Cytochrome P450 1A1 Expression Research Articles

AhR regulates the expression of human cytochrome P450 1A1 (CYP1A1) by recruiting Sp1.

Cytochrome P450 1A1 (CYP1A1) is abundant in the kidney, liver, and intestine and is involved in the phase I metabolism of numerous endogenous and exogenous compounds. Therefore, exploring the regulatory mechanism of its basal expression in humans is particularly important to understand the bioactivation of several procarcinogens to their carcinogenic derivatives. Site-specific mutagenesis and deletion of the transcription factor binding site determined the core cis-acting elements in the human CYP1A1 proximal and distal promoter regions. The proximal promoter region [overlapping xenobiotic-responsive element (XRE) and GC box sequences] determined the basal expression of CYP1A1. In human hepatocellular carcinoma cells (HepG2) with aryl hydrocarbon receptor (AhR) or specificity protein 1 (Sp1) knockdown, we confirmed that AhR and Sp1 are involved in basal CYP1A1 expression. In HepG2 cells overexpressing either AhR or Sp1, AhR determined the proximal transactivation of basal CYP1A1 expression. Via DNA affinity precipitation assays and ChIP, we found that AhR bound to the promoter and recruited Sp1 to transactivate CYP1A1 expression. The coordinated interaction between Sp1 and AhR was identified to be DNA mediated. Our work revealed a basal regulatory mechanism of an interesting human gene by which AhR interacts with Sp1 through DNA and recruits Sp1 to regulate basal CYP1A1 expression.

Guidance for Rifampin and Midazolam Dosing Protocols To Study Intestinal and Hepatic Cytochrome P450 (CYP) 3A4 Induction and De-induction.

Cytochrome P450 3A4 (CYP3A4) catalyses the metabolism of > 30% of clinically used small molecule drugs. Induction of CYP3A4 is often associated with clinically important metabolic drug-drug interactions (DDIs). To collate published data regarding induction of CYP3A4 expression by rifampin and identify an optimal protocol to study DDIs using physiologically based pharmacokinetic (PBPK) modelling. The University of Washington Drug Interaction Database was searched for published data regarding induction of CYP3A4 by rifampin. A verified PBPK model was used to define the optimal dose, duration, timing and route of administration of rifampin and midazolam to assess induction of intestinal and hepatic CYP3A4 by rifampin. Sensitivity analysis was performed to evaluate the impact of participant characteristics including sex, race and age. The maximal induction of intestinal CYP3A4 (9.5-fold) was almost double that of hepatic CYP3A4 (5.5-fold). Maximal induction of intestinal and hepatic CYP3A4 was achieved in > 90% of participants within 5 and 10days, respectively. Intestinal CYP3A4 expression returned to baseline in > 90% of participants within 7days of rifampin cessation, whereas induction of hepatic CYP3A4 persisted for greater than 7days in > 50% of participants. There was a significant difference in magnitude, but not time course, of CYP3A4 induction between males and females. Age and race did not significantly affect either the magnitude or time course of CYP3A4 induction. Maximal induction of intestinal CYP3A4 is achieved faster than hepatic CYP3A4. To assess maximal hepatic CYP3A4 induction, oral rifampin (600mg daily) should be dosed for > 10days.

Effects of absorbents on growth performance, blood profiles and liver gene expression in broilers fed diets naturally contaminated with aflatoxin

ObjectiveThe study was conducted to evaluate the effects of the absorbent (a mixture of activated carbon and hydrated sodium calcium aluminosilicate) on growth performance, blood profiles and hepatic genes expression in broilers fed diets naturally contaminated with aflatoxin.MethodsA total of 1,200 one-day-old male chicks were randomly assigned to 6 treatments with 10 replicate cages per treatment. The dietary treatments were as follows: i) control (basal diets); ii) 50% contaminated corn; iii) 100% contaminated corn; iv) control+1% adsorbent; v) 50% contaminated corn+1% absorbent; vi) 100% contaminated corn+1% absorbent.ResultsDuring d 1 to 21, feeding contaminated diets reduced (p<0.05) body weight (BW), average daily gain (ADG), and average daily feed intake (ADFI), but increased (p<0.05) feed-to-gain ratio (F/G). The absorbent supplementation increased (p<0.05) BW, ADG, and ADFI. There were interactions (p<0.05) in BW, ADG, and ADFI between contaminated corn and absorbent. Overall, birds fed 100% contaminated diets had lower (p<0.05) final BW and ADG, but higher (p<0.05) F/G compared to those fed control diets. The absorbent addition increased (p<0.05) serum albumin concentration on d 14 and 28 and total protein (TP) level on d 28, decreased (p<0.05) alanine transaminase activity on d 14 and activities of aspartate aminotransferase and alkaline phosphatase on d 28. Feeding contaminated diets reduced (p<0.05) hepatic TP content on d 28 and 42. The contaminated diets upregulated (p<0.05) expression of interleukin-6, catalase (CAT), and superoxide dismutase (SOD), but downregulated (p<0.05) glutathione S-transferase (GST) expression in liver. The absorbent supplementation increased (p<0.05) interleukin-1β, CAT, SOD, cytochrome P450 1A1 and GST expression in liver. There were interactions (p<0.05) in the expression of hepatic CAT, SOD, and GST between contaminated corn and absorbent.ConclusionThe results suggest that the naturally aflatoxin-contaminated corn depressed growth performance, while the adsorbent could partially attenuate the adverse effects of aflatoxin on growth performance, blood profiles and hepatic genes expression in broilers.

Diallyl Trisulfide Enhances Benzo[a]pyrene-induced CYP1A1 Expression and Metabolic Activation in Hepatic HepG2 Cells.

Benzo[a]pyrene (BaP), an environmental pollutant produced by combustion processes, induces expression of cytochrome P450 (CYP) 1A1 via the activation of aryl hydrocarbon receptor (AHR). Induced CYP1A1 is involved in BaP metabolism, resulting in either detoxification or metabolic activation in a context-dependent manner. The effect of diallyl trisulfide (DATS), a garlic-derived organosulfur compound, on BaP metabolism has not been investigated. The combined effect of DATS and BaP on BaP metabolism in hepatocyte-derived HepG2 cells was examined. DATS enhanced BaP-induced CYP1A1 and CYP1B1 mRNA expression, BaP hydroxylation and BaP-DNA adduct formation. Combined treatment of BaP and DATS also increased reactive oxygen species levels. DATS enhanced BaP-induced AHR recruitment and histone H3 acetylation on the CYP1A1 promoter. DATS combined treatment enhances BaP metabolic activation through an AHR-modulating mechanism.

Liposome mediated-CYP1A1 gene silencing nanomedicine prepared using lipid film-coated proliposomes as a potential treatment strategy of lung cancer

The occurrence of lung cancer is linked with tobacco smoking, mainly through the generation of polycyclic aromatic hydrocarbons (PAHs). Elevated activity of cytochrome P4501A1 (CYP1A1) plays an important role in the metabolic processing of PAHs and its carcinogenicity. The present work aimed to investigate the role of CYP1A1 gene in PAH-mediated growth and tumor development in vitro and using an in vivo animal model. RNAi strategy was utilized to inhibit the overexpression of CYP1A1 gene using cationic liposomes generated using a lipid film-coated proliposome microparticles. Treatment of PAH-induced human alveolar adenocarcinoma cell line with cationic liposomes carrying CYP1A1 siRNA resulted in down regulation of CYP1A1 mRNA, protein as well as its enzymatic activity, triggering apoptosis and inhibiting multicellular tumor spheroids formation in vitro. Furthermore, silencing of CYP1A1 gene in BALB/c nude xenografts inhibited tumor growth via down regulation of CYP1A1 expression. Altogether, our findings showed that liposome-based gene delivery technology is a viable and stable approach for targeting cancer causing genes such as CY1PA1. This technology facilitated by the use of sugar particles coated with lipid films has demonstrated ability to generate anticancer effects that might be used in the future for therapeutic intervention and treatment of lung cancer.

Biomarker Discovery for Cytochrome P450 1A2 Activity Assessment in Rats, Based on Metabolomics.

Cytochrome P450 1A2 (CYP1A2) is one of the major CYP450 enzymes (CYPs) in the liver, and participates in the biotransformation of various xenobiotics and endogenous signaling molecules. The expression and activity of CYP1A2 show large individual differences, due to genetic and environmental factors. In order to discover non-invasive serum biomarkers associated with hepatic CYP1A2, mass spectrometry-based, untargeted metabolomics were first conducted, in order to dissect the metabolic differences in the serum and liver between control rats and β-naphthoflavone (an inducer of CYP1A2)-treated rats. Real-time reverse transcription polymerase chain reaction and pharmacokinetic analysis of phenacetin and paracetamol were performed, in order to determine the changes of mRNA levels and activity of CYP1A2 in these two groups, respectively. Branched-chain amino acids phenylalanine and tyrosine were ultimately focalized, as they were detected in both the serum and liver with the same trends. These findings were further confirmed by absolute quantification via a liquid chromatography–tandem mass spectrometry (LC-MS/MS)-based targeted metabolomics approach. Furthermore, the ratio of phenylalanine to tyrosine concentration was also found to be highly correlated with CYP1A2 activity and gene expression. This study demonstrates that metabolomics can be a potentially useful tool for biomarker discovery associated with CYPs. Our findings contribute to explaining interindividual variations in CYP1A2-mediated drug metabolism.

Anti-tumor properties of methoxylated analogues of resveratrol in malignant MCF-7 but not in non-tumorigenic MCF-10A mammary epithelial cell lines

Resveratrol is a plant-derived polyphenol that is known for its anti-inflammatory and anti-tumorigenic properties in in vitro and in vivo models. Recent studies show that some resveratrol analogues might be more potent anti-tumor agents, which may partly be attributed to their ability to activate the aryl hydrocarbon receptor (AHR). Here, the anti-tumorigenic properties of resveratrol and structural analogues oxyresveratrol, pinostilbene, pterostilbene and tetramethoxystilbene (TMS) were studied in vitro, using in the malignant human MCF-7 breast cancer cell line and non-tumorigenic breast epithelial cell line MCF-10A.Cell viability and migration assays showed that methoxylated analogues of resveratrol are more potent anti-tumorigenic compounds than resveratrol and its hydroxylated analogue oxyresveratrol, with 2,3’,4,5’-tetramethoxy-trans-stilbene (TMS) being the most potent compound. TMS decreased MCF-7 tumor cell viability with 50% at 3.6 μM and inhibited migration with 37.5 ± 14.8% at 3 μM. In addition, TMS activated the AHR more potently (EC50 in a reporter gene assay 2.0 μM) and induced AHR-mediated induction of cytochrome P450 1A1 (CYP1A1) activity (EC50 value of 0.7 μM) more than resveratrol and the other analogues tested. Cell cycle analysis showed that TMS induced a shift in cell cycle status from the G1 to the G2/M phase causing a cell cycle arrest in the MCF-7 cells, while no effect of TMS was observed in the non-tumorigenic MCF-10A mammary epithelial cell line. Gene expression analysis showed that 3 μM TMS increased gene expression of CYP1A1 (289-fold), CYP1B1 (5-fold) and Nqo1 (2-fold), and decreased gene expression of IL-8 (3-fold) in MCF-7 cells. In MCF-10A cells, 10 μM TMS also increased gene expression of CYP1A1 (5-fold) and CYP1B1 (2-fold), but decreased gene expression of Nqo1 (1.4-fold) in contrast to MCF-7 cells.TMS displays more potent anti-tumorigenic properties and activates the AHR more effectively than resveratrol. In addition, this is the first study to show that TMS, but not resveratrol, selectively inhibits the cell cycle of breast tumor cells and not the non-tumorigenic cells. Our study provides more insight in the anti-tumor properties of the methoxylated analogues of resveratrol in breast cells in vitro.

CYP1A2 contributes to alcohol-induced abnormal lipid metabolism through the PTEN/AKT/SREBP-1c pathway

Abnormal liver lipid metabolism results in a series of liver diseases and contributes to tumourigenesis. The expression and activity of Cytochrome P450 1A2 (CYP1A2) is significantly increased in alcoholic fatty liver according to our previous studies. In this study, we aimed to explore the role of CYP1A2 in lipid metabolism abnormalities induced by alcohol and to investigate the underlying mechanisms. L02 cells were treated with siRNA-CYP1A2 or fluvoxamine and then stimulated with 100 mM ethanol for 24 h. The levels of ALT and TGs in the siRNA-CYP1A2 and fluvoxamine groups were significantly lower than those in the normal control group after ethanol treatment, and the expression of SREBP-1c was decreased when CYP1A2 was inhibited, suggesting that CYP1A2 may contribute to alcohol-induced irregular lipid metabolism by regulating sterol regulatory element-binding protein-1c (SREBP-1c). The expression of phosphatase and tensin homologue deleted on chromosome ten (PTEN) was decreased in normal cells, and it was further reduced when the expression of CYP1A2 was downregulated. Overexpression of PTEN led to decreased expression of p-AKT and SREBP-1c, which were elevated in both the normal and transfected groups after ethanol stimulation. In contrast, when PTEN was inhibited by siRNA-PTEN or bpv, the expression of PTEN was reduced in each group after ethanol treatment, while the expression of p-AKT and SREBP-1c was increased significantly. These results demonstrate that CYP1A2 may participate in the abnormal lipid metabolism caused by alcohol stimulation through the PTEN/AKT/SREBP-1c pathway.

PPM1A expression positively correlates with CYP3A4 expression in the tumor liver tissue of hepatocellular carcinoma patients: A potential novel mechanism for CYP3A4 repression in hepatocellular carcinoma

In most patients with hepatocellular carcinoma (HCC), cytochrome p450 3A4 (CYP3A4) expression has been reported to be significantly reduced in the tumor liver tissue. However, the underlying mechanisms for CYP3A4 repression are not fully understood. We have previously shown that Mg2+/Mn2+‐dependent phosphatase 1A (PPM1A) interacts with human pregnane X receptor (hPXR) and positively regulates hPXR‐mediated CYP3A4 expression in PPM1A expression‐dependent manner. We sought to determine whether PPM1A expression is downregulated in the tumor liver tissue of HCC patients and is positively correlated with CYP3A4 repression. Additionally, we sought to determine whether PPM1A could counteract inhibition of hPXR transactivation function by signaling pathways that are upregulated in HCC. Quantitative RT‐PCR and western blot analyses were performed to study mRNA and protein expression, respectively. Cell‐based reporter gene assays were conducted to examine the hPXR transactivation of CYP3A4 promoter activity. mRNA and protein expression of PPM1A as well as CYP3A4 was found to be significantly repressed in the tumor liver tissues compared to the matched non‐tumor liver tissues. Expression of arginase‐1 mRNA in the tumor and non‐tumor liver tissues, together with upregulation of glypican‐3 mRNA in the tumor liver tissues, verifies that the tumor samples used in our study belong to HCC livers but not non‐hepatocellular tumors metastasized to the liver. In the reporter gene assays, the hPXR‐mediated CYP3A4 promoter activity was inhibited by hepatocyte growth factor, insulin, tumor necrosis factor‐alpha, transforming growth factor‐beta, lipopolysaccharides, cyclin‐dependent kinase 2, protein kinase C activator phorbol 12‐myristate 13‐acetate, and protein kinase A activator dibutyryl‐cAMP. Notably, elevated PPM1A levels counteracted the inhibition of hPXR by these growth factors, cytokines, inflammatory agents, and kinases. The results from the reporter gene assays suggest that decreased PPM1A levels in HCC could not fully counteract the hPXR inhibiting signaling pathways. Together, these results are consistent with the conclusion that PPM1A downregulation in the tumor liver tissue of HCC patients positively correlates with CYP3A4 repression. Downregulation of PPM1A levels in the tumor liver tissue may contribute to reduced hPXR‐mediated CYP3A4 expression, and may provide a novel mechanism for CYP3A4 repression in the tumor liver tissue of HCC patients.Support or Funding InformationThe authors would like to thank Drs. Coleman, Mansour, and Tao for sharing their research facilities. This work was supported by the Auburn University Research Initiative in Cancer Grant and Animal Health and Disease Research Grant to Pondugula SR.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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.

Serotonin Modulates AhR Activation by Interfering with CYP1A1-Mediated Clearance of AhR Ligands.

Background/Aims:Serotonin (5-hydroxytryptamine, 5-HT) is a neurotransmitter and hormone with important physiological functions in many organs, including the intestine. We have previously shown that 5-HT activates the aryl hydrocarbon receptor (AhR) in intestinal epithelial cells (IECs) via a serotonin transporter (SERT)-dependent mechanism. AhR is a nuclear receptor that binds a variety of molecules including tryptophan (TRP) metabolites to regulate physiological processes in the intestine including xenobiotic detoxification and immune modulation. We hypothesized that 5-HT activates AhR indirectly by interfering with metabolic clearance of AhR ligands by cytochrome P450 1A1 (CYP1A1).Methods:Inhibition of CYP1A1 activity by 5-HT was assessed in the human intestinal epithelial cell line Caco-2 and recombinant CYP1A1 microsomes using both luciferase and LC-MS/MS. Degradation of 5-HT by recombinant CYP1A1 was measured by LC-MS/MS. For in vitro studies, CYP1A1 and CYP1B1 mRNA expression levels were measured by RT-PCR and CYP1A1 activity was measured by ethoxyresorufin-O-deethylase (EROD) assays. For in vivo studies, AhR ligands were administered to SERT KO mice and WT littermates and intestinal mucosa CYP1A1 mRNA was measured.Results:We show that 5-HT inhibits metabolism of both the pro-luciferin CYP1A1 substrate Luc-CEE as well as the high affinity AhR ligand 6-formylindolo[3,2-b] carbazole (FICZ). Recombinant CYP1A1 assays revealed that 5-HT is metabolized by CYP1A1 in an NADPH dependent manner. Treatment with 5-HT in TRP-free medium, which is devoid of trace AhR ligands, showed that 5-HT requires the presence of AhR ligands to activate AhR. Cotreatment with 5-HT and FICZ confirmed that 5-HT potentiates induction of AhR target genes by AhR ligands. However, this was only true for ligands which are CYP1A1 substrates such as FICZ. Administration of β-napthoflavone by gavage or indole-3-carbinol via diet to SERT KO mice revealed that lack of SERT impairs intestinal AhR activation.Conclusion:Our studies provide novel evidence of crosstalk between serotonergic and AhR signaling where 5-HT can influence the ability of AhR ligands to activate the receptor in the intestine.

Perfluorinated Alkyl Acids in Hawaiian Cetaceans and Potential Biomarkers of Effect: Peroxisome Proliferator-Activated Receptor Alpha and Cytochrome P450 4A.

Perfluorinated alkyl acids (PFAAs) are persistent in marine biota and are toxic to many species, including marine mammals. We measured the concentrations of 15 PFAAs in liver and kidney samples of 16 species of stranded cetaceans from Hawai'i and other tropical North Pacific regions utilizing high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). Eleven PFAAs in liver and nine PFAAs in kidney were detected, including substantial perfluorooctanesulfonate (PFOS) and perfluoroundecanoic acid (PFUnA). Regression models indicated that phylogenetic family and age class significantly influenced concentrations of certain PFAAs. PFAAs can activate transcription factor peroxisome proliferator-activated receptor alpha (PPARα), which induces transcription of cytochrome P450 4A (CYP4A). Relative expression of PPARα and CYP4A mRNA was quantified using real-time PCR (qPCR) and CYP4A protein expression, using Western blot and then compared to PFAA concentrations in liver and kidney. Concentrations of four PFAA congeners, summation of perfluoroalkyl carboxylic acids (ΣPFCAs), and ΣPFAAs correlated significantly with PPARα mRNA expression and CYP4A protein expression in kidney, suggesting either may be biomarkers of PFAA exposure in cetaceans. This is the first study to quantify PFAAs in marine mammals from this region and the first observation of a direct relationship between PFAA exposure and PPARα and CYP4A expression in cetaceans.

Effect of panaxytriol on cytochrome P450 3A4 via the pregnane X receptor regulatory pathway.

Panaxytriol (PXT) is one of the major effective components of red ginseng and Shenmai injection. The present study aimed to explore the effect of PXT on cytochrome P450 3A4 (CYP3A4) based on the pregnane X receptor (PXR)-CYP3A4 regulatory pathway in HepG2 cells and hPXR-overexpressing HepG2 cells treated with PXT for different time periods using quantitative polymerase chain reaction, Western blot, and dual-luciferase reporter gene assays. PXT could upregulate the levels of PXR and CYP3A4 mRNA in HepG2 cells treated with PXT for 1hr, with no impact on the expression of their protein levels. The expression levels of both PXR and CYP3A4 mRNA and protein in HepG2 cells treated with PXT for 24hr increased in a concentration-dependent manner. The effects of PXT on the expression of PXR and CYP3A4 mRNA and protein in hPXR-overexpressing HepG2 cells were similar to those in HepG2 cells. Moreover, the influence trend of PXT on CYP3A4 was consistent with that of PXR in HepG2 cells and hPXR-overexpressing HepG2 cells. The dual-luciferase reporter gene assay in HepG2 cells further demonstrated that PXT treatment for specific time periods could significantly induce the expression of CYP3A4 mediated by the PXR regulatory pathway.

Effects of fenbendazole and triclabendazole on the expression of cytochrome P450 1A and flavin-monooxygenase isozymes in bovine precision-cut liver slices

Combinations of the anthelmintics fenbendazole (FBZ) and triclabendazole (TCBZ) have shown enhanced efficacy against the liver fluke Fasciola hepatica. This study aimed to measuring the constitutive expression of CYP1A1, CYP1A2, FMO1 and FMO3, thought to be involved in the metabolism of those compounds, by using an absolute quantitative real time (RT)-PCR approach in bovine precision-cut liver slices (PCLS). It also aimed to characterize the effects of FBZ and TCBZ (alone and in combination) on the expression and activity of the aforementioned isozymes.Both FMO1 and FMO3 were equally represented in control PCLS, whereas CYP1A2 was expressed more than CYP1A1 (P<0.05). PCLS cultured in the presence of beta naphthoflavone (β-NF; CYP1A inducer) had higher mRNA levels of CYP1A1, CYP1A2, FMO1 and FMO3 (P<0.05). No clear-cut evidence of transcriptional effects of the anthelmintics were recorded. After incubation of PCLS with FBZ, there was a significant increase (P<0.05) vs. controls and TBCZ was observed for CYP1A1. PCLS treated with FBZ showed a higher (P<0.05) expression of CYP1A2 compared to controls, TCBZ alone, and the combination FBZ+TCBZ. The gene expression profiles of FMO1 and FMO3 were not affected by the presence of the anthelmintics; the only exception was an upregulation of FMO3 by TCBZ alone. The observed transcriptional effects of the xenobiotics were not mirrored by increased enzyme activities using prototypical substrates of the isozymes under study. Although further confirmatory studies are needed, these results suggest that PCLS represent an alternative in vitro tool for studies on the expression, regulation and function of relevant xenobiotic-metabolizing enzymes in cattle.

Utilizing proteomic approach to identify nuclear translocation related serine kinase phosphorylation site of GNMT as downstream effector for benzo[a]pyrene

Glycine N-methyltransferase (GNMT) protein is highly expressed in certain tissues, such as liver, pancreas, and prostate. GNMT serves multiple roles which include a methyl group transfer enzyme and a liver tumor suppressor. Benzo(a)pyrene (BaP), a family member of polycyclic aromatic hydrocarbon (PAH), is a known environmental carcinogen found in coal tar, tobacco smoke, barbecued food and incomplete combustion of auto fuel. BaP recruits cytochrome P450 to transform itself into benzo(a)pyrene-7,8-diol-9,10-epoxide (B(a)PDE), which covalently interacts with DNA causing tumorigenesis. BaP can be detoxified through GNMT and induces GNMT translocation into the cellular nucleus. GNMT translocation is accompanied by phosphorylation, but the role of phosphorylation in GNMT remains to be explored. Using liquid chromatography coupled with tandem mass spectrometry, this study identified serine 9 of GNMT as the phosphorylation site upon BaP treatment. When serine 9 was mutated and lost the capability to be phosphorylated, the occurrence of BaP-induced GNMT nuclear translocation was dramatically decreased. Also, this mutant from of GNMT lost the ability of phosphorylation and increased cytochrome P450 1A1 (Cyp1a) expression upon BaP treatment. In addition, protein kinase C (PKC) and c-Jun NH2-terminal kinase (JNK) may be required for such phosphorylation. Further characterization of phosphorylated GNMT for its link to BaP may bring new insights into chemical detoxification.

Heat Shock Proteins and Cytochrome P450 1a1 Expression as Biomarkers of Exposure to Bisphenol A in Catla catla

Heat Shock Proteins and Cytochrome P450 1a1 Expression as Biomarkers of Exposure to Bisphenol A in Catla catla

Gancao (Glycyrrhizae Radix) provides the main contribution to Shaoyao-Gancao decoction on enhancements of CYP3A4 and MDR1 expression via pregnane X receptor pathway in vitro

BackgroundChinese herbal formula Shaoyao Gancao decoction (SGD) is often used as an adjuvant with chemotherapeutic agents to treat cancer. Due to the herb-drug interactions, the alternations of drug metabolic enzyme and drug transporters induced by SGD deserve to be explored. We aimed to investigate the effect of SGD on the pregnane X receptor (PXR)-mediated transcriptional regulation of cytochrome P450 3A4 (CYP3A4) and drug transporter multidrug resistance protein 1 (MDR1) in vitro. Besides, we assessed the contribution of constituent herbs to SGD on the regulation of CYP3A4 and MDR1.MethodsThe dual luciferase reporter gene system containing the hPXR expression plasmid and the reporter gene plasmid of CYP3A4 or MDR1 was co-transfected to HepG2 and Caco2 cells. Luciferase activities were determined using a Dual-luciferase reporter assay kit. The gene expression of CYP3A4 and MDR1 in the hPXR-transfected LS174T cells were assessed by real-time qPCR. Finally, the contribution of constituent herbs from SGD was evaluated.ResultsSGD, Shaoyao and Gancao concentration-dependently increased promoter activities of CYP3A4 and MDR1 in vitro. Moreover, SGD, Shaoyao and Gancao up-regulated CYP3A4 and MDR1 mRNA in hPXR-transfected LS174T cells. As the herbal constituent of SGD, Gancao possesses significantly higher levels of metabolic enzyme and drug transporters compared with Shaoyao.ConclusionSGD tends to enhance CYP3A4 and MDR1 expression via PXR pathway, especially Gancao provides the main contribution. This study highlights a potential in vitro mechanism for SGD on the regulation of drug metabolic enzymes and drug transporters.

Furanoflavones pongapin and lanceolatin B blocks the cell cycle and induce senescence in CYP1A1-overexpressing breast cancer cells.

Expression of cytochrome P450-1A1 (CYP1A1) is suppressed under physiologic conditions but is induced (a) by polycyclic aromatic hydrocarbons (PAHs) which can be metabolized by CYP1A1 to carcinogens, and (b) in majority of breast cancers. Hence, phytochemicals or dietary flavonoids, if identified as CYP1A1 inhibitors, may help in preventing PAH-mediated carcinogenesis and breast cancer. Herein, we have investigated the cancer chemopreventive potential of a flavonoid-rich Indian medicinal plant, Pongamia pinnata (L.) Pierre. Methanolic extract of its seeds inhibits CYP1A1 in CYP1A1-overexpressing normal human HEK293 cells, with IC50 of 0.6 µg/mL. Its secondary metabolites, the furanoflavonoids pongapin/lanceolatin B, inhibit CYP1A1 with IC50 of 20 nM. Although the furanochalcone pongamol inhibits CYP1A1 with IC50 of only 4.4 µM, a semisynthetic pyrazole-derivative P5b, has ∼10-fold improved potency (IC50, 0.49 μM). Pongapin/lanceolatin B and the methanolic extract of P. pinnata seeds protect CYP1A1-overexpressing HEK293 cells from B[a]P-mediated toxicity. Remarkably, they also block the cell cycle of CYP1A1-overexpressing MCF-7 breast cancer cells, at the G0-G1 phase, repress cyclin D1 levels and induce cellular-senescence. Molecular modeling studies demonstrate the interaction pattern of pongapin/lanceolatin B with CYP1A1. The results strongly indicate the potential of methanolic seed-extract and pongapin/lanceolatin B for further development as cancer chemopreventive agents.

Development of three-dimensional (3D) spheroid cultures of the continuous rainbow trout liver cell line RTL-W1

In vitro experimental systems based on continuous piscine cell lines can be used as an alternative to animal tests for obtaining qualitative and quantitative information on the possible fate and effect of chemicals in fish. However, their capability to reproduce complex metabolic processes and toxic responses as they occur in vivo is limited due to the lack of organ-specific tissue architecture and functions. Here we introduce a three-dimensional (3D) in vitro experimental system based on spheroidal aggregate cultures (spheroids) of the continuous rainbow trout liver cell line RTL-W1 and provide a first description of their structural and functional properties including growth, viability/longevity, metabolic activity, ultrastructure and cytochrome P450 1A (CYP1A) expression determined by bright-field, multi-photon fluorescence and transmission electron microscopy as well as RT-qPCR analysis. Our results show that RTL-W1 cells in 3D spheroids (ø ~ 150 µm) (including those in the interior) were viable, metabolically active and had higher basal and β-naphthoflavone-induced CYP1A expression levels than conventional 2D cell cultures. Furthermore, they displayed ultrastructural characteristics similar to differentiated hepatocytes. The available evidence suggests that 3D RTL-W1 spheroids may have enhanced hepatotypic functions and be a superior in vitro model to assess hepatic biotransformation, bioaccumulation and chronic toxicity compared to conventional cell monolayer cultures.

The impact of p53 function on the metabolic activation of the carcinogenic air pollutant 3-nitrobenzanthrone and its metabolites 3-aminobenzanthrone and N-hydroxy-3-aminobenzanthrone in human cells.

The tumour suppressor p53, encoded by TP53, is a key player in a wide network of signalling pathways. We investigated its role in the bioactivation of the environmental carcinogen 3-nitrobenzanthrone (3-NBA)found in diesel exhaust and its metabolites 3-aminobenzanthrone (3-ABA) and N-hydroxy-3-aminobenzanthrone (N-OH-3-ABA) in a panel of isogenic human colorectal HCT116 cells differing only with respect to their TP53 status [i.e. TP53(+/+), TP53(+/−), TP53(−/−), TP53(R248W/+) or TP53(R248W/−)]. As a measure of metabolic competence, DNA adduct formation was determined using 32P-postlabelling. Wild-type (WT) p53 did not affect the bioactivation of 3-NBA; no difference in DNA adduct formation was observed in TP53(+/+), TP53(+/−) and TP53(−/−) cells. Bioactivation of both metabolites 3-ABA and N-OH-3-ABA on the other hand was WT-TP53 dependent. Lower 3-ABA- and N-OH-3-ABA-DNA adduct levels were found in TP53(+/−) and TP53(−/−) cells compared to TP53(+/+) cells, and p53’s impact was attributed to differences in cytochrome P450 (CYP) 1A1 expression for 3-ABA whereas for N-OH-3-ABA, an impact of this tumour suppressor on sulphotransferase (SULT) 1A1/3 expression was detected. Mutant R248W-p53 protein function was similar to or exceeded the ability of WT-p53 in activating 3-NBA and its metabolites, measured as DNA adducts. However, identification of the xenobiotic-metabolising enzyme(s) (XMEs), through which mutant-p53 regulates these responses, proved difficult to decipher. For example, although both mutant cell lines exhibited higher CYP1A1 induction after 3-NBA treatment compared to TP53(+/+) cells, 3-NBA-derived DNA adduct levels were only higher in TP53(R248W/−) cells but not in TP53(R248W/+) cells. Our results show that p53’s influence on carcinogen activation depends on the agent studied and thereby on the XMEs that mediate the bioactivation of that particular compound. The phenomenon of p53 regulating CYP1A1 expression in human cells is consistent with other recent findings; however, this is the first study highlighting the impact of p53 on sulphotransferase-mediated (i.e. SULT1A1) carcinogen metabolism in human cells.