amines with reactive metabolite of 8-epidiosbulbin E acetate in vitro and in vivo.

Dioscorea bulbifera L. (DBL) is one of traditional Chinese medicines and has been used for the treatment of goiter, tumor and carbuncles. However, clinic application of the herbal medicine has been limited, due to reported severe hepatotoxicity. 8-Epidiosbulbin E acetate (EEA), one of the major components of DBL, can cause severe liver damage. The furan ring of EEA is metabolized by CYP3A4 to a cis-enedial reactive intermediate prone to react amino and/or thiol groups of amino acid residues. In this study, we investigated the interaction of the reactive intermediate with biologic amines. EEA-derived biologic amine adducts, including spermidine, spermine, putrescine, ornithine, lysine and glutamine were detected in cultured mouse primary hepatocytes treated with EEA. Only spermidine adduct was observed in bile of mice given EEA. The detection of the adducts was established by labeling with bromobenzyl mercaptan and LC-MS/MS analysis. Exposure of EEA resulted in concentration dependent cytotoxicity in hepatocytes. Pretreatment with spermidine attenuated the susceptibility of cells to the cytotoxicity of EEA, because of the compensation of the depleted spermidine.

Reactive metabolites of the anticonvulsant drugs and approaches to minimize the adverse drug reaction.

Several generations of antiepileptic drugs (AEDs) are available in the market for the treatment of seizures, but these are amalgamated with acute to chronic side effects. The most common side effects of AEDs are dose-related, but some are idiosyncratic adverse drug reactions (ADRs) that transpire due to the formation of reactive metabolite (RM) after the bioactivation process. Because of the adverse reactions patients usually discontinue the medication in between the treatment. The AEDs such as valproic acid, lamotrigine, phenytoin etc., can be categorized under such types because they form the RM which may prevail with life-threatening adverse effects or immune-mediated reactions. Hepatotoxicity, teratogenicity, cutaneous hypersensitivity, dizziness, addiction, serum sickness reaction, renal calculi, metabolic acidosis are associated with the metabolites of drugs such as arene oxide, N-desmethyldiazepam, 2-(1-hydroxyethyl)-2-methylsuccinimide, 2-(sulphamoy1acetyl)-phenol, E-2-en-VPA and 4-en-VPA and carbamazepine-10,11-epoxide, etc. The major toxicities are associated with the moieties that are either capable of forming RM or the functional groups may itself be too reactive prior to the metabolism. These functional groups or fragment structures are typically known as structural alerts or toxicophores. Therefore, minimizing the bioactivation potential of lead structures in the early phases of drug discovery by a modification to low-risk drug molecules is a priority for the pharmaceutical companies. Additionally, excellent potency and pharmacokinetic (PK) behaviour help in ensuring that appropriate (low dose) candidate drugs progress into the development phase. The current review discusses about RMs in the anticonvulsant drugs along with their mechanism vis-a-vis research efforts that have been taken to minimize the toxic effects of AEDs therapy.

Bioactivation of Isoxazole-Containing Bromodomain and Extra-Terminal Domain (BET) Inhibitors.

The 3,5-dimethylisoxazole motif has become a useful and popular acetyl-lysine mimic employed in isoxazole-containing bromodomain and extra-terminal (BET) inhibitors but may introduce the potential for bioactivations into toxic reactive metabolites. As a test, we coupled deep neural models for quinone formation, metabolite structures, and biomolecule reactivity to predict bioactivation pathways for 32 BET inhibitors and validate the bioactivation of select inhibitors experimentally. Based on model predictions, inhibitors were more likely to undergo bioactivation than reported non-bioactivated molecules containing isoxazoles. The model outputs varied with substituents indicating the ability to scale their impact on bioactivation. We selected OXFBD02, OXFBD04, and I-BET151 for more in-depth analysis. OXFBD's bioactivations were evenly split between traditional quinones and novel extended quinone-methides involving the isoxazole yet strongly favored the latter quinones. Subsequent experimental studies confirmed the formation of both types of quinones for OXFBD molecules, yet traditional quinones were the dominant reactive metabolites. Modeled I-BET151 bioactivations led to extended quinone-methides, which were not verified experimentally. The differences in observed and predicted bioactivations reflected the need to improve overall bioactivation scaling. Nevertheless, our coupled modeling approach predicted BET inhibitor bioactivations including novel extended quinone methides, and we experimentally verified those pathways highlighting potential concerns for toxicity in the development of these new drug leads.

Development of fluorescent-labeled trapping reagents based on cysteine to detect soft and hard electrophilic reactive metabolites.

Trapping assays are conducted at lead optimization stages to detect reactive metabolites (RMs) that can contribute to drug toxicity. The commonly used dansyl glutathione (dGSH) provides a sensitive analysis owing to the fluorescent label, however, it captures only soft electrophilic RMs. TRs for hard electrophilic RMs, few of which are labeled fluorescently, can detect hard electrophilic aldehydes only by forming unstable imine derivatives. In this study, we aimed to develop novel fluorescently labeled TRs that detect both soft and hard electrophilic RMs and form stable ring structures with aldehydes. We designed four dansylated TRs based on cysteine, which has both soft and hard nucleophilic groups. To evaluate the reactivity of the TRs, we incubated them with several substrates and found that one of the TRs (CysGlu-Dan) detected all the soft and hard electrophilic RMs. We also examined the inhibition potential of each TR for seven major CYPs involved in drug metabolism and found that CysGlu-Dan showed an inhibitory profile similar to that of dGSH. In conclusion, CysGlu-Dan can be used to evaluate the risk of RMs in drug discovery.

Metabolomics based comprehensive investigation of Gardeniae Fructus induced hepatotoxicity.

Gardeniae Fructus (Zhizi in Chinese, ZZ in brief), a commonly used herbal medicine, has aroused wide concern for hepatotoxicity, but the mechanism remains to be investigated. This study was aimed at investigating the mechanism of ZZ-induced liver injury in vivo and in vitro based on metabolomics and evaluating the hepatotoxicity prediction ability of the in vitro model. SD rats were administered with extracted ZZ and HepG2 cells were treated with genipin, the major hepatotoxic metabolite of ZZ. Liver, plasma, intracellular and extracellular samples were obtained for metabolomics analysis. As a result, ZZ caused plasma biochemical and liver histopathological alterations in rats, and induced purine and amino acid metabolism disorder in the liver and pyrimidine, primary bile acids, amino acid metabolism and pantothenate and CoA biosynthesis disorder in the plasma. Pyrimidine, purine, amino acid metabolism and pantothenate and CoA biosynthesis were also found to be disturbed in the genipin-treated HepG2 cells, which exhibited similarity with the result in vivo. This study comprehensively illustrates the underlying mechanism involved in ZZ-related hepatotoxicity from the aspect of metabolome, and provides evidence that identifying hepatotoxicity can be achieved in cells, representing a non-animal alternative for systemic toxicology.

Metabolic Activation of Retrorsine may Disrupt Bile Acid Homeostasis in Mice through the Nrf2 Pathway.

BACKGROUND: The hepatotoxic pyrrolizidine alkaloids (PAs) were reported to increase bile acid (BA) levels in the rat. However, it is still unclear whether the production of highly reactive dehydropyrrolizidine through CYP450s is directly relevant to BA changes. OBJECTIVE: To further explore the mechanism by which metabolic activation of PAs induced BA changes, the effect of impaired or enhanced metabolic activation on the BA profiling and BA-related synthesis and to investigate transport genes, and explore the involvement of the Nrf2 pathway. METHODS: Blood and liver samples were collected after intragastrical administration of 35 mg/kg retrorsine or saline for seven days in wild-type (WT) and Nrf2 KO mice. CYP450 inhibitor, 1-aminobenzotriazole (ABT), or gammaglutamylcysteine synthetase inhibitor, L-buthionine-sulfoximine (BSO) were employed in WT mice. Retrorsineinduced hepatotoxicity was evaluated by a biochemical method and H&E staining method. Serum BAs were quantified by high-performance liquid chromatography/triple quadrupole mass spectrometry. Blood pyrrole-protein adducts were semi quantified by high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry. The gene and protein expression of BA-related transporters and enzymes in the liver were measured by a quantitative real-time PCR method and western blotting method. RESULTS: The BA concentrations in serum were increased in the retrorsine-treated WT mice, along with the upregulation of BA transporters, Ostß, Mrp3, Mrp4, and Mrp2. When ABT was co-administered, the altered BA levels and Mrp4 mRNA and protein levels were reversed, accompanied by a 50% reduction of 6,7-dihydro-7-hydroxy-1- hydroxymethyl-5H-pyrrolizine (DHP) formation. When BSO was co-administered, serum BAs were not further increased, but Ostß, Mrp3, Mrp4 mRNA, and Mrp4 protein levels continuously increased. The induction of Mrp4 by retrorsine among the tested BA transporters was the only one that was abolished or enhanced in the presence of ABT or BSO. The Nrf2 protein levels in the nucleus increased in the retrorsine-treated WT mice, which were remarkably repressed by co-administration of ABT and enhanced by co-administration of BSO. In Nrf2 KO mice receiving retrorsine, the bile acids and the mRNA and protein levels of Mrp2, Mrp3, Mrp4, and Ostß were hardly changed, indicating the direct role of Nrf2 in retrorsine-induced BA changes in WT mice. CONCLUSION: The activation of Nrf2 translocation by forming the reactive metabolite of PAs induced the expressions of BA transporters and changed serum BA levels. Mrp4 was a sensitive biomarker for the perturbation of redox status caused by the formation of dehydropyrrolizidine.

Cell-based high-throughput screening for the evaluation of reactive metabolite formation potential.

Here, we established a high-throughput in vitro assay system to predict reactive metabolite (RM) formation. First, we performed the glutathione (GSH) consumption assay to monitor GSH levels as an index of RM formation potential using HepaRG cells pretreated with 500 µM D,L-buthionine-(S,R)-sulfoximine (BSO) and then treated with ticlopidine and diclofenac. Both drugs, under GSH-reduced conditions, significantly decreased relative cellular GSH content by 70% and 34%, respectively, compared with that in cells not pretreated with BSO. Next, we examined the correlation between GSH consumption and covalent binding assays; the results showed good correlation (correlation coefficient = 0.818). We then optimized the test compound concentration for evaluating RM formation potential using 76 validation compound sets, and the highest sensitivity (53%) was observed at 100 µM. Finally, using HepG2 cells, PXB-cells, and human primary hepatocytes, we examined the cell types suitable for evaluating RM formation potential. The expression of CYP3A4 was highest in HepaRG cells, suggesting the highest sensitivity (56.4%) of the GSH consumption assay. Moreover, a co-culture model of PXB-cells and HepaRG cells showed high sensitivity (72.7%) with sufficient specificity (85.7%). Thus, the GSH consumption assay can be used to effectively evaluate RM formation potential in the early stages of drug discovery.