Expression Of Cytochrome P450 Enzymes Research Articles

Methyltransferase Like-3-Mediated N6-Methyladenosine Modification of Long Noncoding RNA Hepatocyte Nuclear Factor 1a Antisense RNA 1/Hepatocyte Nuclear Factor 4a Antisense RNA 1 Regulates Cytochrome P450 Enzyme Expression.

Interindividual variations in the expression and activity of cytochrome P450 enzymes (CYPs) led to lower therapeutic efficacy or adverse drug events. We previously demonstrated that CYPs are regulated by the long noncoding RNAs (lncRNAs) hepatocyte nuclear factor 1a antisense RNA 1 (HNF1A-AS1) and HNF4A-AS1 via transcription factors (TFs) including hepatocyte nuclear factor 1a (HNF1A), hepatocyte nuclear factor 4a (HNF4A), and pregnane X receptor (PXR). However, the upstream mechanisms regulating HNF1A-AS1 and HNF4A-AS1 are poorly understood. N6-methyladenosine (m6A) is a prevalent epitranscriptomic modification in mammalian RNA. Therefore, the aim of this study was to investigate whether m6A modification regulates the expression of HNF1A-AS1 and HNF4A-AS1 and affects CYP expression in HepG2 and Huh7 cells. The methyltransferase-like 3 (METTL3) inhibitor, STM2457, significantly suppressed the expression of HNF1A-AS1 and induced HNF4A-AS1 expression. Consistent with this, a loss-of-function assay of METTL3 in the cell lines resulted in the downregulation of HNF1A-AS1 and its downstream HNF1A, PXR, and CYPs at the RNA level, as well as the downregulation of some CYPs proteins, and upregulation of HNF4A-AS1. The results of gain-of-function experiments showed the opposite trend. Mechanistically, subsequent RNA stability experiments confirmed that METTL3 affected the stability of both lncRNAs, but in opposite ways; that is, METTL3 reduced HNF1A-AS1 stability and increased HNF4A-AS1 stability. Rescue experiments confirmed that the regulation of METTL3 on TFs and CYPs may require the involvement of these two lncRNAs. Altogether, our study demonstrates that METTL3 is involved in TFs-mediated CYP expression by affecting HNF1A-AS1/HNF4A-AS1 stability. SIGNIFICANCE STATEMENT: Although the impact of long noncoding RNAs (lncRNAs) including hepatocyte nuclear factor 1a antisense RNA 1 (HNF1A-AS1) and hepatocyte nuclear factor 4a antisense RNA 1 (HNF4A-AS1) on the downstream transcription factor (TF) and cytochrome P450 enzyme (CYP) expression is well studied, the upstream regulation of these two lncRNAs by methyltransferase-like 3 (METTL3) remains unexplored. This study reveals that METTL3 is involved in the regulation of lncRNA-TF-CYP expression by affecting the stability of HNF1A-AS1 and HNF4A-AS1 in HepG2 and Huh7 cells.

Combinatory Effects of Acrylamide and Deoxynivalenol on In Vitro Cell Viability and Cytochrome P450 Enzymes of Human HepaRG Cells.

Acrylamide (AA) can be formed during the thermal processing of carbohydrate-rich foods. Deoxynivalenol (DON), a mycotoxin produced by Fusarium spp., contaminates many cereal-based products. In addition to potential co-exposure through a mixed diet, co-occurrence of AA and DON in thermally processed cereal-based products is also likely, posing the question of combinatory toxicological effects. In the present study, the effects of AA (0.001-3 mM) and DON (0.1-30 µM) on the cytotoxicity, gene transcription, and expression of major cytochrome P450 (CYP) enzymes were investigated in differentiated human hepatic HepaRG cells. In the chosen ratios of AA-DON (10:1; 100:1), cytotoxicity was clearly driven by DON and no overadditive effects were observed. Using quantitative real-time PCR, about twofold enhanced transcript levels of CYP1A1 were observed at low DON concentrations (0.3 and 1 µM), reflected by an increase in CYP1A activity in the EROD assay. In contrast, CYP2E1 and CYP3A4 gene transcription decreased in a concentration-dependent manner after incubation with DON (0.01-0.3 µM). Nevertheless, confocal microscopy showed comparably constant protein levels. The present study provided no indication of an induction of CYP2E1 as a critical step in AA bioactivation by co-occurrence with DON. Taken together, the combination of AA and DON showed no clear physiologically relevant interaction in HepaRG cells.

Age-Dependent Abundance of CYP450 Enzymes Involved in Metronidazole Metabolism: Application to Pediatric PBPK Modeling.

The expression of cytochrome P450 (CYP) enzymes is highly variable and associated with factors, such as age, genotype, sex, and disease states. In this study, quantification of metronidazole metabolizing CYP isoforms (CYP2A6, CYP2E1, CYP3A4, CYP3A5, and CYP3A7) in human liver microsomes from 115 children and 35 adults was performed using a quantitative proteomics method. The data confirmed age-dependent increase in CYP2A6, CYP2E1, and CYP3A4 abundance, whereas, as expected, CYP3A7 abundance showed postnatal decrease with age. In particular, the fold difference (neonatal to adulthood levels) in the protein abundance of CYP2A6, CYP2E1, and CYP3A4 was 14, 11, and 20, respectively. In contrast, protein abundance of CYP3A7 was > 125-fold higher in the liver microsomes of neonates than of adults. The abundance of CYP2A6 and CYP3A5 was associated with genotypes, rs4803381 and rs776746, respectively. A proteomics-informed physiologically based pharmacokinetic (PBPK) model was developed to describe the pharmacokinetics of metronidazole and its primary metabolite, 2-hydroxymethylmetronidazole. The model revealed an increase in the metabolite-to-parent ratio with age and showed a strong correlation between CYP2A6 abundance and metabolite formation (r 2 = 0.75). Notably, the estimated contribution of CYP3A7 was ~ 75% in metronidazole clearance in neonates. These data suggest that variability in CYP2A6 and CYP3A7 in younger children poses the risk of variable pharmacokinetics of metronidazole and its active metabolite with a potential impact on drug efficacy and safety. No sex-dependent difference was observed in the protein abundance of the studied CYPs. The successful integration of hepatic CYP ontogeny data derived from a large liver bank into the pediatric PBPK model of metronidazole can be extended to other drugs metabolized by the studied CYPs.

Bacterial Influence on Pharmacokinetics of Tacrolimus and Sulfasalazine through Regulation of Host Metabolism

AbstractThe unpredictable oral bioavailability of established drugs like tacrolimus and sulfasalazine presents a significant clinical challenge. This variability can lead to either toxicity or insufficient therapeutic effect. It is known that alterations in drug transporters and metabolic enzymes influence drug bioavailability. Recent evidence suggests that the indirect metabolism by gut microbes could influence transporters and enzymes which can affect the bioavailability of oral drugs. In this study, the pharmacokinetics of tacrolimus and sulfasalazine are modeled under varying host colonic conditions induced by the bacteria E. coli Nissle 1917 and Bifidobacterium adolescentis. Insight is provided into the sensitivity of pharmacokinetics to the bacterial influence on expression of intestinal drug transporters and cytochrome p450 enzymes. Our findings demonstrate that bacterial modulation reduces tacrolimus peak blood concentration compared to healthy renal transplant patients. Conversely, bacterial presence leads to a two‐fold increase in sulfasalazine's peak plasma concentration compared to healthy subjects. These results suggest that incorporating the gut bacteria's influence on colonic transporters and enzymes can improve the explanation of pharmacokinetic variability. This approach has the potential to refine pharmacokinetic models and ultimately address the challenge of variable oral drug bioavailability.

Intein-mediated temperature control for complete biosynthesis of sanguinarine and its halogenated derivatives in yeast

While sanguinarine has gained recognition for antimicrobial and antineoplastic activities, its complex conjugated structure and low abundance in plants impede broad applications. Here, we demonstrate the complete biosynthesis of sanguinarine and halogenated derivatives using highly engineered yeast strains. To overcome sanguinarine cytotoxicity, we establish a splicing intein-mediated temperature-responsive gene expression system (SIMTeGES), a simple strategy that decouples cell growth from product synthesis without sacrificing protein activity. To debottleneck sanguinarine biosynthesis, we identify two reticuline oxidases and facilitated functional expression of flavoproteins and cytochrome P450 enzymes via protein molecular engineering. After comprehensive metabolic engineering, we report the production of sanguinarine at a titer of 448.64 mg L−1. Additionally, our engineered strain enables the biosynthesis of fluorinated sanguinarine, showcasing the biotransformation of halogenated derivatives through more than 15 biocatalytic steps. This work serves as a blueprint for utilizing yeast as a scalable platform for biomanufacturing diverse benzylisoquinoline alkaloids and derivatives.

Circadian time-dependent effects of experimental colitis on theophylline disposition and toxicity.

Drug disposition undergoes significant alteration in patients with inflammatory bowel disease (IBD), yet circadian time-dependency of these changes remains largely unexplored. In this study, we aimed to determine the temporal effects of experimental colitis on drug disposition and toxicity. RNA-sequencing was used to screen genes relevant to colitis induced by dextran sodium sulfate in mice. Liver microsomes and pharmacokinetic analysis were used to analyze the activity of key enzymes. Dual luciferase assays and chromatin immunoprecipitation (ChIP) were employed to elucidate regulatory mechanisms. RNA sequencing analysis revealed that colitis markedly influenced expression of cytochrome P450 (CYP) enzymes. Specifically, a substantial down-regulation of CYP1A2 and CYP2E1 was observed in livers of mice with colitis at Zeitgeber Time 8 (ZT8), with no significant changes detected at ZT20. At ZT8, the altered expression corresponded to diminished metabolism and enhanced incidence of hepato-cardiac toxicity of theophylline, a substrate specifically metabolized by these enzymes. A combination of assays, integrating liver-specific Bmal1 knockout and targeted activation of BMAL1 showed that dysregulation in CYP1A2 and CYP2E1 during colitis was attributable to perturbed BMAL1 functionality. Luciferase reporter and ChIP assays collectively substantiated the role of BMAL1 in regulating Cyp1a2 and Cyp2e1 transcription through its binding affinity to E-box-like sites. Our findings establish a strong link between colitis and chronopharmacology, shedding light on how IBD affects drug disposition and toxicity over time. This research provides a theoretical foundation for optimizing drug dosage in patients with IBD.

Cytochrome P450 1B1 is critical in the development of TNF-α, IL-6, and LPS-induced cellular hypertrophy.

Heart failure (HF) is preceded by cellular hypertrophy (CeH) which alters expression of cytochrome P450 enzymes (CYPs) and arachidonic acid (AA) metabolism. Inflammation is involved in CeH pathophysiology, but mechanisms remain elusive. This study investigates the impacts of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and lipopolysaccharides (LPS) on the development of CeH and the role of CYP1B1. AC16 cells were treated with TNF-α, IL-6, and LPS in the presence and absence of CYP1B1-siRNA or resveratrol. mRNA and protein expression levels of CYP1B1 and hypertrophic markers were determined using PCR and Western blot analysis, respectively. CYP1B1 enzyme activity was determined, and AA metabolites were analyzed using liquid chromatography-tandem mass spectrometry. Our results show that TNF-α, IL-6, and LPS induce expression of hypertrophic markers, induce CYP1B1 expression, and enantioselectively modulate CYP1B1-mediated AA metabolism in favor of mid-chain HETEs. CYP1B1-siRNA or resveratrol ameliorated these effects. In conclusion, our results demonstrate the crucial role of CYP1B1 in TNF-α, IL-6, and LPS-induced CeH.

Identification and Functional Characterization of Alternative Transcripts of LncRNA HNF1A-AS1 and Their Impacts on Cell Growth, Differentiation, Liver Diseases, and in Response to Drug Induction.

The long non-coding RNA (lncRNA) hepatocyte nuclear factor-1 alpha (HNF1A) antisense RNA 1 (HNF1A-AS1) is an important lncRNA for liver growth, development, cell differentiation, and drug metabolism. Like many lncRNAs, HNF1A-AS1 has multiple annotated alternative transcripts in the human genome. Several fundamental biological questions are still not solved: (1) How many transcripts really exist in biological samples, such as liver samples and liver cell lines? (2) What are the expression patterns of different alternative HNF1A-AS1 transcripts at different conditions, including during cell growth and development, after exposure to xenobiotics (such as drugs), and in disease conditions, such as metabolic dysfunction-associated steatotic liver disease (MASLD), alcohol-associated liver disease (ALD) cirrhosis, and obesity? (3) Does the siRNA used in previous studies knock down one or multiple transcripts? (4) Do different transcripts have the same or different functions for gene regulation? The presented data confirm the existence of several annotated HNF1A-AS1 transcripts in liver samples and cell lines, but also identify some new transcripts, which are not annotated in the Ensembl genome database. Expression patterns of the identified HNF1A-AS1 transcripts are highly correlated with the cell differentiation of matured hepatocyte-like cells from human embryonic stem cells (hESC), growth and differentiation of HepaRG cells, in response to rifampicin induction, and in various liver disease conditions. The expression levels of the HNF1A-AS1 transcripts are also highly correlated to the expression of cytochrome P450 enzymes, such as CYP3A4, during HepaRG growth, differentiation, and in response to rifampicin induction.

Systematic engineering pinpoints a versatile strategy for the expression of functional cytochrome P450 enzymes in Escherichia coli cell factories

Production of plant secondary metabolites in engineered microorganisms provides a scalable and sustainable alternative to their sourcing from nature or through chemical synthesis. However, the biosynthesis of many valuable plant-derived products relies on cytochromes P450 – enzymes notoriously difficult to express in microbes. To improve their expression in Escherichia coli, an arsenal of engineering strategies was developed, often paired with an extensive screening of enzyme variants. Here, attempting to identify a broadly applicable strategy, we systematically evaluated six common cytochrome P450 N-terminal modifications and their effect on in vivo activity of enzymes from the CYP79 and CYP83 families. We found that transmembrane domain truncation was the only modification with a significantly positive effect for all seven tested enzymes, increasing their product titres by 2- to 170-fold. Furthermore, when comparing the changes in the protein titre and product generation, we show that higher protein expression does not directly translate to higher in vivo activity, thus making the protein titre an unreliable screening target in the context of cell factories. We propose the transmembrane domain truncation as a first-line approach that enables the expression of wide range of highly active P450 enzymes in E. coli and circumvents the time-consuming screening process. Our results challenge the notion that the engineering strategy must be tailored for each individual cytochrome P450 enzyme and have the potential to simplify and accelerate the future design of E. coli cell factories.

Expanding flavone and flavonol production capabilities in Escherichia coli.

Flavones and flavonols are important classes of flavonoids with nutraceutical and pharmacological value, and their production by fermentation with recombinant microorganisms promises to be a scalable and economically favorable alternative to extraction from plant sources. Flavones and flavonols have been produced recombinantly in a number of microorganisms, with Saccharomyces cerevisiae typically being a preferred production host for these compounds due to higher yields and titers of precursor compounds, as well as generally improved ability to functionally express cytochrome P450 enzymes without requiring modification to improve their solubility. Recently, a rapid prototyping platform has been developed for high-value compounds in E. coli, and a number of gatekeeper (2S)-flavanones, from which flavones and flavonols can be derived, have been produced to high titers in E. coli using this platform. In this study, we extended these metabolic pathways using the previously reported platform to produce apigenin, chrysin, luteolin and kaempferol from the gatekeeper flavonoids naringenin, pinocembrin and eriodictyol by the expression of either type-I flavone synthases (FNS-I) or type-II flavone synthases (FNS-II) for flavone biosynthesis, and by the expression of flavanone 3-dioxygenases (F3H) and flavonol synthases (FLS) for the production of the flavonol kaempferol. In our best-performing strains, titers of apigenin and kaempferol reached 128mgL-1 and 151mgL-1 in 96-DeepWell plates in cultures supplemented with an additional 3mM tyrosine, though titers for chrysin (6.8mgL-1) from phenylalanine, and luteolin (5.0mgL-1) from caffeic acid were considerably lower. In strains with upregulated tyrosine production, apigenin and kaempferol titers reached 80.2mgL-1 and 42.4mgL-1 respectively, without the further supplementation of tyrosine beyond the amount present in the rich medium. Notably, the highest apigenin, chrysin and luteolin titers were achieved with FNS-II enzymes, suggesting that cytochrome P450s can show competitive performance compared with non-cytochrome P450 enzymes in prokaryotes for the production of flavones.

The effect of brain serotonin deficit (TPH2-KO) on the expression and activity of liver cytochrome P450 enzymes in aging male Dark Agouti rats

BackgroundLiver cytochrome P450 (CYP) greatly contributes to the metabolism of endogenous substances and drugs. Recent studies have demonstrated that CYP expression in the liver is controlled by the central nervous system via hormonal pathways. In particular, the expression of hepatic CYPs is negatively regulated by the brain serotoninergic system. The present study aimed to investigate changes in the function of the main liver drug-metabolizing CYP enzymes as a result of serotonin depletion in the brain of aging rats, caused by knockout of brain tryptophan hydroxylase gene (TPH2-KO).MethodsThe hepatic CYP mRNA (qRT-PCR), protein level (Western blotting) and activity (HPLC), and serum hormone levels (ELISA) were measured in Dark Agouti wild-type (WT) male rats (mature 3.5-month-old and senescent 21-month-old) and in TPH2-KO senescent animals.ResultsThe expression/activity of the studied CYPs decreased with age in the liver of wild-type rats. The deprivation of serotonin in the brain of aging males decreased the mRNA level of most of the studied CYPs (CYP1A/2A/2B/3A), and lowered the protein level of CYP2C11 and CYP3A. In contrast, the activities of CYP2C11, CYP3A and CYP2C6 were increased. The expression of cytochrome b5 decreased in aging rats, but increased in TPH2-deficient senescent animals. The serum concentration of growth hormone declined in the aged and further dropped down in TPH2-deficient senescent rats.ConclusionsRat liver cytochrome P450 functions deteriorate with age, which may impair drug metabolism. The TPH2 knockout, which deprives brain serotonin, affects cytochrome P450 expression and activity differently in mature and senescent male rats.

Preclinical Pharmacokinetics and CYP Modulation Activity of Chebulinic Acid: A Potent Molecule Against Metabolic Disease.

Chebulinic acid (CA) is an active constituent of Terminalia chebula fruits with therapeutic potential against multiple metabolic diseases, including dementia, benign prostate hyperplasia, and osteoporosis. The present work intends to explore the preclinical pharmacokinetics, including the absolute bioavailability of CA and its influence on the gene expression of cytochrome P450 enzymes in the liver. Quantifying CA and probe drugs in vitro samples and preclinical serum samples of male SD rats were performed using LC-MS/MS. The influence of CA on the hepatic CYPs and their gene expression was analyzed in rat liver by quantitative real-time polymerase chain reaction. The plasma protein binding was found to be 84.81 ± 7.70 and 96.34 ± 3.12, blood-to-plasma ratio of 0.62 ± 0.16 and 0.80 ± 0.23 at 1 μM and 10 μM concentrations, respectively. Again, the absolute oral bioavailability of CA at 100 mg/kg was found to be 37.56 ± 7.3%. The in-vivo pharmacokinetic profile of probe drugs revealed CA to have significant inducing effects on CYP1A2, 2C11, 2D2, and 2E1 after 14 days, which correlates to both in-vitro rat microsomal data and gene expression results. Altogether, pharmacokinetic parameters reveal CA to have an affinity to distribute across different extravascular tissues and induce rat liver CYP enzymes.

Flucloxacillin is a weak inducer of CYP3A4 in healthy adults and 3D spheroid of primary human hepatocytes.

Flucloxacillin is a widely used antibiotic. It is an agonist to the nuclear receptor Pregnane X Receptor (PXR) that regulates the expression of cytochrome P450 (CYP) enzymes. Treatment with flucloxacillin reduces warfarin efficacy and plasma concentrations of tacrolimus, voriconazole, and repaglinide. We conducted a translational study to investigate if flucloxacillin induces CYP enzymes. We also investigated if flucloxacillin induces its own metabolism as an autoinducer. We performed a randomized, unblinded, two-period, cross-over, clinical pharmacokinetic cocktail study. Twelve healthy adults completed the study. They ingested 1 g flucloxacillin three times daily for 31 days, and we assessed the full pharmacokinetics of the Basel cocktail drugs on days 0, 10, and 28, and plasma concentrations of flucloxacillin on days 0, 9, and 27. 3D spheroid of primary human hepatocytes (PHH) were exposed to flucloxacillin (concentration range: 0.15-250 μM) for 96 hours. Induction of mRNA expression, protein abundance, and enzyme activity of CYP enzymes were assessed. Flucloxacillin treatment reduced the metabolic ratio of midazolam (CYP3A4), (geometric mean ratio (GMR) 10 days (95% confidence interval (CI)): 0.75 (0.64-0.89)) and (GMR 28 days (95% CI): 0.72 (0.62-0.85)). Plasma concentrations of flucloxacillin did not change during 27 days of treatment. Flucloxacillin caused concentration-dependent induction of CYP3A4 and CYP2B6 (mRNA, protein, and activity), CYP2C9 (mRNA and protein), CYP2C19 (mRNA and activity), and CYP2D6 (activity) in 3D spheroid PHH. In conclusion, flucloxacillin is a weak inducer of CYP3A4, which may lead to clinically relevant drug-drug interactions for some narrow therapeutic range drugs that are substrates of CYP3A4.

A cocktail probe approach to evaluate the effect of hormones on the expression and activity of CYP enzymes in human hepatocytes with conditions simulating late stage of pregnancy.

Pregnancy-mediated physiological and biochemical changes contribute to alterations in the pharmacokinetics of certain drugs. There is a paucity of data on the systematic evaluation of the underlying mechanisms. The objective of the current study was to examine the impact of changes in circulating and tissue hormonal concentrationduring the late stage of pregnancy on the activity and expression of hepatic cytochrome P450 (CYP) enzymes using a cocktail probe approach. Freshly isolated primary human hepatocytes were incubated with third trimester physiologic (plasma) and projected liver (ten-fold higher) concentrations of female hormones: progesterone (2µM), estradiol (0.3µM), estriol (0.8µM), estrone (0.2µM), 17α-hydroxyprogesterone (0.1µM), and human growth hormone (0.005µM). The metabolic activity of the hepatocytes was assessed using a cocktail of isozyme-specific P450 probe substrates (CYP1A2 (phenacetin), CYP2C9 (diclofenac), CYP2C19 (S-mephenytoin), CYP2D6 (dextromethorphan), and CYP3A4 (testosterone)). A validated LC-MS/MS assay was used to measure the corresponding metabolite concentrations. CYP450 protein and mRNA levels were measured using western blot and qRT-PCR, respectively. Female hormones at projected third-semester hepatic concentrations significantly enhanced mRNA and protein expression and increased the metabolic activity of CYP3A4. The expression and activity of other CYP450 enzymes studied were not affected by mixtures of female hormones at concentrations used. The increased activity of CYP3A4 is consistent with the clinically observed increase in clearance of CYP3A4 substrates during pregnancy. Overall expression and activity of CYP450 isozymes are differentially regulated during pregnancy.

Okadaic Acid Activates JAK/STAT Signaling to Affect Xenobiotic Metabolism in HepaRG Cells.

Okadaic acid (OA) is a marine biotoxin that is produced by algae and accumulates in filter-feeding shellfish, through which it enters the human food chain, leading to diarrheic shellfish poisoning (DSP) after ingestion. Furthermore, additional effects of OA have been observed, such as cytotoxicity. Additionally, a strong downregulation of the expression of xenobiotic-metabolizing enzymes in the liver can be observed. The underlying mechanisms of this, however, remain to be examined. In this study, we investigated a possible underlying mechanism of the downregulation of cytochrome P450 (CYP) enzymes and the nuclear receptors pregnane X receptor (PXR) and retinoid-X-receptor alpha (RXRα) by OA through NF-κB and subsequent JAK/STAT activation in human HepaRG hepatocarcinoma cells. Our data suggest an activation of NF-κB signaling and subsequent expression and release of interleukins, which then activate JAK-dependent signaling and thus STAT3. Moreover, using the NF-κB inhibitors JSH-23 and Methysticin and the JAK inhibitors Decernotinib and Tofacitinib, we were also able to demonstrate a connection between OA-induced NF-κB and JAK signaling and the downregulation of CYP enzymes. Overall, we provide clear evidence that the effect of OA on the expression of CYP enzymes in HepaRG cells is regulated through NF-κB and subsequent JAK signaling.

Protective effect of compatible herbs in Jin-Gu-Lian formula against Alangium chinense-induced neurotoxicity via oxidative stress, neurotransmitter metabolisms, and pharmacokinetics.

Background: A. chinense frequently used in Miao medicine to treat rheumatic diseases. However, as a famous toxic herb, Alangium chinense and its representative components exhibit ineluctable neurotoxicity, thus creating significant challenges for clinical application. The combined application with compatible herbs in Jin-Gu-Lian formula attenuates such neurotoxicity according to the compatible principle of traditional Chinese medicines. Purpose: We aimed to investigate the detoxification of the compatible herbs in Jin-Gu-Lian formula on A. chinense-induced neurotoxicity and investigate its mechanism. Methods: Neurobehavioral and pathohistological analysis were used to determine the neurotoxicity in rats administered with A. chinense extract (AC), extract of compatible herbs in Jin-Gu-Lian formula (CH) and combination of AC with CH for 14days. The mechanism underlying the reduction of toxicity by combination with CH was assessed by enzyme-linked immunosorbent assays, spectrophotometric assays, liquid chromatography tandem-mass spectrometry and real-time reverse transcription-quantitative polymerase chain reaction. Results: Compatible herbs attenuated the AC-induced neurotoxicity as evidenced by increased locomotor activity, enhanced grip strength, the decreased frequency of AC-induced morphological damage in neurons, as well as a reduction of neuron-specific enolase (NSE) and neurofilament light chain (NEFL) levels. The combination of AC and CH ameliorated AC-induced oxidative damage by modulating the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), and total antioxidant capacity (T-AOC). AC treatment significantly reduced the levels of monoamine and acetylcholine neurotransmitters in the brains of rats, including acetylcholine (Ach), dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), norepinephrine (NE), and serotonin (5-HT). Combined AC and CH treatment regulated the abnormal concentrations and metabolisms of neurotransmitters. Pharmacokinetic studies showed that the co-administration of AC and CH significantly decreased plasma exposure levels of two main components of AC, as evidenced by the reduction of maximum plasma concentration (Cmax), area under the plasma concentration-time curve (AUC) compared to AC. In addition, the AC-induced downregulation in mRNA expression of cytochrome P450 enzymes was significantly reduced in response to combined AC and CH treatment. Conclusion: Compatible herbs in Jin-Gu-Lian formula alleviated the neurotoxicity induced by A. chinense by ameliorating oxidative damage, preventing abnormality of neurotransmitters and modulating pharmacokinetics.

Effects of maternal exposure to procymidone on hepatic metabolism in the offspring of mice.

As an effective fungicide widely used in agricultural production, the excessive procymidone (PRO) residue has been detected in the environment and food. Our previous study demonstrated that PRO could destroy the intestinal barrier in mice and has a joint toxic effect. To explore the cross-generational impact of maternal exposure, 10-week-old C57BL/6 female mice were orally administrated to 10 and 100 mg/kg body weight/day of PRO during pregnancy and lactation. The offspring obtained nutrients from the maternal through the placenta and breast milk, and PRO residues were detected in the liver, intestine, and feces of F1 generation. Fecal examination found that the residual PRO had been completely metabolized when the offspring mice grew to 35 days. The drug residue of F1 generation male mice was higher than that of female mice. We attributed this result to the difference in cytochrome P450 (CYP450) enzyme expression between male and female mice. The transcriptional levels of CYP1A1, CYP1A2, CYP2D9, and CYP3A4, and CYP450 protein expression levels, were higher in female mice. Furthermore, targeted MS of plasma revealed abnormal amino acid levels. In addition, PRO-induced hepatic metabolite changes in F0 and F1-7w mice. KEGG pathway analysis further showed that PRO jointly changed the amino acid biosynthesis pathway of the maternal and offspring. In summary, these results indicated that maternal exposure to PRO during a special period would interfere with self metabolism, and offspring will also have metabolic disorders.

Cytochrome P450 metabolism studies of [6]-gingerol, [8]-gingerol, and [10]-gingerol by liver microsomes of humans and different species combined with expressed CYP enzymes.

Gingerols, mainly [6]-gingerol (6G), [8]-gingerol (8G), and [10]-gingerol (10G), are the functional and specific pungent phytochemicals in ginger. However, poor oral bioavailability limits their applications owing to extensive metabolism. The present study aims to characterize the cytochrome P450 (CYP) metabolic characteristics of 6G, 8G, and 10G by using pooled human liver microsomes (HLM), different animal liver microsomes, and the expressed CYP enzymes. It is shown that NADPH-dependent oxidation and hydrogenation metabolisms of gingerols are the main metabolic types in HLM. With the increase of the carbon chain, the polarity of gingerols decreases and the formation of hydrogenated metabolites is more efficient (CLint: 1.41 μL min-1 mg-1 for 6G, 7.79 μL min-1 mg-1 for 8G and 14.11 μL min-1 mg-1 for 10G), indicating that the phase I metabolism of gingerols by HLM varied with the chemical structure of the substrate. The phase I metabolism of gingerols revealed considerable species variations, and compared to HLM, novel metabolites such as (3S,5S)-gingerdiols and demethylated metabolites are generated in some animal liver microsomes. The primary enzymes involved in the oxidized and demethylated metabolism of these gingerols are CYP1A2 and CYP2C19, but their affinities for gingerols are not the same. CYP2D6 and CYP2B6 contributed significantly to the formation of (3R,5S)-[8]-gingerdiol and (3R,5S)-[10]-gingerdiol, respectively; however, the enzyme responsible for the production of (3R,5S)-[6]-gingerediol is yet to be identified. Some metabolites in microsomes cannot be detected by the 12 investigated CYP enzymes, which may be related to the combined effects of multiple enzymes in microsomes, the different affinity of mixed liver microsomes and CYP enzymes, gene polymorphisms, etc. Overall, this work provides a deeper knowledge of the influence of CYP metabolism on the gingerols, as well as the mode of action and the possibility for drug-herbal interactions.

Effects of Gukang Capsules on activity and protein expression of hepatic cytochrome P450 enzymes in rats

Gukang Capsules are often used in combination with drugs to treat fractures, osteoarthritis, and osteoporosis. Cytochrome P450(CYP450) mainly exists in the liver and participates in the oxidative metabolism of a variety of endogenous and exogenous substances and serves as an important cause of drug-metabolic interactions and adverse reactions. Therefore, it is of great significance to study the effect of Gukang Capsules on the activity and expression of CYP450 for increasing its clinical rational medication and improving the safety of drug combination. In this study, the Cocktail probe method was used to detect the changes in the activities of CYP1A2, CYP3A2, CYP2C11, CYP2C19, CYP2D4, and CYP2E1 in rat liver after treatment with high-, medium-and low-dose Gukang Capsules. The rat liver microsomes were extracted by the calcium chloride method, and protein expression of the above six CYP isoform enzymes was detected by Western blot. The results showed that the low-dose Gukang Capsules could induce CYP3A2 and CYP2D4 in rats, medium-dose Gukang Capsules had no effect on them, and high-dose Gukang Capsules could inhibit them in rats. The high-dose Gukang Capsules did not affect CYP2C11 in rats, but low-and medium-dose Gukang Capsules could induce CYP2C11 in rats. Gukang Capsules could inhibit CYP2C19 in rats and induce CYP1A2 in a dose-independent manner, but did not affect CYP2E1. If Gukang Capsules were co-administered with CYP1A2, CYP2C19, CYP3A2, CYP2C11, and CYP2D4 substrates, the dose should be adjusted to avoid drug interactions.

Challenges in the Heterologous Production of Furanocoumarins in Escherichia coli.

Coumarins and furanocoumarins are plant secondary metabolites with known biological activities. As they are present in low amounts in plants, their heterologous production emerged as a more sustainable and efficient approach to plant extraction. Although coumarins biosynthesis has been positively established, furanocoumarin biosynthesis has been far more challenging. This study aims to evaluate if Escherichia coli could be a suitable host for furanocoumarin biosynthesis. The biosynthetic pathway for coumarins biosynthesis in E. coli was effectively constructed, leading to the production of umbelliferone, esculetin and scopoletin (128.7, 17.6, and 15.7 µM, respectively, from tyrosine). However, it was not possible to complete the pathway with the enzymes that ultimately lead to furanocoumarins production. Prenyltransferase, psoralen synthase, and marmesin synthase did not show any activity when expressed in E. coli. Several strategies were tested to improve the enzymes solubility and activity with no success, including removing potential N-terminal transit peptides and expression of cytochrome P450 reductases, chaperones and/or enzymes to increase dimethylallylpyrophosphate availability. Considering the results herein obtained, E. coli does not seem to be an appropriate host to express these enzymes. However, new alternative microbial enzymes may be a suitable option for reconstituting the furanocoumarins pathway in E. coli. Nevertheless, until further microbial enzymes are identified, Saccharomyces cerevisiae may be considered a preferred host as it has already been proven to successfully express some of these plant enzymes.