CYP1A1 Levels Research Articles

Bupi Yishen formula attenuates kidney injury in 5/6 nephrectomized rats via the tryptophan-kynurenic acid-aryl hydrocarbon receptor pathway

BackgroundBupi Yishen Formula (BYF), a patent traditional Chinese medicine (TCM) formulation, has been used in the clinical treatment of chronic kidney disease (CKD). However, the mechanism of action of BYF has not been fully elucidated.MethodTo investigate the variation in the metabolic profile in response to BYF treatment in a rat model of 5/6 nephrectomy (Nx), rats in the treatment groups received low- or high-dose BYF. At the end of the study, serum and kidney samples were collected for biochemical, pathological, and western blotting analysis. Metabolic changes in serum were analyzed by liquid chromatography-tandem mass spectrometry.ResultsThe results showed that BYF treatment could reduce kidney injury, inhibit inflammation and improve renal function in a dose-dependent manner. In total, 405 and 195 metabolites were identified in negative and positive ion modes, respectively. Metabolic pathway enrichment analysis of differential metabolites based on the Kyoto Encyclopedia of Genes and Genomes database identified 35 metabolic pathways, 3 of which were related to tryptophan metabolism. High-dose BYF reduced the level of kynurenic acid (KA) by more than 50%, while increasing melatonin 25-fold and indole-3-acetic acid twofold. Expression levels of aryl hydrocarbon receptor (AhR), Cyp1A1, and CyP1B1 were significantly reduced in the kidney tissue of rats with high-dose BYF, compared to 5/6 Nx rats.ConclusionBYF has a reno-protective effect against 5/6 Nx-induced CKD, which may be mediated via inhibition of the tryptophan-KA-AhR pathway.

Influence of Bisphenol Compounds at Nanomolar Concentrations on Chromosome Damage Induced by Metabolically Activated Carcinogens in HepG2 Cells

Bisphenol (BP) compounds are endocrine-disrupting organic pollutants. BPs may increase the messenger RNA (mRNA) transcripts of nuclear receptors (NRs) regulating the expression of xenobiotic-metabolizing cytochrome P450 (CYP) enzymes. Their impact on the genotoxicity of metabolically activated carcinogens, however, remains unknown. In this study, effects of the bisphenols A, F, S, and AF on the expression of the aryl hydrocarbon receptor (AhR), the pregnane X receptor (PXR), the constitutive androstane receptor, and individual xenobiotic-metabolizing CYP enzymes in a human hepatoma (HepG2) cell line were investigated, along with in silico binding studies of BPs to each receptor. The results indicated that each BP at 1 to 100 nM concentrations increased the mRNA transcripts and protein levels of AhR, PXR, CYP1A1, 1A2, 1B1, 2E1, and 3A4. The predicted affinities of the BPs for binding AhR were comparable to those of potent agonists. Pretreatment of HepG2 cells with each BP potentiated the induction of micronuclei by benzo[a]pyrene, aflatoxin B1, benzene, and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; this effect was abolished/reduced by inhibitors of NRs and/or CYPs. Our study suggests that BPs at human exposure levels may aggravate chromosome damage by several impactful carcinogens in human cells by inducing relevant CYP enzymes, mostly via NR modulation.

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

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

Toxicity of malathion during Senegalese sole, Solea senegalensis larval development and metamorphosis: Histopathological disorders and effects on type B esterases and CYP1A enzymatic systems.

The toxicity of malathion to Solea senegalensis was studied in a static renewal bioassay during its first month of larval life (between 4 and 30 dph). Through the use of different biomarkers and biochemical, cellular and molecular approaches (inhibition of cholinesterases [ChEs], changes in cytochrome P450-1A [CYP1A] and the study of histopathological alterations), the effects of three concentrations of malathion (1.56, 3.12, and 6.25 μg/L) have been analyzed. In subacute exposure, malathion inhibited cholinesterase activities (AChE, BChE, CbE) in a dose- and time-dependent manner, ranging the inhibition percentage from 20% to 90%. However, the expression levels of CYP1A and AChE transcripts or proteins were not modified. Additionally, exposure to malathion provoked histopathological alterations in several organ systems of Senegalese sole in a time- and dose dependent way, namely disruption of parenchymal architecture in the liver, epithelial desquamation, pyknotic nuclei and steatosis in the intestine, disorganization of supporting cartilage, and sings of hyperplasia and hypertrophy in the gills and degeneration of the epithelial cells from the renal tubules. Malathion exposure also provoked strong disorganization of cardiac fibers from the heart. The findings provide evidence that exposure to sublethal concentrations of malathion that provoked serious injury to the fish S. senegalensis, were below the expected environmental concentrations reported in many other ecosystems and different fish species,revealing a higher sensitivity for Solea senegalensis to malathion exposure, thus reinforcing its use as sentinel species for environmental pollution in coastal and estuarine environments.

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

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

Characterization of a commercially available line of iPSC hepatocytes as models of hepatocyte function and toxicity for regulatory purposes

It has recently become possible to produce hepatocytes from human induced pluripotent stem cells (iPSC-heps), which may offer some advantages over primary human hepatocytes (Prim-heps) in the regulatory environment. The aim of this research was to assess similarities and differences between commercially available iPSC-heps and Prim-heps in preliminary assays of drug metabolism, hepatotoxicity, and drug transport. Hepatocytes were either cultured in collagen-coated 96-well plates (Prim-heps and 2d-iPSC-heps) or in ultra-low adhesion plates as spheroids (3d-iPSC-heps). 3d-iPSC-heps were used to enhance physiological cell-cell contacts, which is essential to maintain the phenotype of mature hepatocytes. Cytochrome P450 (CYP) 3A4, CYP1A2, and CYP2B6 activity levels were evaluated using fluorescent assays. Phase II metabolism was assessed by HPLC measurement of formation of glucuronides and sulfates of 4-methylumbelliferone, 1-naphthol, and estradiol. The toxicity of acetaminophen, amiodarone, aspirin, clozapine, tacrine, tamoxifen, and troglitazone was monitored using a luminescent cell viability assay. Canaliculi formation was monitored by following the fluorescence of 5,6-carboxy-2′,7′-dichlorofluorescein diacetate. All culture models showed similar levels of basal CYP3A4, CYP1A2 and CYP2B6 activity. However, while Prim-heps showed a vigorous response to CYP inducing agents, 2d-iPSC-heps showed no response and 3d-iPSC-heps displayed an inconclusive response. 2d-iPSC-heps showed reduced, yet appreciable, glucuronide and sulfate formation compared to Prim-heps. All culture models showed similar activity in tests of hepatotoxicity, with Prim-heps generally being more sensitive. All models formed canaliculi capable of transporting carboxy-2′,7′-dichlorofluorescein. The iPSC-heps appear to be useful for toxicity and transport studies, but metabolic activity is not optimum, and metabolism studies would benefit from a more mature model.

Neurons expressing the aryl hydrocarbon receptor in the locus coeruleus and island of Calleja major are novel targets of dioxin in the mouse brain

The aryl hydrocarbon receptor (AhR) acts as a receptor that responds to ligands, including dioxin. The AhR–ligand complex translocates from the cytoplasm into the nucleus to induce gene expression. Because dioxin exposure impairs cognitive and neurobehavioral functions, AhR-expressing neurons need to be identified for elucidation of the dioxin neurotoxicity mechanism. Immunohistochemistry was performed to detect AhR-expressing neurons in the mouse brain and confirm the specificity of the anti-AhR antibody using Ahr−/− mice. Intracellular distribution of AhR and expression level of AhR-target genes, Cyp1a1, Cyp1b1, and Ahr repressor (Ahrr), were analyzed by immunohistochemistry and quantitative RT-PCR, respectively, using mice exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The mouse brains were shown to harbor AhR in neurons of the locus coeruleus (LC) and island of Calleja major (ICjM) during developmental period in Ahr+/+ mice but not in Ahr−/− mice. A significant increase in nuclear AhR of ICjM neurons but not LC neurons was found in 14-day-old mice compared to 5- and 7-day-old mice. AhR was significantly translocated into the nucleus in LC and ICjM neurons of TCDD-exposed adult mice. Additionally, the expression levels of Cyp1a1, Cyp1b1, and Ahrr genes in the brain, a surrogate of TCDD in the tissue, were significantly increased by dioxin exposure, suggesting that dioxin-activated AhR induces gene expression in LC and ICjM neurons. This histochemical study shows the ligand-induced nuclear translocation of AhR at the single-neuron level in vivo. Thus, the neurotoxicological significance of the dioxin-activated AhR in the LC and ICjM warrants further studies.

Tumour necrosis factor-α (TNF-α) enhances dietary carcinogen-induced DNA damage in colorectal cancer epithelial cells through activation of JNK signaling pathway

Colorectal cancer (CRC) is the third most common cancer worldwide and the second leading cause of cancer death. Benzo[a]pyrene (BaP) and 2-amino-1-methyl-6-phenylimidazol [4,5-b] pyridine (PhIP) present in cooked meat are pro-carcinogens and considered to be potential risk factors for CRC. Their carcinogenic and mutagenic effects require metabolic activation primarily by cytochrome P450 1 family enzymes (CYPs); the expression of these enzymes can be modulated by aryl hydrocarbon receptor (AhR) activation and the tumour microenvironment, involving mediators of inflammation. In this study, we hypothesized that tumour necrosis factor-α (TNF-α), a key mediator of inflammation, modulates BaP- and PhIP-induced DNA damage in colon cancer epithelial cells. Importantly, we observed that TNF-α alone (0.1–100 pg/ml) induced DNA damage (micronuclei formation) in HCT-116 cells and co-treatment of TNF-α with BaP or PhIP showed higher levels of DNA damage compared to the individual single treatments. TNF-α alone or in combination with BaP or PhIP did not affect the expression levels of CYP1A1 and CYP1B1 (target genes of AhR signaling pathways). The DNA damage induced by TNF-α was elevated in p53 null HTC-116 cells compared to wild type cells, suggesting that TNF-α-induced DNA damage is suppressed by functional p53. In contrast, p53 status failed to affect BaP and PhIP induced micronucleus frequency. Furthermore, JNK and NF-κB signaling pathway were activated by TNF-α treatment but only inhibition of JNK significantly reduced TNF-α-induced DNA damage. Collectively, these findings suggest that TNF-α induced DNA damage involves JNK signaling pathway rather than AhR and NF-κB pathways in colon cancer epithelial cells.

Endogenous ligands of Aryl hydrocarbon receptor may play an important role in DNA damage responses

Aryl hydrocarbon receptor (AhR) was initially discovered as a cellular protein involved in mediating the detoxification of xenobiotic compounds including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Subsequent studies have identified several families of metabolic products as endogenous AhR agonists such as 6-Formylindolo[3,2-b] carbazole (FICZ) and kynurenine and revealed important physiological roles of AhR in the regulation of metabolic homeostasis and immune responses. Recently, we found that treatment with DNA damage agents including etoposide, doxorubicin, and hexavalent chromium resulted in elevated levels of AhR and CYP1A2, the latter being an AhR transcriptional target. Moreover, we found that treatment with the oxidant hydrogen peroxide stimulated AhR expression, which was also accompanied by enhanced expression of CYP1A2. It has been shown that hydrogen peroxide is capable of converting tryptophan into FICZ in vitro. Thus, we speculated that oxidant-induced activation of the AhR axis was mediated through the generation of AhR ligand(s) in vivo. Supporting this, we showed that pre-incubation of tryptophan that was dissolved in PBS with hydrogen peroxide elicited a rapid induction of CYP1A2 expression with an kinetics similar to that of FICZ. Given that a common feature of treatment with etoposide, doxorubicin, and hexavalent chromium is the generation of reactive oxygen species in vivo, we propose that enhanced expression of AhR by these DNA damage agents is primarily due to an elevated level of tryptophan metabolic products including FICZ and that the AhR signaling axis may play a significant role in limiting the extent of DNA damage caused by these compounds.

Detrimental effects of 6 months exposure to very low doses of a mixture of six pesticides associated with chronic vitamin deficiency on rats

This study aimed to evaluate the long-term low-dose effects of exposure to a mixture of 6 pesticide active substances (diquat, imazamox, imazethapyr, tepraloxydin, bentazone, acifluorfen) and to elucidate if chronic vitamin deficiency can influence their toxicity. Two hundred Wistar rats were divided in 4 groups: a vitamin-sufficiency control group, a vitamin-deficiency control group, a vitamin sufficiency test group and a vitamin-deficiency test group. The test groups were treated with the aforementioned pesticides at doses 100 times lower than the corresponding NOAEL. After 6 months, ten rats from each group were sacrificed and a complete evaluation of blood and urine biochemistry, biomarkers of oxidative stress, xenobiotic detoxification enzymes and lysosomal enzymes and organ histopathology was performed. The pesticides mixture and vitamin deficiency determined an increase in alkaline phosphatase levels and urinary calcium levels, abnormal serum lipid profile, and a decrease of total blood proteins levels, red blood cells, haematocrit and haemoglobin. The combination of the two stressors up-regulated CYP1A1, CYP1A2, CYP2B1 and GST levels. This study provides a new proof for the need to move forward from single chemical testing to a more complex approach to account for the multitude of stressors that can challenge the setting of real safety levels.

Trichostatin A sensitizes hepatoma cells to Taxol more than 5-Aza-dC and dexamethasone.

This work was designed to compare the sensitizing effects of epigenetic modifiers on cancer cells vs. that of glucocorticoids. Also, to evaluate their effects on genes involved in epigenetic changes and drug metabolism. Hepatoma cells (HepG2) were treated with the anticancer drug (Taxol), with a histone deacetylase inhibitor (Trichostatin A [TSA]), DNA methyltransferase inhibitor (5-Aza-dC) or dexamethasone (DEX). Cytotoxicity was assessed by MTT assay and the apoptosis was determined by Annexin V-FITC. The expression levels of HDAC1, HDAC3, Dnmt1, Dnmt3α, CYP1A2, CYP3A4, CYP2B6, CYP2C19 and CYP2D6 were monitored by qRT-PCR. TSA, synergistically enhanced cells sensitivity with the anticancer effect of Taxol more than 5-Aza-dC and DEX. This was evidenced by the relative decrease in IC50 in cells cotreated with Taxol+TSA, Taxol+5-Aza-dC or Taxol+DEX. Apoptosis was induced in 51.2, 16.9 and 41.3% of cells, respectively. In presence of Taxol, TSA induced four-fold increase in the expression of HDAC1 and downregulated Dnmt1&3α genes. CYP2D6 demonstrated progressive expression (up to 28-fold) with the increasing number of drugs. Moreover, the isoform overexpressed in cells treated with TSA+Taxol>DEX+Taxol>5-Aza-dC+Taxol (6.4, 4.6 and 2.99, respectively). The investigated genes were clustered in two distinct subsets, where no coregulation was observed between HDAC1 and HDAC3. However, tight pairwise correlation-based cluster was seen between (CYP3A4/Dnmt3α and CYP2D6/CYP2C19). The data reflects the sensitizing effect of acetylation modification by TSA on the responsiveness of hepatoma cells to anticancer therapy. The effect of histone deacetylase inhibition was more than hypomethylation and glucocorticoid effects. TSA exerts its role through its modulatory role on epigenetics and drugs metabolizing genes. Other modifiers (5-Aza-dC and DEX), however may adopt different mechanisms.

Trichostatin A sensitizes hepatoma cells to Taxol more than 5-Aza-dC and dexamethasone.

This work was designed to compare the sensitizing effects of epigenetic modifiers on cancer cells vs. that of glucocorticoids. Also, to evaluate their effects on genes involved in epigenetic changes and drug metabolism. Hepatoma cells (HepG2) were treated with the anticancer drug (Taxol), with a histone deacetylase inhibitor (Trichostatin A [TSA]), DNA methyltransferase inhibitor (5-Aza-dC) or dexamethasone (DEX). Cytotoxicity was assessed by MTT assay and the apoptosis was determined by Annexin V-FITC. The expression levels of HDAC1, HDAC3, Dnmt1, Dnmt3α, CYP1A2, CYP3A4, CYP2B6, CYP2C19 and CYP2D6 were monitored by qRT-PCR. TSA, synergistically enhanced cells sensitivity with the anticancer effect of Taxol more than 5-Aza-dC and DEX. This was evidenced by the relative decrease in IC50 in cells cotreated with Taxol+TSA, Taxol+5-Aza-dC or Taxol+DEX. Apoptosis was induced in 51.2, 16.9 and 41.3% of cells, respectively. In presence of Taxol, TSA induced four-fold increase in the expression of HDAC1 and downregulated Dnmt1&3α genes. CYP2D6 demonstrated progressive expression (up to 28-fold) with the increasing number of drugs. Moreover, the isoform overexpressed in cells treated with TSA+Taxol>DEX+Taxol>5-Aza-dC+Taxol (6.4, 4.6 and 2.99, respectively). The investigated genes were clustered in two distinct subsets, where no coregulation was observed between HDAC1 and HDAC3. However, tight pairwise correlation-based cluster was seen between (CYP3A4/Dnmt3α and CYP2D6/CYP2C19). The data reflects the sensitizing effect of acetylation modification by TSA on the responsiveness of hepatoma cells to anticancer therapy. The effect of histone deacetylase inhibition was more than hypomethylation and glucocorticoid effects. TSA exerts its role through its modulatory role on epigenetics and drugs metabolizing genes. Other modifiers (5-Aza-dC and DEX), however may adopt different mechanisms.

Anti-androgenic effect of astaxanthin in LNCaP cells is mediated through the aryl hydrocarbon-androgen receptors cross talk.

The aim of this study was to investigate the anti-androgenic effects of astaxanthin (AST) on human prostatic cancer cell growth, and its impact on androgen receptor (AR) signaling using prostate cancer (PCa) cell line LNCaP. LNCaP cells were treated with AST alone and in combination with CH223191 and flutamide (Flu) in the presence and absence of testosterone. MTT assay, cellular prostate-specific antigen (PSA) and dihydrotestosterone (DHT) production, mRNA levels of CYP1A1, PSA, Kallikrein-Related Peptidase 2 (KLK2), Transmembrane Serine Protease 2 (TMPRSS2), and AR genes were measured as endpoints. The expression of CYP1A1, PSA, KLK2, TMPRSS2, and AR mRNA levels was decreased which results in reducing the production of PSA and DHT in the presence of testosterone. Our data clearly demonstrate that AST has a potential ability to suppress the human prostate LNCaP cells growth at high concentrations. AST was able to repress the testosterone-induced transcription of AR-target genes. PRACTICAL APPLICATIONS: Astaxanthin is a natural compound with the most potent antioxidant activity among other antioxidants. In the current study, ASX suppressed the LNCaP cells at high concentrations. Furthermore, AST inhibited testosterone-induced transcriptional activation of androgen-related genes. AST induced the expression of CYP1A1, which is able to metabolize the steroid hormones. It seems that AST can act as AhR exogenous ligand by induction of CYP1A1, which results in testosterone metabolism and consequent suppression of AR genes. So that, AST could prevent the growth of testosterone-dependent PCa cells, downregulate downstream genes in testosterone pathways, and enhance the metabolism of testosterone via AhR pathway. Collectively, AST could be considered as a potential candidate for the treatment of PCa.

The Long Noncoding RNA Hepatocyte Nuclear Factor 4α Antisense RNA 1 Negatively Regulates Cytochrome P450 Enzymes in Huh7 Cells via Histone Modifications.

The maintenance of homeostasis of cytochromes P450 enzymes (P450s) under both physiologic and xenobiotic exposure conditions is ensured by the action of positive and negative regulators. In the current study, the hepatocyte nuclear factor 4α (HNF4A) antisense RNA 1 (HNF4A-AS1), an antisense long noncoding RNA of HNF4A, was found to be a negative regulator of the basal and rifampicin (RIF)-induced expression of nuclear receptors and downstream P450s. In Huh7 cells, knockdown of HNF4A-AS1 resulted in elevated expression of HNF4A, pregnane X receptor (PXR), and P450s (including CYP3A4) under both basal and RIF-induced conditions. Conversely, overexpression of HNF4A-AS1 led to decreased basal expression of constitutive androstane receptor, aryl hydrocarbon receptor, PXR, and all studied P450s. Of note, significantly diminished induction levels of PXR and CYP1A2, 2C8, 2C19, and 3A4 by RIF were also observed in HNF4A-AS1 plasmid-transfected Huh7 cells. Moreover, the negative feedback of HNF4A on HNF4A-AS1-mediated gene expression was validated using a loss-of-function experiment in this study. Strikingly, our data showed that increased enrichment levels of histone 3 lysine 4 trimethylation and HNF4A in the CYP3A4 promoter contribute to the elevated CYP3A4 expression after HNF4A-AS1 knockdown. Overall, the current study reveals that histone modifications contribute to the negative regulation of nuclear receptors and P450s by HNF4A-AS1 in basal and drug-induced levels. SIGNIFICANCE STATEMENT: Utilizing loss-of-function and gain-of-function experiments, the current study systematically investigated the negative regulation of HNF4A-AS1 on the expression of nuclear receptors (including HNF4A, constitutive androstane receptor, aryl hydrocarbon receptor, and pregnane X receptor) and P450s (including CYP1A2, 2E1, 2B6, 2D6, 2C8, 2C9, 2C19, and 3A4) in both basal and rifampicin-induced levels in Huh7 cells. Notably, this study is the first to reveal the contribution of histone modification to the HNF4A-AS1-mediated expression of CYP3A4 in Huh7 cells.

Chrysene, a four-ring polycyclic aromatic hydrocarbon, induces hepatotoxicity in mice by activation of the aryl hydrocarbon receptor (AhR)

Polycyclic aromatic hydrocarbons (PAHs) are a group of persistent organic global environmental pollutants and cause harmful effects on human health. Here, we evaluated adverse effects of chrysene, which is a four-ring PAH and an important member of 16 priority PAHs, on the liver. Chrysene was detected in some common raw and cooked Chinese food samples. Hepatotoxicity including increased relative liver weight, hepatocyte swelling and degeneration, and elevated serum alanine aminotransferase (ALT) levels were observed in chrysene-exposed C57BL/6 mice. Glutamine treatment effectively ameliorated chrysene-induced mice liver injury by decreasing serum ALT levels. Chrysene induced mice hepatic glutathione depletion and oxidative DNA damage with increased 8-hydroxy-2′-deoxyguanosine (8-OHdG) levels. Hepatic expression levels of the aryl hydrocarbon receptor (AhR), AhR-related target genes including CYP1A1, CYP1A2 and CYP1B1, and AhR nuclear translocator (ARNT) were significantly increased in chrysene-exposed C57BL/6 mice. Chrysene induced mice hepatic mRNA levels of the nuclear factor erythroid 2–related factor 2 (Nrf2) and Nrf2-mediated phase II detoxifying and antioxidant enzymes including NQO1, UGT1A1, UGT1A6, SULT1A1, GSTm1, GSTm3, Catalase (CAT), GPx1, and SOD2. We found that chrysene had toxic effects including increased relative liver weight and elevated serum ALT levels on AhR+/+ mice but not AhR−/- mice. Chrysene significantly induced hepatic mRNA levels of CYP1A1 and CYP1A2 in AhR+/+ mice but not AhR−/- mice. To our knowledge, this study is the first to demonstrate that hepatotoxicity causes by chrysene is dependent on AhR, and Nrf2 plays an important regulation role in protection against oxidative liver injury induced by chrysene.

The Effectiveness of Dietary Byproduct Antioxidants on Induced CYP Genes Expression and Histological Alteration in Piglets Liver and Kidney Fed with Aflatoxin B1 and Ochratoxin A.

The purpose of this study was to investigate the potential of a byproduct mixture derived from grapeseed and sea buckthorn oil industry to mitigate the harmful damage produced by ochratoxin A and aflatoxin B1 at hepatic and renal level in piglets after weaning. Forty cross-bred TOPIGS-40 hybrid piglets after weaning were assigned to three experimental groups (E1, E2, E3) and one control group (C), and fed with experimental diets for 30 days. The basal diet was served as a control and contained normal compound feed for starter piglets without mycotoxins. The experimental groups were fed as follows: E1—basal diet plus a mixture (1:1) of two byproducts (grapeseed and sea buckthorn meal); E2—the basal diet experimentally contaminated with mycotoxins (479 ppb OTA and 62ppb AFB1); and E3—basal diet containing 5% of the mixture (1:1) of grapeseed and sea buckthorn meal and contaminated with the mix of OTA and AFB1. After 4 weeks, the animals were slaughtered, and tissue samples were taken from liver and kidney in order to perform gene expression and histological analysis. The gene expression analysis showed that when weaned piglets were fed with contaminated diet, the expression of most analyzed genes was downregulated. Among the CYP450 family, CYP1A2 was the gene with the highest downregulation. According to these results, in liver, we found that mycotoxins induced histomorphological alterations in liver and kidney and had an effect on the expression level of CYP1A2, CYP2A19, CYP2E1, and CYP3A29, but we did not detect important changes in the expression level of CY4A24, MRP2 and GSTA1 genes.

663 Specific polycyclic aromatic hydrocarbons elicit differential expression of CYP1A1 in trophoblast cells

Prenatal exposures to polycyclic aromatic hydrocarbons (PAHs) are associated with adverse outcomes including small for gestational age infants. We have previously reported differential levels of PAHs in the placenta associated with preterm delivery. We found that levels of a specific PAH, dibenzoanthracene (DBA), was significantly higher than levels of either benzo[a]pyrene (BaP) or Benzo[b]fluoranthene (BbF). Furthermore, we reported that placental DBA levels are significantly and negatively correlated with gestational age at delivery. In this study we aimed to determine if there is a differential response in trophoblast cells with distinct PAH treatments. Immortalized choriocarcinoma cells (BeWo) were treated with 10 μM of either BaP, BbF or DBA for 24 hours. DMSO treatment served as a control. Media was collected and measured to determine levels of hCG production using an ELISA assay. RNA was extracted from cells after 24 hours of treatment, and used to make cDNA. TaqMan primers and probes were used to measure expression levels of CYP1A1, a gene involved in the processing of xenobiotic compounds, using RT-qPCR. GAPDH served as a control. While treatment with each PAH increased CYP1A1 expression compared with DMSO, DBA elicited the largest response, with a 54-fold increase in CYP1A1 expression (p<0.001). CYP1A1 expression was increased 16.5-fold with BbF treatment (p=0.001), and 7.4-fold with BaP treatment (p=0.006). Levels of hCG were not altered with any PAH treatment compared with DMSO, consistent with previous publications in trophoblast cells. In placenta tissue, levels of DBA are found at higher levels compared with BaP and BbF, and DBA levels are increased in placentae from preterm birth. In this study we find that DBA elicits a higher upregulation of CYP1A1 compared with BaP and BbF. Further experimentation is necessary using RNA-Seq analyses to help yield insight into pathways differentially responsive to DBA, yielding insight into its association with preterm delivery.

Elevated levels of expression of cytochrome P450 3A4 in a human liver epithelial cell line in differentiation-inducing conditions.

Cytochrome P450 (CYP) enzymes, especially CYP3A4 play a major role in the metabolism of xenobiotics in human liver. CYP3A4-expressing human liver or hepatoma cell lines may be good cell substitutes of human hepatocytes for drug metabolism studies. However, there are only a few cell lines expressing high levels of CYP3A4. The aim of this study is to investigate the expression of CYP3A4 and its mechanism in an immortalized non-tumorigenic human liver epithelial cell line, THLE-5b in differentiation-inducing conditions. When THLE-5b cells were cultivated in culture medium supplemented with hepatocytic differentiation-inducing factors, they showed hepatocytic morphology. In addition, elevated levels of expression not only of α1-antitrypsin (AAT) and albumin (ALB) mRNAs, but also of CYP3A4 mRNA, which are functional hepatocyte markers, were observed compared with the control. Among hepatocytic differentiation-inducing factors, dexamethasone (DEX) and insulin-transferrin-sodium selenite (ITS) seemed to be involved in elevation of expression of CYP3A4 mRNA. The mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) inhibitor U0126 or the phosphatidylinositol-3-kinase (PI3K) inhibitor LY294002 reduced CYP3A4 mRNA levels of THLE-5b cells. Furthermore, the CpG site of the CYP3A4 promoter region in THLE-5b cells was found to be unmethylated, although in low CYP3A4-expressing HepG2 cells, the site was methylated. In conclusion, THLE-5b cells, which are unmethylated at the CpG site of the CYP3A4 promoter region, express CYP3A4 mRNA through the MEK/ERK1/2 and PI3K/Akt signaling pathways and acquire hepatocytic functions in differentiation-inducing conditions. Thus, THLE-5b cells could be a useful cell system for the study of drug metabolism.

Effects of Curcuma comosa Roxb. Extract on the Expression of CYP450s in Immortalized Hepatocyte-like Cells (imHC)

Hepatocytes are the most abundant liver cells that produce biotransformation enzymes, including phase I metabolism enzymes and cytochrome P450s (CYP450s). These enzymes are induced or suppressed by several drugs and chemicals. Here, immortalized hepatocyte-like cells (imHC) were applied as a novel hepatocyte to study the CYP450 enzyme expression induced by a Thai folk herb, Curcuma comosa Roxb. (C. comosa). The results demonstrated that the mRNA expression levels were dependent on cell cultivation conditions, the extract's concentration, and the duration of treatment. In the 3-day cultivation, 1 µg/mL C. comosa extract upregulated the mRNA expression levels of CYP1A1, CYP1A2, CYP2B6, CYP2C9, CYP2D6, CYP2E1 and CYP3A4 in imHC, after treatment for 48 h, which was comparable to the expression levels of those genes in HepG2 cells. In addition, C. comosa increased CYP3A4 protein expression in both imHC and HepG2 cells. Furthermore, 1 µg/mLC. comosa could neutralize erythromycin's inhibitory effect, a specific CYP3A4 inhibitor, on CYP3A4 enzyme activity. In conclusion, imHC exhibited several detoxification enzymes, and this cell could be used to replace hepatic carcinoma cells in hepatotoxic studies. The function of C. comosa as a CYP enzyme-inducing agent indicates a promising treatment for the prevention of drug-induced hepatotoxicity by activating detoxification enzymes.

Benzo(a)pyrene induces MUC5AC expression through the AhR/mitochondrial ROS/ERK pathway in airway epithelial cells

ObjectivesBenzo(a)pyrene (BaP) is a ubiquitous air pollutants, and BaP exposure leads to a risk of respiratory diseases. The oversecretion of airway mucus and high expression of mucin 5AC (MUC5AC) are associated with common respiratory disorders caused by air pollution. We aimed to investigate the effect of BaP on MUC5AC expression, especially the mechanisms by which BaP induces MUC5AC gene expression. MethodsThe human airway epithelial cell NCI-H292 was used to test the effects of BaP on the expression of MUC5AC in vitro. MUC5AC mRNA and protein expression were assessed with real-time quantitative PCR, immunochemistry, and western blotting. A luciferase assay was conducted to detect the activity of the promoter. The total cellular ROS and mitochondrial ROS were measured by corresponding probes. Small-interfering RNAs were used for gene silencing. AhR-overexpressing cell lines were constructed by transfection with AhR overexpression lentivirus. ResultsWe found that BaP stimulation upregulated the MUC5AC mRNA and protein levels and activated the ERK pathway. Suppressing ERK with U0126 (an ERK inhibitor) or knocking down ERK with siRNA decreased BaP-induced MUC5AC expression. The luciferase activity transfected with the MUC5AC promoter and cAMP-response element (CRE) was increased after BaP treatment, whereas CREB siRNA suppressed the BaP-induced overexpression of MUC5AC. In addition, BaP increased mitochondrial ROS production, and Mito-TEMP, a mitochondrial ROS inhibitor, inhibited BaP-induced MUC5AC expression and ERK activation. BaP increased the mRNA levels of CYP1A1 and CYP1B1, while Alizarin, a CYP1s inhibitor, suppressed the effects of BaP, including the MUC5AC overexpression, ERK activation and mitochondrial ROS generation. BaP induced the translocation of aryl hydrocarbon receptor (AhR) from the cytoplasm to the nucleus. SiRNA-mediated knockdown or chemical inhibition of AhR decreased the BaP-induced expression of MUC5AC, while the overexpression of AhR significantly enhanced the BaP-induced expression of MUC5AC. ITE, an endogenous AhR ligand, also upregulated the mRNA and protein expression of MUC5AC. Furthermore, resveratrol treatment inhibited the BaP-induced MUC5AC overexpression, AhR translocation, mitochondrial ROS production and ERK pathway activation. ConclusionHere, we highlighted the crucial role of AhR/mitochondrial ROS/ERK pathway activation in BaP-induced MUC5AC overexpression and identified resveratrol as a promising drug to reduce BaP-induced MUC5AC overexpression.