CYP1A1 Protein Levels Research Articles

Gene expression of MTATP6 and cytochrome P450 in MCF-7 and MDA-MB -231 breast cancer cell lines with juglone and curcumin supplemented

It is aimed to determine the effects of naphthoquinones as juglone and curcumin application on cell viability and expression analyzes of CYP3A4 and MTATP6 genes in MCF-7 and MDA-MB-231 human breast cancer cell lines. MCF-7 and MDA-MB-231 cells were incubated, were replaced with containing various concentrations of 5, 10, 15 μM curcumin and 5, 10, 15 μM juglone for MCF-7 and 1, 5, 10 μM curcumin and 1, 2, 3 μM juglone for MDA-MB-231 for 24 h. CYP3A4 and MTATP6 gene expression levels in both cell lines were determined by quantitative real-time polymerase chain reaction (qPCR) method and western blot method. IC50 values for 24 h were found as 22.41 μM for curcumin, and 16.27 μM for juglone in MCF-7, and 10.43 μM for curcumin, and 3.42 μM for juglone in MDA-MB-231 cells. Curcumin showed anti-proliferative, and antioxidant effects. CYP3A4 and MTATP6 gene expressions were decreased in MCF-7 breast cancer cell line when the cells treated with juglone or curcumin. CYP3A4 and MTATP6 gene expressions were decreased at all application doses of juglone in MDA-MB-231 cells whereas CYP3A4 and MTATP6 protein levels were only decreased at 10 μM curcumin compared with the control group.

Inhibition of CYP1A1 expression enhances diabetic wound healing by modulating inflammation and oxidative stress in a rat model

ObjectiveWound therapies utilizing gene delivery to the skin offer considerable promise owing to their localized treatment benefits and straightforward application. This study investigated the impact of skin electroporation of CYP1A1 shRNA lentiviral particles on diabetic wound healing in a streptozotocin (STZ)-induced rat model. MethodsMale Sprague Dawley (SD) rats were made diabetic by injecting STZ and subsequently creating foot skin wounds. The rats were randomly divided into four groups: normal, diabetic foot ulcers (DFU), DFU + control shRNA (electroporation of control shRNA lentiviral particles), and DFU + CYP1A1 shRNA (electroporation of CYP1A1 shRNA lentiviral particles). Wound healing progress was monitored at multiple time points (0, 1, 3, 5, 7, 10, 14 days). On day 14, wound tissue specimens were collected for histological examination. Wound samples collected at days 7 and 14 were used for gene expression analysis via qRT-PCR, assessment of CYP1A1 protein levels using western blotting, and evaluation of oxidative stress markers. ResultsTreatment with CYP1A1 shRNA significantly enhanced diabetic wound healing rates compared to untreated controls over the observation period. Histological analysis revealed improved wound characteristics in the CYP1A1 shRNA-treated group, including enhanced epithelial regeneration, reduced inflammation, and increased collagen deposition, indicative of improved tissue repair. Furthermore, suppression of CYP1A1 corresponded with decreased expression levels of pro-inflammatory cytokines (interleukin-1β, tumor necrosis factor-α, and interleukin-6) and diminished oxidative stress markers (malondialdehyde, superoxide dismutase) within wound tissues. ConclusionTargeted suppression of CYP1A1 represents a promising therapeutic strategy to enhance diabetic wound healing by modulating inflammation and oxidative stress.

New insights into the regulation of bile acids synthesis during the early stages of liver regeneration: A human and experimental study

Background and aims: Liver regeneration is essential for the preservation of homeostasis and survival. Bile acids (BAs)-mediated signaling is necessary for liver regeneration, but BAs levels need to be carefully controlled to avoid hepatotoxicity. We studied the early response of the BAs-fibroblast growth factor 19 (FGF19) axis in healthy individuals undergoing hepatectomy for living donor liver transplant. We also evaluated BAs synthesis in mice upon partial hepatectomy (PH) and acute inflammation, focusing on the regulation of cytochrome-7A1 (CYP7A1), a key enzyme in BAs synthesis from cholesterol.Methods: Serum was obtained from twelve human liver donors. Mice underwent 2/3-PH or sham-operation. Acute inflammation was induced with bacterial lipopolysaccharide (LPS) in mice fed control or antoxidant-supplemented diets. BAs and 7α-hydroxy-4-cholesten-3-one (C4) levels were measured by HPLC-MS/MS; serum FGF19 by ELISA. Gene expression and protein levels were analyzed by RT-qPCR and western-blot.Results: Serum BAs levels increased after PH. In patients with more pronounced hypercholanemia, FGF19 concentrations transiently rose, while C4 levels (a readout of CYP7A1 activity) dropped 2 h post-resection in all cases. Serum BAs and C4 followed the same pattern in mice 1 h after PH, but C4 levels also dropped in sham-operated and LPS-treated animals, without marked changes in CYP7A1 protein levels. LPS-induced serum C4 decline was attenuated in mice fed an antioxidant-supplemented diet.Conclusions: In human liver regeneration FGF19 upregulation may constitute a protective response from BAs excess during liver regeneration. Our findings suggest the existence of post-translational mechanisms regulating CYP7A1 activity, and therefore BAs synthesis, independent from CYP7A1/Cyp7a1 gene transcription.

Pharmacokinetic evaluation of Sinisan containing vinegar-processed products in depressive rats, a comprehensive perspective of ‘individual herb, herb-pair, and herbal formula’

Ethnopharmacological relevanceAs a classical formula for the treatment of depression, the clinical application of vinegar-processed products of Bupleuri Radix (Bupleurum chinense DC., BR) and Paeoniae Radix Alba (Paeonia lactiflora Pall., PRA) contained in Sinisan (SNS) is still controversial. Aim of the studyThree levels of ‘individual herb, herb-pair, and herbal formula’ were employed to investigate whether and how the processing of main drugs affected the active constituents of pharmacokinetics in SNS, as well as their impacts on the hepatic CYP450 enzyme. Materials and methodsRats were subjected to construct a chronic unpredictable mild stimulation (CUMS) model. A rapid and sensitive ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS/MS) analytical method was developed and validated for simultaneously quantitative evaluation of thirteen potential active compounds of SNS in depressive rat plasma, and successfully applied to a holistic comparison of pharmacokinetics. The differences in pharmacokinetic parameters based on three different forms of drug composition from BR and PRA before and after vinegar-processing were compared. Meanwhile, qRT-PCR and Western Blot were utilized to explore the metabolic activity of three isoforms of CYP450 enzyme scattered in the livers of depressive rats. ResultsThe characteristic pharmacokinetics profiles of thirteen representative constituents in CUMS rats were influenced by vinegar-processing of BR and PRA and/or the compatibility. In detail, there were significant differences in the Cmax, AUC0-24, AUC0-∞, t1/2, and MRT0-24 of most constituents among the three different forms of drug composition from BR and PRA before and after vinegar-processing, with the most obvious changes in six constituents from the adjuvant and mediating guide drugs. And also, the pharmacokinetic parameters of seven constituents from BR and PRA in SNS containing vinegar-processed products obviously changed after compatibility. Additionally, the mRNA and protein levels of CYP1A2, CYP2E1, and CYP3A1 were observed to increase significantly with the processing of BR and PRA and the combination/formulation. ConclusionsIn conclusion, SNS containing vinegar-processed products was more conducive to the absorption of most activated constituents compared to the original formula in vivo. The vinegar-processing of BR and PRA and the compatibility co-contribute to the pharmacokinetic variability of active compounds of SNS in CUMS rats, and the extent of contribution varies among drugs, which might be related to the regulation of the hepatic drug metabolizing enzymes. The finding of the investigation could help to better understand how active compounds metabolized in vivo, which might be helpful for guiding the clinical application of SNS containing vinegar-processed products.

Sex- and lifestyle-related factors are associated with altered hepatic CYP protein levels in people diagnosed with mental disorders.

In this study, we used human postmortem tissue to investigate hepatic protein expression levels of CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 by LC‒MS/MS in a population of people suffering from mental disorders (n=171). We report hepatic protein levels of these six CYP isoforms in 171 individuals in total, and define a focused population dataset of 116 individuals after excluding 55 samples due to low Microsomal Protein Per Gram of Liver (MPPGL) yield. Postmortem decay was most likely the reason for the low MPPGL yield in the 55 samples. In the focused population, we found women to have significantly higher protein levels of CYP3A4 than men in addition to decreased CYP3A4 protein levels among obese individuals. Furthermore, MPPGL was negatively correlated with body mass index (BMI). An increase in CYP1A2 protein levels was observed among smokers and increased CYP2E1 protein levels were observed among individuals with a history of alcohol abuse. Finally, individuals who received phenobarbital (CYP3A4 inducer) had significantly higher CYP3A4 levels. In conclusion, lifestyle-related factors prevalent among people suffering from mental disorders are associated with altered CYP protein levels which may alter drug metabolism and affect the efficacy of commonly prescribed drugs. Furthermore, this investigation demonstrates that postmortem hepatic tissue can be used to study how lifestyle and effectors affect hepatic CYP-levels in a large cohort of patients. Significance Statement Using a large number of postmortem hepatic tissue specimens (n=116) originating from the autopsy of individuals diagnosed with mental disorders, we were able to show that hepatic CYP-levels were affected by alcohol, smoking, BMI, and sex and that MPPGL was affected by BMI. Theses lifestyle-related changes may alter drug metabolism and affect the efficacy of commonly prescribed drugs. It is a novel approach to use a large postmortem cohort to investigate how lifestyle and effectors affect hepatic CYP-levels.

Investigating the Contribution of Major Drug-Metabolising Enzymes to Possum-Specific Fertility Control.

The potential to improve the effectiveness and efficiency of potential oestrogen-based oral contraceptives (fertility control) for possums was investigated by comparing the inhibitory potential of hepatic CYP3A and UGT2B catalytic activity using a selected compound library (CYP450 inhibitor-based compounds) in possums to that of three other species (mouse, avian, and human). The results showed higher CYP3A protein levels in possum liver microsomes compared to other test species (up to a 4-fold difference). Moreover, possum liver microsomes had significantly higher basal p-nitrophenol glucuronidation activity than other test species (up to an 8-fold difference). However, no CYP450 inhibitor-based compounds significantly decreased the catalytic activity of possum CYP3A and UGT2B below the estimated IC50 and 2-fold IC50 values and were therefore not considered to be potent inhibitors of these enzymes. However, compounds such as isosilybin (65%), ketoconazole (72%), and fluconazole (74%) showed reduced UGT2B glucuronidation activity in possums, mainly at 2-fold IC50 values compared to the control (p < 0.05). Given the structural features of these compounds, these results could provide opportunities for future compound screening. More importantly, however, this study provided preliminary evidence that the basal activity and protein content of two major drug-metabolising enzymes differ in possums compared to other test species, suggesting that this could be further exploited to reach the ultimate goal: a potential target-specific fertility control for possums in New Zealand.

Ginsenoside Rb1 for overcoming cisplatin-insensitivity of A549/DDP cells in vitro and vivo through the dual-inhibition on two efflux pumps of ABCB1 and PTCH1

BackgroundThe multi-drug resistance is an inherent weakness in the chemotherapeutics of non-small cell lung cancer occurring frequently all over the world. Clinically, ginseng and Chinese medicinal prescriptions including ginseng usually used as anti-tumor adjuncts due to its characteristic of qi-invigorating, which could improve the curative effect of chemotherapy drugs and reduce their toxic side effects. Triterpenoid saponins are the crucial active ingredients in Panax ginseng, and Ginsenoside Rb1 is of the highest quantities. However, the research on the tumor drug-resistance reversal effect and mechanism of ginsenoside Rb1 is still not clear. PurposeThis study aimed to systematically estimate the reversal activity of Ginsenoside Rb1 on cisplatin-insensitivity of A549/DDP cells and to reveal its prospective molecular mechanism. MethodsMTT assay were conducted to evaluate the reversal activity on cisplatin-insensitivity of A549/DDP cells of Ginsenoside Rb1in vitro, and the behavior was also studied by establishing a subcutaneous transplanted tumor model of A549/DDP in BALB/c-nu mice. In addition, P-gp ATPase activity assay, cisplatin accumulation assay, Annexin V-FITC apoptosis assay, real-time qPCR analysis and western blotting analysis were used to clarify the potential mechanism. ResultsGinsenoside Rb1 could effectively reverse the cisplatin-resistance of A549/DDP in vitro and vivo. And after the co-treatment of Ginsenoside Rb1 plus cisplatin, the accumulation of cisplatin increased in A549/DDP cells, which was accompanied with the down-regulation of the mRNA and protein expression levels of ABCB1, SHH, PTCH1 and GLI2. Besides, the apoptosis-inducing ability of cisplatin improved by the relative regulation on the protein expression level of Bax and Bcl-2. Far more importantly, the changes of CYP3A4 mRNA and protein levels were not significant. ConclusionGinsenoside Rb1 could increase the concentration of intracellular cisplatin and improve the insensitivity for cisplatin on A549/DDP cells. Even better, there was perhaps no unpredictable CYP3A4-mediated pharmacokinetic interactions after the combination of Ginsenoside Rb1 plus cisplatin. Ginsenoside Rb1 was a probable reversal agent for the cisplatin-insensitivity of A549/DDP cells, with a bifunction of inhibiting the efflux of two drug pumps (P-gp and PTCH1) by targeting ABCB1 and Hedgehog (Hh) pathway. In general, this research laid the groundwork for the development of a new reversal agent for the cisplatin-insensitivity of NSCLC.

Control of Arterial Hypertension by the AhR Blocker CH-223191: A Chronopharmacological Study in Chronic Intermittent Hypoxia Conditions.

Chronic intermittent hypoxia (CIH) is a major contributor to the development of hypertension (HTN) in obstructive sleep apnea (OSA). OSA subjects frequently display a non-dipping pattern of blood pressure (BP) and resistant HTN. After discovering that AHR-CYP1A1 axis is a druggable target in CIH-HTN, we hypothesized that CH-223191 could control BP in both active and inactive periods of the animals, recovering the BP dipping profile in CIH conditions.We evaluated the chronopharmacology of the antihypertensive efficacy of the AhR blocker CH-223191 in CIH conditions (21% to 5% of O2, 5.6cycles/h, 10.5h/day, in inactive period of Wistar rats). BP was measured by radiotelemetry, at 8am (active phase) and at 6pm (inactive phase) of the animals. The circadian variation of AhR activation in the kidney in normoxia was also assessed, measuring the CYP1A1 (hallmark of AhR activation) protein levels.Despite drug administration before starting the inactive period of the animals, CH-223191 was not able to decrease BP during the inactive phase, in CIH conditions, therefore not reverting the non-dipping profile. These results suggest that a higher dose or different time of administration of CH-223191 might be needed for an antihypertensive effect throughout the 24-h cycle.

Focal adhesion alterations in G0-positive melanoma cells.

Melanoma is a highly heterogeneous malignant tumor that exhibits various forms of drug resistance. Recently, reversal transition of cancer cells to the G0 phase of the cell cycle under the influence of therapeutic drugs has been identified as an event associated with tumor dissemination. In the present study, we investigated the ability of chemotherapeutic agent dacarbazine to induce a transition of melanoma cells to the G0 phase as a mechanism of chemoresistance. We used the flow cytometry to analyze cell distribution within cell cycle phases after dacarbazine treatment as well as to identifyG0 -positive cells population. Transcriptome profiling was provided to determine genes associated with dacarbazine resistance. We evaluated the activity of β-galactosidase in cells treated with dacarbazine by substrate hydrolysis. Cell adhesion strength was measured by centrifugal assay application with subsequent staining of adhesive cells with Ki-67 monoclonal antibodies. Ability of melanoma cells to metabolize dacarbazine was determined by expressional analysis of CYP1A1, CYP1A2, CYP2E1 followed by CYP1A1 protein level evaluation by the ELISA method. The present study determined that dacarbazine treatment of melanoma cells could induce an increase in the percentage of cells in G0 phase without alterations of β-galactosidase positive cells which corresponded to the fraction of the senescent cells. Transcriptomic profiling of cells under dacarbazine induction of G0 -positive cells percentage revealed that 'VEGFA-VEGFR2 signaling pathway' and 'Cell cycle' signaling were mostly enriched by dysregulated genes. 'Focal adhesion' signaling was also found to be triggered by dacarbazine. In melanoma cells treated with dacarbazine, an increase in G0 -positive cells among adherent cells was found. Dacarbazine induces the alteration in a percentage of melanoma cells residing in G0 phase of a cell cycle. The altered adhesive phenotype of cancer cells under transition in the G0 phase may refer to a specific intercellular communication pattern of quiescent/senescent cancer cells.

Artemisia extracts differ from artemisinin effects on human hepatic CYP450s 2B6 and 3A4 in vitro

Ethnopharmacological relevanceThe Chinese medicinal herb, Artemisia annua L., has been used for >2,000 yr as traditional tea infusions to treat a variety of infectious diseases including malaria, and its use is spreading globally (along with A. afra Jacq. ex Willd.) mainly through grassroots efforts. Aim of the studyArtemisinin is more bioavailable delivered from the plant, Artemisia annua L. than the pure drug, but little is known about how delivery via a hot water infusion (tea) alters induction of hepatic CYP2B6 and CYP3A4 that metabolize artemisinin. Materials and methodsHepaRG cells were treated with 10 μM artemisinin or rifampicin (positive control), and teas (10 g/L) of A. annua SAM, and A. afra SEN and MAL with 1.6, 0.05 and 0 mg/g DW artemisinin in the leaves, respectively; qPCR and Western blots were used to measure CYP2B6 and CYP3A4 responses. Enzymatic activity of these P450s was measured using human liver microsomes and P450-Glo assays. ResultsAll teas inhibited activity of CYP2B6 and CYP3A4. Artemisinin and the high artemisinin-containing tea infusion (SAM) induced CYP2B6 and CYP3A4 transcription, but artemisinin-deficient teas, MAL and SEN, did not. Artemisinin increased CYP2B6 and CYP3A4 protein levels, but none of the three teas did, indicating a post-transcription inhibition by all three teas. ConclusionsThis study showed that Artemisia teas inhibit activity and artemisinin autoinduction of CYP2B6 and CYP3A4 post transcription, a response likely the effect of other phytochemicals in these teas. Results are important for understanding Artemisia tea posology.

Impact of Interindividual Variability in CYP3A Activity on Ibrutinib and Venetoclax Metabolism In Vitro

Ibrutinib and venetoclax are orally administered active agents approved for the treatment of chronic lymphocytic leukemia (CLL), the most common form of adult leukemia in the Western world. Ibrutinib is an irreversible inhibitor of Bruton’s tyrosine kinase (BTK), whereas venetoclax is a B‐cell lymphoma 2 (BCL2) inhibitor. Although these drugs are effective in CLL treatment, managing life‐threatening side effects remains a clinical challenge. Previous studies have shown that ibrutinib and venetoclax are primarily metabolized by cytochrome P450 (CYP) 3A4. However, the influence of interindividual variation in CYP3A activity on the metabolism of ibrutinib and venetoclax has not been addressed. The objectives of this study were to 1) examine ibrutinib and venetoclax metabolism in single‐donor human liver microsomes and cryopreserved human hepatocytes, and 2) assess the correlation between metabolite formation and CYP3A activity markers. Ibrutinib (5 μM) or venetoclax (2.5 μM) were incubated with human liver microsomes from 20 individual donors. Relative levels of ibrutinib metabolites (PCI45227 and PCI45752) and venetoclax metabolites (M5, M4, and M3) were measured by LC‐MS/MS analysis. Formation of metabolites varied widely between the donors. Metabolite formation was highly correlated with CYP3A activity, as measured by midazolam 1’‐hydroxylation. Pearson correlation coefficients (r) for PCI45227, PCI45752, M5, M4, and M3 were 0.97, 0.85, 0.92, 0.93, and 0.95, respectively. These findings indicate that CYP3A phenotype may have a significant influence on the pharmacokinetics of ibrutinib and venetoclax in CLL treatment. Future studies will be conducted in human hepatocytes to evaluate the effect of CYP3A activity and protein levels on ibrutinib and venetoclax metabolism.

The Selective NMDA Receptor GluN2B Subunit Antagonist CP-101,606 with Antidepressant Properties Modulates Cytochrome P450 Expression in the Liver.

Recent research indicates that selective NMDA receptor GluN2B subunit antagonists may become useful for the treatment of major depressive disorders. We aimed to examine in parallel the effect of the selective NMDA receptor GluN2B subunit antagonist CP-101,606 on the pituitary/serum hormone levels and on the regulation of cytochrome P450 in rat liver. CP-101,606 (20 mg/kg ip. for 5 days) decreased the activity of CYP1A, CYP2A, CYP2B, CYP2C11 and CYP3A, but not that of CYP2C6. The alterations in enzymatic activity were accompanied by changes in the CYP protein and mRNA levels. In parallel, a decrease in the pituitary growth hormone-releasing hormone, and in serum growth hormone and corticosterone (but not T3 and T4) concentration was observed. After a 3-week administration period of CP-101,606 less changes were found. A decrease in the CYP3A enzyme activity and protein level was still maintained, though no change in the mRNA level was found. A slight decrease in the serum concentration of corticosterone was also maintained, while GH level returned to the control value. The obtained results imply engagement of the glutamatergic system in the neuroendocrine regulation of cytochrome P450 and potential involvement of drugs acting on NMDA receptors in metabolic drug–drug interactions.

119 Xenobiotic metabolism is triggered in atopic dermatitis

Atopic dermatitis (AD) is a chronic inflammatory skin disease whose detailed pathogenesis remains elusive despite a clear involvement of epidermal barrier impairment and immune dysregulation. Xenobiotic metabolism is an important detoxification mechanism utilized by organs, including skin, to eliminate noxious exogenous and endogenous compounds and includes phase I and phase II enzymes as well as membrane transporters. Transcriptional control of xenobiotic metabolism is mediated via aryl hydrocarbon receptor (AhR), pregnane X receptor (PXR), constitutive androstane receptor (CAR) and peroxisome proliferator-activated receptors (PPARs). Previous work from our laboratory suggested that AD patients might have increased xenobiotic metabolism in the epidermis. Furthermore, transgenic mice overexpressing the constitutively activated human PXR displayed a Th2/Th17 skin inflammation and barrier dysfunction resembling AD. The goal of the current study is to decipher the role of xenobiotic metabolism in AD pathogenesis. Results show an increased expression of phase I and II enzymes, such as CYP1A1, CYP1B1 and CYP3A4, UGT1A1, UGT1A6, UGT1A9 and UGT1A10 in AD human epidermal equivalents (HEEs) when compared to control (CTL) HEEs. This is in line with the upregulation of upstream receptors namely AHR, PPARA and PPARG. In contrast, the transcription of membrane transporters (ABCC1, ABCC2, ABCB1) is downregulated. Immunofluorescence staining confirmed increased CYP1A1, CYP3A and UGT1A1 protein levels in AD HEEs. These results suggest a constitutive activation of the xenobiotic metabolism in AD, which can cause oxidative stress and inflammation. Accordingly, SOD2 is upregulated in AD HEEs when compared to CTL HEEs, indicating mitochondrial stress. Thus, upregulation of xenobiotic metabolism in nonlesional AD might contribute to disease pathogenesis.

Aryl Hydrocarbon Receptor and Cysteine Redox Dynamics Underlie (Mal)adaptive Mechanisms to Chronic Intermittent Hypoxia in Kidney Cortex.

We hypothesized that an interplay between aryl hydrocarbon receptor (AhR) and cysteine-related thiolome at the kidney cortex underlies the mechanisms of (mal)adaptation to chronic intermittent hypoxia (CIH), promoting arterial hypertension (HTN). Using a rat model of CIH-HTN, we investigated the impact of short-term (1 and 7 days), mid-term (14 and 21 days, pre-HTN), and long-term intermittent hypoxia (IH) (up to 60 days, established HTN) on CYP1A1 protein level (a sensitive hallmark of AhR activation) and cysteine-related thiol pools. We found that acute and chronic IH had opposite effects on CYP1A1 and the thiolome. While short-term IH decreased CYP1A1 and increased protein-S-thiolation, long-term IH increased CYP1A1 and free oxidized cysteine. In addition, an in vitro administration of cystine, but not cysteine, to human endothelial cells increased Cyp1a1 expression, supporting cystine as a putative AhR activator. This study supports CYP1A1 as a biomarker of obstructive sleep apnea (OSA) severity and oxidized pools of cysteine as risk indicator of OSA-HTN. This work contributes to a better understanding of the mechanisms underlying the phenotype of OSA-HTN, mimicked by this model, which is in line with precision medicine challenges in OSA.

Stability investigations of cytochrome P450 (CYP) enzymes immediately after death in a pig model support the applicability of postmortem hepatic CYP quantification.

Quantification of drug‐metabolizing cytochrome P450 (CYP) isoforms using LC–MS/MS has been proposed as a potential way of estimating antemortem CYP levels using postmortem tissue, but the postmortem stability of CYP proteins is incompletely investigated. If one can use data obtained from the analysis of postmortem specimens to inform physiologically based pharmacokinetic (PBPK) models this greatly increases the access to rare specimens among special subpopulations. In this study, we developed and validated an LC–MS/MS method for targeted CYP protein quantification in a porcine animal model to study postmortem stability. We measured 19.9–28.3 pmol CYP1A2, 50.3–66.2 pmol CYP2D25, 132.9–142.7 pmol CYP2E1, and 16.8–48 pmol CYP3A29 protein per mg PLM in nondegraded tissue. In tissue stored at 4°C, we found that the CYP protein levels were unaffected by degradation after 72 h. At 21°C CYP1A2, CYP2D25, and CYP2E1 protein levels were nearly unaffected by degradation after 24 h, whereas a loss of approximately 50% was seen after 48 h. At 21°C CYP3A29 had a loss of 50% at 24 h and 70% at 48 h exhibiting less postmortem stability. In vitro enzyme activity measurements in the same tissue stored at 21°C showed a 50% decrease after 24 h and a complete loss of enzyme activity after 48 h. When stored at 4°C, the in vitro enzyme activity decreased to 50% activity after 96 h. In conclusion, measuring CYP levels by an LC–MS/MS approach was clearly less affected by postmortem changes than an activity‐based approach. The found postmortem stability for 24 h at 21°C for 3 out of 4 CYP isoforms supports the use of properly stored postmortem tissue to inform PBPK models.

The Influence of Long-Term Treatment with Asenapine on Liver Cytochrome P450 Expression and Activity in the Rat. The Involvement of Different Mechanisms.

Therapy of schizophrenia requires long-term treatment with a relevant antipsychotic drug to achieve a therapeutic effect. The aim of the present study was to investigate the influence of prolonged treatment with the atypical neuroleptic asenapine on the expression and activity of rat cytochrome P450 (CYP) in the liver. The experiment was carried out on male Wistar rats. Asenapine (0.3 mg/kg s.c.) was administered for two weeks. The levels of CYP mRNA protein and activity were determined in the liver and hormone concentrations were measured in the pituitary gland and blood serum. Asenapine significantly decreased the activity of CYP1A (caffeine 8-hydroxylation and 3-N-demethylation), CYP2B, CYP2C11 and CYP3A (testosterone hydroxylation at positions 16β; 2α and 16α; 2β and 6β, respectively). The neuroleptic did not affect the activity of CYP2A (testosterone 7α-hydroxylation), CYP2C6 (warfarin 7-hydroxylation) and CYP2E1 (chlorzoxazone 6-hydroxylation). The mRNA and protein levels of CYP1A2, CYP2B1, CYP2C11 and CYP3A1 were decreased, while those of CYP2B2 and CYP3A2 were not changed. Simultaneously, pituitary level of growth hormone-releasing hormone and serum concentrations of growth hormone and corticosterone were reduced, while that of triiodothyronine was enhanced. In conclusion, chronic treatment with asenapine down-regulates liver cytochrome P450 enzymes, which involves neuroendocrine mechanisms. Thus, chronic asenapine treatment may slow the metabolism of CYP1A, CYP2B, CYP2C11 and CYP3A substrates (steroids and drugs). Since asenapine is metabolized by CYP1A and CYP3A, the neuroleptic may inhibit its own metabolism, therefore, the plasma concentration of asenapine in patients after prolonged treatment may be higher than expected based on a single dose.

Saikosaponins and the deglycosylated metabolites exert liver meridian guiding effect through PXR/CYP3A4 inhibition

Ethnopharmacological relevanceRadix Bupleuri (RB), traditionally used to treat inflammatory disorders and infectious diseases, represents one of the most successful and widely used herbal drugs in Asia over the past 2000 years. Being realized the role in regulating metabolism and controlling Yin/Yang, RB is not only chosen specifically for treating liver meridian and the corresponding organs, but also believed to have liver meridian guiding property and help potentiate the therapeutic effects of liver. However, the ingredients in RB with liver meridian guiding property and the underly mechanism have not been comprehensively investigated. Aim of studyConsidering the important role of CYP3A4 in first-pass metabolism and the liver exposure of drugs, the present study aimed to determine whether saikosaponins (SSs) and the corresponding saikogenins (SGs) have a role in inhibiting the catalytic activity of CYP3A4 in human liver microsomes and HepG2 hepatoma cells and whether they could suppress CYP3A4 expression by PXR-mediated pathways in HepG2 hepatoma cells. Materials and methodsThe effect of SSs and SGs on CYP3A4-mediated midazolam1′-hydroxylation activities in pooled human liver microsomes (HLMs) was first studied. Dose-dependent experiments were performed to obtain the half inhibit concentration (IC50) values. HepG2 cells were used to assay catalytic activity of CYP3A4, reporter function, mRNA levels, and protein expression. The inhibitory effects of SSa and SSd on CYP3A4 activity are negligible, while the corresponding SGs (SGF and SGG) have obvious inhibitory effects on CYP3A4 activity, with IC50 values of 0.45 and 1.30 μM. The similar results were obtained from testing CYP3A4 catalytic activity in HepG2 cells, which correlated well with the suppression of the mRNA and protein levels of CYP3A4. Time-dependent testing of CYP3A4 mRNA and protein levels, as well as co-transfection experiments using the CYP3A4 promoter luciferase plasmid, further confirmed that SSs and SGs could inhibit the expression of CYP3A4 at the transcription level. Furthermore, PXR protein expression decreased in a concentration- and time-dependent manner after cells were exposed to SSs and SGs. PXR overexpression and RNA interference experiments further showed that SSs and SGs down-regulate the catalytic activity and expression of CYP3A4 in HepG2 may be mainly through PXR-dependent manner. ConclusionSSs and SGs inhibit the catalytic activity and expression of CYP3A4 in a PXR-dependent manner, which may be highly related to the liver meridian guiding property of RB.

Effects of Shengmai San on key enzymes involved in hepatic and intestinal drug metabolism in rats

Ethnopharmacological relevanceShengmai San (SMS) has been commonly used as a traditional Chinese medicine for the treatment of cardiovascular disorders, of which drug interactions need to be assessed for the safety concern. There is little evidence for the alterations of hepatic and intestinal drug-metabolizing enzymes after repeated SMS treatments to assess drug interactions. Aim of the studyThe studies aim to illustrate the effects of repeated treatments with SMS on cytochrome P450s (CYPs), reduced nicotinamide adenine dinucleotide (phosphate)-quinone oxidoreductase (NQO), uridine diphosphate-glucuronosyltransferase (UGT), and glutathione S-transferase (GST) using in vivo rat model. Materials and methodsThe SMS was prepared using Schisandrae Fructus, Ginseng Radix, and Ophiopogonis Radix (OR) (1:2:2). Chromatographic analyses of decoctions were performed using ultra-performance liquid chromatography (UPLC) and LC-mass spectrometry. Sprague-Dawley rats were orally treated with the SMS and its component herbal decoctions for 2 or 3 weeks. Hepatic and intestinal enzyme activities were determined. CYP3A expression and the kinetics of intestinal nifedipine oxidation (NFO, a CYP3A marker reaction) were determined. ResultsSchisandrol A, schisandrin B, ginsenoside Rb1 and ophiopogonin D were identified in SMS. SMS selectively suppressed intestinal, but not hepatic, NFO activity in a dose- and time-dependent manner. Hepatic and intestinal UGT, NQO and GST activities were not affected. A 3-week SMS treatment decreased the maximal velocity of intestinal NFO by 50%, while the CYP3A protein level remained unchanged. Among SMS component herbs, the decoction of OR decreased intestinal NFO activity. ConclusionsThese findings demonstrate that 3-week treatment with SMS and OR suppress intestinal, but not hepatic CYP3A function. It suggested that the potential interactions of SMS with CYP 3A drug substrates should be noticed, especially the drugs whose bioavailability depends heavily on intestinal CYP3A.

Stress can attenuate hepatic lipid accumulation via elevation of hepatic β-muricholic acid levels in mice with nonalcoholic steatohepatitis

AbstractStress can affect our body and is known to lead to some diseases. However, the influence on the development of nonalcohol fatty liver disease (NAFLD) remains unknown. This study demonstrated that chronic restraint stress attenuated hepatic lipid accumulation via elevation of hepatic β-muricholic acid (βMCA) levels in the development of nonalcoholic steatohepatitis (NASH) in mice. Serum cortisol and corticosterone levels, i.e., human and rodent stress markers, were correlated with serum bile acid levels in patients with NAFLD and methionine- and choline-deficient (MCD) diet-induced mice, respectively, suggesting that stress is related to bile acid (BA) homeostasis in NASH. In the mouse model, hepatic βMCA and cholic acid (CA) levels were increased after the stress challenge. Considering that a short stress enhanced hepatic CYP7A1 protein levels in normal mice and corticosterone increased CYP7A1 protein levels in primary mouse hepatocytes, the enhanced Cyp7a1 expression was postulated to be involved in the chronic stress-increased hepatic βMCA level. Interestingly, chronic stress decreased hepatic lipid levels in MCD-induced NASH mice. Furthermore, βMCA suppressed lipid accumulation in mouse primary hepatocytes exposed to palmitic acid/oleic acid, but CA did not. In addition, Cyp7a1 expression seemed to be related to lipid accumulation in hepatocytes. In conclusion, chronic stress can change hepatic lipid accumulation in NASH mice, disrupting BA homeostasis via induction of hepatic Cyp7a1 expression. This study discovered a new βMCA action in the liver, indicating the possibility that βMCA is available for NAFLD therapy.Chronic stress can change hepatic lipid accumulation via elevation of hepatic β-muricholic acid levels in nonalcoholic steatohepatitis mice, disrupting bile acid homeostasis by induction of hepatic Cyp7a1 expression. This study describes a new role βMCA in the liver, indicating its potential usefulness for nonalcohol fatty liver disease therapy.

Pregnancy-Related Hormones Increase Nifedipine Metabolism in Human Hepatocytes by Inducing CYP3A4 Expression

Pregnancy-related hormones (PRH) have emerged as key regulators of hepatic cytochrome P450 (CYP) enzyme expression and function. The impact of PRH on protein levels of CYP3A4 and other key CYP enzymes, and the metabolism of nifedipine (a CYP3A4 substrate commonly prescribed during pregnancy), was evaluated in primary human hepatocytes. Sandwich-cultured human hepatocytes (SCHH) from female donors were exposed to PRH (estradiol, estriol, estetrol, progesterone, and cortisol), individually or in combination as a cocktail. Absolute protein concentrations of twelve CYP isoforms in SCHH membrane fractions were quantified by nanoLC-MS/MS, and metabolism of nifedipine to dehydronifedipine in SCHH was evaluated. PRH significantly increased CYP3A4 protein concentrations and nifedipine metabolism to dehydronifedipine in a concentration-dependent manner. CYP3A4 mRNA levels in hepatocyte-derived exosomes positively correlated with CYP3A4 protein levels and dehydronifedipine formation in SCHH. PRH also increased CYP2B6, CYP2C8 and CYP2A6 levels. Our findings demonstrate that PRH increase nifedipine metabolism in SCHH by inducing CYP3A4 expression and alter expression of other key CYP proteins in an isoform-specific manner, and suggest that hepatocyte-derived exosomes warrant further investigation as biomarkers of hepatic CYP3A4 metabolism. Together, these results offer mechanistic insight into the increases in nifedipine metabolism and clearance observed in pregnant women.