Short-term inhalation exposure to cigarette smoke induces oxidative stress and inflammation in lungs without systemic oxidative stress in mice.

Smoking is a well-established risk factor for various pathologies, including pulmonary diseases, cardiovascular disorders, and cancers. The toxic effects of cigarette smoke (CS) are mediated through multiple pathways and diverse mechanisms. A key pathogenic factor is oxidative stress, primarily induced by excessive formation of reactive oxygen species. However, it remains unclear whether smoking directly induces systemic oxidative stress or if such stress is a secondary consequence. This study aimed to determine whether short-term inhalation exposure to CS induces oxidative stress in extrapulmonary organs in addition to the lung in a murine model. In the experiment, 3R4F reference cigarettes were used to generate CS, and 8-week-old male BALB/c mice were exposed to CS at a total particulate matter concentration of either 0 or 800 µg/L for four consecutive days. CS exposure led to an increase in neutrophils, eosinophils, and total cell counts in bronchoalveolar lavage fluid. It also elevated levels of lactate dehydrogenase and malondialdehyde (MDA), markers indicative of tissue damage and oxidative stress, respectively. Conversely, no significant changes were observed in systemic oxidative stress markers such as total oxidant scavenging capacity, MDA, glutathione (GSH), and the GSH/GSSG ratio in blood samples. In line with these findings, CS exposure elevated NADPH oxidase (NOX)-dependent superoxide generation in the lung but not in other organs like the liver, kidney, heart, aorta, and brain. Collectively, our results indicate that short-term exposure to CS induces inflammation and oxidative stress in the lung without significantly affecting oxidative stress in extrapulmonary organs under the current experimental conditions. NOX may play a role in these pulmonary-specific events.

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into the intricate mechanisms underlying drug-induced liver injury (DILI) with a specific focus on bromfenac, the withdrawn nonsteroidal anti-inflammatory drug.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.

Development of human pluripotent stem cell-derived hepatic organoids as an alternative model for drug safety assessment.

Human in vitro hepatic models that faithfully recapitulate liver function are essential for successful basic and translational research. A limitation of current in vitro models, which are extensively used for drug discovery and toxicity testing, is the loss of drug metabolic function due to the low expression and activity of cytochrome P450 (CYP450) enzymes. Here, we aimed to generate human pluripotent stem cell-derived hepatic organoids (hHOs) with a high drug metabolic ability. We established a two-step protocol to produce hHOs from human pluripotent stem cells for long-term expansion and drug testing. Fully differentiated hHOs had multicellular composition and exhibited cellular polarity and hepatobiliary structures. They also displayed remarkable CYP450 activity and recapitulated the metabolic clearance, CYP450-mediated drug toxicity, and metabolism. Furthermore, hHOs successfully modeled Wilson's disease in terms of Cu metabolism, drug responses, and diagnostic marker expression and secretion. In conclusion, hHOs exhibit high capacity for drug testing and disease modeling. Hence, this hepatic model system provides an advanced tool for studying hepatic drug metabolism and diseases.

Heterogeneity of glutamine metabolism in acquired-EGFR-TKI-resistant lung cancer.

AIMS: The purpose of this study was to evaluate the heterogeneities of glutamine metabolism in EGFR-TKI-resistant lung cancer cells and its potential as a therapeutic target. MAIN METHODS: Cell proliferation and cell cycle assays was performed by IncuCyte real-time analysis and flow cytometry, respectively. Tumor growth was assessed in xenografts implanted with HCC827 GR. An isotopologue analysis was conducted by LC-MS/MS using 13C-(U)-glutamine labeling to determine the amounts of metabolites. Cellular ATP and mitochondrial oxidative phosphorylation were determined by XFp analysis. KEY FINDINGS: We found that the cell growth of the two acquired EGFR-TKI-resistant lung cancer cells lines (HCC827 GR and H292 ER) depends on glutamine. In HCC827 GR, glutamine deficiency caused reduced GSH synthesis and, subsequently, enhanced ROS generation relative to their parental cells, HCC827. On the other hand, in H292 ER, glutamine mainly acted as a carbon source for TCA-cycle intermediates, and its depletion led to reduced mitochondrial ATP production. CB-839, a specific GLS inhibitor, inhibited the latter's conversion of glutamine to glutamate and exerted enhanced anti-proliferating effects on the two acquired EGFR-TKI-resistant lung cancer cell lines versus their parental cell lines. Moreover, oral administration of CB-839 significantly suppressed HCC827 GR tumor growth in the xenograft model. SIGNIFICANCE: These findings suggest that glutamine dependency in acquired EGFR-TKI-resistant lung cancer is heterogeneous and that inhibition of glutamine metabolism by CB-839 may serve as a therapeutic tool for acquired EGFR-TKI-resistant lung cancer.

TNP and its analogs: Modulation of IP6K and CYP3A4 inhibition.

Inositol hexakisphosphate kinase (IP6K) is an important mammalian enzyme involved in various biological processes such as insulin signalling and blood clotting. Recent analyses on drug metabolism and pharmacokinetic properties on TNP (N2-(m-trifluorobenzyl), N6-(p-nitrobenzyl)purine), a pan-IP6K inhibitor, have suggested that it may inhibit cytochrome P450 (CYP450) enzymes and induce unwanted drug-drug interactions in the liver. In this study, we confirmed that TNP inhibits CYP3A4 in type I binding mode more selectively than the other CYP450 isoforms. In an effort to find novel purine-based IP6K inhibitors with minimal CYP3A4 inhibition, we designed and synthesised 15 TNP analogs. Structure-activity relationship and biochemical studies, including ADP-Glo kinase assay and quantification of cell-based IP7 production, showed that compound 9 dramatically reduced CYP3A4 inhibition while retaining IP6K-inhibitory activity. Compound 9 can be a tool molecule for structural optimisation of purine-based IP6K inhibitors.

Generation of uniform liver spheroids from human pluripotent stem cells for imaging-based drug toxicity analysis.

Recent advances in pluripotent stem cell technology provide an alternative source of human hepatocytes to overcome the limitations of current toxicity tests. However, this approach requires optimization and standardization before it can be used as a fast and reliable toxicity screening system. Here, we designed and tested microwell culture platforms with various diameters. We found that large quantities of uniformly-sized hepatocyte-like cell (HLC) spheroids (3D-uniHLC-Ss) could be efficiently and reproducibly generated in a short period time from a small number of differentiating human pluripotent stem cells (hPSCs). The hPSC-3D-uniHLC-Ss that were produced in 500-µm diameter microwells consistently exhibited high expressions of hepatic marker genes and had no significant signs of cell death. Importantly, a hepatic master gene hepatocyte nuclear factor 4α (HNF4α) was maintained at high levels, and the epithelial-mesenchymal transition was significantly attenuated in hPSC-3D-uniHLC-Ss. Additionally, when compared with 3D-HLC-Ss that were produced in other 3D platforms, hPSC-3D-uniHLC-Ss showed significantly higher hepatic gene expressions and drug-metabolizing activity of the enzyme, CYP3A4. Imaging-based drug toxicity studies demonstrated that hPSC-3D-uniHLC-Ss exhibited enhanced sensitivity to various hepatotoxicants, compared to HLCs, which were differentiated under 2D conditions. Precise prediction of drug-induced hepatotoxicity is a crucial step in the early phases of drug discovery. Thus, the hPSC-3D-uniHLC-Ss produced using our microwell platform could be used as an imaging-based toxicity screening system to predict drug hepatotoxicity.

Quasi-Irreversible Inhibition of CYP2D6 by Berberine.

In our previous study, Hwang-Ryun-Hae-Dok-Tang, which contains berberine (BBR) as a main active ingredient, inhibited cytochrome P450 (CYP) 2D6 in a quasi-irreversible manner. However, no information is available on the detailed mechanism of BBR-induced CYP2D6 inhibition. Thus, the present study aimed to characterize the inhibition mode and kinetics of BBR and its analogues against CYP2D6 using pooled human liver microsomes (HLM). BBR exhibited selective quasi-irreversible inhibition of CYP2D6 with inactivation rate constant (kinact) of 0.025 min-1, inhibition constant (KI) of 4.29 µM, and kinact/KI of 5.83 mL/min/µmol. In pooled HLM, BBR was metabolized to thalifendine (TFD), demethyleneberberine (DMB), M1 (proposed as demethylene-TFD), and to a lesser extent berberrubine (BRB), showing moderate metabolic stability with a half-life of 35.4 min and a microsomal intrinsic clearance of 7.82 µL/min/mg protein. However, unlike BBR, those metabolites (i.e., TFD, DMB, and BRB) were neither selective nor potent inhibitors of CYP2D6, based on comparison of half-maximal inhibitory concentration (IC50). Notably, TFD, but not DMB, exhibited metabolism-dependent CYP2D6 inhibition as in the case of BBR, which suggests that methylenedioxybenzene moiety of BBR may play a critical role in the quasi-irreversible inhibition. Moreover, the metabolic clearance of nebivolol (ß-blocker; CYP2D6 substrate) was reduced in the presence of BBR. The present results warrant further evaluation of BBR-drug interactions in clinical situations.

Live-cell screening platform using human-induced pluripotent stem cells expressing fluorescence-tagged cytochrome P450 1A1.

Human-induced pluripotent stem cells (hiPSCs) are invaluable sources for drug screening and toxicity tests because of their differentiation potential and proliferative capacity. Recently, the CRISPR-Cas9-mediated homologous recombination system has enabled reporter knock-ins at desired loci in hiPSCs, and here, we generated a hiPSC reporter line expressing mCherry-tagged cytochrome P450 1A1 (CYP1A1), which can be utilized to screen for the modulators of aryl hydrocarbon receptor (AHR) in live cells. CYP1A1-mCherry hiPSCs exhibited typical characteristics of pluripotent stem cells such as marker expression, differentiation potential, and normal karyotype. After differentiation into hepatocyte-like cells (HLCs), CYP1A1-mCherry fusion protein was expressed and localized at the endoplasmic reticulum, and induced by AHR agonists. We obtained 23 hits modulating CYP1A1 expression from high-content screening with 241 hepatotoxicity chemicals and nuclear receptor ligands, and identified three upregulating chemicals and two downregulating compounds. Responses of hiPSC-HLCs against an AHR agonist were more similar to human primary hepatocytes than of HepG2 hepatocellular carcinoma cells. This platform has the advantages of live-cell screening without sacrificing cells (unlike previously available CYP1A1 reporter cell lines), as well as an indefinite supply of cells, and can be utilized in a wide range of screening related to AHR- and CYP1A1-associated diseases in desired cell types.

Short-term regulation of the hepatic activities of cytochrome P450 and glutathione S-transferase by nose-only cigarette smoke exposure in mice.

The present study aimed to determine the effects of cigarette smoke on the regulation of hepatic cytochrome P450 (CYP) and glutathione S-transferase (GST) enzymes in male BALB/c mice exposed to nose-only cigarette smoke for 4 days. There were no significant increases in serum liver injury markers (alanine aminotransferase and aspartate aminotransferase) or oxidative stress (total antioxidant capacity, malondialdehyde, and glutathione disulfide/reduced glutathione) following cigarette smoke exposure, but malondialdehyde was elevated in the bronchoalveolar lavage fluid of smoke-exposed mice. Additionally, the hepatic microsomal protein levels of Cyp1a and Cyp2b, and the activities of ethoxyresorufin O-deethylase, pentoxyresorufin O-depenylase, and chlorzoxazone 6-hydrxylase, were elevated in smoke-exposed mice. Interestingly, the hepatic activities of GST toward 1-chloro-2,4-dinitrobenzene, 1,2-dichloro-4-nitrobenzene, and ethacrynic acid, but not cumene hydroperoxide were enhanced by cigarette smoke exposure, which was consistent with the increased expression levels of mu- and pi-class GSTs, but not alpha-class GSTs, observed in immunoblot analyses. These findings indicate that the short-term inhalation of cigarette smoke induces drug-metabolizing enzymes such as CYP1A, CYP2B, and mu/pi-class GSTs in the absence of hepatic injury and oxidative stress. Furthermore, smoking may alter hepatic drug metabolism, as well as the disposition and toxicity of xenobiotics, including some therapeutic drugs and cigarette smoke constituents.

Age-Related Changes in Sulfur Amino Acid Metabolism in Male C57BL/6 Mice.

Alterations in sulfur amino acid metabolism are associated with an increased risk of a number of common late-life diseases, which raises the possibility that metabolism of sulfur amino acids may change with age. The present study was conducted to understand the age-related changes in hepatic metabolism of sulfur amino acids in 2-, 6-, 18- and 30-month-old male C57BL/6 mice. For this purpose, metabolite profiling of sulfur amino acids from methionine to taurine or glutathione (GSH) was performed. The levels of sulfur amino acids and their metabolites were not significantly different among 2-, 6- and 18-month-old mice, except for plasma GSH and hepatic homocysteine. Plasma total GSH and hepatic total homocysteine levels were significantly higher in 2-month-old mice than those in the other age groups. In contrast, 30-month-old mice exhibited increased hepatic methionine and cysteine, compared with all other groups, but decreased hepatic S-adenosylmethionine (SAM), S-adenosylhomocysteine and homocysteine, relative to 2-month-old mice. No differences in hepatic reduced GSH, GSH disulfide, or taurine were observed. The hepatic changes in homocysteine and cysteine may be attributed to upregulation of cystathionine ß-synthase and down-regulation of γ-glutamylcysteine ligase in the aged mice. The elevation of hepatic cysteine levels may be involved in the maintenance of hepatic GSH levels. The opposite changes of methionine and SAM suggest that the regulatory role of SAM in hepatic sulfur amino acid metabolism may be impaired in 30-month-old mice.

Role of the CYP3A4-mediated 11,12-epoxyeicosatrienoic acid pathway in the development of tamoxifen-resistant breast cancer.

Epoxyeicosatrienoic acid (EET) production via cytochrome P450 (CYP) epoxygenases closely correlates with the progression of breast cancer. However, its role in the development of chemoresistant breast cancers has yet to be elucidated. Here, we found that CYP3A4 expression and its epoxy-product, 11,12-epoxyeicosatrienoic acid (11,12-EET) was enhanced in tamoxifen (TAM)-resistant MCF-7 (TAMR-MCF-7) breast cancer cells compared to control MCF-7 cells. Treatment of TAMR-MCF-7 cells with ketoconazole and azamulin (selective CYP3A4 inhibitors) or 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE, an EET antagonist) inhibited cellular proliferation and recovered the sensitivity to 4-hydroxytamoxifen. Chick chorioallantoic membrane and trans-well migration analyses revealed that the enhanced angiogenic, tumorigenic, and migration intensities of TAMR-MCF-7 cells were also significantly suppressed by ketoconazole and 14,15-EEZE. We previously reported that Pin1, a peptidyl prolyl isomerase, is a crucial regulator for higher angiogenesis and epithelial-mesenchymal transition characteristics of TAMR-MCF-7 cells. EET inhibition suppressed E2F1-dependent Pin1 gene transcription, and Pin1 silencing also blocked cell proliferation, angiogenesis, and migration of TAMR-MCF-7 cells. Our findings suggest that the CYP3A4-mediated EET pathway represents a potential therapeutic target for the treatment of tamoxifen-resistant breast cancer.

Development, validation, and application of ELISA for detection of anti-HD105 antibodies in pre-clinical safety evaluation using monkeys.

Unwanted immunogenicity of protein therapeutics can result in severe side effects and should be assessed in animals before applying the treatment to humans. Monkeys are the most relevant choice for pre-clinical toxicity testing of antibody-based therapeutics. To assess the immunogenicity of HD105, a novel antibody therapeutic that targets both vascular endothelial growth factor and Delta-like-ligand 4, a bridging enzyme-linked immunosorbent assay was developed as an anti-drug antibody (ADA) assay and validated for use in pre-clinical studies using non-human primates. This method was found to have suitable assay sensitivity, intra- and inter-assay precision, confirmation, drug tolerance, recovery, and sample stability for measuring ADA in monkey serum samples. The results showed that ADA elevation occurred following repeated doses of HD105, and that ADA production was negatively associated with serum HD105 concentration. These results suggest that intravenous administration of HD105 induces production of ADA in monkeys and that the detection of ADA may be negatively influenced by free HD105 in serum.

Hepatic expression of cytochrome P450 in Zucker diabetic fatty rats.

In this study, the hepatic expression of cytochrome P450 (CYP) enzymes, including CYP1A1/2, 2B1, 2C11, 2E1, 3A1/2, and 4A, was investigated in 5-week-old (insulinresistant state) and 11-week-old (diabetic) Zucker diabetic fatty (ZDF) rats. Serum glucose and glycated hemoglobin levels were increased in 11-week-old ZDF rats, but not in 5-weekold ZDF rats. Hyperinsulinemia was observed in both age groups. The microsomal protein, total CYP, CYP reductase, CYP1A1/2, and CYP3A1 levels did not differ between 5- and 11-week-old ZDF rats and their respective control rats, while CYP4A was up-regulated in both groups. Hepatic levels of cytochrome b5, CYP2B1, CYP2C11, CYP2E1, and CYP3A2 were decreased in 5-week-old ZDF rats, but not in 11-week-old ZDF rats. Similarly, pentoxyresorufin O-depentylase, testosterone 2α- and 16α-hydroxylase, chlorzoxazone 6- hydroxylase, and midazolam 1'- and 4-hydroxylase activities were decreased only in 5-weekold ZDF rats. Based on these results, the 5-week-old ZDF rats exhibited down-regulation of the major CYP enzymes. These results suggest that hepatic expression of CYP enzymes may be dysregulated during development in ZDF rats. With the exception of CYP2B1 and CYP4A, the hepatic levels and activities of CYP were comparable between 11-week-old ZDF and control rats, suggesting that xenobiotic metabolism is normally regulated in the early diabetic state.

Discovery of enantioselectivity of urea inhibitors of soluble epoxide hydrolase.

Soluble epoxide hydrolase (sEH) hydrolyzes epoxyeicosatrienoic acids (EETs) in the metabolic pathway of arachidonic acid and has been considered as an important therapeutic target for chronic diseases such as hypertension, diabetes and inflammation. Although many urea derivatives are known as sEH inhibitors, the enantioselectivity of the inhibitors is not highlighted in spite of the stereoselective hydrolysis of EETs by sEH. In an effort to explore the importance of enantioselectivity in the urea scaffold, a series of enantiomers with the stereocenter adjacent to the urea nitrogen atom were prepared. The selectivity of enantiomers of 1-(α-alkyl-α-phenylmethyl)-3-(3-phenylpropyl)ureas showed wide range differences up to 125 fold with the low IC50 value up to 13 nM. The S-configuration with planar phenyl and small alkyl groups at α-position is crucial for the activity and selectivity. However, restriction of the free rotation of two α-groups with indan-1-yl or 1,2,3,4-tetrahydronaphthalen-1-yl moiety abolishes the selectivity between the enantiomers, despite the increase in activity up to 13 nM. The hydrophilic group like sulfonamido group at para position of 3-phenylpropyl motif of 1-(α-alkyl-α-phenylmethyl-3-(3-phenylpropyl)urea improves the activity as well as enantiomeric selectivity. All these ureas are proved to be specific inhibitor of sEH without inhibition against mEH.

Prediction of Drug-Induced Liver Injury in HepG2 Cells Cultured with Human Liver Microsomes.

Drug-induced liver injury (DILI) via metabolic activation by drug-metabolizing enzymes, especially cytochrome P450 (CYP), is a major cause of drug failure and drug withdrawal. In this study, an in vitro model using HepG2 cells in combination with human liver microsomes was developed for the prediction of DILI. The cytotoxicity of cyclophosphamide, a model drug for bioactivation, was augmented in HepG2 cells cultured with microsomes in a manner dependent on exposure time, microsomal protein concentration, and NADPH. Experiments using pan- or isoform-selective CYP inhibitors showed that CYP2B6 and CYP3A4 are responsible for the bioactivation of cyclophosphamide. In a metabolite identification study employing LC-ESI-QTrap and LC-ESI-QTOF, cyclophosphamide metabolites including phosphoramide mustard, a toxic metabolite, were detected in HepG2 cells cultured with microsomes, but not without microsomes. The cytotoxic effects of acetaminophen and diclofenac were also potentiated by microsomes. The potentiation of acetaminophen cytotoxicity was dependent on CYP-dependent metabolism, and the augmentation of diclofenac cytotoxicity was not mediated by either CYP- or UDP-glucuronosyltransferase-dependent metabolism. The cytotoxic effects of leflunomide, nefazodone, and bakuchiol were attenuated by microsomes. The detoxication of leflunomide by microsomes was attributed to mainly CYP3A4-dependent metabolism. The protective effect of microsomes against nefazodone cytotoxicity was dependent on both CYP-mediated metabolism and nonspecific protein binding. Nonspecific protein binding but not CYP-dependent metabolism played a critical role in the attenuation of bakuchiol cytotoxicity. The present study suggests that HepG2 cells cultured with human liver microsomes can be a reliable model in which to predict DILI via bioactivation by drug metabolizing enzymes.

Metabolic characterization of meso-dihydroguaiaretic acid in liver microsomes and in mice.

meso-Dihydroguaiaretic acid (MDGA) is a major component of Myristica fragrans and Machilus thunbergii that is traditionally used as a spice and for medicinal purposes. Despite reports of various biological activities exerted by MDGA, there is no information regarding its metabolic properties. The purpose of this study was to determine the metabolic stability and cytochrome P450 (CYP) inhibitory potential of MDGA, using pooled human liver microsomes (HLMs) to characterize its metabolic properties. In addition, pharmacokinetic analysis was performed in mice treated intravenously (5 mg/kg) or orally (20 mg/kg) with MDGA for comparison with our in vitro results. The half-life of MDGA in HLMs and mouse liver microsomes incubated with NADPH, UDPGA or NADPH plus UDPGA was 25.41 and 22.74, 0.39 and 0.20 or 0.28 and 0.22 min, respectively. In our pharmacokinetic study, MDGA rapidly declined in plasma and had low bioavailability, which was attributable to extensive metabolism by UDP-glucuronosyltransferases and CYPs. Among CYP isoforms, CYP2E1 activity was selectively inhibited by MDGA through a competitive inhibitory mode, with an inhibitory constant (Ki) value of 13.1 µM. These results suggest that MDGA can be used as a selective CYP2E1 inhibitor in vitro, which warrants evaluation of the pharmacological significance of MDGA-induced CYP2E1 inhibition.