Habitual coffee consumption and risk of dementia in older persons: modulation by CYP1A2 polymorphism.

Higher coffee consumption has been associated with reduced dementia risk, yet with inconsistencies across studies. CYP1A2 polymorphisms, which affects caffeine metabolism, may modulate the association between coffee and the risk of dementia and Alzheimer's disease (AD). We included 5964 participants of the Three-City Study (mean age 74 years-old), free of dementia at baseline when they reported their daily coffee consumption, with available genome-wide genotyping and followed for dementia over a median of 9.0 (range 0.8-18.7) years. In Cox proportional-hazards models, the relationship between coffee consumption and dementia risk was modified by CYP1A2 polymorphism at rs762551 (p for interaction = 0.034). In multivariable-adjusted models, coffee intake was linearly associated with a decreased risk of dementia among carriers of the C allele only ("slower caffeine metabolizers"; HR for 1-cup increased [95% CI] 0.90 [0.83-0.97]), while in non-carriers ("faster caffeine metabolizers"), there was no significant association but a J-shaped trend toward a decrease in dementia risk up to 3 cups/day and increased risk beyond. Thus, compared to null intake, drinking ≥ 4 cups of coffee daily was associated with a reduced dementia risk in slower but not faster metabolizers (HR [95% CI] for ≥ 4 vs. 0 cup/day = 0.45 [0.25-0.80] and 1.32 [0.89-1.96], respectively). Results were similar when studying AD and another CYP1A2 candidate polymorphism (rs2472304), but no interaction was found with CYP1A2 rs2472297 or rs2470893. In this cohort, a linear association of coffee intake to lower dementia risk was apparent only among carriers of CYP1A2 polymorphisms predisposing to slower caffeine metabolism.

Dihydrotanshinone I-Induced CYP1 Enzyme Inhibition and Alteration of Estradiol Metabolism.

Dihydrotanshinone I (DHTI) is a pharmacologically active component occurring in the roots of the herbal medicine Salvia miltiorrhiza Bunge. This study investigated DHTI-induced inhibition of CYP1A1, CYP1A2, and CYP1B1 with the aim to determine the potential effects of DHTI on the bioactivation of estradiol (E2), possibly related to preventive/therapeutic strategy for E2-associated breast cancer. Ethoxyresorufin as a specific substrate for CYP1s was incubated with human recombinant CYP1A1, CYP1A2, or CYP1B1 in the presence of DHTI at various concentrations. Enzymatic inhibition and kinetic behaviors were examined by monitoring the formation of the corresponding product. Molecular docking was further conducted to define the interactions between DHTI and the three CYP1s. The same method and procedure were employed to examine the DHTI-induced alteration of E2 metabolism. DHTI showed significant inhibition of ethoxyresorufin O-deethylation activity catalyzed by CYP1A1, CYP1A2 and CYP1B1 in a concentration-dependent manner (IC50 = 0.56, 0.44, and 0.11 µM, respectively). Kinetic analysis showed that DHTI acted as a competitive type of inhibitor of CYP1A1 and CYP1B1, whereas it noncompetitively inhibited CYP1A2. The observed enzyme inhibition was independent of NADPH and time. Molecular docking analysis revealed hydrogen bonding interactions between DHTI and Asp-326 of CYP1B1. Moreover, DHTI displayed preferential activity to inhibit 4-hydroxylation of E2 (a genotoxic pathway) mediated by CYP1B1. Exposure to DHTI could reduce the risk of genotoxicity induced by E2. SIGNIFICANCE STATEMENT: CYP1A1, CYP1A2, and CYP1B1 enzymes are involved in the conversion of estradiol (E2) into 2-hydroxyestradiol (2-OHE2) and 4-hydroxyestradiol (4-OHE2) through oxidation. 2-OHE2 is negatively correlated with breast cancer risk, and 4-OHE2 may be a significant initiator and promoter of breast cancer. The present study revealed that dihydrotanshinone I (DHTI) competitively inhibits CYP1A1/CYP1B1 and noncompetitively inhibits CYP1A2. DHTI exhibits a preference for inhibiting the genotoxicity associated with E2 4-hydroxylation pathway mediated by CYP1B1, potentially reducing the risk of 4-OHE2-induced genotoxicity.

Role of circadian clock in the chronoefficacy and chronotoxicity of clopidogrel.

BACKGROUND AND PURPOSE: The role of circadian locomotor output cycles kaput (CLOCK) in regulating drug chronoefficacy and chronotoxicity remains elusive. Here, we aimed to uncover the impact of CLOCK and dosing time on clopidogrel efficacy and toxicity. EXPERIMENTAL APPROACH: The antiplatelet effect, toxicity and pharmacokinetics experiments were conducted with Clock-/- mice and wild-type mice, after gavage administration of clopidogrel at different circadian time points. The expression levels of drug-metabolizing enzymes were determined by quantitative polymerase chain reaction (qPCR) and western blotting. Transcriptional gene regulation was investigated using luciferase reporter and chromatin immunoprecipitation assays. KEY RESULTS: The antiplatelet effect and toxicity of clopidogrel in wild-type mice showed a dosing time-dependent variation. Clock ablation reduced the antiplatelet effect of clopidogrel, but increased clopidogrel-induced hepatotoxicity, with attenuated rhythms of clopidogrel active metabolite (Clop-AM) and clopidogrel, respectively. We found that Clock regulated the diurnal variation of Clop-AM formation by modulating the rhythmic expression of CYP1A2 and CYP3A1, and altered clopidogrel chronopharmacokinetics by regulation of CES1D expression. Mechanistic studies revealed that CLOCK activated Cyp1a2 and Ces1d transcription by directly binding to the enhancer box (E-box) elements in their promoters, and promoted Cyp3a11 transcription through enhancing the transactivation activity of albumin D-site-binding protein (DBP) and thyrotroph embryonic factor (TEF). CONCLUSIONS AND IMPLICATIONS: CLOCK regulates the diurnal rhythmicity in clopidogrel efficacy and toxicity through regulation of CYP1A2, CYP3A11 and CES1D expression. These findings may contribute to optimizing dosing schedules for clopidogrel and may deepen understanding of the circadian clock and chronopharmacology.

Selective inhibitory effects of suberosin on CYP1A2 in human liver microsomes.

Suberosin is a natural phytoconstituent isolated from Citropsis articulata, especially employed for its anticoagulant properties. Although metabolic studies assessing suberosin have been conducted, it is possible interactions with drugs and food have not yet been investigated. In the present study, we analyzed the selective inhibitory effects of suberosin on cytochrome P450 (CYP) enzymes using a cocktail probe assay. Various concentrations of suberosin (0-50 µM) were incubated with isoform-specific CYP probes in human liver microsomes (HLMs). We found that suberosin significantly inhibited CYP1A2-catalyzed phenacetin O-deethylation, exhibiting IC50 values of 9.39 ± 2.05 and 3.07 ± 0.45 µM with and without preincubation in the presence of ß-NADPH, respectively. Moreover, suberosin showed concentration-dependent, but not time-dependent, CYP1A2 inhibition in HLMs, indicating that suberosin acts as a substrate and reversible CYP1A2 inhibitor. Using a Lineweaver-Burk plot, we found that suberosin competitively inhibited CYP1A2-catalyzed phenacetin O-deethylation. Furthermore, suberosin showed similar inhibitory effects on recombinant human CYP1A1 and 1A2. In conclusion, suberosin may elicit herb-drug interactions by selectively inhibiting CYP1A2 during the concurrent administration of drugs that act as CYP1A2 substrates.

Yeast polysaccharide mitigated oxidative injury in broilers induced by mixed mycotoxins via regulating intestinal mucosal oxidative stress and hepatic metabolic enzymes.

This study was aimed to investigate the effects of yeast polysaccharides (YPS) on growth performance, intestinal health, and aflatoxin metabolism in livers of broilers fed diets naturally contaminated with mixed mycotoxins (MYCO). A total of 480 one-day-old Arbor Acre male broilers were randomly allocated into a 2 × 3 factorial arrangement of treatments (8 replicates with 10 birds per replicate) for 6 wk to assess the effects of 3 levels of YPS (0, 1, or 2 g/kg) on the broilers fed diets contaminated with or without MYCO (95 µg/kg aflatoxin B1, 1.5 mg/kg deoxynivalenol, and 490 µg/kg zearalenone). Results showed that mycotoxins contaminated diets led to significant increments in serum malondialdehyde (MDA) and 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels, mRNA expressions of TLR4 and 4EBP1 associated with oxidative stress, mRNA expressions of CYP1A1, CYP1A2, CYP2A6, and CYP3A4 associated with hepatic phase Ⅰ metabolizing enzymes, mRNA expressions of p53 associated with hepatic mitochondrial apoptosis, and AFB1 residues in the liver (P < 0.05); meanwhile dietary MYCO decreased the jejunal villus height (VH), villus height/crypt depth (VH/CD), the activity of serum total antioxidant capacity (T-AOC), mRNA expressions of jejunal HIF-1α, HMOX, and XDH associated with oxidative stress, mRNA expressions of jejunal CLDN1, ZO1, and ZO2, and mRNA expression of GST associated with hepatic phase Ⅱ metabolizing enzymes of broilers (P < 0.05). Notably, the adverse effects induced by MYCO on broilers were mitigated by supplementation with YPS. Dietary YPS supplementation reduced the concentrations of serum MDA and 8-OHdG, jejunal CD, mRNA expression of jejunal TLR2, and 4EBP1, hepatic CYP1A2, and p53, and the AFB1 residues in the liver (P < 0.05), and elevated the serum T-AOC and SOD, jejunal VH, and VH/CD, and mRNA expression of jejunal XDH, hepatic GST of broilers (P < 0.05). There were significant interactions between MYCO and YPS levels on the growth performance (BW, ADFI, ADG, and F/G) at d 1 to 21, d 22 to 42, and d 1 to 42, serum GSH-Px activity, and mRNA expression of jejunal CLDN2 and hepatic ras of broilers (P < 0.05). In contrast with MYCO group, the addition of YPS increased BW, ADFI, and ADG, the serum GSH-Px activity (14.31%-46.92%), mRNA levels of jejunal CLDN2 (94.39%-103.02%), decreased F/G, and mRNA levels of hepatic ras (57.83%-63.62%) of broilers (P < 0.05). In conclusion, dietary supplements with YPS protected broilers from mixed mycotoxins toxicities meanwhile keeping normal performance of broilers, presumably via reducing intestinal oxidative stress, protecting intestinal structural integrity, and improving hepatic metabolic enzymes to minimize the AFB1 residue in the liver and enhance the performance of broilers.

Cytochrome P450-mediated herb-drug interaction (HDI) of Polygonum multiflorum Thunb. based on pharmacokinetic studies and in vitro inhibition assays.

BACKGROUND: Polygonum multiflorum Thunb. (PM) is well known both in China and other countries of the world for its tonic properties, however, it has lost its former glory due to liver toxicity incidents in recent years. PURPOSE: The purpose of this study is to determine whether the occurrence of herb-drug interaction (HDI) caused by PM is associated with cytochrome P450 (CYP450) based on pharmacokinetic studies and in vitro inhibition assays. The objective was to provide a reference for the rational and safe use of drugs in clinical practice. METHODS: In this study, raw PM (R), together with its two processed products which included PM by Chinese Pharmacopoeia (M) and PM by "nine cycles of steaming and sunning (NCSS)" ("9"), were prepared as the main research objects. A method based on fluorescence technology was used to evaluate the inhibition levels of raw and processed PMs, as well as corresponding characteristic compounds on seven recombinant human cytochrome P450s (rhCYP450s). The pharmacokinetics of sulindac (a representative of commonly used nonsteroidal anti-inflammatory drugs) and psoralen (a major compound of Psoralea in combination with PM) in rat plasma were studied when combined with raw and different processed products of PM. RESULTS: The inhibitory level order of the three extracts on major different subtypes of CYP450 (CYP1A2, CYP2B6, CYP2C8, CYP2C19, CYP2D6, and CYP3A4) was: R > M > "9". However, the inhibition level of R and "9" is higher than that of M on CYP2C9. Further studies showed that trans-THSG and emodin could selectively inhibit CYP3A4 and CYP1A2, respectively. Epicatechin gallate mainly inhibited CYP3A4 and CYP1A2, followed by CYP2C8 and CYP2C9. Genistein mainly inhibited CYP3A4, followed by CYP2C9 and CYP2C8. CYP3A4 and CYP2C9 were also inhibited by daidzein. The inhibitory effects of all the PM extracts were associated with their characteristic compounds. The results of HDI showed that R increased sulindac exposure to rat blood, and R and M increased psoralen exposure to rat blood, which were consistent with corresponding metabolic enzymes. Overall, the in vitro and in vivo results indicated that PM, especially R, would be at high risk to cause toxicity and drug interactions via CYP450 inhibition. CONCLUSION: This study not only elucidates the scientific connotation of "efficiency enhancement and toxicity reduction" of PM by NCSS from the perspective of metabolic inhibition but also contributes to HDI prediction and appropriate clinical medication of PM.

Interaction between Changan Granule and its main components in the plasma and CYP450 enzymes.

ETHNOPHARMACOLOGICAL RELEVANCE: Changan Granule (CAG) is a Chinese patent drug developed based on an empirical prescription in accordance with the formulation theory of Traditional Chinese Medicine. The prescription is composed of eight herbal drugs which have been traditionally used by Chinese people for a long history. It has effects of invigorating spleen and supplementing qi, as well as regulating liver and ceasing diarrhea, and is indicated for the treatment of irritable bowel syndrome (IBS). AIM OF THE STUDY: This study was aimed to investigate the interaction between CAG and its main components and cytochrome P450 (CYP450) enzymes so as to characterize the major metabolites and metabolic enzymes and evaluate the safety concerns to its clinical use. MATERIALS AND METHODS: Both in vivo and in vitro experiments using such as diarrhea-predominant IBS (IBS-D) rat model, HepG2 cells, and human liver microsomes (HLM) were carried out to investigate the interaction between CAG and its main components and CYP450 enzymes. Real-time quantitative PCR (qPCR), ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), and cocktail probes were employed to qualitatively or quantitatively measure the metabolites and metabolic enzymes. RESULTS: CAG inhibited the enzyme activities of CYP1A2, CYP2E1, CYP2D6, CYP2C9, and CYP3A4 and the mRNA expressions of CYP2E1, CYP2C9, CYP3A4, and CYP2D6 in vitro. CAG down-regulated the increased expression of CYP1A2 and up-regulated the decreased expression of CYP3A1 in vivo. Twenty-two metabolites were characterized from the main components of CAG after incubation with HLM in vitro. CYP2D6, CYP2E1, CYP3A4 and CYP2C9 were identified as the characteristic metabolic enzymes. CONCLUSIONS: This study provides a reference for clinical application of CAG in safety. CAG and CYP450 enzymes are interacted. CAG is mainly metabolized by CYP2E1 and CYP2D6. The expression of CYP2E1 and CYP2D6 are more susceptible to be influenced by CAG in comparison with that of CYP3A4, CYP2C9 and CYP1A2. It implies the potential risk of interaction when CAG is taken together with the drugs metabolized by CYP2E1 and CYP2D6.

Coffee Intake, Caffeine Metabolism Genotype, and Survival Among Men with Prostate Cancer.

BACKGROUND: Coffee intake may lower prostate cancer risk and progression, but postdiagnosis outcomes by caffeine metabolism genotype are not well characterized. OBJECTIVE: To evaluate associations between coffee intake, caffeine metabolism genotype, and survival in a large, multicenter study of men with prostate cancer. DESIGN, SETTING, AND PARTICIPANTS: Data from The PRACTICAL Consortium database for 5727 men with prostate cancer from seven US, Australian, and European studies were included. The cases included had data available for the CYP1A2 -163C>A rs762551 single-nucleotide variant associated with caffeine metabolism, coffee intake, and >6 mo of follow-up. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Multivariable-adjusted Cox proportional hazards models across pooled patient-level data were used to compare the effect of coffee intake (categorized as low [reference], high, or none/very low) in relation to overall survival (OS) and prostate cancer-specific survival (PCSS), with stratified analyses conducted by clinical disease risk and genotype. RESULTS AND LIMITATIONS: High coffee intake appeared to be associated with longer PCSS (hazard ratio [HR] 0.85, 95% confidence interval [CI] 0.68-1.08; p = 0.18) and OS (HR 0.90, 95% CI 0.77-1.07; p = 0.24), although results were not statistically significant. In the group with clinically localized disease, high coffee intake was associated with longer PCSS (HR 0.66, 95% CI 0.44-0.98; p = 0.040), with comparable results for the group with advanced disease (HR 0.92, 95% CI 0.69-1.23; p = 0.6). High coffee intake was associated with longer PCSS among men with the CYP1A2 AA (HR 0.67, 95% CI 0.49-0.93; p = 0.017) but not the AC/CC genotype (p = 0.8); an interaction was detected (p = 0.042). No associations with OS were observed in subgroup analyses (p > 0.05). Limitations include the nominal statistical significance and residual confounding. CONCLUSIONS: Coffee intake was associated with longer PCSS among men with a CYP1A2 -163AA (*1F/*1F) genotype, a finding that will require further replication. PATIENT SUMMARY: It is likely that coffee intake is associated with longer prostate cancer-specific survival in certain groups, but more research is needed to fully understand which men may benefit and why.

Evaluation of the Herb-Drug Interaction Potential of Commonly Used Botanicals on the US Market with Regard to PXR- and AhR-Mediated Influences on CYP3A4 and CYP1A2.

In this study, hydroethanolic extracts of 30 top-selling botanicals (herbs) commonly used as ingredients of herbal dietary supplements in the US were screened for their potential to activate the human pregnane X receptor (hPXR) and human aryl hydrocarbon receptor (hAhR) and to increase the activities of hPXR- and hAhR-regulated drug metabolizing cytochrome P450 enzymes (i.e., CYP3A4 and CYP1A2, respectively). Of the 30 botanicals tested, 21 induced PXR and 29 induced AhR transcriptional activities. Out of the 21 botanicals that induced hPXR transcriptional activity, 14 yielded >50% induction in CYP3A4 activity at concentrations ranging from 6 to 60 µg/mL and 16 out of the 29 botanicals that activated hAhR yielded >50% induction in CYP1A2 activity at concentrations ranging from 3 to 30 µg/mL. Moreover, eight botanicals (G. gummi-gutta [garcinia], Hemp [low and high CBD content], H. perforatum [St. John's wort], M. vulgare [horehound], M. oleifera [moringa], O. vulgare [oregano], P. johimbe [yohimbe] and W. somnifera [ashwagandha]) yielded >50% induction in both CYP3A4 and CYP1A2 activity. Herbal products are mixtures of phytoconstituents, any of which could modulate drug metabolism. Our data reveals that several top-selling botanicals may pose herb-drug interaction (HDI) risks via CYP450 induction. While in vitro experiments can provide useful guidance in assessing a botanical's HDI potential, their clinical relevance needs to be investigated in vivo. Botanicals whose effects on hPXR/CYP3A4, and hAhR/CYP1A2 activity were most pronounced will be slated for further clinical investigation.

[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.

Atractylodes lancea for cholangiocarcinoma: Modulatory effects on CYP1A2 and CYP3A1 and pharmacokinetics in rats and biodistribution in mice.

Atractylodes lancea (Thunb.) DC. (A. lancea: AL) is a promising candidate for the treatment of cholangiocarcinoma (bile duct cancer). The study investigated (i) the propensity of capsule formulation of the standardized extract of AL (formulated AL) to modulate mRNA and protein expression and activities of CYP1A2 and CYP3A1 in rats after long- and short-term exposure, (ii) the pharmacokinetics of atractylodin (ATD: active constituent) after long-term administration of formulated AL, and (iii) the biodistribution of atractylodin-loaded polylactic-co-glycolic acid (ATD-PLGA-NPs) in mice. To investigate CYP1A2 and CYP3A1 modulatory activities following long-term exposure, rats of both genders received oral doses of the formulated AL at 1,000 (low dose), 3,000 (medium dose), and 5,000 (high dose) mg/kg body weight daily for 12 months. For short-term effects, male rats were orally administered the formulated AL at the dose of 5,000 mg/kg body weight daily for 1, 7, 14 and 21 days. The pharmacokinetic study was conducted in male rats after administration of the formulated AL at the dose of 5,000 mg/kg body weight daily for 9 months. The biodistribution study was conducted in a male mouse receiving ATD-PLGA-NPs at the equivalent dose to ATD of 100 mg/kg body weight. The high dose of formulated AL produced an inducing effect on CYP1A2 but an inhibitory effect on CYP3A1 activities in male rats. The low dose, however, did not inhibit or induce the activities of both enzymes in male and female rats. ATD reached maximum plasma concentration (Cmax) of 359.73 ng/mL at 3 h (tmax). Mean residence time (MRT) and terminal phase elimination half-life (t1/2z) were 3.03 and 0.56 h, respectively. The extent of biodistribution of ATD in mouse livers receiving ATD-PLGA-NPs was 5-fold of that receiving free ATD. Clinical use of low-dose AL should be considered to avoid potential herb-drug interactions after long-term use. ATD-PLGA-NPs is a potential drug delivery system for cholangiocarcinoma treatment.

Functional single-nucleotide polymorphism (rs762551) in CYP1A2 gene affects white coffee intake in healthy 20- to 40-year-old adults.

Coffee is one of the most consumed beverages in the world, but the extent to which it is consumed is affected by both environmental and genetic factors. Genome-wide association studies and candidate date association studies have identified several gene variants associated with increased consumption of coffee. Functional single-nucleotide polymorphisms in rs762551 (cytochrome P450 1A2 [CYP1A2]) and rs5751876 (adenosine receptor A2A [ADORA2A]) has been linked to individual caffeine response. Coffee intake has been shown to affect lipid metabolism. We thus hypothesize that rs762551 (CYP1A2) A allele carriers consume more coffee than C allele carriers and that rs5751876 (ADORA2A) C allele carriers consume less coffee than T allele carriers. Additionally, we hypothesize that CYP1A2 genotype can modulate serum glucose concentrations and lipid profile. A total of 421 participants aged 20 to 40 years were recruited from 2016 to 2018 in Poznan, Poland. Genotyping of CYP1A2 and ADORA2A was performed using TaqMan probes. Individuals with AA CYP1A2 genotype consumed relatively more coffee with milk (72.81 ± 10.15 mL/1000 kcal vs 43.38 ± 6.42 mL/1000 kcal, P = .008) and with milk or cream than did C allele carriers, whereas the rs5751876 ADORA2A polymorphism was not associated with coffee or tea intake. Additionally, subjects with AA CYP1A2 genotype had 10% higher serum triacylglycerol (TG) concentrations than C allele carriers. This study suggests that CYP1A2 rs762551 polymorphism is associated with coffee intake and serum TG concentrations in healthy 20- to 40-year-old adults.

Psoralen and isopsoralen from Psoraleae Fructus aroused hepatotoxicity via induction of aryl hydrocarbon receptor-mediated CYP1A2 expression.

ETHNOPHARMACOLOGICAL RELEVANCE: Psoraleae Fructus (PF), a traditional Chinese medicine, has long been used to treat diseases such as cancer, osteoporosis and leukoderma. Psoralen and isopsoralen are main bioactive ingredients of PF with anti-tumor, anti-inflammatory, estrogen-like neuroprotection, etc., meanwhile they are also representative hepatotoxic components of PF. Hepatic CYP1A2 has been reported to be the important metabolic enzymes involved in psoralen and isopsoralen-induced hepatotoxicity. However, the relationship between the hepatotoxicity and CYP1A2 expression, and the underlying mechanism of regulating CYP1A2 expression remain unclear. AIM OF STUDY: The aim of this study was to explore the associated mechanism between psoralen or isopsoralen induced hepatotoxicity and activated aryl hydrocarbon receptor (AhR)-mediated transcriptional induction of CYP1A2 in vitro and in vivo. MATERIALS AND METHODS: Psoralen and isopsoralen at different doses were treated on HepG2 cells (10, 25, 50, 100, 200 µM for 2, 12, 24, 36, 48 h) and mice (20, 80, 160 mg/kg for 3, 7, 14 days) for different time, to assess the correlation of induced hepatotoxicity and CYP1A2 mRNA and protein expression in vivo and in vitro, as well as the effect on CYP1A2 enzyme activity evaluated by phenacetin metabolism. In addition, the potential mechanism of the regulation of CYP1A2 expression mediated by AhR was explored through nucleocytoplasmic shuttling, immunofluorescence, cellular thermal shift assay and molecular docking, etc. RESULTS: Psoralen and isopsoralen induced cytotoxicity in HepG2 cells, and hepatomegaly, biochemicals disorder and tissue pathological impairment in mice, respectively in dose- and time-dependent manners. Simultaneously accompanied with elevated levels of CYP1A2 mRNA and protein in the same trend, and the CYP1A2 activity was remarkably inhibited in vitro but significantly elevated overall in vivo. Besides, psoralen and isopsoralen bound to AhR and activated translocation of AhR from the cytoplasm to the nucleus, leading to the transcriptional induction of target gene CYP1A2. CONCLUSIONS: Hepatotoxicities in HepG2 cells and mice aroused by psoralen and isopsoralen were related to the induction of CYP1A2 expression and activity, whose underlying mechanism might be psoralen or isopsoralen activated AhR translocation and induced increase of CYP1A2 transcriptional expression. Hopefully, these finding are conductive to propose an alert about the combined usage of psoralen or isopsoralen and AhR ligands or CYP1A2 substrates in clinical practice.

Cytochrome P450 inhibition activities of non-standardized botanical products.

ETHNOPHARMACOLOGICAL RELEVANCE: R-tab, H-tab and E-cap botanical products are used for the treatment of various ailments. R-tab is traditionally prescribed for improving urination, H-tab is for relieving piles, hemorrhoids, fissures, and rectal inflammation and E-cap is for regulating menstruation. AIMS OF THE STUDY: To extract the botanical products and determine their potential interaction with the cytochrome P450 (CYP1A2, CYP2D6 and CYP3A4) enzymes. MATERIALS AND METHODS: R-tab, H-tab and E-cap botanical products were first extracted using solvents and analyzed using HPLC and LC-MS/MS. The effects of methanol extracts on the cytochrome induction and inhibition activities were determined using a series of in vitro assays, including multiplex RT-qPCR, CYP activity assays (P450-Glo™) and LC-MS/MS-based assays. For the CYP induction assay, omeprazole, rifampicin and dexamethasone were used as CYP1A2, CYP2D6 and CYP3A4 inducers, respectively. Ketoconazole and acetaminophen were used as positive and negative controls for the CYP3A4 inhibition assay, whereas furafylline and ketoconazole were used as positive and negative controls for the CYP1A2 inhibition assay. RESULTS: All three botanical products did not show any significant induction in CYP1A2, CYP2D6 and CYP3A4 mRNA expression. By contrast, R-tab inhibited the mRNA expression of CYP1A2 significantly from the lowest concentration of 0.01 µg/mL, while, H-tab inhibited the mRNA expression of CYP1A2 and CYP3A4 from 0.1 µg/mL. Based on the P450 Glo assays, E-cap extract inhibited the metabolic activity of CYP1A2 with an IC50 value of 37.24 µg/mL. On the other hand, R-tab, H-tab and E-cap showed inhibitory effects on the CYP3A4 enzymatic activity with IC50 values of 17.42, 18.20 and 20.60 µg/mL, respectively. However, using the LC-MS/MS-based methods, the concentration-dependent effects of R-tab and H-tab extracts on the metabolism of testosterone appeared to be more prominent, with IC50 values of 51.90 and 56.90 µg/mL as compared with the rest of the results, which were all above 100 µg/mL CONCLUSION: The CYP3A4 mRNA and enzymatic activity were moderately inhibited by R-tab and H-tab. Methanol extract of botanical products in solid dosage forms can be evaluated for their herb-drug interaction risks using in vitro assays and may provide the minimum data required for safety labeling.

Schisandra chinensis Oil Attenuates Aristolochic Acid I-Induced Nephrotoxicity in vivo and in vitro.

OBJECTIVE: To investigate the protective effects of Schisandra chinensis oil (SCEO) against aristolochic acid I (AA I)-induced nephrotoxicity in vivo and in vitro and elucidate the underlying mechanism. METHODS: C57BL/6 mice were randomly divided into 5 groups according to a random number table, including control group, AA I group, and AA I +SCEO (0.25, 0.5 and 1 g/kg) groups (n=5 per group). Pretreatment with SCEO was done for 2 days by oral administration, while the control and AA I groups were treated with sodium carboxymethyl cellulose. Mice of all groups except for the control group were injected intraperitoneally with AA I (5 mg/kg) from day 3 until day 7. Histopathological examination and apoptosis of kidney tissue were observed by hematoxylin and eosin and TdT-mediated dUTP nick-end labeling (TUNEL) staining, respectively. The levels of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and serum creatinine (SCr), as well as renal malondialdehyde (MDA), glutathione, r-glutamyl cysteingl+glycine (GSH), and superoxide dismutase (SOD) were analyzed using enzyme-linked immunosorbent assay (ELISA). Expressions of hepatic cytochrome P450 1A1 (CYP1A1), CYP1A2, and nad(p)hquinonedehydrogenase1 (NQO1) were analyzed using ELISA, quantitative real-time polymerase chain reaction (qPCR) and Western blot, respectively. In vitro, SCEO (40 µ g/mL) was added 12 h before treatment with AA I (40 µ mol/mL for 48 h) in human renal proximal tubule cell line (HK-2), then apoptosis and reactive oxygen species (ROS) were analyzed by flow cytometry. RESULTS: SCEO 0.5 and 1 g/kg ameliorated histopathological changes and TUNEL+ staining in the kidney tissues of mice with AA I-induced nephrotoxicity, and reduced serum levels of ALT, AST, BUN and SCr (P<0.01 or P<0.05). SCEO 0.5 and 1 g/kg alleviated the ROS generation in kidney, containing MDA, GSH and SOD (P<0.01 or P<0.05). SCEO 1 g/kg increased the expressions of CYP1A1 and CYP1A2 and decreased NQO1 level in the liver tissues (P<0.01 or P<0.05). Besides, in vitro studies also demonstrated that SCEO 40 µ g/mL inhibited apoptosis and ROS generation (P<0.05 or P<0.01). CONCLUSIONS: SCEO can alleviate AA I-induced kidney damage both in vivo and in vitro. The protective mechanism may be closely related to the regulation of metabolic enzymes, thereby inhibiting apoptosis and ROS production.

Metabolic Activation of Perampanel Mediated by CYP1A2.

Perampanel (PRP), a noncompetitive α-amino-3-hydroxy-5-methyl-4-isoxazolepropanoic acid (AMPA) receptor antagonist with high selectivity, has been used as a new adjuvant for the treatment of fractional seizures with or without primary generalized tonic-clonic seizures and secondary generalized seizures in epilepsy patients over the age of 12. Adverse events such as liver injury have been reported during the clinical application of PRP. The purpose of the study is to explore the in vitro and in vivo metabolic activation of PRP. Two GSH conjugates were detected in rat liver microsomal incubations containing PRP, GSH, and NADPH. The two GSH conjugates were both obtained from the bile of rats and rat primary hepatocytes after exposure to PRP. Similar microsomal incubations complemented with N-acetylcysteine (NAC) in place of GSH offered two NAC conjugates. As expected, the NAC conjugates were detected in the urine of PRP-treated rats. One of the two NAC conjugates was identified as NAC conjugate 12 verified by chemical synthesis. The individual human recombinant P450 enzyme incubation assay demonstrated that CYP1A2 dominated the catalysis for the metabolic activation of PRP. Pretreatment with α-naphthoflavone (NTF) decreased the formation of PRP-derived GSH conjugates in both livers of rats and cultured primary hepatocytes after being treated with PRP. Additionally, NTF was found to decrease the susceptibility of primary hepatocytes to the cytotoxicity of PRP. The findings indicate that PRP was metabolized to the corresponding epoxide, which could participate in PRP-induced cytotoxicity.

CYP1A2 polymorphisms modify the association of habitual coffee consumption with appetite, macronutrient intake, and body mass index: results from an observational cohort and a cross-over randomized study.

BACKGROUND/OBJECTIVES: Evidence regarding the influence of coffee on appetite and weight control is equivocal and the influence of covariates, such as genetic variation in caffeine metabolism, remains unknown. Herein, we addressed the novel hypothesis that genetic variation in CYP1A2, a gene responsible for more than 95% of caffeine metabolism, differentially impacts the association of coffee consumption with appetite and BMI among individuals with different genetic predispositions to obesity. SUBJECTS/METHODS: A cross-over randomized intervention study involving 18 volunteers assessed the effects of coffee consumption on dietary intake, appetite, and levels of the appetite-controlling hormones asprosin and leptin. Data on habitual coffee intake, BMI, and perceived appetite were obtained from an observational cohort of 284 volunteers using validated questionnaires. Participants were stratified according to a validated genetic risk score (GRS) for obesity and to the -163C > A (rs762551) polymorphism of CYP1A2 as rapid (AA), intermediate (AC), or slow (CC) caffeine metabolizers. RESULTS: Coffee consumption led to lower energy and dietary fat intake and circulating asprosin levels (P for interaction of rs762551 genotype*coffee consumption=0.056, 0.039, and 0.043, respectively) as compared to slow/intermediate metabolizers. High coffee consumption was more prevalent in rapid compared to slow metabolizers (P = 0.008 after adjustment for age, sex, and BMI) and was associated with lower appetite perception and lower BMI only in rapid metabolizers (P for interaction of rs762551 genotype*coffee consumption = 0.002 and 0.048, respectively). This differential association of rs762551 genotype and coffee consumption with BMI was more evident in individuals at higher genetic risk of obesity (mean adjusted difference in BMI = -5.82 kg/m2 for rapid versus slow/intermediate metabolizers who consumed more than 14 cups of coffee per week). CONCLUSIONS: CYP1A2 rs762551 polymorphism modifies the association of habitual coffee consumption with BMI, in part by influencing appetite, energy intake and circulating levels of the orexigenic hormone asprosin. This association is more evident in subjects with high genetic predisposition to obesity. ClinicalTrials.gov: registered Clinical Trial NCT04514588.

Bulbine natalensis (currently Bulbine latifolia) and select bulbine knipholones modulate the activity of AhR, CYP1A2, CYP2B6, and P-gp.

Bulbine natalensis, an emerging medicinal herb on the global market with androgenic properties, is often formulated in dietary supplements that promote perceived sexual enhancement. However, to date, comprehensive safety studies of B. natalensis are lacking, particularly those related to its herb-drug interaction potential. The purpose of this study was to assess the inductive and inhibitory effects of extracts and pure compounds of B. natalensis on human cytochrome P-450 isozymes in vitro. Our findings demonstrated that both water and methanolic extracts of B. natalensis as well as knipholone, bulbine-knipholone, and 6'-O-methylknipholone dose-dependently increased mRNA expression encoded by CYP2B6, CYP1A2, and ABCB1 genes. Functional analyses showed that water (60 to 2.20 µg/mL) and methanolic (30 to 3.75 µg/mL) extracts and knipholones (10 to 0.33 µM) increased CYP2B6 and CYP1A2 activity in a dose-dependent manner. Additionally, water extract (60 µg/mL), methanolic extract (30 µg/mL), and knipholone (10 µM) caused activation of the aryl hydrocarbon receptor up to 11.1 ± 0.7, 8.9 ± 0.6, and 7.1 ± 2.0-fold, respectively. Furthermore, inhibition studies revealed that methanolic extract attenuated the activity of metabolically active CYP1A2 (IC50, 22.6 ± 0.4 µg/mL) and CYP2B6 (IC50, 34.2 ± 6.6 µg/mL) proteins, whereas water extracts had no inhibitory effect on either isoform. These findings suggest that chronic consumption of B. natalensis may affect normal homeostasis of select CYPs with subsequent risks for HDIs when concomitantly ingested with conventional medications that are substrates of CYP2B6 and CYP1A2. However, more in-depth translational studies are required to validate our current findings and their clinical relevance.

Schisandra chinensis Oil Attenuates Aristolochic Acid I-Induced Nephrotoxicity in vivo and in vitro / 中国结合医学杂志

OBJECTIVE@#To investigate the protective effects of Schisandra chinensis oil (SCEO) against aristolochic acid I (AA I)-induced nephrotoxicity in vivo and in vitro and elucidate the underlying mechanism.@*METHODS@#C57BL/6 mice were randomly divided into 5 groups according to a random number table, including control group, AA I group, and AA I +SCEO (0.25, 0.5 and 1 g/kg) groups (n=5 per group). Pretreatment with SCEO was done for 2 days by oral administration, while the control and AA I groups were treated with sodium carboxymethyl cellulose. Mice of all groups except for the control group were injected intraperitoneally with AA I (5 mg/kg) from day 3 until day 7. Histopathological examination and apoptosis of kidney tissue were observed by hematoxylin and eosin and TdT-mediated dUTP nick-end labeling (TUNEL) staining, respectively. The levels of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and serum creatinine (SCr), as well as renal malondialdehyde (MDA), glutathione, r-glutamyl cysteingl+glycine (GSH), and superoxide dismutase (SOD) were analyzed using enzyme-linked immunosorbent assay (ELISA). Expressions of hepatic cytochrome P450 1A1 (CYP1A1), CYP1A2, and nad(p)hquinonedehydrogenase1 (NQO1) were analyzed using ELISA, quantitative real-time polymerase chain reaction (qPCR) and Western blot, respectively. In vitro, SCEO (40 µ g/mL) was added 12 h before treatment with AA I (40 µ mol/mL for 48 h) in human renal proximal tubule cell line (HK-2), then apoptosis and reactive oxygen species (ROS) were analyzed by flow cytometry.@*RESULTS@#SCEO 0.5 and 1 g/kg ameliorated histopathological changes and TUNEL+ staining in the kidney tissues of mice with AA I-induced nephrotoxicity, and reduced serum levels of ALT, AST, BUN and SCr (P<0.01 or P<0.05). SCEO 0.5 and 1 g/kg alleviated the ROS generation in kidney, containing MDA, GSH and SOD (P<0.01 or P<0.05). SCEO 1 g/kg increased the expressions of CYP1A1 and CYP1A2 and decreased NQO1 level in the liver tissues (P<0.01 or P<0.05). Besides, in vitro studies also demonstrated that SCEO 40 µ g/mL inhibited apoptosis and ROS generation (P<0.05 or P<0.01).@*CONCLUSIONS@#SCEO can alleviate AA I-induced kidney damage both in vivo and in vitro. The protective mechanism may be closely related to the regulation of metabolic enzymes, thereby inhibiting apoptosis and ROS production.

Momordica charantia L. Extract Protects Hippocampal Neuronal Cells against PAHs-Induced Neurotoxicity: Possible Active Constituents Include Stigmasterol and Vitamin E.

Polycyclic aromatic hydrocarbons (PAHs) have been recognized to cause neurobehavioral dysfunctions and disorder of cognition and behavioral patterns in childhood. Momordica charantia L. (MC) has been widely known for its nutraceutical and health-promoting properties. To date, the effect of MC for the prevention and handling of PAHs-induced neurotoxicity has not been reported. In the current study, the neuroprotective effects of MC and its underlying mechanisms were investigated in mouse hippocampal neuronal cell line (HT22); moreover, in silico analysis was performed with the phytochemicals MC to decipher their potential function as neuroprotectants. MC was demonstrated to possess neuroprotective effect by reducing reactive oxygen species' (ROS') production and down-regulating cyclin D1, p53, and p38 mitogen-activated protein kinase (MAPK) protein expressions, resulting in the inhibition of cell apoptosis and the normalization of cell cycle progression. Additionally, 28 phytochemicals of MC and their competence on inhibiting cytochrome P450 (CYP: CYP1A1, CYP1A2, and CYP1B1) functions were resolved. In silico analysis of vitamin E and stigmasterol revealed that their binding to either CYP1A1 or CYP1A2 was more efficient than the binding of each positive control (alizarin or purpurin). Together, MC is potentially an interesting neuroprotectant including vitamin E and stigmasterol as probable active components for the prevention for PAHs-induced neurotoxicity.