Specific CYP Research Articles

Specific human CYP enzymes-dependent mutagenicity of tris(2-butoxyethyl) phosphate (an organophosphorus flame retardant) in human and hamster cell lines

Tris(2-butoxyethyl) phosphate (TBOEP) is an organophosphorus flame retardant ubiquitously present in the environment and even the human body. TBOEP is toxic in multiple tissues, which forms dealkylated and hydroxylated metabolites under incubation with human hepatic microsomes; however, the impact of TBOEP metabolism on its toxicity, particularly mutagenicity (typically requiring metabolic activation), is left unidentified. In this study, the mutagenicity of TBOEP in human hepatoma cell lines (HepG2 and C3A) and the role of specific CYPs were studied. Through molecular docking, TBOEP bound to human CYP1A1, 1B1, 2B6 and 3A4 with energies and conformations favorable for catalyzing reactions, while the conformations of its binding with human CYP1A2 and 2E1 appeared unfavorable. In C3A cells (endogenous CYPs being substantial), TBOEP exposing for 72 h (2-cell cycle) at low micromolar levels induced micronucleus, which was abolished by 1-aminobenzotriazole (inhibitor of CYPs); in HepG2 cells (CYPs being insufficient) TBOEP did not induce micronucleus, whose effect was however potentiated by pretreating the cells with PCB126 (CYP1A1 inducer) or rifampicin (CYP3A4 inducer). TBOEP induced micronucleus in Chinese hamster V79-derived cell lines genetically engineered for stably expressing human CYP1A1 and 3A4, but not in cells expressing the other CYPs. In C3A cells, TBOEP selectively induced centromere protein B-free micronucleus (visualized by immunofluorescence) and PIG-A gene mutations, and elevated γ-H2AX rather than p-H3 (by Western blot) which indicated specific double-strand DNA breaks. Therefore, this study suggests that TBOEP may induce DNA/chromosome breaks and gene mutations in human cells, which requires metabolic activation by CYPs, primarily CYP1A1 and 3A4.

The susceptibility of humans to neurodegenerative and neurodevelopmental toxicities caused by organophosphorus pesticides.

The toxicology field is concerned with the impact of organophosphorus (OP) compounds on human health. These compounds have been linked to an increased risk of neurological disorders, including neurodegenerative and neurodevelopmental diseases. This article aims to review studies on the role of OP compounds in developing these neurological disorders and explore how genetic variations can affect susceptibility to the neurotoxicity of these pesticides. Studies have shown that exposure to OP compounds can lead to the development of various neurological disorders, such as Alzheimer's disease (AD), Parkinson's disease (PD), attention deficit hyperactivity disorder (ADHD), autism, intellectual disability, and other developmental neurotoxicities. Apart from inhibiting the cholinesterase enzyme, OP compounds are believed to cause other pathological mechanisms at both the extracellular level (cholinergic, serotonergic, dopaminergic, glutamatergic, and GABAergic synapses) and the intracellular level (oxidative stress, mitochondrial dysfunction, inflammation, autophagy, and apoptosis) that contribute to these disorders. Specific genetic polymorphisms, including PON1, ABCB1, NOS, DRD4, GST, CYP, and APOE, have increased the risk of developing OP-related neurological disorders.

Safety profile of lorlatinib: correction of adverse events

Lorlatinib is a new third-generation tyrosine kinase (TKI) inhibitor of ALK/ROS1, which has antitumour activity against most of the known mutations of resistance to crizotinib and second-generation TKI, as well as high intracranial efficacy. The safety of lorlatinib was evaluated in a multi-cohort phase I study involving 295 patients receiving the recommended dose of lorlatinib 100 mg once a day. Adverse events of lorlatinib were mainly mild to moderate severity. The most frequent complications – hypercholesterolemia (82.4%), hypertriglyceridemia (60.7%), edema (51.2%), peripheral neuropathy (43.7%) and side effects from the central nervous system (39.7%), were reversible and well controlled by dose modification and concomitant therapy, as evidenced by the low frequency of discontinuation of therapy due to adverse reactions. The majority of patients (81.0%) required the appointment of at least one hypolipidemic drug. When prescribing concomitant therapy, the possibility of drug interaction with lorlatinib, whose metabolism is carried out with the participation of specific CYP450 enzymes, should also be taken into account. Based on the presented results, an expert consensus opinion was developed on the correction of the main adverse reactions, including hyperlipidemia, complications from the central nervous system, weight gain, edema, peripheral neuropathy and others. No new adverse events were reported in the CROWN Phase III study conducted later. Lorlatinib has a characteristic toxicity profile, which must be taken into account for successful long-term targeted therapy while maintaining a good quality of life for patients. In the Russian Federation, the drug is approved for use in a wide clinical practice both for the treatment of patients with ALK-positive metastatic non-small cell lung cancer (NSCLC) in the first line, and after progression to second-generation TKI. The article presents recommendations for the correction of the main adverse events of lorlatinib, as well as their own experience in managing patients.

Metabolism of ropinirole is mediated by several canine CYP enzymes.

Ropinirole has been shown to provoke vomiting in dogs by activating the dopamine D2-like receptors in the chemoreceptor trigger zone. In humans, ropinirole is metabolized primarily by CYP1A2. Corresponding dog CYP1A2 is known to be a polymorphic enzyme which can cause variation in pharmacokinetics of compounds metabolised via this enzyme. The aim of this study was to understand metabolic clearance of ropinirole in dogs, the enzymes involved in ropinirole metabolism and specially to estimate whether the clearance can be sensitive towards the polymorphism of canine CYP1A2. Metabolism of ropinirole was studied in dog hepatocytes and specific recombinant canine CYP isoforms. Metabolite identification and metabolite formation were evaluated using LC-mass spectrometry. Ropinirole was moderately stable in dog hepatocytes with Clint of 16.3μL/min/million cells, and the metabolites detected were 7-hydroxy ropinirole and respective glucuronide conjugate as well as despropyl ropinirole. Regarding recombinant CYPs either 7-hydroxy ropinirole, despropyl ropinirole or both were detected for each CYP isoform studied. The highest rates of metabolite formation were observed in CYP2B11, CYP2C21, CYP2D15, CYP1A2 and CYP1A1. Fluvoxamine, a fairly selective human CYP1A/CYP2C19 inhibitor inhibited ropinirole metabolism by CYP1A1, CYP1A2, CYP2B11, CYP2C21 and CYP2D15 for 65.8%-100% showing no selectivity towards canine CYP isoforms. Although human metabolism of ropinirole is mainly mediated through CYP1A2, the current study shows that several canine CYP isoforms are able to contribute to the clearance of ropinirole in dogs. This is expected to reduce a possible impact of canine CYP1A2 polymorphism on ropinirole pharmacokinetics.

Metabolism of Benzalkonium Chloride by CYP‐Mediated ω‐Oxidation and C‐C Cleavage Followed by β‐Oxidation in Humans

IntroductionBenzalkonium chlorides (BACs) are widely used quaternary ammonium disinfectants. A recent study found that median levels of BACs in human blood have increased 174% during COVID from pre‐COVID levels, with BACs being detected in 98% of blood samples. Although the parent BACs are not readily excreted into urine, we have identified several BAC metabolites in anonymous human urine samples. Formation of these metabolites requires ω‐oxidation and oxidative C‐C cleavage of BAC alkyl chains followed by β‐oxidation to produce a series of chain‐shortened carboxylic acid (COOH) BAC metabolites.Here, we aim to elucidate the metabolic route(s) that can account for the observed urinary metabolites of BACs. Our approach includes LC‐MS‐based investigation of metabolite profiles in microsomes, hepatocytes, and recombinant CYPs in comparison with those observed in human urine. BACs of the alkyl chain lengths found in commercial mixtures (C12, C14, and C16) served as substrates. Authentic metabolite standards were synthesized and confirmed by NMR and LC‐MS/MS. Selective CYP inhibitors were employed to assign specific CYP isoform contributions to metabolite formation.ResultsAs an extension of our previous work, we confirmed that NADPH‐dependent ω‐oxidation in microsomes and recombinant CYPs produced ω‐OH and ω‐COOH metabolites, as well as ω‐1 oxidized products, of all BAC chain lengths. We further found that the responsible CYPs (2D6, 4F2, 4F11, and 4F12) are all capable of performing C‐C cleavage of BAC side chains’ terminal carbons. The resulting products were chain‐shortened ω‐COOH BACs having lost 1 or 2 carbons (‐CH2 or ‐C2H4). In hepatocytes, subsequent β‐oxidation of the ω‐COOH C10, C12, C14, and C16 BAC metabolites (having lost 0 or 2 carbons during CYP metabolism) produces a series of even‐carbon ω‐COOH products (C4, C6, and C8) given the successive 2 carbon removal (‐C2H4) from the even‐carbon starting chain lengths. Additionally, hepatocytes produce a series of odd‐carbon ω‐COOH products (C5, C7, and C9) that logically originate from β‐oxidation of ω‐COOH C11, C13, and C15 BAC metabolites (having lost 1 carbon during CYP metabolism). The ω‐COOH BAC metabolites produced in hepatocytes were the same as those detected in human urine samples, and the relative abundance among the various chain lengths of ω‐COOH products was similar between human hepatocytes and urine. In most urine samples, C8, C10, and C12 were the most abundant COOH metabolites of even‐carbon length, and C9 was most abundant among the odd‐carbon series. In hepatocytes, addition of HET0016 and quinidine to inhibit CYP4F/CYP2D6 abolished all metabolite formation, including the β‐oxidation products.ConclusionThe above data confirm that CYP4F and/or CYP2D6 are required to initiate BAC metabolism. The relative abundance of ω‐ and β‐oxidized products (ω‐COOH BACs C4 ‐ C16) observed in urine was consistent with the human cellular metabolism of BACs observed in hepatocytes. While parent BACs are commonly found in human blood, they are not readily excreted to urine. Thus, we propose to expand monitoring of human urine samples for chain‐shortened ω‐COOH BAC metabolites as a practical surrogate for assessing BAC exposure in the general population.

Prediction of CYP‐mediated DDIs involving inhibition: Approaches to address the requirements for system qualification of the Simcyp Simulator

Physiologically‐based pharmacokinetic (PBPK) modeling is being increasingly used in drug development to avoid unnecessary clinical drug–drug interaction (DDI) studies and inform drug labels. Thus, regulatory agencies are recommending, or indeed requesting, more rigorous demonstration of the prediction accuracy of PBPK platforms in the area of their intended use. We describe a framework for qualification of the Simcyp Simulator with respect to competitive and mechanism‐based inhibition (MBI) of CYP1A2, CYP2D6, CYP2C8, CYP2C9, CYP2C19, and CYP3A4/5. Initially, a DDI matrix, consisting of a range of weak, moderate, and strong inhibitors and substrates with varying fraction metabolized by specific CYP enzymes that were susceptible to different degrees of inhibition, were identified. Simulations were run with 123 clinical DDI studies involving competitive inhibition and 78 clinical DDI studies involving MBI. For competitive inhibition, the overall prediction accuracy was good with an average fold error (AFE) of 0.91 and 0.92 for changes in the maximum plasma concentration (Cmax) and area under the plasma concentration (AUC) time profile, respectively, as a consequence of the DDI. For MBI, an AFE of 1.03 was determined for both Cmax and AUC. The prediction accuracy was generally comparable across all CYP enzymes, irrespective of the isozyme and mechanism of inhibition. These findings provide confidence in application of the Simcyp Simulator (V19 R1) for assessment of the DDI potential of drugs in development either as inhibitors or victim drugs of CYP‐mediated interactions. The approach described herein and the identified DDI matrix can be used to qualify subsequent versions of the platform.

Towards the Elucidation of the Pharmacokinetics of Voriconazole: A Quantitative Characterization of Its Metabolism.

The small-molecule drug voriconazole (VRC) shows a complex and not yet fully understood metabolism. Consequently, its in vivo pharmacokinetics are challenging to predict, leading to therapy failures or adverse events. Thus, a quantitative in vitro characterization of the metabolism and inhibition properties of VRC for human CYP enzymes was aimed for. The Michaelis–Menten kinetics of voriconazole N-oxide (NO) formation, the major circulating metabolite, by CYP2C19, CYP2C9 and CYP3A4, was determined in incubations of human recombinant CYP enzymes and liver and intestine microsomes. The contribution of the individual enzymes to NO formation was 63.1% CYP2C19, 13.4% CYP2C9 and 29.5% CYP3A4 as determined by specific CYP inhibition in microsomes and intersystem extrapolation factors. The type of inhibition and inhibitory potential of VRC, NO and hydroxyvoriconazole (OH–VRC), emerging to be formed independently of CYP enzymes, were evaluated by their effects on CYP marker reactions. Time-independent inhibition by VRC, NO and OH–VRC was observed on all three enzymes with NO being the weakest and VRC and OH–VRC being comparably strong inhibitors of CYP2C9 and CYP3A4. CYP2C19 was significantly inhibited by VRC only. Overall, the quantitative in vitro evaluations of the metabolism contributed to the elucidation of the pharmacokinetics of VRC and provided a basis for physiologically-based pharmacokinetic modeling and thus VRC treatment optimization.

التأثيرات الناتجة عن استخدام عقاري الديكلوفيناك والسيبروفلوكساسين على الكبد والكلى لدى الجرذان

The toxic effect of diclofenac (DCF) sodium and Ciprofloxacin (CIP) on gene expression of cytochrome P450 oxidase (CYPs) and the histology of liver and kidney of male albino rat has been evaluated in this study. DCF and CIP were chosen since they are inhibitors for specific CYP enzymes. Thirty-five adult male albino rats were divided into 7 groups of 5 animals each (A, B, C, D, E, F and G) and were treated orally with drugs for 21 consecutive days. Group A served as the control while B and C were treated with 5.3, 10.6 mg/kg body weight (bw) DCF sodium and groups D and E were treated with 40 and 80 mg/kg bw CIP, respectively. Groups F and G were treated with a mixture of the low and the high doses of both drugs, respectively. Both drugs significantly downregulated the mRNA expression of CYP1a2, CYP3a4 and CYP2c9. They caused hepatorenal histological changes. In the liver, massive fibrosis, necrosis, inflammatory cell infiltration with hemorrhages and hydrophilic degeneration have been observed. A massive tissue injury with glomerular and tubular damages due to sever necrosis, degeneration of concomitant inflammatory cells and blood vessels congestion have been shown in renal tissues. Although DCF and CIP are still used as therapeutic drugs, their use should be limited as their chronic administration induces a toxic effect on human health.

Liver proteome profiling in dairy cows during the transition from gestation to lactation: Effects of supplementation with essential fatty acids and conjugated linoleic acids as explored by PLS-DA

This study aimed at investigating the synergistic effects of essential fatty acids (EFA) and conjugated linoleic acids (CLA) on the liver proteome profile of dairy cows during the transition to lactation. 16 Holstein cows were infused from 9 wk. antepartum to 9 wk. postpartum into the abomasum with either coconut oil (CTRL) or a mixture of EFA (linseed + safflower oil) and CLA (EFA + CLA). Label-free quantitative proteomics was performed in liver tissue biopsied at days −21, +1, +28, and + 63 relative to calving. Differentially abundant proteins (DAP) between treatment groups were identified at the intersection between a multivariate and a univariate analysis. In total, 1680 proteins were identified at each time point, of which between groups DAP were assigned to the metabolism of xenobiotics by cytochrome P450, drug metabolism - cytochrome P450, steroid hormone biosynthesis, glycolysis/gluconeogenesis, and glutathione metabolism. Cytochrome P450, as a central hub, enriched with specific CYP enzymes comprising: CYP51A1 (d − 21), CYP1A1 & CYP4F2 (d + 28), and CYP4V2 (d + 63). Collectively, supplementation of EFA + CLA in transition cows impacted hepatic lipid metabolism and enriched several common biological pathways at all time points that were mainly related to ω-oxidation of fatty acids through the Cytochrome p450 pathway. SignificanceIn three aspects this manuscript is notable. First, this is among the first longitudinal proteomics studies in nutrition of dairy cows. The selected time points are critical periods around parturition with profound endocrine and metabolic adaptations. Second, our findings provided novel information on key drivers of biologically relevant pathways suggested according to previously reported performance, zootechnical, and metabolism data (already published elsewhere). Third, our results revealed the role of cytochrome P450 that is hardly investigated, and of ω-oxidation pathways in the metabolism of fatty acids with the involvement of specific enzymes.

Long-term maintenance of functional primary human hepatocytes in 3D gelatin matrices produced by solution blow spinning

Solution blow spinning (SBS) has recently emerged as a novel method that can produce nano- and microfiber structures suitable for tissue engineering. Gelatin is an excellent precursor for SBS as it is derived mainly from collagens that are abundant in natural extracellular matrices. Here we report, for the first time the successful generation of 3D thermally crosslinked preforms by using SBS from porcine gelatin. These SBS mats were shown to have three-dimensional fibrous porous structure similar to that of mammalian tissue extracellular matrix. In pharma industry, there is an urgent need for adequate 3D liver tissue models that could be used in high throughput setting for drug screening and to assess drug induced liver injury. We used SBS mats as culturing substrates for human hepatocytes to create an array of 3D human liver tissue equivalents in 96-well format. The SBS mats were highly cytocompatible, facilitated the induction of hepatocyte specific CYP gene expression in response to common medications, and supported the maintenance of hepatocyte differentiation and polarization status in long term cultures for more than 3 weeks. Together, our results show that SBS-generated gelatin scaffolds are a simple and efficient platform for use in vitro for drug testing applications.

Herb-drug interactions and toxicity: Underscoring potential mechanisms and forecasting clinically relevant interactions induced by common phytoconstituents via data mining and computational approaches

Herbals in the form of medicine are employed extensively around the world. Herbal and conventional medicine combination is a potentially dangerous practice mainly in comorbid, hepato insufficient and frail patients leading to perilous herb-drug interactions (HDI) and toxicity. This study features potential HDI of 15 globally famous plant species through data mining and computational methods. Several plant species were found to mimic warfarin. Phytochemicals from M. charantia induced hypoglycemica. M. chamomila and G. biloba possessed anticoagulant activities. S. hispanica reduces postprandial glycemia. R. officinalis has been reported to inhibit the efflux of anticancer substrates while A. sativum can boost the clearance of anticancer agents. P. ginseng can alter blood coagulation. A cross link of the biological and in silico data revealed that a plethora of herbal metabolites such as ursolic and rosmarinic acid among others are possible/probable inhibitors of specific CYP450 enzymes. Consequently, plant species/metabolites with a given pharmacological property/metabolizing enzyme should not be mixed with drugs having the same pharmacological property/metabolizing enzyme. Even if combined with drugs, herbal medicines must be used at low doses for a short period of time and under the supervision of a healthcare professional to avoid potential adverse and toxic effects.

Development of Saccharomyces cerevisiae Y486–based biosensor carrying both CPR-CYP3A4 and DIN7-GFP constructs for mutagenicity testing of procarcinogens

In the world and Vietnam, a great number of toxic substances from industrial and agricultural activities, food production, and healthcare services are daily released into the environment. Many exogenous harmful substances are procarcinogens, but become carcinogens by the bioactivation of human cytochrome P450 enzymes (CYPs). Thus, development of analytical testing for rapid detection of procarcinogens plays a crucial role in food safety and environmental monitoring. This study aims to establish a biosensor basing on Saccharomyces cerevisiae Y486 cotransformed with two promoter–gene constructs, CYP3A4–CPR and DIN7–GFP. The results showed that all recombinant proteins were coexpressed in Y486 cells. The molecular weight of recombinant CPR and CYP3A4 were 75 kDa and 56 kDa, respectively. CYP3A4 enzyme only showed its catalytic activity in biotransformation of the specific substance as coexpressed with CPR. Kinetic constants, Km, Vmax, and Vmax/Km, of this CPR–CYP3A4 enzyme complex were 3.2 µM, 3.5 pmol/pmol CYP/min, and 1.1 μL/pmol CYP/min, respectively. Coexpressing constructs of CPR–CYP3A4 and DIN7-GFP in Y486 strain was able to identify aflatoxin B1 in the range of 0.1 - 0.4 µM; benzo(c)pyrene in the range of 10 - 40 µM. However, this system could not detect other procacinogens, such as, N-Nitrosodimethylamine, at any investigated concentrations. These findings were the first trial for further development of other biosensors to determine diverse procarcinogens in the enviroment by redesign of coexpressing constructs or replacement of the specific CYPs and inducible promoters.

Inhibition of 20-hydroxyeicosatetraenoic acid biosynthesis by vitamin E analogs in human and bovine cytochrome P450 microsomes.

Dairy cattle are predisposed to disease around the time of calving due to dysfunctional inflammatory responses. Oxylipids are lipid-derived mediators that regulate all aspects of the inflammatory response, and shifts in oxylipid profiles are correlated with disease risk. For example, 20-hydroxyeicosatetraenoic acid (HETE) is an oxylipid derived from cytochrome P450 enzymes (CYP450) found at significantly greater concentrations around calving and during clinical disease. Biosynthesis of 20-HETE occurs almost exclusively from two specific CYP450 of which CYP450 family four sub-family F member two (CYP4F2) is the major contributor to 20-HETE production in humans. To further study the activities of 20-HETE and potentially reduce its production in vivo, mitigation methods must be explored. Additional substrates of CYP4F2, such as vitamin E, are known to both increase and decrease the metabolism of other CYP4F2 substrates. This study aimed to determine whether vitamin E analogs may reduce the production of 20-HETE through competition for CYP4F2 activity in human CYP4F2, bovine-kidney and bovine-mammary microsomes. Gamma-tocopherol reduced 20-HETE production from human and bovine-kidney microsomes (35.3% and 27.5%, respectively) whereas γ-tocotrienol only reduced 20-HETE production from human microsomes (40.1%). Finally, bovine-mammary microsomes did not produce a quantifiable amount of 20-HETE, suggesting basal mammary CYP4F2 activity may not be a significant contributor to 20-HETE found in milk. Together, these data show that analogs of vitamin E can reduce the production of 20-HETE, potentially through competition with arachidonic acid for metabolism by CYP4F2, posing a potential means for limiting 20-HETE production during clinical diseases of dairy cattle.

The genotoxicity potential of luteolin is enhanced by CYP1A1 and CYP1A2 in human lymphoblastoid TK6 cells

Luteolin (5,7,3′,4′-tetrahydroxyflavone) belongs to the flavone subclass of flavonoids. Luteolin and its glycosides are present in many botanical families, including edible plants, fruits, and vegetables. While the beneficial properties of luteolin have been widely studied, fewer studies have investigated its toxicity. In the present study, using human lymphoblastoid TK6 cells and our newly developed TK6-derived cell lines that each stably express a single human cytochrome P450 (CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C18, 2C9, 2C19, 2D6, 2E1, 3A4, 3A5, and 3A7), we systematically evaluated luteolin-induced cytotoxicity and genotoxicity, and the role of specific CYPs in the bioactivation of luteolin. Treatments with luteolin for 4−24 h induced cytotoxicity, apoptosis, DNA damage, and chromosome damage in a concentration-dependent manner. Subsequently, we observed that luteolin-induced cytotoxicity and genotoxicity, measured by the high-throughput micronucleus assay, were significantly increased in TK6 cells transduced with CYP1A1 and 1A2. In addition, key apoptosis and DNA damage biomarkers, including cleaved PARP-1, cleaved caspase-3, and phosphorylated histone 2AX (γH2A.X), were all significantly increased in the CYP1A1- and 1A2-expressing cells compared with the empty vector controls. Analysis by LC–MS/MS revealed that TK6 cells biotransformed the majority of luteolin into diosmetin, a less toxic O-methylated flavone, after 24 h; the presence of CYP1A1 and 1A2 partially reversed this process. Altogether, these results indicate that metabolism by CYP1A1 and 1A2 enhanced the toxicity of luteolin in vitro. Our results further support the utility of our TK6 cell system for identification of the specific CYPs responsible for chemical bioactivation and toxicity potential.

Anatomical distribution and expression of CYP in humans: Neuropharmacological implications

The cytochrome P450 (CYP450) superfamily is responsible for the metabolism of most xenobiotics and pharmacological treatments generally used in clinical settings. Genetic factors as well as environmental determinants acting through fine epigenetic mechanisms modulate the expression of CYP over the lifespan (fetal vs. infancy vs. adult phases) and in diverse organs. In addition, pathological processes might alter the expression of CYP. In this selective review, we sought to summarize the evidence on the expression of CYP focusing on three specific aspects: (a) the anatomical distribution of the expression in body districts relevant in terms of drug pharmacokinetics (liver, gut, and kidney) and pharmacodynamics, focusing for the latter on the brain, since this is the target organ of psychopharmacological agents; (b) the patterns of expression during developmental phases; and (c) the expression of CYP450 enzymes during pathological processes such as cancer. We showed that CYP isoforms show distinct patterns of expression depending on the body district and the specific developmental phases. Of particular relevance for neuropsychopharmacology is the complex regulatory mechanisms that significantly modulate the complexity of the pharmacokinetic regulation, including the concentration of specific CYP isoforms in distinct areas of the brain, where they could greatly affect local substrate and metabolite concentrations of drugs.

The fibrotic response of primary liver spheroids recapitulates in vivo hepatic stellate cell activation

A major obstacle in the development of efficient therapies for progressive liver fibrosis is the lack of representative in vitro models of liver fibrosis to aid in understanding the mechanisms of the disease and to promote the development of pharmaceuticals.Our aim was to develop a relevant in vitro mouse liver fibrosis model, based on the central hypothesis that liver fibrosis in vitro cannot be studied using only hepatic stellate cells (HSCs)–the main producer of scar tissue during fibrosis–, but requires cultures in which at least hepatocytes are integrated.We established robust methods to generate co-culture spheroids from freshly isolated mouse hepatocytes and HSCs. Characteristics and functionality of these spheroids were analyzed by qPCR of cell-type specific markers, CYP induction and immunohistochemistry. Compound toxicity was determined by ATP-assays.Hepatocytes and HSCs maintained their cell-type specific marker expression over a 15-day culture period without major hepatocyte dedifferentiation or HSC activation. Exposure of spheroids to TGFβ can directly activate HSCs, while acetaminophen exposure mounts a hepatocyte damage dependent activation of HSCs. Pharmaceuticals with known anti-fibrotic properties, such as Valproic acid and Verteporfin, reduce HSC activation in response to hepatocyte damage in these cultures. A comparison between the fibrotic response of the spheroid co-cultures and in vivo activated HSCs showed that these 3D co-cultures are more representative than the commonly used 2D HSC monocultures. Finally, we showed that the 3D cultures can be integrated in microfluidic chips.We conclude that our hepatocyte-stellate cell-spheroid cultures are a robust in vitro model of liver fibrosis. This model could be used to further unravel the mechanism of HSC activation and facilitate the discovery of, or testing for novel anti-fibrotic compounds, as these spheroids better reproduce HSC in vivo activation compared to the more traditional 2D mono-culture models.

Evaluation of pyrrolizidine alkaloid-induced genotoxicity using metabolically competent TK6 cell lines

Pyrrolizidine alkaloid (PA)-containing plants are among the most common poisonous plants affecting humans, livestock, and wildlife worldwide. A large number of PAs are known to induce genetic damage after metabolic activation. In the present study, using a battery of fourteen newly developed TK6 cell lines, each expressing a single human cytochrome P450 (CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C18, 2C9, 2C19, 2D6, 2E1, 3A4, 3A5, and 3A7), we identified specific CYPs responsible for bioactivating three PAs – lasiocarpine, riddelliine, and senkirkine. Among the fourteen cell lines, cells expressing CYP3A4 showed significant increases in PA-induced cytotoxicity, evidenced by decreased ATP production and cell viability, and increased caspase 3/7 activities. LC-MS/MS analysis revealed the formation of 1-hydroxymethyl-7-hydroxy-6,7-dihydropyrrolizine (DHP), the main reactive metabolite of PAs, in CYP3A4-expressing TK6 cells. DHP was also detected in CYP3A5- and 3A7-expressing cells after PA exposure, but to a much lesser extent. Subsequently, using a high-throughput micronucleus assay, we demonstrated that PAs induced concentration-dependent increases in micronuclei and G2/M phase cell cycle arrest in three CYP3A variant-expressing TK6 cell lines. Using Western blotting, we observed that PA-induced apoptosis, cell cycle changes, and DNA damage were primarily mediated by CYP3A4. Benchmark dose (BMD) modeling demonstrated that lasiocarpine, of the three PAs, was the most potent inducer of micronuclei, with a BMD100 of 0.036 μM. These results indicate that our TK6 cell system holds promise for genotoxicity screening of compounds requiring metabolic activation, identifying specific CYPs involved in bioactivation, and discriminating the genotoxic compounds that have different chemical structures.

Binding and Metabolism of Brominated Flame Retardant β-1,2-Dibromo-4-(1,2-dibromoethyl)cyclohexane in Human Microsomal P450 Enzymes: Insights from Computational Studies.

The emerging brominated flame retardant, 1,2-dibromo-4-(1,2-dibromoethyl)cyclohexane (TBECH), has recently attracted strong interest due to its extensive detection in the environment and potential toxicological effects on humans. Previous in vitro experiments have shown that the technical mixture of TBECH and the pure β-isomer (β-TBECH) can be metabolized by cytochrome P450 enzymes (CYPs) into multiple metabolites, but the specific CYP isoforms involved in TBECH metabolism and the relevant metabolic regioselectivity remain unknown. Here, we, for the first time, investigated the binding patterns and affinities of β-TBECH in human CYPs 1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, and 3A4, through molecular dynamics (MD) simulations. The binding affinities of β-TBECH in CYPs, which are estimated by the calculated binding free energies, follow the order of 2A6 > 2C9 > 2B6 > 2E1 > 3A4 ≈ 2C19 ≈ 1A2 > 2D6. Although all CYPs are important β-TBECH receptors, only 2A6, 2C19, 2E1, and 3A4 are responsible for metabolizing β-TBECH. Specially, 2A6 and 2E1 may selectively hydroxylate the C1 and C7 sites of β-TBECH, while 2C19 and 3A4 show metabolic preference for C7- and C8-hydroxylations, respectively. The three hydroxylation routes proposed by the further density functional theory (DFT) calculations generate C1-, C7-, and C8-hydroxylated metabolites, while the latter two may further undergo debromination to yield the respective ketone and aldehyde as additional metabolites. The results provide meaningful insight into the binding and metabolism of β-TBECH by human CYPs, which is helpful for understanding the metabolic fate and toxicity mechanism of this chemical.

Assessment of Inhibition of Bovine Hepatic Cytochrome P450 by 43 Commercial Bovine Medicines Using a Combination of In Vitro Assays and Pharmacokinetic Data from the Literature.

There has been a lack of information about the inhibition of bovine medicines on bovine hepatic CYP450 at their commercial doses and dosing routes. The aim of this work was to assess the inhibition of 43 bovine medicines on bovine hepatic CYP450 using a combination of in vitro assay and Cmax values from pharmacokinetic studies with their commercial doses and dosing routes in the literature. Those drugs were first evaluated through a single point inhibitory assay at 3 μM in bovine liver microsomes for six specific CYP450 metabolisms, phenacetin o-deethylation, coumarin 7- hydroxylation, tolbutamide 4-hydroxylation, bufuralol 1-hydroxylation, chlorzoxazone 6-hydroxylation and midazolam 1'-hydroxylation. When the inhibition was greater than 20% in the assay, IC50 values were then determined. The potential in vivo bovine hepatic CYP450 inhibition by those drugs was assessed using a combination of the IC50 values and in vivo Cmax values from pharmacokinetic studies at their commercial doses and administration routes in the literature. Fifteen bovine medicines or metabolites showed in vitro inhibition on one or more bovine hepatic CYP450 metabolisms with different IC50 values. Desfuroylceftiour (active metabolite of ceftiofur), nitroxinil and flunixin have the potential to inhibit one of the bovine hepatic CYP450 isoforms in vivo at their commercial doses and administration routes. The rest of the bovine medicines had low risks of in vivo bovine hepatic CYP450 inhibition. This combination of in vitro assay and in vivo Cmax data provides a good approach to assess the inhibition of bovine medicines on bovine hepatic CYP450.

Signature profile of cyclooxygenase-independent F2 series prostaglandins in C. elegans and their role in sperm motility

We previously discovered that Caenorhabditis elegans synthesizes Cox-independent F-series prostaglandins (PGs). To delineate the Cox-independent prostaglandin pathways and evaluate their role in sperm motility in C. elegans, we developed a novel biochemical method for the rapid production of F-series PGs using arachidonic acid as the substrate and worm lysate as source of enzyme(s). Among the four F2-series PGs produced in the reaction, three of them were identified as 8-isoPGF2α, 5iPF2 VI, and PGF2α based on their retention times and MS/MS spectral comparison with standards using LC-MS/MS. PG production was not markedly affected by specific antioxidants, or Cox, Lox, and Cyp inhibitors, suggesting that these PGs are formed through a novel, biologically regulated mechanism in C. elegans. This study also assessed the ability of 8-isoPGF2α, 5iPF2 VI, PGF2α, and a mixture containing these PGs in a 0.5/0.08/1 ratio that reflects their synthetic composition to modulate sperm motility in fat-2 mutants. PGF2α and the PG mixture at 25 μM concentration significantly stimulated sperm velocity by 28% and 38%, whereas 8-isoPGF2α and 5iPF2 VI reduced the velocity by 21% and 30%, respectively, compared to vehicle control. These results indicate that the sperm motility effects of PGs are structure- and composition-dependent in C. elegans.