Microsomal Incubations Research Articles

Research on the metabolites and key metabolic enzymes of allocryptopine in chicken liver microsomes via stable isotope tracing technology.

Allocryptopine (ALL), a principal active component of the novel veterinary medicine Bopu Powder®, has gained widespread application in the poultry farming sector for the effective management of Escherichia coli (E. coli) diarrhea. In order to explore the metabolites and the pivotal enzymes associated with ALL, this study was conducted employing an in vitro chicken liver microsomal incubation. The metabolites of ALL were analyzed and identified by combining isotope tracing technology with the application of mass spectrometry fragmentation patterns. The key metabolic enzymes involved in the biotransformation of ALL were explored using the CYP450 recombinant enzyme method, which facilitated the identification of the enzymes contributing to ALL's metabolic pathway. The liver microsomal metabolism investigation revealed a total of five metabolites, with the predominant being M2 (harmol or 3-hydroxy-4-methoxy-6-methyl-5,7,8,15-tetrahydro-[1,3]dioxolo[4',5':4,5]benzo[1,2-g]benzo[c]azecin-14(6 H)-one). The recombinant enzyme analysis conclusively identified CYP2D6 as the pivotal CYP450 isoenzyme that plays a central role in the metabolic pathway of the principal ALL metabolite, M2. This research not only expands our comprehension of the biotransformation process of ALL but also provides significant scientific evidence for the clinical safety of ALL, which was of great importance for guiding the application of ALL in the field of veterinary medicine.

Stereoselective In Vitro Metabolism, Hepatotoxicity, and Cytotoxic Effects of Four Enantiomers of the Fungicide Propiconazole.

Propiconazole (PRO) is a chiral triazole fungicide that has been widely used for several years. However, its metabolic characteristics and hepatotoxicity in the chiral level environment remain unclear. In this study, the stereoselective behavior of PRO was investigated by using liver microsome incubation, cell viability assay, inhalation exposure, and molecular docking. Our results demonstrated that the isomers trans (-)-2R,4R-PRO and cis (+)-2R,4S-PRO exhibited slower metabolic rates in rat liver microsomes. The cytochrome P450 family 1 subfamily A polypeptide 2 enzyme was found to play a key role in the metabolism of PRO, contributing to its stereoselective behavior. Histopathological and cell viability results showed that exposure to rac-PRO could induce severe hepatotoxicity in mice. This effect might be related to the accumulation of cis (+)-2R,4S-PRO in the liver, which has a slow metabolism and is highly toxic. Our findings indicate that avoiding the application of cis (+)-2R,4S-PRO in agriculture can significantly reduce adverse effects on nontarget organisms.

Metabolic Activation and Cytotoxicity of Donepezil Induced by CYP3A4.

Donepezil (DNP) is a selective cholinesterase inhibitor widely used for the therapy of Alzheimer's disease. Instances of liver injury correlated with DNP treatment have been reported, yet the underlying hepatotoxic mechanism remains to be elucidated. This study aimed to explore the contribution of metabolic activation to the hepatotoxicity of DNP. The structure of 6-O-desmethyl DNP (M1), the oxidative metabolite of DNP, was characterized by chemical synthesis, LC-MS/MS, and nuclear magnetic resonance. A reactive quinone methide resulting from the metabolism of DNP was captured by glutathione (GSH) fortified in liver microsomal incubations after exposure to DNP, and the resulting GSH conjugate (M2) was detected in the bile of rats receiving DNP. Recombinant human P450 enzyme incubation studies demonstrated that CYP3A4 was the principal enzyme responsible for the production of M1 and M2. The generation of M2 declined in rat primary hepatocytes pretreated with ketoconazole, an inhibitor of CYP3A4, which also decreased the vulnerability of rat primary hepatocytes to DNP-caused cytotoxicity. These findings suggest that the quinone methide metabolite may contribute to the cytotoxicity and hepatotoxicity caused by the DNP.

Species specific kinetics of imidacloprid and carbendazim in mouse and rat and consequences for biomonitoring

This study aimed to develop physiologically based kinetic (PBK) models to predict the blood concentrations of imidacloprid and carbendazim and their primary metabolites 5-hydroxy-imidacloprid and 2-aminobenzimidazole after single or repeated oral exposure in mouse (Mus musculus), and compare this to corresponding kinetic data in rat (Rattus norvegicus). PBK model constants for conversion of imidacloprid and carbendazim and formation and clearance of their selected primary metabolites were quantified by in vitro mouse liver microsomal and S9 incubations. The performance of the newly developed PBK models was evaluated, based on a comparison to available literature data, showing that the models performed well. Predictions made were also compared to results from PBK model simulations for rats reported previously to obtain insight in species dependent differences in kinetics of these pesticides. The results thus obtained revealed substantial species differences in kinetics for these two pesticides between mouse and rat, especially for imidacloprid and to a lesser extent for carbendazim. Repeated dose PBK model simulations revealed that the models can facilitate estimation of external exposure levels under wildlife conditions based on internal blood concentrations of the parent compound. The rate of conversion and liver volume fraction were shown to influence the accuracy of these predictions with lower values providing less variable outcomes. It is concluded that PBK modeling provides a new approach methodology of use for wildlife biomonitoring studies and that results of the present study facilitate benchmarking of the species and compounds for which kinetics enable this with sufficient accuracy.

Metabolic profile characterization of voxelotor in human urine based on in vivo and in vitro models for doping control.

Voxelotor was approved for the treatment of sickle cell anemia as a potent hemoglobin S polymerization inhibitor. Owing to its ability to affect blood components and its potential to enhance athletic performance, voxelotor was included in the prohibited list issued by the World Anti-Doping Agency in 2023, banning its use both in and out of competition. This study aimed to comprehensively investigate the metabolic profile of voxelotor in human urine and identify suitable metabolites for long-term analytical retrospectivity in doping control. A novel strategy for metabolite identification was established by combining in vivo human administration with isotope labeling-based in vitro metabolism analysis. A single microdose of voxelotor was administered orally to five volunteers, and urine samples were collected for up to 28 days post-administration. Concurrently, in vitro incubation of human liver microsomes with voxelotor and D3-voxelotor was conducted, and the microsomal incubates were analyzed via liquid chromatography-high-resolution mass spectrometry. Targeted metabolite searches in human urine samples and automated nontargeted screening of isotope metabolite ion pairs in incubation samples led to the discovery of 9 phase I metabolites and 23 phase II metabolites. Analysis of the urine excretion curves revealed that 4 metabolites, along with voxelotor, were suitable for long-term anti-doping monitoring, with a detection window exceeding 20 days. Using both in vivo and in vitro metabolic models, this study provides comprehensive insight into the metabolic profile of voxelotor in human urine for the first time, enhancing the capacity for doping screening and extending the retrospectivity of voxelotor detection.

Research progress on in vitro effects of traditional Chinese medicine ingredients on liver UDP-glucuronosyltransferases

UDP-glucuronosyltransferases(UGTs) are the main phase Ⅱ metabolizing enzymes in the human body, participating in the glucuronidation of various chemicals in the body. Traditional Chinese medicine(TCM) ingredients affect the activities of UGTs involved in drug metabolism, and the fluctuations in the blood concentrations of drugs metabolized by UGTs will lead to the risk of TCM-TCM or TCM-chemical drug interaction, which will cause drug safety problems and affect drug efficacy. Therefore, it is essential to explore the effect of TCM ingredients on the activities of UGTs. The recombinant human UGTs method and liver microsomal incubation method are reliable and effective, and they are thus often used to study the effects of TCM ingredients on UGTs in the liver. Studies have reported that different TCM ingredients can induce or inhibit the activities of UGTs, while the systematical review remains to be carried out. This paper reviews the effects of different ingredients and extracts of TCM and Chinese patent medicines on recombinant human UGTs or liver microsomal UGTs, aiming to provide reference for the rational and safe use of TCM in clinical practice. From the perspective of drug-drug interaction mediated by UGTs, the exacting classical drug pairs and incompatibility principles of traditional Chinese medicine were clarified, which laid the foundation for exploring new drug compatibility.

Human phase-I metabolism of three synthetic cannabinoids bearing a cumyl moiety and a cyclobutyl methyl or norbornyl methyl tail: Cumyl-CBMEGACLONE, Cumyl-NBMEGACLONE, and Cumyl-NBMINACA.

Synthetic cannabinoid receptor agonists (SCRAs) continue to show high prevalence on the new psychoactive substances drug market. Around 2019-2020, new SCRAs bearing a cumyl moiety emerged: Cumyl-CBMEGACLONE and Cumyl-NBMEGACLONE, carrying a cyclobutyl methyl (CBM) and a norbornyl methyl moiety (NBM) attached to the γ-carbolinone core. These were followed by Cumyl-NBMINACA, the indazole carboxamide analog of Cumyl-NBMEGACLONE. The study aimed at evaluating the human phase-I metabolism of these compounds and at identifying suitable urinary markers to prove their consumption. After enzymatic hydrolysis, 14 authentic urine samples (eight for Cumyl-CBMEGACLONE, four for Cumyl-NBMEGACLONE, and two for Cumyl-NBMINACA) were analyzed by liquid chromatography-quadrupole time-of-flight mass spectrometry. Results were compared with in vitro metabolites generated by pooled human liver microsomes incubation. Fifteen human phase-I metabolites were identified for Cumyl-CBMEGACLONE, nine for Cumyl-NBMEGACLONE, and thirteen for Cumyl-NBMINACA. The main in vivo metabolites were built by monohydroxylation, dihydroxylation, or trihydroxylation. The following urinary biomarkers are suggested for detecting the consumption of the investigated SCRAs: products of monohydroxylation at the CBM and at the core for Cumyl-CBMEGACLONE; two products of monohydroxylation at the norbonyl methyl tail for Cumyl-NBMEGACLONE; and metabolites built by dihydroxylation at the NBM substructure and by an additional hydroxylation at the cumyl moiety for Cumyl-NBMINACA.

Distinct Species-Specific and Toxigenic Metabolic Profiles for 6PPD and 6PPD Quinone by P450 Enzymes: Insights from In Vitro and In Silico Studies.

The tire rubber antioxidant N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD) and its quinone product (6PPDQ) are prevalent emerging contaminants, yet their biotransformation profiles remain poorly understood, hampering the assessment of environmental and health risks. This study investigated the phase-I metabolism of 6PPD and 6PPDQ across aquatic and mammalian species through in vitro liver microsome (LM) incubations and in silico simulations. A total of 40 metabolites from seven pathways were identified using the highly sensitive nano-electrospray ionization mass spectrometry. Notably, 6PPDQ was consistently detected as a 6PPD metabolite with an approximate 2% yield, highlighting biotransformation as a neglected indirect exposure pathway for 6PPDQ in organisms. 6PPDQ was calculated to form through a facile two-step phenyl hydroxylation of 6PPD, catalyzed by cytochrome P450 enzymes. Distinct species-specific metabolic kinetics were observed, with fish LM demonstrating retarded biotransformation rates for 6PPD and 6PPDQ compared to mammalian LM, suggesting the vulnerability of aquatic vertebrates to these contaminants. Intriguingly, two novel coupled metabolites were identified for 6PPD, which were predicted to exhibit elevated toxicity compared to 6PPDQ and result from C-N oxidative coupling by P450s. These unveiled metabolic profiles offer valuable insights for the risk assessment of 6PPD and 6PPDQ, which may inform future studies and regulatory actions.

PBK models to predict internal and external dose levels following oral exposure of rats to imidacloprid and carbendazim

Monitoring oral exposure to pesticides in wildlife is crucial for assessing environmental risks and preventing adverse effects on non-target species. Traditionally, this requires invasive tissue sampling, raising ethical, regulatory, and economic concerns. To address this gap, our study aims to develop a method for assessing external oral dose levels in rats using physiologically-based kinetic (PBK) modeling based on blood concentration levels of two pesticides, imidacloprid and carbendazim, and one of their primary metabolites. We utilized in vitro metabolic kinetic data from hepatic microsomal and S9 incubations to inform our models. These models were then evaluated by comparing their predictions with existing in vivo experimental data from the literature. Our results demonstrate that the models provide accurate predictions, presenting a novel in vitro and in silico approach for environmental exposure and risk assessment of pesticides. This methodology has the potential for application in wildlife species, advancing the frontier of knowledge in non-invasive pesticide exposure assessment.

CYP3A4-Mediated Metabolic Activation and Cytotoxicity of Chlortoluron.

Chlortoluron (CTU) is an herbicide extensively used in agricultural settings for crop cultivation. Its presence in water has been identified as a pollutant detrimental to aquatic species. The objective of the present study was to explore the metabolic activation and hepatotoxicity of CTU. Through human and rat liver microsomal incubations supplemented with CTU, nicotinamide adenine dinucleotide phosphate (NADPH), and either glutathione or N-acetyl cysteine, a benzylic alcohol metabolite (M1) was discerned, alongside a phenol metabolite (M2), a glutathione conjugate (M3), and an N-acetyl cysteine conjugate (M4). In rats exposed to CTU, biliary M3 and urinary M4 were detected in their bile and urine, respectively. The generation of M1 was detected in the presence of NADPH. The observation of M3 and M4 suggests the formation of an iminoquinone methide intermediate arising from the oxidation of M1. CYP3A4 was found to be the principal enzyme catalyzing the metabolic activation of CTU. Furthermore, CTU exhibited cytotoxic properties in cultured rat primary hepatocytes in a concentration-dependent pattern. Concomitant treatment of hepatocytes with ketoconazole mitigated their susceptibility to the cytotoxic effects of CTU.

In vitro and in vivo studies on the metabolism and pharmacokinetics of the selective gut microbial β-glucuronidase targeting compound Inh 1

In vitro studies using rat, mouse, and human microsomes and hepatocytes on the bacterial β-glucuronidase inhibitor 1-((6,8-dimethyl-2-oxo-1,2-dihydroquinolin-3-yl)methyl)-3-(4-ethoxyphenyl)-1-(2-hydroxyethyl)thiourea) (Inh 1) revealed extensive metabolism in all species. The intrinsic clearances of Inh 1 in human, mouse, and rat hepatic microsomes were 30.9, 67.8, and 201 µL/min/mg, respectively. For intact hepatocytes intrinsic clearances of 21.6, 96.0, and 129 µL/min/106 cells were seen for human, mouse and rat, respectively. The metabolism of Inh 1 involved an uncommon desulphurisation reaction in addition to oxidation, deethylation, and conjugation reactions at multiple sites. Six metabolites were detected in microsomal incubations in human and rat, and seven for the mouse. With hepatocytes, 18 metabolites were characterised, 9 for human, and 11 for mouse and rat. Following IV administration to mice (3 mg/kg), plasma concentrations of Inh 1 exhibited a monophasic decline with a terminal elimination half-life of 0.91 h and low systemic clearance (11.8% of liver blood flow). After PO dosing to mice (3 mg/kg), peak observed Inh 1 concentrations of 495 ng/mL were measured 0.5 h post dose, declining to under 10 ng/mL at 8 h post dose. The absolute oral bioavailability of Inh 1 in the mouse was ca. 26%.

Arsenite-Induced Drug-Drug Interactions in Rats.

Arsenite is an important heavy metal. Some Chinese traditional medicines contain significant amounts of arsenite. The aim of this study was to investigate subacute exposure of arsenite on activities of cytochrome P450 enzymes and pharmacokinetic behaviors of drugs in rats. Midazolam, tolbutamide, metoprolol, omeprazole, caffeine, and chlorzoxazone, the probe substrates for cytochrome P450 (CYP) s3A, 2C6, 2D, 2C11, 1A, and 2E, were selected as probe drugs for the pharmacokinetic study. Significant decreases in areas under the curves of probe substrates were observed in rats after consecutive 30-day exposure to As at 12 mg/kg. Microsomal incubation study showed that the subacute exposure to arsenite resulted in little change in effects on the activities of P450 enzymes examined. However, everted gut sac study demonstrated that such exposure induced significant decreases in intestinal absorption of these drugs by both passive diffusion and carrier-mediated transport. In addition, in vivo study showed that the arsenite exposure decreased the rate of peristaltic propulsion. The decreases in intestinal permeability of the probe drugs and peristaltic propulsion rate most likely resulted in the observed decreases in the internal exposure of the probe drugs. Exposure to arsenite may lead to the reduction of the efficiencies of pharmaceutical agents coadministered resulting from the observed drug-drug interactions. SIGNIFICANCE STATEMENT: Exposure to arsenite may lead to the reduction of the efficiencies of pharmaceutical agents coadministered resulting from the observed drug-drug interactions. The present study, we found that P450 enzyme probe drug exposure was reduced in arsenic-exposed animals (areas under the curve) and the intestinal absorption of the drug was reduced in the animals. Subacute arsenic exposure tends to cause damage to intestinal function, which leads to reduced drug absorption.

Identification of an Epoxide Metabolite of Amitriptyline In Vitro and In Vivo.

Amitriptyline (ATL), a tricyclic antidepressant, has been reported to cause various adverse effects, particularly hepatotoxicity. The mechanisms of ATL-induced hepatotoxicity remain unknown. The study was performed to identify the olefin epoxidation metabolite of ATL and determine the possible toxicity mechanism. Two glutathione (GSH) conjugates (M1 and M2) and two N-acetylcysteine (NAC) conjugates (M3 and M4) were detected in rat liver microsomal incubations supplemented with GSH and NAC, respectively. Moreover, M1/M2 and M3/M4 were respectively found in ATL-treated rat primary hepatocytes and in bile and urine of rats given ATL. Recombinant P450 enzyme incubations demonstrated that CYP3A4 was the primary enzyme involved in the olefin epoxidation of ATL. Treatment of hepatocytes with ATL resulted in significant cell death. Inhibition of CYP3A attenuated the susceptibility to the observed cytotoxicity of ATL. The metabolic activation of ATL most likely participates in the cytotoxicity of ATL.

Identification of UDP-glucuronosyltransferase (UGT) isoforms involved in the metabolism of Chlorophenols (CPs)

Chlorophenols (CPs) are a group of pollutants that pose a great threat to the environment, they are widely used in industrial and agricultural wastes, pesticides, herbicides, textiles, pharmaceuticals and plastics. Among CPs, pentachlorophenol was listed as one of the persistent organic pollutants (POPs) by the Stockholm convention. This study aims to identify the UDP-glucosyltransferase (UGT) isoforms involved in the metabolic elimination of CPs. CPs’ mono-glucuronide was detected in the human liver microsomes (HLMs) incubation mixture with co-factor uridine-diphosphate glucuronic acid (UDPGA). HLMs-catalyzed glucuronidation metabolism reaction equations followed Michaelis-Menten or substrate inhibition type. Recombinant enzymes and chemical reagents inhibition experiments were utilized to phenotype the main UGT isoforms involved in the glucuronidation of CPs. UGT1A6 might be the major enzyme in the glucuronidation of mono-chlorophenol isomer. UGT1A1, UGT1A6, UGT1A9, UGT2B4 and UGT2B7 were the most important five UGT isoforms for metabolizing the di-chlorophenol and tri-chlorophenol isomers. UGT1A1 and UGT1A3 were the most important UGT isoforms in the catalysis of tetra-chlorophenol and pentachlorophenol isomers. Species differences were investigated using rat liver microsomes (RLMs), pig liver microsomes (PLMs), dog liver microsomes (DLMs), and monkey liver microsomes (MyLMs). All these results were helpful for elucidating the metabolic elimination and toxicity of CPs.

Metabolic Activation and Cytotoxicity of Gramine Mediated by CYP3A in Rats.

Gramine (GRM), which occurs in Gramineae plants, has been developed to be a biological insecticide. Exposure to GRM was reported to induce elevations of serum ALT and AST in rats, but the mechanisms of the observed hepatotoxicity have not been elucidated. The present study aimed to identify reactive metabolites that potentially participate in the toxicity. In rat liver microsomal incubations fortified with glutathione or N-acetylcysteine, one oxidative metabolite (M1), one glutathione conjugate (M2), and one N-acetylcysteine conjugate (M3) were detected after exposure to GRM. The corresponding conjugates were detected in the bile and urine of rats after GRM administration. CYP3A was the main enzyme mediating the metabolic activation of GRM. The detected GSH and NAC conjugates suggest that GRM was metabolized to a quinone imine intermediate. Both GRM and M1 showed significant toxicity to rat primary hepatocytes.

Rapid investigating of phase I metabolites of SR9009 in vitro horse liver microsomes via feature-based molecular networking approach: Potential applications in doping control

SR9009, a peroxisome proliferator-activated receptor δ (PPARδ) agonist, is known for its potential benefits in energy homeostasis. It failed to receive the United States Food and Drug Administration (USFDA) approval and its illegal distribution has raised concerns. As a result, it has been classified as a prohibited substance by the World Anti-Doping Agency and the International Federation of Horseracing Authorities (IFHA). This study emphasizes the application of the in-silico molecular networking technology to analyze phase I drug metabolites in horses, distinguishing it from conventional methodologies in forensic science. Feature-based molecular networking (FBMN) analysis identified 15 metabolites, with novel major N-dealkylated metabolite (-C8H7NO4S), indicative of diverse metabolic modifications in horse liver microsomes incubation assay. Additionally, a proposed metabolic pathway of SR9009 in the in vitro assay was outlined, including the previously known dehydroxylated metabolite. Finally, the metabolic pathways included in this study were as follows: hydroxylation, dehydrogenation, N-dealkylation dihydroxylation, and combinations. Molecular networking provided insights into MS spectra connectivity, facilitating rapid interpretation and accurate detection of previously undiscovered metabolites. In conclusion, this study contributes to the understanding of SR9009 metabolism in horses and underscores the importance of advanced analytical techniques, such as molecular networking, in enhancing the accuracy and efficiency of metabolite analysis for forensic and doping control purposes.

Abstract 1823: In vitro evaluation of novel pyrimidine nucleosides for the treatment of pancreatic cancer

Abstract The purpose of the study is to synthesize and characterize novel 5-FU analogs (AGY1 and AGY2) and investigate their anticancer activity against pancreatic cancer cell lines. Methods: 5-FU derivatives were synthesized and characterized using Nuclear magnetic resonance (NMR), high-performance liquid chromatography (HPLC), and elemental analysis to determine the presence of new bond formation and the purity of the derivatives or analogs. 2D cell viability, 3D models, cell migration, and cell cycle studies were performed on MiaPaCa-2 (MP-2) cells and PANC-1 cells. Apoptosis was performed on PANC-1 cells by staining with Acridine Orange and Ethidium Bromide. The metabolic stability of the analogs was determined by using human liver microsomes. PARP, p53, and BAX expressions in PANC-1 were determined by Western Blot. Results: NMR data showed the conjugation of alkyl and tetrahydrofuran groups to the 5-FU ring confirming the formation of AGY1 and AGY2. The elemental analysis of AGY1 and AGY2 showed 99.6% and 99.5 % purity respectively. The IC50 value of the AGY1 against 2D MP-2 was found to be 12.2 ± 1 µM, while the IC50 value of AGY2 was determined to be 2.9 ± 0.2 µM. Both AGY1 and AGY2 anticancer activities were significantly higher than 5-FU (18.5 ± 1.1 µM). However, AGY2 was more effective compared to AGY1. A similar trend was observed for AGY2 analog where the IC50 value for treated 2D PANC-1 was 3.1 ± 0.29 µM and demonstrated a higher anticancer activity compared to AGY1 (6.4 ± 0.38 µM) or 5-FU (7.5 ± 1.1 µM). For cell migration studies, AGY2 (43 ± 5.5) exposed MP-2 cells showed significant arrest towards the wound gap compared to AGY1 (74 ± 18.5) and 5-FU (86 ± 15.6). A similar result of cell migration arrests AGY2 was observed in PANC-1 treated-AGY1, AGY2, and 5-FU. Organoid and spheroid models demonstrated high and comparable anticancer activity of AGY1 and AGY2 compared to 5-FU treated organoid and spheroid models. The cell-cycle analysis showed that AGY2-treated MP-2 cells at 1μΜ concentration showed a higher G1 population arrest (64.25%) compared to AGY1 (54.2%) and 5-FU (55.75%). Similarly, in PANC-1 cells, AGY2 at 1μΜ concentration showed a higher S-phase arrest (53.86%) compared to AGY1 (50.76%) and 5-FU (36.14%). Metabolic stability data showed higher stability of AGY2 (95% intact) compared to AGY1 (85% intact) compared to 5-FU (55% intact) after 2 hour of human liver microsomal incubation. The expressions of p53 and BAX proteins in PANC-1 cells were significantly upregulated and PARP was downregulated at higher concentration when treated with AGY1 and AGY2 compared to 5-FU. Conclusion: We demonstrate that AGY2 showed more promising anticancer activity compared to AGY1 and 5-FU against pancreatic cancer cells. AGY2 has the potential to improve 5-FU’s metabolic stability and enhance its therapeutic efficacy. Citation Format: Raviteja Bulusu, Joy Okoro, Esther Frimpong, Bo Han, Sherise Rogers, Xue Zhu, Edward Agyare. In vitro evaluation of novel pyrimidine nucleosides for the treatment of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1823.

Evaluation on the Potential for Hepatotoxic Components from Herba Epimedii to Induce Apoptosis in HepG2 Cells and the Analysis of the Influence of Metabolism in Liver Microsomes.

The potential hepatotoxicity of Herba Epimedii is a focal point in traditional Chinese medicine security applications. As determined in our previous study, the flavonoid constituents of Herba Epimedii, sagittatoside A, icariside I, baohuoside I and icaritin, are related to the hepatotoxicity of this herb. However, the hepatotoxic mechanism of these components needs to be clarified further, and whether these components can maintain their injury action following liver metabolism needs to be confirmed. Herein, the effects of sagittatoside A, icariside I, baohuoside I and icaritin on the apoptosis of HepG2 cells and the expression of key proteins, including Bax, Bcl-2, Caspase-3 and Caspase-9, were evaluated. Moreover, with liver microsome incubation, the influences of metabolism on the apoptotic activities of these components were investigated. Then, by HPLC-MS/MS analyses, the in vitro metabolic stability of these components was determined after incubation with different kinds of liver microsomes to explain the reason for the influence. The results suggested that sagittatoside A, baohuoside I and icaritin could induce apoptosis, which is likely to be closely related to the induction of the intrinsic apoptosis pathway. After metabolic incubation, the sagittatoside A and icaritin metabolism mixture could still induce apoptosis due to less metabolic elimination, while the icariside I and baohuoside I metabolism mixtures respectively got and lost the ability to induce apoptosis, probably due to quick metabolism and metabolic transformation. The findings of this study may provide important references to explore the material basis and mechanism of the hepatotoxicity of Herba Epimedii.

Exposure to Synthetic Psychoactive Substances: A Potential Cause for Increased Human Hepatotoxicity Markers.

Approximately 30 million people worldwide consume new psychoactive substances (NPS), creating a serious public health issue due to their toxicity and potency. Drug-induced liver injury is the leading cause of liver disease, responsible for 4% of global deaths each year. A systematic literature search revealed 64 case reports, in vitro and in vivo studies on NPS hepatotoxicity. Maximum elevated concentrations of aspartate aminotransferase (136 to 15 632 U/L), alanine transaminase (121.5 to 9162 U/L), total bilirubin (0.7 to 702 mg/dL; 0.04 to 39.03 mmol/L), direct (0.2-15.1 mg/dL; 0.01-0.84 mmol/L) and indirect (5.3 mg/dL; 0.29 mmol/L) bilirubin, alkaline phosphatase (79-260 U/L), and gamma-glutamyltransferase (260 U/L) were observed as biochemical markers of liver damage, with acute and fulminant liver failure the major toxic effects described in the NPS case reports. In vitro laboratory studies and subsequent in vivo NPS exposure studies on rats and mice provide data on potential mechanisms of toxicity. Oxidative stress, plasma membrane stability, and cellular energy changes led to apoptosis and cell death. Experimental studies of human liver microsome incubation with synthetic NPS, with and without specific cytochrome P450 inhibitors, highlighted specific enzyme inhibitions and potential drug-drug interactions leading to hepatotoxicity. Mild to severe hepatotoxic effects following synthetic NPS exposure were described in case reports. In diagnosing the etiology of liver damage, synthetic NPS exposure should be considered as part of the differential diagnosis. Identification of NPS toxicity is important for educating patients on the dangers of NPS consumption and to suggest promising treatments for observed hepatotoxicity.

In vitro and in vivo metabolic activation and hepatotoxicity of chlorzoxazone mediated by CYP3A.

Chlorzoxazone (CZX), a benzoxazolone derivative, has been approved for the treatment of musculoskeletal disorders to relieve localized muscle spasm. However, its idiosyncratic toxicity reported in patients brought attention, particularly for hepatotoxicity. The present study for the first time aimed at the relationship between CZX-induced hepatotoxicity and identification of oxirane intermediate resulting from metabolic activation of CZX. Two N-acetylcysteine (NAC) conjugates (namely M1 and M2) and two glutathione (GSH) conjugates (namely M3 and M4) were detected in rat & human microsomal incubations with CZX (200μM) fortified with NAC or GSH, respectively. The formation of M1-M4 was NADPH-dependent and these metabolites were also observed in urine or bile of SD rats given CZX intragastrically at 10mg/kg or 25mg/kg. NAC was found to attach at C-6' of the benzo group of M1 by sufficient NMR data. CYPs3A4 and 3A5 dominated the metabolic activation of CZX. The two GSH conjugates were also observed in cultured rat primary hepatocytes after exposure to CZX. Inhibition of CYP3A attenuated the susceptibility of hepatocytes to the cytotoxicity of CZX (10-400μM). The in vitro and in vivo studies provided solid evidence for the formation of oxirane intermediate of CZX. This would facilitate the understanding of the underlying mechanisms of toxic action of CZX.