Adverse effects of the 5-alpha-reductase inhibitor finasteride on Daphnia magna: Endocrine system and lipid metabolism disruption.

Finasteride, a steroid 5-alpha reductase inhibitor, is commonly used for the treatment of benign prostatic hyperplasia and hair loss. However, despite continued use, its environmental implications have not been thoroughly investigated. Thus, we investigated the acute and chronic adverse impacts of finasteride on Daphnia magna, a crucial planktonic crustacean in freshwater ecosystems selected as bioindicator organism for understanding the ecotoxicological effects. Chronic exposure (for 23 days) to finasteride negatively affected development and reproduction, leading to reduced fecundity, delayed first brood, reduced growth, and reduced neonate size. Additionally, acute exposure (< 24 h) caused decreased expression levels of genes crucial for reproduction and development, especially EcR-A/B (ecdysone receptors), Jhe (juvenile hormone esterase), and Vtg2 (vitellogenin), with oxidative stress-related genes. Untargeted lipidomics/metabolomic analyses revealed lipidomic alteration, including 19 upregulated and 4 downregulated enriched lipid ontology categories, and confirmed downregulation of metabolites. Pathway analysis implicated significant effects on metabolic pathways, including the pentose phosphate pathway, histidine metabolism, beta-alanine metabolism, as well as alanine, aspartate, and glutamate metabolism. This comprehensive study unravels the intricate molecular and metabolic responses of D. magna to finasteride exposure, underscoring the multifaceted impacts of this anti-androgenic compound on a keystone species of freshwater ecosystems. The findings emphasize the importance of understanding the environmental repercussions of widely used pharmaceuticals to protect biodiversity in aquatic ecosystems.

A combined in vitro-in silico method for assessing the androgenic activities of bisphenol A and its analogues.

Interactions between endocrine-disruptor chemicals (EDCs) and androgen receptor (AR) have adverse effects on the endocrine system, leading to human reproductive dysfunction. Bisphenol A (BPA) is an EDC that can damage both the environment and human health. Although numerous BPA analogues have been produced as substitutes for BPA, few studies have evaluated their endocrine-disrupting abilities. We assessed the (anti)-androgenic activities of BPA and its analogues using a yeast-based reporter assay. The BPA analogues tested were bisphenol S (BPS), 4-phenylphenol (4PP), 4,4'-(9-fluorenyliden)-diphenol (BPFL), tetramethyl bisphenol F (TMBPF), and tetramethyl bisphenol A (TMBPA). We also conducted molecular docking and dynamics simulations to assess the interactions of BPA and its analogues with the ligand-binding domain of human AR (AR-LBD). Neither BPA nor its analogues had androgenic activity; however, all except BPFL exerted robust anti-androgenic effects. Consistent with the in vitro results, anti-androgenic analogues of BPA formed hydrogen bonding patterns with key residues that differed from the patterns of endogenous hormones, indicating that the analogues display in inappropriate orientations when interacting with the binding pocket of AR-LBD. Our findings indicate that BPA and its analogues disrupt androgen signaling by interacting with the AR-LBD. Overall, BPA and its analogues display endocrine-disrupting activity, which is mediated by AR.

Investigating Endocrine Disrupting Impacts of Nine Disinfection Byproducts on Human and Zebrafish Estrogen Receptor Alpha.

BACKGROUND: Disinfection byproducts (DBPs) cause endocrine disruption via estrogenic or anti-estrogenic effects on estrogen receptors. However, most studies have focused on human systems, with little experimental data being presented on aquatic biota. This study aimed to compare the effects of nine DBPs on zebrafish and human estrogen receptor alpha (zERα and hERα). METHODS: In vitro enzyme response-based tests, including cytotoxicity and reporter gene assays, were performed. Additionally, statistical analysis and molecular docking studies were employed to compare ERα responses. RESULTS: Iodoacetic acid (IAA), chloroacetonitrile (CAN), and bromoacetonitrile (BAN) showed robust estrogenic activity on hERα(maximal induction ratios of 108.7%, 50.3%, and 54.7%, respectively), while IAA strongly inhibited the estrogenic activity induced by 17ß-estradiol (E2) in zERα (59.8% induction at the maximum concentration). Chloroacetamide (CAM) and bromoacetamide (BAM) also showed robust anti-estrogen effects in zERα (48.1% and 50.8% induction at the maximum concentration, respectively). These dissimilar endocrine disruption patterns were thoroughly assessed using Pearson correlation and distance-based analyses. Clear differences between the estrogenic responses of the two ERαs were observed, whereas no pattern of anti-estrogenic activities could be established. Some DBPs strongly induced estrogenic endocrine disruption as agonists of hERα, while others inhibited estrogenic activity as antagonists of zERα. Principal coordinate analysis (PCoA) showed similar correlation coefficients for estrogenic and anti-estrogenic responses. Reproducible results were obtained from computational analysis and the reporter gene assay. CONCLUSIONS: Overall, the effects of DBPs on both human and zebrafish highlight the importance of controlling their differences in responsiveness for estrogenic activities including the water quality monitoring and endocrine disruption, as DBPs have species-specific ligand-receptor interactions.

In vitro metabolic characterization of the SARS-CoV-2 papain-like protease inhibitors GRL0617 and HY-17542.

The SARS-CoV-2 pandemic requires a new therapeutic target for viral infection, and papain-like protease (Plpro) has been suggested as a druggable target. This in-vitro study was conducted to examine the drug metabolism of the GRL0617 and HY-17542, Plpro inhibitors. Metabolism of these inhibitors was studied to predict the pharmacokinetics in human liver microsomes. The hepatic cytochrome P450 (CYP) isoforms responsible for their metabolism were identified using recombinant enzymes. The drug-drug interaction potential mediated by cytochrome P450 inhibition was estimated. In human liver microsomes, the Plpro inhibitors had phase I and phase I + II metabolism with half-lives of 26.35 and 29.53 min, respectively. Hydroxylation (M1) and desaturation (-H2, M3) of the para-amino toluene side chain were the predominant reactions mediated with CYP3A4 and CYP3A5. CYP2D6 is responsible for the hydroxylation of the naphthalene side ring. GRL0617 inhibits major drug-metabolizing enzymes, including CYP2C9 and CYP3A4. HY-17542 is structural analog of GRL0617 and it is metabolized to GRL0617 through non-cytochrome P450 reactions in human liver microsomes without NADPH. Like GRL0617 and HY-17542 undergoes additional hepatic metabolism. The in-vitro hepatic metabolism of the Plpro inhibitors featured short half-lives; preclinical metabolism studies are needed to determine therapeutic doses for these inhibitors.

Thyroid-Friendly Soft Materials as 3D Cell Culture Tool for Stimulating Thyroid Cell Function.

The disruption of thyroid hormones because of chemical exposure is a significant societal problem. Chemical evaluations of environmental and human health risks are conventionally based on animal experiments. However, owing to recent breakthroughs in biotechnology, the potential toxicity of chemicals can now be evaluated using 3D cell cultures. In this study, the interactive effects of thyroid-friendly soft (TS) microspheres on thyroid cell aggregates are elucidated and their potential as a reliable toxicity assessment tool is evaluated. Using state-of-the-art characterization methods coupled with cell-based analysis and quadrupole time-of-flight mass spectrometry, it is shown that TS-microsphere-integrated thyroid cell aggregates exhibit improved thyroid function. Specifically, the responses of zebrafish embryos, which are used for thyroid toxicity analysis, and the TS-microsphere-integrated cell aggregates to methimazole (MMI), a known thyroid inhibitor, are compared. The results show that the thyroid hormone disruption response of the TS-microsphere-integrated thyroid cell aggregates to MMI is more sensitive compared with those of the zebrafish embryos and conventionally formed cell aggregates. This proof-of-concept approach can be used to control cellular function in the desired direction and hence evaluate thyroid function. Thus, the proposed TS-microsphere-integrated cell aggregates may yield new fundamental insights for advancing in vitro cell-based research.

Endocrine-disrupting potential and toxicological effect of para-phenylphenol on Daphnia magna.

Several phenol derivatives are suspected endocrine disruptors and have received attention in risk assessment studies for several decades owing to the structural similarity between estrogens and phenolic compounds. We assessed the endocrine disrupting effect of the phenolic compound para-phenylphenol (PPP) through acute tests and evaluating chronic endpoints in an invertebrate model, Daphnia magna. Exposure of D. magna to PPP induced substantial adverse effects, namely, reduced fecundity, slowed growth rate, delayed first brood, and a reduction in neonate size. Furthermore, we investigated the mRNA expression of relevant genes to elucidate the mechanism of endocrine disruption by PPP. Exposure of D. magna to PPP induced the substantial downregulation of genes and markers related to reproduction and development, such as EcR-A, EcR-B, Jhe, and Vtg. Consequently, we demonstrated that PPP has an endocrine disrupting effect on reproduction and development in D. magna.

Species Differences in Response to Binding Interactions of Bisphenol A and its Analogs with the Modeled Estrogen Receptor 1 and In Vitro Reporter Gene Assay in Human and Zebrafish.

Adverse impacts associated with the interactions of numerous endocrine-disruptor chemicals (EDCs) with estrogen receptor 1 play a pivotal role in reproductive dysfunction. The predictive studies on these interactions thus are crucial in the risk assessment of EDCs but rely heavily on the accuracy of specific protein structure in three dimensions. As the three-dimensional (3D) structure of zebrafish estrogen receptor 1 (zEsr1) is not available, the 3D structure of zEsr1 ligand-binding domain (zEsr1-LBD) was generated using MODELLER and its quality was assessed by the PROCHECK, ERRAT, ProSA, and Verify-3D tools. After the generated model was verified as reliable, bisphenol A and its analogs were docked on the zEsr1-LBD and human estrogen receptor 1 ligand-binding domain (hESR1-LBD) using the Discovery Studio and Autodock Vina programs. The molecular dynamics followed by molecular docking were simulated using the Nanoscale Molecular Dynamics program and compared to those of the in vitro reporter gene assays. Some chemicals were bound with an orientation similar to that of 17ß-estradiol in both models and in silico binding energies showed moderate or high correlations with in vitro results (0.33 ≤ r2 ≤ 0.71). Notably, hydrogen bond occupancy during molecular dynamics simulations exhibited a high correlation with in vitro results (r2 ≥ 0.81) in both complexes. These results show that the combined in silico and in vitro approaches is a valuable tool for identifying EDCs in different species, facilitating the assessment of EDC-induced reproductive toxicity. Environ Toxicol Chem 2022;41:2431-2443. © 2022 SETAC.

Integration of Bioinspired Fibrous Strands with 3D Spheroids for Environmental Hazard Monitoring.

Numerous methods have been introduced to produce 3D cell cultures that can reduce the need for animal experimentation. This study presents a unique 3D culture platform that features bioinspired strands of electrospun nanofibers (BSeNs) and aquatic cell lines to compensate for shortcomings in the current cell spheroid generation techniques. The use of BSeNs in 3D zebrafish liver cell cultures is found to improve liver and reproductive functions through spheroid-based in vitro assays such as whole transcriptome sequencing and reproductive toxicity testing, with optimized properties exhibiting results similar to those obtained for fish embryo acute toxicity (FET, OECD TG 236) following exposure to environmental endocrine-disrupting chemicals (17ß-Estradiol (E2), 4-hydroxytamoxifen (4-HT), and bisphenol compounds (bisphenol A (BPA) and 9,9-Bis(4-hydroxyphenyl)fluorene (BPFL)). These findings indicate that the beneficial effects of bioinspired materials that closely mimic ECM environments can yield efficient zebrafish cells with intrinsic functions and xenobiotic metabolism similar to those of zebrafish embryos. As a closer analog for the in vivo conditions that are associated with exposure to potentially hazardous chemicals, the straightforward culture model introduced in this study shows promise as an alternative tool that can be used to further eco-environmental assessment.

Mono(2-ethylhexyl) phthalate modulates lipid accumulation and reproductive signaling in Daphnia magna.

Mono(2-ethylhexyl) phthalate (MEHP) is a primary metabolite of di(2-ethylhexyl) phthalate (DEHP), which is widely used in industry as a plasticizer. Both DEHP and MEHP have been identified as endocrine disruptors affecting reproduction systems in natural aquatic environments. However, the effects of MEHP exposure on aquatic invertebrates such as Daphnia magna are still poorly understood. In the present study, lipid alterations caused by MEHP in D. magna were identified by analyzing lipid accumulation and nontarget metabolomics. In addition, reproductive endpoints were investigated. MEHP exposure under any conditions upto 2 mg/L was not associated with mortality of D. magna; yet, the number of lipid droplets and the adult female daphnids reproduction rates increased after 96 h of exposure and 21 days of exposure, respectively. MEHP also enhanced lipid metabolism, as evident from 283 potential lipid metabolites, including glycerolipids, glycerophospholipids, and sphingolipids, identified following 48 h of exposure. The MEHP-treated group exhibited significantly higher ecdysone receptor (EcR) and vitellogenin 2 (Vtg2) expression levels at 6 and 24 h. At 48 h, EcR and Vtg2 expression levels were downregulated in the 1 and 2 mg/L MEHP exposure groups. Our data reveal that the EcR pathway changes over MEHP exposure could be associated with lipid accumulation, owing to increased lipid levels and the subsequent increase in the reproduction of MEHP-exposed D. magna.

Transcriptomic and physiological analysis of endocrine disrupting chemicals Impacts on 3D Zebrafish liver cell culture system.

In recent decades, extensive efforts have focused on developing in vitro platforms mimicking fish livers to better understand the acute or chronic effects of toxicants on lower aquatic vertebrates. Fish liver cell lines have emerged as a promising culture system for these in vitro platforms because they complement the currently limited in vitro tools that mostly consist of mammalian cell lines and adhere to the 3Rs: replacement, reduction, and refinement of living animal tests. However, monolayer cell lines have lower transcriptional and physiological responses upon exposure to toxic chemicals than freshly isolated primary cells. To overcome this challenge, we utilized a three-dimensional (3D) spheroid-based in vitro platform, in which hepatocyte cells had self-organized into spheroid forms via E-cadherin bonds. This platform exhibited augmented transcriptomic and phenotypic regulation of liver cells in comparison to monolayer cells. We examined the organoid platform using the zebrafish liver (ZFL) cell line as a model system. ZFL cells spontaneously clustered into 3D spheroids with long-term viability by optimizing cell seeding density on a non-adherent substrate. Interestingly, 3D ZFL spheroids treated with estrogenic chemicals were activated to synthesize a higher level of vitellogenin (Vtg) than monolayer cells. Whole-transcriptome sequencing analysis confirmed that 3D ZFL spheroids had greater transcriptional regulation of genes related to reproductive toxicological response and liver functions, such as the urea cycle, estrogen receptors, and vitellogenin, compared to monolayer cells. These results may contribute to the engineering of novel 3D in vitro platforms for screening harmful chemicals and improving understanding of the underlying liver toxicity mechanisms at the molecular and cellular levels.

LC-MS-based assay of granisetron 7-hydroxylation activity for the evaluation of CYP1A1 induction from diesel particulate matter-exposed hepatic and respiratory cell lines.

Particulate matter (PM) generally consists of aggregated particles containing trace metals and polycyclic aromatic hydrocarbons (PAHs). Cytochrome P450 (CYP) 1A1, one of the extensively investigated biomarkers, is highly inducible when PAHs activate the aryl hydrocarbon receptor (AhR). The present study focused on developing a LC-MS/MS-based assay to evaluate CYP1A1 induction potential following PM exposure. This assay adapted a CYP1A1 selective reaction of granisetron 7-hydroxylation in response to an AhR inducer, 6-formylindolo[3,2-b]carbazole (FICZ), in HepaRG and A549 cell lines. Exposure to FICZ (10 nM) increased the levels of granisetron 7-hydroxylation significantly, whereas no elevation of ethoxyresorufin-O-deethylation (EROD) activity was found in HepaRG cells. In A549 cells, granisetron 7-hydroxylation showed a better dose-response from 0 to 10000 nM FICZ treatment than EROD. EROD Additionally, the application of the assay with diesel PM exposure showed a concentration-dependent induction of CYP1A1 in HepaRG, A549, and human nasal epithelial cells. The granisetron assay has better selectivity for CYP1A1 than the conventional EROD assay, which is overlapped reaction with CYP1A2 and CYP1B1, with high correlations between AhR activation and CYP1A1 mRNA levels. Accompanying the great application potential to different organs and cell culture systems, future studies will implement the granisetron assay for the respiratory toxicity evaluation.

Development of a Liquid Chromatography/Mass Spectrometry-Based Inhibition Assay for the Screening of Steroid 5-α Reductase in Human and Fish Cell Lines.

Steroid 5-α reductase (5AR) is responsible for the reduction of steroids to 5-α reduced metabolites, such as the reduction of testosterone to 5-α dihydrotestosterone (DHT). A new adverse outcome pathway (AOP) for 5AR inhibition to reduce female reproduction in fish (AOP 289) is under development to clarify the antiestrogenic effects of 5AR inhibitors in female fish. A sensitive method for the DHT analysis using chemical derivatization and liquid chromatography-tandem mass spectrometry was developed. A cell-based 5AR inhibition assay that utilizes human cell lines, a transient overexpression system, and fish cell lines was developed. The measured IC50 values of two well-known 5AR inhibitors, finasteride and dutasteride, were comparable in the different systems. However, the IC50 of dutasteride in the fish cell lines was lower than that in the human cell lines. Finasteride showed a higher IC50 against the RTG-2 cell line. These results demonstrated that 5ARs inhibition could differ in terms of structural characteristics among species. The assay has high sensitivity and reproducibility and is suitable for the application in 5AR inhibition screening for various endocrine disruption chemicals (EDCs). Future studies will continue to evaluate the quantitative inhibition of 5AR by EDCs to compare the endocrine-disrupting pathway in different species.

Mono-(2-ethylhexyl) phthalate induces oxidative stress and lipid accumulation in zebrafish liver cells.

The purpose of the present study was to examine the antioxidant and oxidative stress changes in zebrafish liver (ZFL) cells in the presence of mono-(2-ethylhexyl) phthalate (MEHP). When reactive oxygen species (ROS) and antioxidant levels were measured by immunoassay, significant differences were observed between MEHP-treated and control cells, while catalase levels did not change in any group. MEHP-treated cells had higher levels of ROS, glutathione peroxidase (GPx), glutathione S-transferase (GST), glutathione, and superoxide dismutase (SOD) than control cells. However, lower levels of lipid peroxidation were observed in MEHP-treated cells compared to control cells. After 24 h of MEHP treatment, ROS, SOD, GPx, and GST activity increased in a dose-dependent manner. Cellular lipid droplet formation and endoplasmic reticulum stress were both induced in the presence of MEHP. These findings demonstrated the potential impacts of the association of MEHP with adverse outcomes in fish liver. Future studies will focus on clarifying the molecular mechanism of phthalate toxicity via oxidative stress and peroxisome proliferator activated receptor as the major mechanistic pathway.

Potential adverse effect of tyrosinase inhibitors on teleosts:A review.

Coloration plays a crucial role in the social communication and survival of organisms. Multidisciplinary studies have been conducted to elucidate the correlation between coloration and melanin biosynthesis (referred as melanogenesis). The multi-copper enzyme tyrosinase catalyzes the first two steps of melanogenesis for coloration in teleosts. Due to the increasing demand of tyrosinase inhibitors for the production of skin whitening cosmetics, hypopigmentation pharmaceuticals, and anti-browning agents, a large number of natural and synthetic inhibitors have been developed over the past few decades. Although a number of previous studies have focused on human use and toxicity, such as the increased cytotoxic effects of ROS-generating compounds, their ecotoxicological impacts on aquatic organisms are still poorly understood. Hence, the focus of the present review is to describe the role of coloration in teleosts as well as potential ecotoxicological effects elicited by exposure to tyrosinase inhibitors. Furthermore, this review introduces our recently registered adverse outcome pathway (AOP) related to tyrosinase inhibition and population decline in teleosts.

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.

Role of cytochrome P450 enzymes in fimasartan metabolism in vitro.

Fimasartan (FMS), an angiotensin II receptor antagonist, is metabolized to FMS S-oxide, FMS N-glucuronide, oxidative desulfurized FMS (BR-A-557), and hydroxy-n-butyl FMSs. The purpose of this study was to characterize enzymes involved in NADPH-dependent FMS metabolism using recombinant enzymes such as cytochrome P450 (CYP) and flavin-containing monooxygenase (FMO), as well as selective chemical inhibitors. The results showed that CYP, but not FMO, plays a major role in FMS metabolism. CYP2C9, CYP3A4, and CYP3A5 were involved in the formation of FMS S-oxide, which was further metabolized to BR-A-557 by CYP3A4/5. CYP2C9 played an exclusive role in n-butyl hydroxylation. The specificity constant (kcat/Km) values for S-oxidation by CYP2C9, CYP3A4, and CYP3A5 were 0.21, 0.34, and 0.19 µM-1∙min-1, respectively. The kcat/Km values of hydroxylation at the 1-, 2-/3-, and 4-n-butyl group in CYP2C9 were 0.0076, 0.041, and 0.035 µM-1∙min-1, respectively. The kcat and Km values provide information for the prediction of FMS metabolism in vivo. In addition, simultaneous determination of the FMS metabolites may be used to evaluate CYP2C9 and CYP3A4/5 activity.

Cytochrome P450 4A11 inhibition assays based on characterization of lauric acid metabolites.

This study was designed to characterize lauric acid metabolism to facilitate the establishment of cytochrome P450 4A11 (CYP4A11) inhibition assay. Three metabolites (2-, 11-, and 12-hydroxylauric acids) were identified in pooled human liver microsomes based on comparisons with authentic standards. Reaction phenotyping using 14 recombinant CYPs showed that ω-hydroxylation was mediated dominantly by CYP4A11 and marginally by CYP4F3B. CYP2B6 played an exclusive role in the formation of 2-hydroxylauric acid. The production of 11-hydroxylauric acid was mediated by CYP2E1, CYP2C9, CYP2B6, CYP1A2, CYP3A4, and CYP4A11. The IC50 values of HET0016, a well-known pan-CYP4 inhibitor, against the formation of 12-, 11-, and 2-hydroxylauric acid were 1.0, 1.0, and 0.009 µM, respectively. Among the 50 natural compounds examined, plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone) inhibited the formation of 12-, 11-, and 2-hydroxylauric acid with IC50 values of 1.7, 2.3, and 2.7 µM, respectively. In the selectivity study, HET0016 inhibited CYP2B6 with an IC50 of 9.2 nM, as well as CYP1A2, CYP2C19, and CYP2E1 with IC50 values of 1-2 µM. Plumbagin inhibited all CYP enzymes tested with IC50 values of 1.7-3.0 µM. These methods can be used as tools to develop CYP4A11 inhibitors; simultaneous determination of the hydroxylauric acid metabolites provides further information on selectivity.

Inhibition of Cytochrome P450 by Propolis in Human Liver Microsomes.

Although propolis is one of the most popular functional foods for human health, there have been no comprehensive studies of herb-drug interactions through cytochrome P450 (CYP) inhibition. The purpose of this study was to determine the inhibitory effects of propolis on the activities of CYP1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1 and 3A4 using pooled human liver microsomes (HLMs). Propolis inhibited CYP1A2, CYP2E1 and CYP2C19 with an IC50 value of 6.9, 16.8, and 43.1 µg/mL, respectively, whereas CYP2A6, 2B6, 2C9, 2D6, and 3A4 were unaffected. Based on half-maximal inhibitory concentration shifts between microsomes incubated with and without nicotinamide adenine dinucleotide phosphate, propolis-induced CYP1A2, CYP2C19, and CYP2E1 inhibition was metabolism-independent. To evaluate the interaction potential between propolis and therapeutic drugs, the effects of propolis on metabolism of duloxetine, a serotonin-norepinephrine reuptake inhibitor, were determined in HLMs. CYP1A2 and CYP2D6 are involved in hydroxylation of duloxetine to 4-hydroxy duloxetine, the major metabolite, which was decreased following propolis addition in HLMs. These results raise the possibility of interactions between propolis and therapeutic drugs metabolized by CYP1A2.

Characterization of fimasartan metabolites in human liver microsomes and human plasma.

1. The metabolites of fimasartan (FMS), a new angiotensin II receptor antagonist, were characterized in human liver microsomes (HLM) and human subjects. 2. We developed a method for a simultaneous quantitative and qualitative analysis using predictive multiple reaction monitoring information-dependent acquisition-enhanced product ion scanning. To characterize metabolic reactions, FMS metabolites were analyzed using quadrupole-time of flight mass spectrometer in full-scan mode. 3. The structures of metabolites were confirmed by comparison of chromatographic retention times and mass spectra with those of authentic metabolite standards. 4. In the cofactor-dependent microsomal metabolism study, the half-lives of FMS were 56.7, 247.9 and 53.3 min in the presence of NADPH, UDPGA and NADPH + UDPGA, respectively. 5. The main metabolic routes in HLM were S-oxidation, oxidative desulfuration, n-butyl hydroxylation and N-glucuronidation. 6. In humans orally administered with 120 mg FMS daily for 7 days, the prominent metabolites were FMS S-oxide and FMS N-glucuronide in the 0-8-h pooled plasma sample of each subject. 7. This study characterizes, for the first time, the metabolites of FMS in humans to provide information for its safe use in clinical medicine.

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.