Cytochrome P450 1A1 mRNA Levels Research Articles

Delayed clearance of the pro-carcinogen benzo[a]pyrene in PLHC-1 cells when co-exposed to the antifungal drug clotrimazole and effects on the CYP1A biomarker

Cytochrome P450 1 A (CYP1A) is a key enzyme in the metabolism of the polycyclic aromatic hydrocarbon (PAH) benzo[a]pyrene (BaP) in animals, and a biomarker for environmental PAH exposure. The common antimycotic imidazole drug clotrimazole (CLO) has been detected in the aquatic environment and likely co-exists with BaP. Like BaP, CLO can bind to CYP1A enzymes and can act as a CYP1A inhibitor. Co-exposure of BaP with CLO significantly delayed BaP elimination in a fish liver cell line (PLHC-1). Intracellular BaP concentration was 2.4 times higher after 6 h in co-exposed cells, compared to cells exposed to BaP alone. Higher BaP concentrations in cells co-exposed to CLO positively correlated with CLO dose, indicating CLO-mediated delays in BaP clearance. After 24 h, BaP was undetectable irrespective of CLO co-exposure. In contrast, intracellular CLO concentrations remained constant over the 72 h experimental period. Co-exposure of BaP with CLO caused synergistic and time-dependent increases on the CYP1A biomarker both on CYP1A mRNA levels and on CYP1A enzyme activity, in accordance with an apparent delayed BaP elimination in the presence of CLO. These results indicate a toxicokinetic interaction between BaP and CLO on the CYP1A enzyme that delays metabolic clearance of BaP.

Risk assessment of drug-drug interaction potential for bintrafusp alfa with cytochrome P4503A4 substrates: A totality of evidence approach.

Bintrafusp alfa, a first-in-class bifunctional fusion protein composed of the extracellular domain of TGF-βRII (a TGF-β "trap") fused to a human IgG1 mAb blocking PD-L1, is being evaluated for efficacy and safety in solid tumor indications as monotherapy and in combination with small-molecule drugs. We evaluated the perpetrator drug-drug interaction (DDI) potential of bintrafusp alfa via cytochrome P4503A4 (CYP3A4) enzyme modulation, which is responsible for the metabolism of a majority of drugs. The holistic approach included (1) evaluation of longitudinal profiles of cytokines implicated in CYP3A4 modulation and serum 4β-hydroxycholesterol, an endogenous marker of CYP3A4 activity, in a phase I clinical study, and (2) transcriptomics analysis of the CYP3A4 mRNA levels vs the TGFB gene expression signature in normal hepatic tissues. Bintrafusp alfa was confirmed not to cause relevant proinflammatory cytokine modulation or alterations in 4β-hydroxycholesterol serum concentrations in phase I studies. Transcriptomics analyses revealed no meaningful correlations between TGFB gene expression and CYP3A4 mRNA expression, supporting the conclusion that the risk of CYP3A4 enzyme modulation due to TGF-β neutralization by bintrafusp alfa is low. Thus, bintrafusp alfa is not expected to have DDI potential as a perpetrator with co-administered drugs metabolized by CYP3A4; this information is relevant to clinical evaluations of bintrafusp alfa in combination settings.

The UVR Filter Octinoxate Modulates Aryl Hydrocarbon Receptor Signaling in Keratinocytes via Inhibition of CYP1A1 and CYP1B1.

Ultraviolet radiation (UVR) is a consistent part of the environment that has both beneficial and harmful effects on human health. UVR filters in the form of commercial sunscreens have been widely used to reduce the negative health effects of UVR exposure. Despite their benefit, literature suggests that some filters can penetrate skin and have off-target biological effects. We noted that many organic filters are hydrophobic and contain aromatic rings, making them potential modulators of Aryl hydrocarbon Receptor (AhR) signaling. We hypothesized that some filters may be able to act as agonists or antagonists on the AhR. Using a luciferase reporter cell line, we observed that the UVR filter octinoxate potentiated the ability of the known AhR ligand, 6-formylindolo[3,2-b]carbazole (FICZ), to activate the AhR. Cotreatments of keratinocytes with octinoxate and FICZ lead to increased levels of cytochrome P4501A1 (CYP1A1) and P4501B1 (CYP1B1) mRNA transcripts, in an AhR-dependent fashion. Mechanistic studies revealed that octinoxate is an inhibitor of CYP1A1 and CYP1B1, with IC50 values at approximately 1 µM and 586 nM, respectively. In vivo topical application of octinoxate and FICZ also elevated CYP1A1 and CYP1B1 mRNA levels in mouse skin. Our results show that octinoxate is able to indirectly modulate AhR signaling by inhibiting CYP1A1 and CYP1B1 enzyme function, which may have important downstream consequences for the metabolism of various compounds and skin integrity. It is important to continue studying the off-target effects of octinoxate and other UVR filters, because they are used on skin on a daily basis world-wide.

CYP1A1, GSTT1, IL-6 and IL-8 transcription and IL-6 secretion on umbilical endothelial cells from hypertensive pregnant women: Preliminary results.

The impact of pregnancy hypertension in the offspring endothelia remains unknown. We evaluated the transcriptional expression of four genes that participate in the process of endothelial dysfunction using umbilical vein endothelial cell cultures (HUVEC) from healthy pregnant women (PW) and those with hypertensive disorders (HD). The cytochrome P450 1A1 (CYP1A1), gluthathione S-transferase subtype T1 (GSTT1), interleukin 6 (IL-6) and 8 (IL-8) mRNA and IL-6 protein levels were assessed. IL-6 and IL-8 transcripts were significantly reduced in HUVEC obtained from HD women. Our results suggest that a hypertensive environment in utero modifies the transcriptional expression of key inflammatory molecules in the newborn.

Cytotoxicity of hexahelicene and its effect on the aryl hydrocarbon receptor pathway

Carbohelicenes are a group of helical-shaped polycyclic aromatic hydrocarbons. This study examined the effect of hexahelicene (or [6]helicene) and of its imidazolium derivative, 1-butyl-3-(2-methyl[6]helicenyl)-imidazolium bromide (I[6]H), on the activity of the aryl hydrocarbon receptor (AhR) and expression of cytochrome P450 1A1 (CYP1A1) in human hepatoma HepG2 cells. An MTT viability assay showed that both [6]helicene and I[6]H were cytotoxic to HepG2 cells after 24 h of exposure, with IC50 values of 0.9 and 8.4 μM, respectively. Using a gene reporter assay performed in transiently transfected HepG2 cells, we found that 1 μM [6]helicene, unlike I[6]H, significantly increased the activity of AhR to 2.1-fold compared to the control after 24 h of exposure. Moreover, [6]helicene induced a small but significant increase in the level of CYP1A1 mRNA. On the other hand, neither the protein level nor activity of CYP1A1 were affected by [6]helicene in HepG2 cells. The effect of [6]helicene on the AhR pathway was thus much lower than that of 2,3,7,8-tetrachlorodibenzo-p-dioxin, a potent AhR activator. We conclude that [6]helicene is a poor activator of the AhR pathway in HepG2 cells, and that the possible activation of the AhR pathway in vivo remains to be investigated.

Urolithin A Is a Dietary Microbiota-Derived Human Aryl Hydrocarbon Receptor Antagonist.

Urolithins (e.g., UroA and B) are gut microbiota-derived metabolites of the natural polyphenol ellagic acid. Urolithins are associated with various health benefits, including attenuation of inflammatory signaling, anti-cancer effects and repression of lipid accumulation. The molecular mechanisms underlying the beneficial effects of urolithins remain unclear. We hypothesize that some of the human health benefits of urolithins are mediated through the aryl hydrocarbon receptor (AHR). Utilizing a cell-based reporter system, we tested urolithins for the capacity to modulate AHR activity. Cytochrome P450 1A1 (CYP1A1) mRNA levels were assessed by real-time quantitative polymerase chain reaction. Competitive ligand binding assays were performed to determine whether UroA is a direct ligand for the AHR. Subcellular AHR protein levels were examined utilizing immunoblotting analysis. AHR expression was repressed in Caco-2 cells by siRNA transfection to investigate AHR-dependency. UroA and B were able to antagonize 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced AHR-mediated transcriptional activity. Furthermore, UroA and B attenuated TCDD-mediated stimulation of CYP1A1 mRNA levels. In addition, competitive ligand binding assays characterized UroA as a direct AHR ligand. Consistent with other AHR antagonists, UroA failed to induce AHR retention in the nucleus. AHR is necessary for UroA-mediated attenuation of cytokine-induced interleukin 6 (IL6) and prostaglandin-endoperoxide synthase 2 (PTGS2) expression in Caco-2 cells. Here we identified UroA as the first dietary-derived human selective AHR antagonist produced by the gut microbiota through multi-step metabolism. Furthermore, previously reported anti-inflammatory activity of UroA may at least in part be mediated through AHR.

Activating the pregnane X receptor by imperatorin attenuates dextran sulphate sodium-induced colitis in mice.

Activation of the human pregnane X receptor (PXR; NR1I2) has potential therapeutic uses for inflammatory bowel disease (IBD). Imperatorin (IMP), a naturally occurring coumarin, is the main bioactive ingredient of Angelica dahurica Radix, which is regularly used to treat the common cold and intestinal disorders. However, there are no data on the protective effects of IMP against IBD. The effects of IMP on PXR-modulated cytochrome P450 3A4 (CYP3A4) expression were assessed using a PXR transactivation assay, a mammalian two-hybrid assay, a competitive ligand-binding assay, analysis of CYP3A4 mRNA and protein expression levels and measurement of CYP3A4 activity using a cell-based reporter gene assay and in vitro model. The inhibitory effects of IMP on NF-κB activity were evaluated by a reporter assay and NF-κB p65 nuclear translocation. The anti-IBD effects of IMP were investigated in a dextran sulphate sodium (DSS)-induced colitis mouse model. Colon inflammatory cytokines were assessed by elisa. IMP activated CYP3A4 promoter activity, recruited steroid receptor coactivator 1 to the ligand-binding domain of PXR and increased the expression and activity of CYP3A4. PXR knockdown substantially reduced IMP-induced increase in CYP3A4 expression. Furthermore, IMP-mediated PXR activation suppressed the nuclear translocation of NF-κB and down-regulated LPS-induced expression of pro-inflammatory genes. Nevertheless, PXR knockdown partially reduced the IMP-mediated inhibition of NF-κB. IMP ameliorated DSS-induced colitis by PXR/NF-κB signalling. IMP acts as a PXR agonist to attenuate DSS-induced colitis by suppression of the NF-κB-mediated pro-inflammatory response in a PXR/NF-κB-dependent manner.

Effects of Rhizoma Alismatis extract on biochemical indices and adipose gene expression in oleic acid-induced hepatocyte injury in Jian carp (Cyprinus carpio var. Jian)

Fatty liver is an increasingly serious disease of fish in aquaculture. However, the mechanisms responsible for the occurrence of fatty liver remain unclear, and no effective methods for the prevention and treatment of this disease have yet been found. In the present study, we aimed to develop an in vitro model of hepatocyte injury using oleic acid as hepatotoxicant and evaluate the protective effects of Rhizoma Alismatis extract (RAE) in Jian carp using this model. Primary hepatocytes from Jian carp were isolated and purified and cultured in vitro. The result indicated that 0.4mmolL-1 oleic acid and 48h could be the optimal conditions to induce hepatocyte injury model in cultured hepatocytes. Hepatocytes were exposed to oleic acid, followed by the addition of RAE at 0, 1, 5, 10, 20, or 50μgmL-1. The hepatocytes and supernatant were then analyzed. RAE suppressed oleic acid-induced elevations in aspartate aminotransferase, alanine aminotransferase, triglycerides, total cholesterol, lactate dehydrogenase, alkaline phosphatase, cholinesterase, malondialdehyde, γ-glutamyl transferase, cytochrome P450 1A, cytochrome P450 2E1, liver-type fatty acid binding protein, free fatty acid, fatty acid synthetase, and tumor necrosis factor-α (P<0.01 or P<0.05); reduced protein levels of cytochrome P450 1A, nuclear factor (NF)-κB p65, and NF-κB c-Rel; and inhibited cytochrome P4503A, NF-κB c-Rel, nuclear factor erythroid-related factor 2, peroxisome proliferator-activated receptor-α, and cytochrome P4501A mRNA levels. In conclusion, RAE exhibited a protective effect against hepatocyte injury in Jian carp. Further in vivo studies are needed to provide more evidence for the use of RAE as a hepatoprotective agent for the treatment of hepatocyte injury.

Interactive effects of hypoxia and PCB co-exposure on expression of CYP1A and its potential regulators in Atlantic croaker liver.

Although marine and coastal environments which are contaminated with xenobiotic organic compounds often become hypoxic during the summer, the interactive effects of hypoxia and xenobiotic exposure on marine species such as teleost fishes remain poorly understood. The expression and activity of monooxygenase enzyme cytochrome P450-1A (CYP1A) in fishes are upregulated by exposure to polychlorinated biphenyls (PCBs), whereas they are down-regulated during hypoxia exposure. We investigated the interactive effects of hypoxia and PCB co-exposure on hepatic CYP1A expression in Atlantic croaker and on potential regulators of CYP1A. Croaker were exposed to hypoxia (1.7 mg/L dissolved oxygen), 3,3',4,4'-tetrachlorobiphenyl (PCB 77, dose: 2 and 8 µg/g body weight), and Aroclor 1254 (a common PCB mixture, dose: 0.5 and 1 µg/g body weight), alone and in combination for 4 weeks. PCB 77 exposure markedly increased hepatic CYP1A mRNA and protein expression, and ethoxyresorufin-O-deethylase (EROD, an indicator of CYP1A enzyme) activity and increased endothelial nitric oxide synthase (eNOS) protein expression. PCB 77 treatment also increased interleukin-1β (IL-1β, a cytokine) mRNA levels and protein carbonyl (PC, an indicator of reactive oxygen species, ROS) contents. These marked PCB 77- and Aroclor 1254-induced increases in CYP1A mRNA levels and EROD activity were significantly attenuated by co-exposure to hypoxia, whereas the increases in hepatic eNOS protein and IL-1β mRNA expression, and PC contents were augmented by hypoxia co-exposure. The results suggest that biotransformation of organic xenobiotics by CYP1A is reduced in fish during co-exposure to hypoxia and is accompanied by alterations in eNOS, ROS, and IL-1β levels.

Enhanced intrinsic CYP3A4 activity in human hepatic C3A cells with optically controlled CRISPR/dCas9 activator complex.

Human hepatic C3A cells have been applied in bioartificial liver development, although these cells display low intrinsic cytochrome P450 3A4 (CYP3A4) enzyme activity. We aimed to enhance CYP3A4 enzyme activity of C3A cells utilizing CRISPR gene editing technology. We designed two CYP3A4 expression enhanced systems applying clustered regularly interspaced short palindromic repeats (CRISPR) gene technology: a CRISPR-on activation system including dCas9-VP64-GFP and two U6-sgRNA-mCherry elements, and a light-controlled CRISPR-on activation system combining our CRISPR-on activation system with an optical control system to facilitate regulation of CYP3A4 expression for various applications. Results of enzymatic activity assays displayed increased CYP3A4 activity in C3A cells expressing the CRISPR-on activation system compared with C3A cells. In addition, CYP3A4 activity increased in C3A cells expressing the light-controlled CRISPR-on activation system under blue light radiation compared with C3A cells. Notably, there was no statistical difference in the increase of CYP3A4 protein amounts induced by these two methods. After expansion in culture, C3A cells with the light-controlled CRISPR-on activation system exhibited no statistical difference in CYP3A4 mRNA levels between generations. Our findings provide a method to stably enhance functional gene expression in bioartificial liver cells with the potential for large-scale cell expansion.

Protective action of the phyllanthin against carbon tetrachloride-induced hepatocyte damage in Cyprinus carpio.

The present study was to investigate the protective effect and possible mechanism of phyllanthin against carbon tetrachloride (CCl4)-induced hepatocyte damage in carp. Phyllanthin (5, 10, and 15 μg/ml) was added to carp primary hepatocytes before (pre-treatment) and after (post-treatment) incubation of the hepatocytes in medium containing CCl4 at 8 mM; supernatant and cell were collected for the analyses of cell viability, biochemical parameters, and gene expression. The results showed that phyllanthin at the concentration of 15 μg/ml significantly suppressed the elevation of glutamate pyruvate transaminase (GPT), glutamate oxalate transaminase (GOT), lactate dehydrogenase (LDH), and malondialdehyde (MDA), and the reduction of cell viability, superoxide dismutase (SOD) activity, cytochrome P450 1a (CYP1A), and cytochrome P450 3a (CYP3A) messenger RNA (mRNA) levels expect LDH in the post-treatment. The levels of GPT, GOT, and CYP1A mRNA were also effectively restored in the pretreatment with phyllanthin (10 μg/ml). Overall, our results suggested that phyllanthin may be used as a hepatoprotective agent to prevent liver diseases in fish.

Responses of the CYP1A biomarker in Jenynsia multidentata and Phalloceros caudimaculatus and evaluation of a CYP1A refractory phenotype

The level of cytochrome P450 1A (CYP1A) in fish is used as a typical environmental biomarker for the presence of organic contaminants. We used RT-qPCR to investigate CYP1A mRNA levels in the liver, gill and gonopodium of guppies Jenynsia multidentata and Phalloceros caudimaculatus in wetlands within the Rio Grande city (RG) which is under the influence of the Patos Lagoon Estuary (RS, Brazil). The CYP1A mRNA levels evaluated in fish liver from two locations that receive non-treated wastewater effluents (S3 and S4) and another locations near an oil refinery (S6) and an industrial complex (S7), were higher than in locations remote from those sites (S1, S2 and S5). The sum of 16 priority PAHs in sediment confirmed high levels in S4 and S6 (3914.0 and 4414.0 ng g−1 dw, respectively) comparing to S7>S2>S3>S5>S1 (119.3, 66.3, 62.8, 16.4 and 1.7 ng g−1 dw). J. multidentata from sites S1 to S4 that were transferred to the laboratory exhibited CYP1A induction after 24 h waterborne exposure to 1 µM betanaphtoflavone (BNF) in all organs compared to controls, except in the liver of fish from site S4. This lack of CYP1A induction by BNF indicates a CYP1A refractory phenotype in guppy. Although this characteristic possibly involves the alteration in AHR signaling or control, the mechanism of resistance is unknown. The present study provides information about the use of the use of CYP1A in South American guppies as an useful biomarker tool for environmental contamination studies.

Mixture effects between different azoles and β-naphthoflavone on the CYP1A biomarker in a fish cell line

The cytochrome P450 1A (CYP1A) biomarker response was studied in the Poeciliopsis lucida hepatocellular carcinoma (PLHC-1) cell line, which represents a good model for studies on aryl hydrocarbon receptor (AhR) – CYP1A signaling. The PLHC-1 cells were exposed to the prototypical CYP1A inducer and AhR agonist β-naphthoflavone (BNF) in combination with different azoles. Two imidazoles (clotrimazole and prochloraz) and two benzimidazoles (nocodazole and omeprazole) were used. Exposure to clotrimazole, prochloraz and nocodazole resulted in 2–4 fold induction of the CYP1A-mediated ethoxyresorufin-O-deethylase (EROD) activities at 24 and 48h, whereas exposure to the omeprazole for 48h had no effect on the EROD activity. Clotrimazole, nocodazole and prochloraz also acted as inhibitors of EROD activities in situ in PLHC-1 cells (IC50=1.3–7.7μM), whereas omeprazole had no effect on this activity (IC50=72μM). Exposure to 10μM prochloraz resulted in 3-fold induction of CYP1A mRNA and exposure to 10μM nocodazole resulted in 16-fold induction of CYP1A mRNA levels at 24h compared to controls. In the mixture experiments, more-than-additive mixture effects between BNF and the azoles clotrimazole, prochloraz and nocodazole on EROD activities were evident, with nocodazole showing the strongest mixture effect. The presence of nocodazole increased the response to BNF up to 200-fold on CYP1A mRNA and up to 16-fold on EROD activities and prolonged the effect of BNF exposure on EROD activities by 24h or longer. This suggests that azoles that are inhibitors and/or competing substrates for the CYP1A enzymes can cause increased sensitivity to exposures to chemicals that depend on CYP1A metabolism for their elimination in situations of mixed chemical exposures. The results also suggest that the EROD biomarker response can be significantly affected in azole-contaminated areas. The responsiveness of the EROD biomarker to BNF exposure was studied in PLHC-1 that had been pre-treated with nocodazole for 5 or 24h at concentrations that are known to disassemble microtubules at 24h in these cells. Pre-treatment of PLHC-1 cells with nocodazole for either 5 or 24h had no effect on the responsiveness to BNF exposure, which implies that the EROD activity can be induced in cells with disassembled microtubules.

Beta-naphthoflavone-inducedCYP1A expression in the guppy Jenynsia multidentata: Time-dependent response, anesthetic MS-222 effect and fin analysis

Cytochrome P450 1A (CYP1A) expression in fish is used as a biomarker of exposure to organic contaminants, such PAHs, PCBs and dioxins, in the aquatic environment. South American guppy fish Jenynsia multidentata were exposed to the prototypical aryl hydrocarbon receptor (AHR) agonist beta-naphthoflavone (BNF; 1μM) and the fins were biopsied to characterize different aspects of CYP1A induction. RTq-PCR was used to quantify CYP1A mRNA levels in fish tissues. CYP1A induction in the gill, liver and anal fin (gonopodium) occurred within the first hour of waterborne exposure to BNF and persisted throughout 2, 4, 8, 24, 48 and 96h compared to controls (DMSO vehicle; p<0.05). The organ-specific temporal pattern of induction was marked by mRNA levels consistently augment as duration of exposure increases and tend to a sustained induction from 24h to 96h for gill and liver (∼15-fold and ∼50-fold over control, respectively). In gonopodium, there was a maximum CYP1A mRNA level at 4h (∼34-fold over control). Basal CYP1A mRNA levels and its induction following BNF exposure were not affected by administration of a chemical anesthetic (fish immersion in 100mgl−1 MS-222 for 2–5min) in the gill, liver, gonopodium, dorsal or tail fin (p<0.05). In an ex vivo assay, in which small pieces of biopsied fins were exposed to BNF for 4h, high CYP1A induction was observed in the tail and gonopodium (∼49-fold and ∼69-fold, respectively) but not in the dorsal fin compared to controls. To our knowledge, this is the first study to show that a 1h waterborne exposure to an AHR agonist is sufficient to cause CYP1A induction in fish organs and fins. The present study added new information to the field regarding the use of MS-222 as an anesthetic on fish and the analysis of biopsied fins as an alternative non-lethalex vivo assay for evaluating the CYP1A biomarker in fish. This observation could be useful for planning fish toxicological bioassays and biomonitoring studies on the aquatic environments in South America.

Aryl hydrocarbon receptor repressor and TiPARP (ARTD14) use similar, but also distinct mechanisms to repress aryl hydrocarbon receptor signaling.

The aryl hydrocarbon receptor (AHR) regulates the toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The AHR repressor (AHRR) is an AHR target gene and functions as a ligand-induced repressor of AHR; however, its mechanism of inhibition is controversial. Recently, we reported that TCDD-inducible poly (ADP-ribose) polymerase (TiPARP; ARTD14) also acts as a repressor of AHR, representing a new player in the mechanism of AHR action. Here we compared the ability of AHRR- and TiPARP-mediated inhibition of AHR activity. TCDD increased AHRR mRNA levels and recruitment of AHRR to cytochrome P450 1A1 (CYP1A1) in MCF7 cells. Knockdown of TiPARP, but not AHRR, increased TCDD-induced CYP1A1 mRNA and AHR protein levels. Similarly, immortalized TiPARP−/− mouse embryonic fibroblasts (MEFs) and AHRR−/− MEFs exhibited enhanced AHR transactivation. However, unlike TiPARP−/− MEFs, AHRR−/− MEFs did not exhibit increased AHR protein levels. Overexpression of TiPARP in AHRR−/− MEFs or AHRRΔ8, the active isoform of AHRR, in TiPARP−/− MEFs reduced TCDD-induced CYP1A1 mRNA levels, suggesting that they independently repress AHR. GFP-AHRRΔ8 and GFP-TiPARP expressed as small diffuse nuclear foci in MCF7 and HuH7 cells. GFP-AHRRΔ8_Δ1-49, which lacks its putative nuclear localization signal, localized to both the nucleus and the cytoplasm, while the GFP-AHRRΔ8_Δ1-100 mutant localized predominantly in large cytoplasmic foci. Neither GFP-AHRRΔ8_Δ1-49 nor GFP-AHRRΔ8_Δ1-100 repressed AHR. Taken together, AHRR and TiPARP repress AHR transactivation by similar, but also different mechanisms.

Effects of Decreased Vitamin D and Accumulated Uremic Toxin on Human CYP3A4 Activity in Patients with End-Stage Renal Disease

In patients with end-stage renal disease, not only renal clearance but also hepatic clearance is known to be impaired. For instance, the concentration of erythromycin, a substrate of cytochrome P450 3A4 (CYP3A4), has been reported to be elevated in patients with end-stage renal disease. The purpose of this study is to elucidate the reason for the decrease in hepatic clearance in patients with end-stage renal disease. Deproteinized pooled sera were used to assess the effects of low-molecular-weight uremic toxins on CYP3A4 activity in human liver microsomes and human LS180 cells. Four uremic toxins (3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid, hippuric acid, indole-3-acetic acid, and 3-indoxyl sulfate) present at high concentrations in uremic serum were also studied. Simultaneous treatment of uremic serum (less than 10%) or uremic toxins did not affect testosterone 6β-hydroxylation in human liver microsomes. On the other hand, pretreatment of each serum activates CYP3A4 in LS180 cells, and the increased CYP3A4 activity in uremic serum-treated cells was smaller than normal serum-treated cells. In addition, CYP3A4 and CYP24A1 mRNA levels also increased in LS180 cells exposed to normal serum, and this effect was reduced in uremic serum-treated cells and in cells exposed to uremic serum added to normal serum. Furthermore, addition of 1,25-dihydroxyvitamin D to uremic serum partially restored the serum effect on CYP3A4 expression. The present study suggests that the decrease of 1,25-dihydroxyvitamin D and the accumulation of uremic toxins contributed to the decreased hepatic clearance of CYP3A4 substrates in patients with end-stage renal disease.

Effect of Chinese herbs on CYP3A4 activity and expression in vitro

Ethnopharmacological relevanceTraditional Chinese Medicine (TCM) has become more popular among cancer patients in the Western world, who often use Chinese herbs as adjuvant therapy to reduce the adverse effects of conventional chemotherapy. However, pharmacokinetic (PK) interactions between Chinese herbs and anticancer drugs can occur and have dramatic consequences for these patients. Currently, only a few possible PK interactions between Chinese herbs and conventional Western drugs have been documented. Aim of the studySince the drug-metabolizing enzyme cytochrome P450 3A4 (CYP3A4) contributes to most of the PK interactions with (anticancer) drugs, the effect of four Chinese herbs (Oldenlandia diffusa, Codonopsis tangshen, Rehmannia glutinosa and Astragalus propinquus) on the activity and expression of CYP3A4 was investigated in vitro. Materials and methodsEthanol and water–ethanol extracts of the four Chinese herbs were prepared from raw material. CYP3A4 inhibition was assessed by the use of Supersomes™ in a fluorescence assay. Furthermore, CYP3A4 induction was evaluated in a human pregnane X receptor (hPXR)-mediated CYP3A4 reporter gene assay and a quantitative real time PCR assay, both in human colon adenocarcinoma-derived LS180 cells (LS180). ResultsExtracts of Oldenlandia diffusa, Codonopsis tangshen, Rehmannia glutinosa and Astragalus propinquus inhibited CYP3A4 in human CYP3A4 Supersomes™ (IC50 values: 17–83µg/mL). Oldenlandia diffusa and Rehmannia glutinosa significantly induced PXR-mediated CYP3A4 (p<0.001). Oldenlandia diffusa also significantly induced CYP3A4 mRNA levels (p<0.001 at 250µg/mL). ConclusionsConcomitant use of Oldenlandia diffusa and Rehmannia glutinosa could result in induction of CYP3A4, leading to a reduced efficacy of drugs that are CYP3A4 substrates and have a narrow therapeutic window. Because of the possible enhanced toxicity caused by CYP3A4 inhibition, clinical effects of CYP3A4 inhibition by Astragalus propinquus and Codonopsis tangshen must also be taken into account. In conclusion, herb–drug interactions between Chinese herbs and various CYP3A4 substrates can occur. Further research to investigate the clinical relevance of the interactions caused by Oldenlandia diffusa, Codonopsis tangshen, Rehmannia glutinosa and Astragalus propinquus is required.

Pregnane X receptor dependent up-regulation of CYP2C9 and CYP3A4 in tumor cells by antitumor acridine agents, C-1748 and C-1305, selectively diminished under hypoxia

Induction of proteins involved in drug metabolism and in drug delivery has a significant impact on drug–drug interactions and on the final therapeutic effects. Two antitumor acridine derivatives selected for present studies, C-1748 (9-(2′-hydroxyethylamino)-4-methyl-1-nitroacridine) and C-1305 (5-dimethylaminopropylamino-8-hydroxy-triazoloacridinone), expressed high and low susceptibility to metabolic transformations with liver microsomes, respectively. In the current study, we examined the influence of these compounds on cytochrome P450 3A4 (CYP3A4) and 2C9 (CYP2C9) enzymatic activity and gene expression in HepG2 tumor cells. Luminescence and HPLC examination, real-time RT-PCR and western blot analyses along with transfection of pregnane X receptor (PXR) siRNA and CYP3A4 reporter gene assays were applied. We found that both compounds strongly induced CYP3A4 and CYP2C9 activity and expression as well as expression of UGT1A1 and MDR1 in a concentration- and time-dependent manner. C-1748-mediated CYP3A4 and CYP2C9 mRNA induction equal to rifampicin occurred at extremely low concentrations (0.001 and 0.01μM), whereas 10μM C-1305 induced three-times higher CYP3A4 and CYP2C9 mRNA levels than rifampicin did. CYP3A4 and CYP2C9 expressions were shown to be PXR-dependent; however, neither compound influenced PXR expression. Thus, the observed drug-mediated induction of isoenzymes occurs on a PXR-mediated regulatory level. Furthermore, C-1748 and C-1305 were demonstrated to be selective PXR agonists. These effects are hypoxia-inhibited only in the case of C-1748, which is sensitive to P450 metabolism. In summary, PXR was found to be a new target of the studied compounds. Thus, possible combinations of these compounds with other therapeutics might lead to the PXR-dependent enzyme-mediated drug–drug interactions.

Chemopreventive Role of Metformin during DMBA–induced Breast Carcinogenesis in MCF10A Cells through CYP1A1‐AhR Signaling Pathway

Recent studies have established that metformin (MET), an oral hypoglycemic drug, possesses antioxidant activity and effective against different types of cancer. Although several postulated mechanisms were reported, the role of the cytochrome P450 1A1 (CYP1A1)‐aryl hydrocarbon receptor (AhR) signaling pathway in the protective effect of MET against breast cancer has not been reported before. Therefore, we examined the capacity of MET to inhibit CYP1A1 gene expression and DNA adducts formation in human breast cells MCF10A by dimethylbenz[a]anthracene (DMBA), a well‐known breast chemical carcinogen. Our results showed that DMBA‐induced DNA adduct was completely prevented by MET as evidenced by decrease in DNA repair genes and 8‐hydroxy‐2′‐deoxyguanosine level. Importantly, MET inhibited the induction of CYP1A1 mRNA, protein and activity levels by DMBA, suggesting a direct role of CYP1A1. Mechanistically, this was strongly correlated with MET ability to inhibit AhR‐dependent luciferase activity, but there was no effect on AhR activation, as evidenced by binding assay, suggesting that MET is not an AhR ligand. In conclusion, the present work provides the first evidence that MET inhibits the DMBA‐mediated carcinogenicity by inhibiting the expression of CYP1A1 through an AhR ligand‐independent mechanism. This work was supported by the Deanship of Scientific Research grant no. RGP‐VPP‐141.

Posttranslational mechanisms modulating the expression of the cytochrome P450 1A1 gene by methylmercury in HepG2 cells: A role of heme oxygenase-1

Recently we demonstrated the ability of mercuric chloride (Hg2+) in human hepatoma HepG2 cells to significantly decrease the TCDD-mediated induction of Cytochrome P450 1A1 (CYP1A1) mRNA, protein, and catalytic activity levels. In this study we investigated the effect of methylmercury (MeHg) on CYP1A1 in HepG2 cells. For this purpose, cells were co-exposed to MeHg and TCDD and the expression of CYP1A1 mRNA, protein, and catalytic activity levels were determined. Our results showed that MeHg did not alter the TCDD-mediated induction of CYP1A1 mRNA, or protein levels; however it was able to significantly decrease CYP1A1 catalytic activity levels in a concentration-dependent manner. Importantly, this inhibition was specific to CYP1A1and was not radiated to other aryl hydrocarbon receptor (AhR)-regulated genes, as MeHg induced NAD(P)H:quinone oxidoreductase 1 mRNA and protein levels. Mechanistically, the inhibitory effect of MeHg on the induction of CYP1A1 coincided with an increase in heme oxygenase-1 (HO-1) mRNA levels. Furthermore, the inhibition of HO-1 activity, by tin mesoporphyrin, caused a complete restoration of MeHg-mediated inhibition of CYP1A1 activity, induced by TCDD. In addition, transfection of HepG2 cells with siRNA targeting the human HO-1 gene reversed the MeHg-mediated inhibition of TCDD-induced CYP1A1. In conclusion, this study demonstrated that MeHg inhibited the TCDD-mediated induction of CYP1A1 through a posttranslational mechanism and confirms the role of HO-1 in a MeHg-mediated effect.