Pig Liver Microsomes Research Articles

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.

Comparative Analysis of the Gelsemium Alkaloids Metabolism in Human, Pig, Goat, and Rat Liver Microsomes.

The aim of this study was to investigate the metabolism of Gelsemium elegans in human, pig, goat and rat liver microsomes and to elucidate the metabolic pathways and cleavage patterns of the Gelsemium alkaloids among different species. A human, goat, pig and rat liver microsomes were incubated in vitro. After incubating at 37°C for 1 hour and centrifuging, the processed samples were detected by HPLC/Qq-TOFMS was used to detect alcohol extract of Gelsemium elegans and its metabolites. Forty-six natural products were characterized from alcohol extract of Gelsemium elegans and 13 metabolites were identified. These 13 metabolites belong to the gelsemine, koumine, gelsedine, humantenine, yohimbane, and sarpagine classes of alkaloids. The metabolic pathways included oxidation, demethylation and dehydrogenation. After preliminary identification, the metabolites detected in the four species were different. All 13 metabolites were detected in pig and rat microsomes, but no oxidative metabolites of Gelsedine-type alkaloids were detected in goat and human microsomes. In this study, Gelsemium elegans metabolic patterns in different species are clarified and the in vitro metabolism of Gelsemium elegans is investigated. It is of great significance for its clinical development and rational application.

P62 | In‐feed medication with fenbendazole: Induction of cytochrome P450‐dependent metabolism in pig liver microsomes

P62 | In‐feed medication with fenbendazole: Induction of cytochrome <scp>P450</scp>‐dependent metabolism in pig liver microsomes

Newly Identified Tree Shrew Cytochrome P450 2A13 is Expressed in Liver and Lung and Encodes a Functional Drug-Metabolizing Enzyme Similar to Dog Cytochrome P450 2A13 and Pig Cytochrome P450 2A19.

The tree shrew, a non-rodent primate-like species, is used in various fields of biomedical research, including hepatitis virus infection, myopia, depression, and toxicology. Recent genome analysis found that the numbers of cytochrome P450 (P450 or CYP) genes are similar in tree shrews and humans and their sequence identities are high. Although the P450s are a family of important drug-metabolizing enzymes, they have not yet been fully investigated in tree shrews. In the current study, tree shrew CYP2A13 cDNA was isolated from liver, and its characteristics were compared with those of pig, dog, and human CYP2As. Tree shrew CYP2A13 amino acid sequences were highly identical (87-92%) to the human CYP2As and contained sequence motifs characteristic of P450s. Phylogenetic analysis revealed that tree shrew CYP2A13 was more closely related to human CYP2As than to rat CYP2As, similar to dog and pig CYP2As. Among the tissue types analyzed, tree shrew CYP2A13 mRNA was preferentially expressed in liver and lung, similar to dog CYP2A13 mRNA, whereas dog CYP2A25 and pig CYP2A19 mRNAs were predominantly expressed in liver. Tree shrew liver microsomes and tree shrew CYP2A13 proteins heterologously expressed in Escherichia coli catalyzed coumarin 7-hydroxylation and phenacetin O-deethylation, just as human, dog, and pig CYP2A proteins and liver microsomes do. These results demonstrate that tree shrew CYP2A13 is expressed in liver and lung and encodes a functional drug-metabolizing enzyme. SIGNIFICANCE STATEMENT: Novel tree shrew cytochrome P450 2A13 (CYP2A13) was identified and characterized in comparison with human, dog, and pig CYP2As. Tree shrew CYP2A13 isolated from liver had high sequence identities and close phylogenetic relationships to its human homologs and was abundantly expressed in liver and lung at the mRNA level. Tree shrew CYP2A13 metabolized coumarin and phenacetin, human selective CYP2A6 and CYP2A13 substrates, respectively, similar to dog and pig CYP2As, and is a functional drug-metabolizing enzyme likely responsible for drug clearances.

Plasma Protein Binding Rate and Pharmacokinetics of Lekethromycin in Rats.

Lekethromycin (LKMS), a novel macrolide lactone, is still unclear regarding its absorption. Thus, we conducted this study to investigate the characteristics of LKMS in rats. We chose the ultrafiltration method to measure the plasma protein binding rate of LKMS. As a result, LKMS was characterized by quick absorption, delayed elimination, and extensive distribution in rats following intramuscular (im) and subcutaneous (sc) administration. Moreover, LKMS has a high protein binding rate (78–91%) in rats at a concentration range of 10–800 ng/mL. LKMS bioavailability was found to be approximately 84–139% and 52–77% after im and sc administration, respectively; however, LKMS was found to have extremely poor bioavailability after oral administration (po) in rats. The pharmacokinetic parameters cannot be considered linearly correlated with the administered dose. Additionally, LKMS and its corresponding metabolites were shown to be metabolically stable in the liver microsomes of rats, dogs, pigs, and humans. Notably, only one phase I metabolite was identified during in vitro study, suggesting most of drug was not converted. Collectively, LKMS had quick absorption but poor absorption after oral administration, extensive tissue distribution, metabolic stability, and slow elimination in rats.

A high-resolution mass spectrometric approach to a qualitative and quantitative comparative metabolism of the humantenine-type alkaloid rankinidine.

Rankinidine belongs to the humantenine-type alkaloids isolated from Gelsemium. Currently, the mechanism behind the toxicity differences of rankinidine has not been explained. In this study, our purpose was to elucidate the major in vitro metabolic pathways of rankinidine and to compare the formation of metabolites of rankinidine in human (HLMs), rat (RLMs), goat (GLMs) and pig (PLMs) liver microsomes. This is the first study to compare the in vitro metabolism of rankinidine with high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (LC/QTOF). The MS/MS data and LC/MS peak area acquired in positive ion mode were used to analyze metabolite structures and compare metabolism. We identified 11 metabolites (M1-M11) in total and found five main metabolic pathways, consisting of demethylation (M1), reduction (M2), oxidation at different positions (M3-M5), oxidation and reduction (M6-M10) and demethylation and oxidation (M11). The metabolism of rankinidine has qualitative and quantitative species-specific differences in vitro. In PLMs and GLMs, the main metabolic pathway of rankinidine was oxidation. Notably, among the four species, the oxidation ability of rankinidine was highest in pigs and goats, and the demethylation and reduction abilities of rankinidine were highest in humans and rats. The interspecific metabolic differences of rankinidine in HLMs, PLMs, GLMs and RLMs were compared and studied for the first time using LC/QTOF. These findings will certainly support future studies of rankinidine metabolism in vivo and will contribute to elucidating the cause of species-specific differences behind Gelsemium toxicity.

Metabolic conjugation reduces in vitro toxicity of the flavonoid nevadensin

The present study focuses on the association between metabolic capacity and toxicity of the natural occurring flavonoid nevadensin in vitro. Human colon (HT29), liver (HepG2) and bone marrow (KG1) carcinoma cells were used and strong cell line dependent differences in toxic effect strength were found. HepG2 and KG1 cells were more sensitive against nevadensin treatment in comparison to HT29 cells. High resolution mass spectrometry experiments showed that nevadensin is rapidly glucuronidated in HT29 cells, whereas KG1 cells do not metabolize nevadensin, thus glucuronidation was supposed to be a crucial metabolic pathway in vitro. To proof this suggestion, nevadensin glucuronides were isolated from pig liver microsomes und structurally elucidated via NMR spectroscopy. In HepG2 cells a cellular enrichment of nevadensin itself as well as nevadensin-7-O-glucuronide was determined by tandem mass spectrometry. A proteomic screening of uridine 5′-diphospho (UDP)-glucuronosyltransferase (UGT) in HT29 and HepG2 cells provided first hints that the isoforms UGT1A6 and UGT1A1 are responsible for nevadensin glucuronidation. Additionally, nevadensin was found to be a potent SULT inhibitor in HepG2 cells. In sum, the present study clearly illustrates the importance of obtaining detailed information about metabolic competence of cell lines which should be considered in the evaluation of toxic endpoints.

In vitro Metabolism of Humantenine in Liver Microsomes from Human, Pig, Goat and Rat.

Gelsemium elegans Benth (G. elegans) is a well-known toxic plant. Alkaloids are the main active components of G. elegans. Currently, the metabolism of several alkaloids, such as gelsenicine, koumine, and gelsemine, has been widely studied. However, as one of the most important alkaloids in G. elegans, the metabolism of humantenine has not been studied yet. In order to elaborate on the in vitro metabolism of humantenine, a comparative analysis of its metabolic profile in human, pig, goat and rat liver microsomes was carried out using high performance chromatography/ quadrupole time-of-flight mass spectrometry (HPLC/QqTOF-MS) for the first time. Totally, ten metabolites of humantenine were identified in liver microsomes from human (HLMs), pig (PLMs), goat (GLMs) and rat (RLMs) based on the accurate MS/MS spectra. Five metabolic pathways of humantenine, including demethylation, dehydrogenation, oxidation, dehydrogenation and oxidation, and demethylation and oxidation, were proposed in this study. There were qualitative and quantitative species differences in the metabolism of humantenine among the four species. The in vitro metabolism of humantenine in HLMs, PLMs, GLMs and RLMs was studied by a sensitive and specific detection method based on HPLC/QqTOF-MS. The results indicated that there were species-related differences in the metabolism of humantenine. This work might be of great significance for the further research and explanation of species differences in terms of toxicological effects of G. elegans.

The metabolism of gelsevirine in human, pig, goat and rat liver microsomes.

Gelsemium is a small genus of flowering plants from the family Loganiaceae comprising five species, three of which, Gelsemium sempervirens (L.) J. St.‐Hil., G. elegans Benth and G. rankinii Small, are particularly popular. Compared with other alkaloids from G. elegans, gelsemine, gelsevirine and koumine exhibit equally potent anxiolytic effects and low toxicity. Although the pharmacological activities and metabolism of koumine and gelsemine have been reported in previous studies, the species differences of gelsevirine metabolism have not been well studied. In this study, the metabolism of gelsevirine was investigated by using liver microsomes of humans, pigs, goats and rats by means of HPLC‐QqTOF/MS. The results indicated that the metabolism of gelsevirine in liver microsomes had qualitative and quantitative species differences. Based on the results, the possible metabolic pathways of gelsevirine in liver microsomes were proposed. Investigation of the metabolism of gelsevirine will provide a basis for further studies of the in vivo metabolism of this drug.

Novel amide and imidazole compounds as potent hematopoietic prostaglandin D2 synthase inhibitors

In seeking novel and potent small molecule hematopoietic prostaglandin D2 synthase (H-PGDS) inhibitors as potential therapies for PGD2-mediated diseases and conditions, we explored a series comprising multiple aryl/heteroaryl rings attached in a linear arrangement. Each compound incorporates an amide or imidazole “linker” between the pyrimidine or pyridine “core” ring and the “tail” ring system. We synthesized and screened twenty analogs by fluorescence polarization binding assay, thermal shift assay, glutathione S-transferase inhibition assay, and a cell-based assay measuring suppression of LPS-induced PGD2 stimulation. Amide analogs show ten-fold greater shift in the thermal shift assay in the presence of glutathione (GSH) versus the same assay run in the absence of GSH. The imidazole analogs did not produce a significant change in thermal shift between the two assay conditions, suggesting a possible stabilization effect of the amide linker in the synthase-GSH-inhibitor complex. Imidazole analog 23, (KMN-010034) demonstrates superior potency across the in vitro assays and good in vitro metabolic stability in both human and guinea pig liver microsomes.

A study of in vitro metabolism and cytotoxicity of mephedrone and methoxetamine in human and pig liver models using GC/MS and LC/MS analyses

AbstractIn the current study, the metabolism of two novel psychoactive substances (NPSs), mephedrone and methoxetamine (MXE), was studied in vitro in pig liver microsomes to determine potential metabolites by liquid chromatography-mass spectrometry (LC-MS). Later, in vitro studies were performed using HepaRG™ cells to determine the human metabolites of these drugs using gas chromatography-mass spectrometry (GC-MS). The aim of the study was to detect metabolites from the metabolic mixture in the human cell lines using GC-MS, since this is a more readily available technique within forensic laboratories. Microsomes were prepared through a conventional ultracentrifugation method and incubated under optimized conditions with the drugs for 3 h. Subsequently, the samples were investigated using LC-MS. A similar methodology was then applied in the HepaRG™ cells, and the GC-MS conditions were optimized using N,O-bis(trimethylsilyl)trifluoroacetamide as a derivatization agent. The analysis showed two molecules from a successful in vitro metabolism, namely, hydroxytoly-mephedrone and nor-dihydro mephedrone. For MXE, two metabolites are presented produced by the O-demethylation and reduction of the ketone moiety to the corresponding alcohol, respectively. Using the human HepaRG™ cells, only nor-dihydro mephedrone could be identified by GC-MS. Since hydroxytoly-mephedrone and the MXE metabolites are more polar, it is suggested that GC-MS even with derivatization may not be suitable. In addition, cytotoxicity was studied utilizing HepaRG™ cell lines. The drugs show cytotoxic effects causing in vitro cell death, within the specified range of EC50 0.3211 mM (79 μg/mL) and 0.6297 mM (111 μg/mL) for mephedrone and MXE, respectively. These drugs were able to cause 73–84% cell death.

Differences in xanthotoxin metabolites in seven mammalian liver microsomes.

Xanthotoxin, abundantly occurring in fruits, vegetables, grapefruit juice and oils, is widely used in medicine for the treatment of psoriasis and vitiligo. Xanthotoxin possesses the ability to inhibit mechanism-based cytochrome P450 (CYP450)-mediated activities in rats and mice. Furthermore, it time-dependently obstructs a number of CYP450-mediated functions in humans. CYP450 enzymes are most abundant in the liver and induce metabolic activation of numerous xenobiotic compounds. The present study aimed to identify the similarities and differences in xanthotoxin metabolism in liver microsomes of 7 mammalian species, including human liver microsomes (HLM), Rhesus monkey liver microsomes (RMLM), Cynomolgus monkey liver microsomes (CMLM), Sprague Dawley rat liver microsomes (RLM), mouse liver microsomes (MLM), Dunkin Hartley guinea pig liver microsomes (PLM) and Beagle dog liver microsomes (DLM). Ultra-high performance liquid chromatography/quadrupole time-of-flight mass spectrometric analysis was used to determine the metabolites. A total of 3 metabolites were detected in RMLM, CMLM and RLM. Furthermore, two metabolites were observed in MLM, HLM, PLM and DLM. By analyzing the type and quantity of metabolites, the metabolism of xanthotoxin in MLM was indicated to be most similar to that in HLM. The metabolic transformations of xanthotoxin in the liver microsomes of the 7 species were analyzed in further detail. On the whole, the results of the present study provide a deeper understanding of the metabolic patterns of xanthotoxin in liver microsomes of different species, which may prove to be advantageous regarding the metabolic mechanisms of action of xanthotoxin. Further insight into drug metabolism with respect to different species will also aid in the selection of appropriate animal models for further research.

Are pigs a suitable animal model for in vivo metabolism studies of new psychoactive substances? A comparison study using different in vitro/in vivo tools and U-47700 as model drug

Being highly potent, New Synthetic Opioids (NSO) have become a public health concern. Little is known though about the metabolism and toxicokinetics (TK) of many of the non fentanyl NSO such as U-47700. Obtaining such data in humans is challenging and so we investigated if pigs were a suitable model species as TK model for U-47700. The metabolic fate of U-47700 was elucidated after intravenous administration to one pig in vivo and results were compared to metabolic patterns formed by different other in vitro systems (human and pig liver microsomes, human liver S9 fraction) and compared to rat and human in vivo data. Furthermore, monooxygenase isozymes responsible for the major metabolic steps were elucidated.In total, 12 phase I and 8 phase II metabolites of U-47700 could be identified. The predominant reactions were N-demethylation, hydroxylation, and combination of them followed by glucuronidation or sulfation. The most predominant monooxygenase catalyzed conversions were N-demethylation, and hydroxylation by CYP3A4 and 2B6, and FMO3 catalyzed N-oxidation. Similar main phase I metabolites were found in vitro as compared to in vivo (pig/human). The metabolic pattern elucidated in the pig was comparable to human in vivo data. Thus, pigs seem to be a suitable animal model for metabolism and further TK of U-47700.

In vitro Metabolism studies of Catechol‐Containing Natural Products and Analogues

Many biologically active natural products contain phenol groups that are prone to rapid phase‐II metabolism. In order to better understand the structural basis for these metabolic transformations, we have investigated the in vitro metabolism of rooperol, a bis‐catechol with anti‐cancer activity, and caffeic acid phenethyl amide (CAPA), a synthetic analog of a natural product with antioxidant and cytoprotective activities. In the presence of pig liver microsomes supplemented with UDP‐glucuronic acid, both of these catechols undergo rapid phase‐II metabolism to form mono‐ (CAPA and rooperol) and bis‐ (rooperol) glucuronide metabolites. The identity of these metabolites was determined by HPLC‐MS. We followed the kinetics of these in vitro metabolism reactions using HPLC. In both cases, the disappearance of the compound over time corresponded to a first‐order process with half‐life of 4.0 ± 0.5 min for rooperol and 5.1 ± 0.8 min for CAPA. In the case of rooperol, this data is in agreement with previously published pharmacokinetic (PK) studies in humans, in which only phase‐II metabolites were detected after oral administration, as well as PK studies in baboons which also report rapid phase‐II metabolism following IV administration. While human or non‐human primate PK data for CAPA have not been reported, our data suggest that this compound will also suffer from rapid phase‐II metabolism, and thus more metabolically stable analogs of both rooperol and CAPA are required.Support or Funding InformationThis research was supported by the U.S. Department of Education HSI STEM program (841.031c), Award #P021C160036The Robert and Ella Owens Medical Research Foundation

In vitro investigation of permeability and metabolism of licoricidin

AimLicoricidin has multiple pharmacological activities. The present study was designed to investigate the permeability and pharmacokinetic behavior of licoricidin using in vitro models. Material and methodsFirst, human liver microsomes and recombinant human supersomes were used to investigate the interactions between metabolic enzymes and licoricidin. In addition, rat, minipig, rabbit, dog, monkey, and human liver microsomes were used to determine metabolic differences among species. The parallel artificial membrane permeability assay (PAMPA) was used to explore licoricidin permeability behavior. Key findingsAt 100 μM, licoricidin strongly inhibited CYP2C9, CYP2C19, CYP3A4, UGT1A3, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT2B4, UGT2B7, UGT2B15, and UGT2B17. Licoricidin metabolism exhibited considerable differences among species; dog, pig, and rat liver microsomes showed higher metabolic capacity than the other species. Seven licoricidin metabolites were identified by liquid chromatography-tandem mass spectrometry, and hydration and hydroxylation were the major metabolic pathways. The PAMPA permeability of licoricidin was moderate at both pH 4.0 and 7.4. SignificanceThe present study will support further pharmacological or toxicological research on licoricidin.

Metabolism of Scoparone in Experimental Animals and Humans.

Scoparone, a major constituent of the Chinese herbal medicine Yin Chen Hao, expresses beneficial effects in experimental models of various diseases. The intrinsic doses and effects of scoparone are dependent on its metabolism, both in humans and animals. We evaluated in detail the metabolism of scoparone in human, mouse, rat, pig, dog, and rabbit liver microsomes in vitro and in humans in vivo. Oxidation of scoparone to isoscopoletin via 6-O-demethylation was the major metabolic pathway in liver microsomes from humans, mouse, rat, pig and dog, whereas 7-O-demethylation to scopoletin was the main reaction in rabbit. The scoparone oxidation rates in liver microsomes were 0.8 - 1.2 µmol/(min*g protein) in mouse, pig, and rabbit, 0.2 - 0.4 µmol/(min*g protein) in man and dog, and less than 0.1 µmol/(min*g) in rat. In liver microsomes of all species, isoscopoletin was oxidized to 3-[4-methoxy-ρ-(3, 6)-benzoquinone]-2-propenoate and esculetin, which was formed also in the oxidation of scopoletin. Human CYP2A13 exhibited the highest rate of isoscopoletin and scopoletin oxidation, followed by CYP1A1 and CYP1A2. Glucuronidation of isoscopoletin and scopoletin was catalyzed by the human UGT1A1, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, and UGT2B17. Dog was most similar to man in scoparone metabolism. Isoscopoletin glucuronide and sulfate conjugates were the major scoparone in vivo metabolites in humans, and they were completely excreted within 24 h in urine. Scoparone and its metabolites did not activate key nuclear receptors regulating CYP and UGT enzymes. These results outline comprehensively the metabolic pathways of scoparone in man and key preclinical animal species.

Effects of enrofloxacin on antioxidant system, microsomal enzymatic activity, and proteomics in porcine liver.

Enrofloxacin (EF) is a widely used fluoroquinolone, usually regarded as a safe and effective treatment for bacterial infections. Adverse effects of EF have previously been demonstrated in some species, but so far there have been no studies looking specifically at the impact of EF on pigs. In this study, three different doses of EF (5, 25 and 125mgkgbw-1 ) were administrated to Bama pigs. The results showed that lipid peroxidation of pig liver tissue occurred with all EF doses. The 125mgkg dose of EF induced catalase (CAT) and glutathione peroxidase (GSH-px) and increased CYP450 content in pig liver microsomes. The activity of microsomal NADPH-cytochrome C reductase (NCCR) was elevated at both the 25 and 125mgkg doses of EF. Microsomal erythromycin N-demethylase (ERND) and aminopyrin N-demethylase (AND) were inhibited by high doses of EF, while aniline-4-hydroxylase (AH) was unaffected. None of the EF treatments affected superoxide dismutase (SOD) or cytochrome b5 content. Antioxidases and microsomal enzymes may work together to resist the adverse effects of EF. Proteomic analysis revealed increased protein expression of carboxylesterase (CES) and alpha-enolase (ENO1) in microsomes as a stress response to EF. These results provide new information about the adverse effect of fluoroquinolones and help guide their usage more effectively in the clinic or animal breeding.

Comparison of In Vitro Hepatic Scoparone 7-O-Demethylation between Humans and Experimental Animals.

Scoparone is a natural bioactive compound in Chinese herbal medicines. It has numerous pharmacological actions, including liver protective, hypolipidemic, antitumor, and anti-inflammatory effects. The primary metabolism route of scoparone is O-demethylation to scopoletin or isoscopoletin catalyzed by CYP enzymes. The aims of our study were to identify the human CYP enzymes catalyzing scoparone 7-O-demethylation to scopoletin and to compare this oxidation reaction in liver microsomes among different species. A high throughput fluorescent-based assay method was developed to determine the scoparone 7-O-demethylation to scopoletin rate. The rate was 100 - 400 nmol/(min×g protein) in mouse and rabbit liver microsomes, 10 - 20 nmol/(min×g protein) in pig microsomes, 1 - 3 nmol/(min×g protein) in human and less than 1 nmol/(min×g protein) in rat liver microsomes. Human CYP1A1 (Km13 µM and Vmax0.8 min-1), CYP1A2 (Km48 µM and Vmax0.3 min-1), and CYP2A13 (Km10 µM and Vmax22 min-1) were the most efficient catalysts of the reaction. The CYP2A6 selective inhibitor pilocarpine and an antibody against mouse CYP2A5 inhibited scoparone 7-O-demethylation to scopoletin in rabbit, mouse, and pig liver microsomes, indicating involvement of CYP2A enzymes in the reaction. Hepatic scoparone 7-O-demethylation to scopoletin differed between species both with respect to the rate of reaction and catalyzing enzymes. These species differences need to be taken into account when testing scoparone pharmacokinetics in animals and humans.

Biotransformation of NSAIDs by pig liver microsomes in vitro: Kinetics, metabolites identification and toxicity prediction

Non-steroidal anti-inflammatory drugs (NSAIDs) are one of the most frequently used pharmaceuticals in animals. In the current study, the biotransformation of five NSAIDs by pig liver microsomes (PLMs) was studied. The pseudo-first-order kinetics mode was obtained for the metabolization of the studied NSAIDs by PLMs in vitro. The metabolites were identified by high performance liquid chromatography with a high-resolution LTQ-Orbitrap mass spectrometry. The hydroxylation of benzene was confirmed to be the dominating metabolic pathway. Finally, the toxicity of the metabolites was predicted by the Estimation Programs Interface Suite software based on quantitative structure-activity relationships. Decreased toxicity was expected for the most metabolites of the studied NSAIDs except flurbiprofen, whose main metabolite exhibited slightly more toxicity. The present study provided a preliminary foundation to understand the metabolites of some NSAIDs and their toxicity, which was of great significance in animal food safety.

Biotransformation of OH-PBDEs by pig liver microsomes: Investigating kinetics, identifying metabolites, and examining the role of different CYP isoforms

Hydroxylated polybrominated diphenyl ethers (OH-PBDEs) are of great concern due to their potential risk to animal and human health. The biotransformation potential of OH-PBDEs in organisms is important for the understanding of their health risk. In the present study, the biotransformation of 3′-OH-2,4-di-BDE (3′-OH-BDE-7), 4′-OH-2,2′,4-tri-BDE (4′-OH-BDE-17) and 3-OH-2,2′,4,4′-tetra-BDE (3-OH-BDE-47) by pig liver microsomes was studied. Compared with their precursor PBDEs, the three OH-PBDEs were more readily biotransformed by pig liver microsomes, and the biotransformation rate followed the order: 3′-OH-BDE-7 > 4′-OH-BDE-17 > 3-OH-BDE-47. These results revealed that the biotransformation rate of OH-PBDEs was decreased with an increase in the number of bromine substituents. Cleavage of the diphenyl ether bond was the dominant pathway for biotransformation of the three OH-PBDEs by pig liver microsomes, while debromination and hydroxylation were found to be of less importance. CYP3A4 was suggested to be the specific enzyme responsible for the biotransformation of OH-PBDEs via associated inhibition assay. These findings may enrich our understanding of health risk associated with OH-PBDEs in mammals and human beings.