Cytochrome P450 mRNA Expression Research Articles

Annona Muricata L. extract restores renal function, oxidative stress, immunohistochemical structure, and gene expression of TNF-α, IL-β1, and CYP2E1 in the kidney of DMBA-intoxicated rats.

Introduction: 7,12-dimethylbenz (a) anthracene (DMBA) is a harmful polycyclic aromatic hydrocarbon derivative known for its cytotoxic, carcinogenic, and mutagenic effects in mammals and other species. Annona muricata, L. (Graviola; GRV) is a tropical fruit tree traditionally well-documented for its various medicinal benefits. This investigation is the first report on the potential antioxidant and antinfammatory reno-protective impact of GRV against DMBA-induced nephrotoxicity in rats. Methods: Forty male albino rats were allocated into four equal groups (n = 10). The 1st group served as the control, the 2nd group (GRV) was gastro-gavaged with GRV (200mg/kg b.wt), the 3rd group (DMBA) was treated with a single dose of DMBA (15mg/kg body weight), and the 4th group (DMBA + GRV) was gastro-gavaged with a single dose of DMBA, followed by GRV (200mg/kg b.wt). The GRV administration was continued for 8weeks. Results and Discussion: Results revealed a significant improvement in renal function, represented by a decrease in urea, creatinine, and uric acid (UA) in the DMBA + GRV group. The antioxidant potential of GRV was confirmed in the DMBA + GRV group by a significant decline in malondialdehyde (MDA) and a significant increase in catalase (CAT), superoxide dismutase (SOD), glutathione S transferase (GST), and reduced glutathione (GSH) compared to DMBA-intoxicated rats; however, it was not identical to the control. Additionally, the antiinflammatory role of GRV was suggested by a significant decline in mRNA expression of cytochrome P450, family 2, subfamily e, polypeptide 1 (CYP2E1), tumor necrosis factor-alpha (TNF-α), and interleukin 1 beta (IL-1β) in the DMBA + GRV group. Moreover, GRV improved the histopathologic and immunohistochemical expression of TNF-α, CYP450, and IL1β in DMBA-intoxicated kidney tissue. Conclusively, GRV is a natural medicinal product that can alleviate the renal injury resulting from environmental exposure to DMBA. The reno-protective effects of GRV may involve its anti-inflammatory and/or antioxidant properties, which are based on the presence of phytochemical compounds such as acetogenins, alkaloids, and flavonoids.

Upregulation of Beta 1 and Arachidonic Acid Metabolizing Enzymes in the Mouse Hearts and Kidneys after Sub Chronic Administration of Rofecoxib.

An imbalance in the levels of arachidonic acid (ARA) metabolites in cardiovascular disorders and drug-induced cardiotoxicity have been previously described. This study aimed to investigate the influence of cyclooxygenase-2 (COX-2) selective inhibitors on the gene expression of ARA-metabolizing genes and beta1 gene in the hearts and kidneys of experimental mice. Thirty-five balb/c mice were divided into five groups with seven mice per group. The groups were then given two distinct types of COX-2 selective inhibitors, rofecoxib and celecoxib, in two different doses equivalent to those used in human treatment for 30 days. The mRNA expression of beta1, ace2, and ARA-metabolizing genes, coxs, lipoxygenases (aloxs), and cytochrome p450 (cyp450s) in mice heart and kidneys were assessed. Genes were analyzed using real-time polymerase chain reaction analysis. In addition, rofecoxib-induced histological alterations were examined. It was found that only the high dose of rofecoxib (5 mg/kg) caused toxicological alterations, a finding that was indicated by a significant increase (P < 0.05) in the relative weight of the mouse hearts and increase in the ventricle wall thickness as observed through pathohistological examination. This increase was associated with a significant increase in the mRNA expression level of the beta1 receptor in both the heart and kidneys of the mice (53- and 12-fold, respectively). The expression of both cox1 and 2 genes was increased 4-fold in the kidneys. In addition, the expression of the alox12 gene increased significantly (by 67-fold in the heart and by 21-fold in the kidney), while alox15 gene expression was upregulated in the heart by 8-fold and 5-fold in the kidney. The genes responsible for synthesizing 20- Hydroxyeicosatetraenoic acid (cyp4a12 and cyp1a1) were significantly upregulated (P < 0.05) in the hearts of high-dose rofecoxib-treated mice by 7- and 17 -fold, respectively. In addition, the expression of epoxyeicosatrienoic acid-synthesizing genes, cyp2c29 and cyp2j5, was increased significantly (P < 0.05) in the hearts of high-dose rofecoxib-treated mice by 4- and 16-fold, respectively. Rofecoxib caused upregulation of the mRNA expression of the beta 1 gene in association with increased expression of ARA-metabolizing genes in mouse hearts and kidneys. These findings may help us understand the molecular cardiotoxic mechanism of rofecoxib.

6-Formylindolo[3,2-b]carbazole Protects Against Angiotensin II-Induced Cellular Hypertrophy through the Induction of Cytochrome P450 1A1 and Its Associated 19(S)-HETE Metabolite In Vitro.

Aryl hydrocarbon receptor (AhR) is a multifunctional receptor that regulates cytochrome P450 1A1 (CYP1A1), an arachidonic acid (AA) metabolizing enzyme producing 19-hydroxyeicosatetraenoic acid (HETE). 6-formylindolo[3,2-b]carbazole (FICZ) demonstrates great affinity toward the AhR. Recently, we have shown that 19(S)-HETE is preferentially cardioprotective. This study investigates the role of FICZ on AhR and cytochrome P450 (CYP) 1A1-mediated AA metabolism and whether it attenuates angiotensin (Ang) II-induced cardiac hypertrophy. Adult human ventricular cardiomyocytes cell line treated with FICZ in the presence and absence of Ang II 10 μM. Protein levels of AhR and CYPs were determined by Western blot analysis and the mRNA expression of cardiac hypertrophic markers and CYPs were determined by real-time polymerase chain reaction. CYP1A1 enzyme activity and proteasomal degradation were determined by 7-ethoxyresorufin O-deethylase and proteasome 20S activity assays, respectively. Liquid chromatography tandem mass spectrometry was used to measure AA metabolites. Our results show that Ang II-induced cardiac hypertrophy modulates AA metabolites in an enantioselective manner, and that FICZ activates AhR in a time-dependent manner, inhibits AhR proteasomal degradation, induces CYP1A1, increases the concentration of 19(S)-HETE, and attenuates Ang II-induced cardiac hypertrophy by inhibiting the hypertrophic markers and decreasing cell surface area through midchain-HETE-dependent mechanism. In conclusion, the results demonstrate the ability of FICZ to protect against Ang II-induced cardiac hypertrophy by increasing the concentration of 19(S)-HETE through AhR regulated enzyme induction and inhibition of midchain-HETEs metabolites. SIGNIFICANCE STATEMENT: This study shows that 6-formylindolo[3,2-b]carbazole attenuate angiotensin II-induced cellular hypertrophy. The novel findings of our investigation are in characterizing the aryl hydrocarbon receptor involvement and the enantioselective differences in arachidonic acid metabolism in cardiac hypertrophy, which opens a new pathway to tackle and eventually treat heart failure.

Congener-Specific Uptake and Metabolism of Bisphenols in Carrot Cells: Dissipation Kinetics, Biotransformation, and Enzyme Responses.

Food consumption has been considered a key pathway of bisphenol compound (BP) exposure for humans. However, there is a lack of evidence concerning their congener-specific behavior and metabolism in plants. Herein, we examined the uptake and metabolism of five BPs in plants using carrot cells. Bisphenol S (BPS) and bisphenol AF (BPAF) exhibited substantially lower dissipation rates in the cells than the other BPs, indicating a strong selectivity in the uptake and metabolism among bisphenol congeners. For a total of 23 metabolites of BPs, the predominant biotransformation pathways were found to be glycosylation, methoxylation, and conjugation, while hydroxylation, methylation, and glutathionylation were only observed for some BPs. The changes in the mRNA expression of cytochrome P450 (P450) and the activities of glycosyltransferase and glutathione S-transferase were remarkably higher in cells exposed to bisphenol F, bisphenol A, and bisphenol B than in cells exposed to BPS and BPAF, indicating congener specificity in their effects on enzymes and the associated biotransformation processes. Consequently, the potential congener-specific differences in plant uptake, metabolism, and accumulation must be considered when assessing the environmental risks posed by these commonly used plasticizers.

Biochanin A in murine Schistosoma mansoni infection: effects on inflammation, oxidative stress and fibrosis.

Biochanin A (BCA) is a multifunctional natural compound that possesses anti-infective, anti-inflammatory, anti-oxidative and hepatoprotective effects. The aim of the study was to assess the therapeutic efficacy of BCA on Schistosoma mansoni-infected mice. Fifty mice were divided into six different groups as non-infected, non-infected BCA-treated, infected untreated, early infected BCA-treated (seven days post-infection (dpi)), late infected BCA-treated 60 dpi and infected praziquantel (PZQ)-treated groups. Parasitological, histopathological examination and immunohistochemical staining of transforming growth factor (TGF)-β, inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX-2) were investigated in liver sections. Cytochrome P450 (CYP450) gene expression of S. mansoni was evaluated by quantitative real-time polymerase chain reaction (RT-qPCR). A single dose of BCA significantly reduced worm burden in early (82.14%) and late infection (77.74%), mean tissue egg load in early (7.27 ± 0.495) and late BCA administration (7.63 ± 0.435) and decreased granuloma size. CYP450 mRNA expression was significantly reduced in early BCA treatment as compared to late treatment which emphasizes that early administration of BCA had more pronounced effects on worms than late administration. Both early and late BCA administration led to significant reduction in inflammatory cytokines as TGF and iNOS. Although the reduction of TGF and iNOS in BCA-treated mice was superior to PZQ, no statistically significant differences were noted. However, a significant downregulation of COX2 was noted in hepatocytes as compared to both infected control and PZQ-treated mice. BCA has schistosomicidal, anti-inflammatory, antioxidant and anti-fibrotic effects and could be regarded as a potential drug in schistosomiasis treatment.

Garcinia mangostana and α-Mangostin Revive Ulcerative Colitis-Modified Hepatic Cytochrome P450 Profiles in Mice.

&lt;b&gt;Background and Objective:&lt;/b&gt; Ulcerative colitis (UC) is inflammation of the large intestine with ulceration but can also cause extraintestinal manifestations (EIM) by damaging surrounding organs such as the liver. &lt;i&gt;Garcinia mangostana&lt;/i&gt; (GM) pericarp and α-mangostin (MGS) have been reported to have anti-inflammatory activity. This study evaluated the effects of GM pericarp extract and MGS on the expression of hepatic cytochrome P450 (CYP) enzymes as an EIM of UC. &lt;b&gt;Materials and Methods:&lt;/b&gt; Male ICR mice were orally administered GM pericarp extract (40, 200 and 1000 mg/kg/day), MGS (30 mg/kg/day) or sulfasalazine (SUL) (100 mg/kg/day) daily for 7 days. On days 4-7, UC was induced by dextran sulfate sodium (DSS 40 kDa, 6 g/kg/day). Profiles of CYP mRNA expression were determined by RT/qPCR. Alkoxyresorufin &lt;i&gt;O&lt;/i&gt;-dealkylation (including ethoxy-, methoxy-, pentoxy- and benzyloxy-resorufin), aniline hydroxylation and erythromycin &lt;i&gt;N&lt;/i&gt;-demethylation CYP responsive activities were also examined. &lt;b&gt;Results:&lt;/b&gt; The DSS-induced UC mice showed suppressed expression&lt;i&gt; &lt;/i&gt;of &lt;i&gt;Cyp1a1&lt;/i&gt;, &lt;i&gt;Cyp1a2&lt;/i&gt;, &lt;i&gt;Cyp2b9/10&lt;/i&gt;, &lt;i&gt;Cyp2e1&lt;/i&gt;, &lt;i&gt;Cyp2c29&lt;/i&gt;, &lt;i&gt;Cyp2d9&lt;/i&gt;, &lt;i&gt;Cyp3a11&lt;/i&gt; and &lt;i&gt;Cyp3a13&lt;/i&gt; mRNAs. The GM pericarp extract and MGS restored expression of all investigated CYPs and their responsive enzyme activities in DSS-induced UC mice to levels comparable to the control and parallel to the effects of the anti-inflammatory control SUL. &lt;b&gt;Conclusion:&lt;/b&gt; The GM is a promising therapy to restore UC-modified hepatic CYP profiles.

Long-term oral administration of Paederia foetida decreases cytochrome P450 mRNA expression: The predictive approaches in a rat model

Long-term oral administration of Paederia foetida decreases cytochrome P450 mRNA expression: The predictive approaches in a rat model

Salinomycin induces cell cycle arrest and apoptosis and modulates hepatic cytochrome P450 mRNA expression in HepG2/C3a cells

Salinomycin (SAL) is a monocarboxylic polyether ionophore antibiotic isolated from Streptomyces albus. It exhibits an effective antitumor potential against numerous human cancer cells. This study aimed to assess the antiproliferative effects of SAL in human hepatocellular carcinoma HepG2/C3a cell line. We investigated the effects of SAL on cell growth, DNA damage induction, cell cycle changes and apoptosis; and relative changes in expression of cell cycle-related, apoptosis-related, and CYP450 genes. SAL induced cell cycle arrest in the G2/M phase, upregulation of CDKN1A and GADD45A and downregulation of cyclin genes including CCNB1 and CCNA2. SAL effectively suppressed mRNA levels of CTNNB1 gene, an important oncogene that promotes tumorigenesis. The decrease of HepG2/C3A cells’ survival can also be due to downregulation of antiapoptotic BCL-2 expression, thus promoting the induction of apoptosis by SAL. This study also demonstrated the ability of SAL in modulating hepatic cytochrome P450 (CYP) mRNA expression, such that SAL caused the upregulation of CYP1A members and CYP3A5; and downregulation of CYP3A4. Taken together, these data contribute to the understanding of the mechanism of action of SAL, highlighting that metabolizing enzymes modulated by SAL can interfere with chemotherapy treatment and it must be considered in associated treatments.

Study on the effects of Zhuanggu Guanjie Pill, a modern Chinese medicine formula, on the activities and mRNA expression of seven CYP isozymes in rats

Ethnopharmacological relevanceZhuanggu Guanjie Pill (ZGGJP), a modern Chinese medicine formula, is composed of 12 herbs and has been used to treat osteoporosis in China for almost 30 years. However, no in vivo study of the influences of ZGGJP on the cytochrome P450 (CYP) activities have been reported. Aim of the studyThe aim of this study was to evaluate the effects of ZGGJP on the activities and the mRNA expression of CYP enzymes (CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A) and their corresponding nuclear receptor levels in rats. Materials and methodsAfter 7 days oral treatment of ZGGJP at low- and high-dose, cocktail solution was given to rats. Blood samples were collected at series of time points. The plasma concentrations of probe drugs and their corresponding metabolites were determined by UPLC-MS/MS. The influence of ZGGJP on the activities of seven CYPs were evaluated the metabolic ratios (Cmax and AUC0-t) for metabolites/probe drugs. In addition, the effects of ZGGJP on the mRNA expression of CYPs and their corresponding nuclear receptors in rat liver were evaluated by real-time PCR. ResultsZGGJP showed significant inductive effects on CYP1A2 and CYP2B6 of both male and female rats. The influence of ZGGJP on CYP2C9 and CYP3A showed gender difference. ZGGJP could induce the activities of CYP2C9 and CYP3A in female rats, but have no influence on the activities in male rats. ZGGJP had no effects on CYP2D6, CYP2C19 and CYP2E1. The mRNA expression results of CYPs were in accordance with the pharmacokinetic results. The mRNA expression levels of constitutive androstane receptor (CAR) and vitamin D receptor (VDR) were increased significantly in female rats at high dosage, but no significant changes were observed in male rats. ConclusionZGGJP had inductive effects on CYP1A2 and CYP2B6 in both male and female rats. The results showed that ZGGJP could induce the activities of CYP2C9 and CYP3A in female rats, but had no effect in male rats. This may suggest that the influence of ZGGJP on CYP2C9 and CYP3A exhibit gender difference. The inductive effects of ZGGJP on the activities of CYPs, exhibiting gender difference, may be regulated by CAR and VDR. Therefore, co-administration of ZGGJP with other drugs, especially using CYP2C9 and CYP3A substrates in females, may need dose adjustment to avoid herb-drug interaction.

Effects of paeoniflorin on the activities and mRNA expression of rat CYP1A2, CYP2C11 and CYP3A1 enzymes in vivo

Paeoniflorin is the major constituent in extracts of the paeony root, the purpose of the present study was to assess the effects of paeoniflorin on the activities and mRNA expression of the rat hepatic drug-metabolizing enzymes cytochrome P450 (CYP1A2), CYP2C11 and CYP3A1 in vivo. Sprague-Dawley (SD) male rats were treated with paeoniflorin at the dosage of 25, 50 and 100 mg/kg or 0.9% sodium chloride solution by intragastric administration for 7 days, then were given probe drugs phenacetin (CYP1A2), tolbutamide (CYP2C11), or midazolam (CYP3A1) orally on the eighth day. Blood samples were collected at various times, and the plasma concentrations of the probe drugs were estimated with ultra-high-performance liquid chromatography. The mRNA expression levels of rat hepatic CYP1A2, CYP2C11 and CYP3A1 were analysed with real-time PCR. The pharmacokinetic results indicated that paeoniflorin inhibits the activities of CYP1A2, CYP2C11 and CYP3A1 in vivo. The effect was most pronounced on CYP3A1, according to the United States Food and Drug Administration classification of inhibitors of CYP3A, it reached the category of moderate inhibition. The mRNA expression levels of 3 CYP enzymes were also tended to be inhibited. We conclude that paeoniflorin can inhibit the activities of CYP1A2, CYP2C11 and CYP3A1 in vivo, which may affect the metabolism of drugs that are primarily dependent on these pathways.

Commiphora myrrha (Nees) Engl. resin extracts induce phase-I cytochrome P450 2C8, 2C9, 2C19, and 3A4 isoenzyme expressions in human hepatocellular carcinoma (HepG2) cells

Commiphora myrrha (Nees) Engl. (C. myrrha) resin is the most Middle Eastern herbal medicine used against numerous diseases. After being decocted or macerated, this resin is widely consumed among Saudi Arabian patients who are already under prescribed medication. Despite its popularity, no studies have been reported on potential modulation effects of these resin extracts on drug metabolism. Therefore, we studied C. myrrha resin extracts on the expression of cytochrome P450 (CYP) drug-metabolizing isoenzyme in human hepatocellular carcinoma cell line HepG2. The C. myrrha extracts were prepared by sonication and boiling, resembling the most popular traditional preparations of maceration and decoction, respectively. Both boiled and sonicated aqueous extracts were fingerprinted using high-performance liquid chromatography equipped with ultra-violet detector (HPLC-UVD). The viability of HepG2 cells treated with these aqueous extracts was determined using CellTiter-Glo® assay in order to select the efficient and non-toxic resin extract concentrations for phase-I metabolic CYP isoenzyme expression analysis. The isoenzyme gene and protein expression levels of CYP 2C8, 2C9, 2C19, and 3A4 were assessed using reverse transcription-quantitative polymerase chain reaction and Western blot technologies. The HPLC-UVD fingerprinting revealed different chromatograms for C. myrrha boiled and sonicated aqueous extracts. Both aqueous extracts were toxic to HepG2 cells when tested at concentrations exceeding 150 µg/ml of the dry crude extract. The CYP 2C8, 2C9, and 2C19 mRNA expression levels increased up to 4.0-fold in HepG2 cells treated with either boiled or sonicated C. myrrha aqueous extracts tested between 1 and 30 µg/ml, as compared with the untreated cells. However, CYP3A4 mRNA expression level exceeded the 2.0-fold cutoff when the cells were exposed to 30 µg/ml of C. myrrha extracts. The up-regulation of CYP mRNA expression levels induced by both boiled and sonicated C. myrrha aqueous extracts was confirmed at the CYP protein expression levels. In conclusion, both sonicated and boiled C. myrrha aqueous extracts modulate CYP 2C8, 2C9, 2C19, and 3A4 gene expression at clinically-relevant concentrations regardless of preparation methods. Further in vitro and in vivo experiments are required for CYP isoenzyme activity assessment and the establishment of herb-drug interaction profile for these traditional medicinal resin extracts.

Curcumin ameliorates duodenal toxicity of AFB1 in chicken through inducing P-glycoprotein and downregulating cytochrome P450 enzymes

It has been reported that oral intake of aflatoxin B1 (AFB1)-contaminated feed could cause acute, sub-chronic, or chronic toxicity in livestock and poultry. However, the harmful effect of AFB1 on the small intestine is still controversial. Therefore, blocking the entry of AFB1 into the body through the digestive tract is one of the important methods to prevent its toxicity. In the present study, 1-day-old Arbor Acres broilers were randomly divided into 6 groups including control group, curcumin control group (450 mg curcumin/kg feed), curcumin low-, medium-, and high-dose group (150, 300, and 450 mg curcumin/kg feed + 5 mg AFB1/kg feed), and AFB1 group (5 mg AFB1/kg feed). After 28 d, the samples of chickens' duodenums were collected for further analyses. AFB1 caused abnormal functional and morphological changes in the duodenum, including histological lesions, increased the length of the duodenum and depth of crypt, decreased the unit weight of the duodenum, height of villus, and the value of villus height/crypt depth. Meanwhile, AFB1 administration enhanced malonaldehyde activity, 8-HOdG level, and the mRNA expression of cytochrome P450 (CYP450) enzymes, and reduced superoxide dismutase, catalase, adenosine triphosphatase (ATPase) activity and the mRNA expression of Abcb1. Importantly, curcumin supplementation partially ameliorated AFB1-induced abnormal functional and morphological signs of the duodenum, alleviated AFB1-induced oxidative stress, and decreased the mRNA expression of CYP450 enzymes. Furthermore, curcumin ameliorated AFB1-induced decrease in the Abcb1 mRNA expression, P-glycoprotein (P-gp) level, and ATPase activities. It has been suggested from these results that curcumin supplementation in the feed could ameliorate AFB1-induced duodenal toxicity and damage through downregulating CYP450 enzymes, promoting ATPase activities, and inducing P-gp in chickens.

Development of Caco-2 cells expressing four CYPs via a mammalian artificial chromosome

BackgroundOral administration is the most common way to deliver drugs to the systemic circulation or target organs. Orally administered drugs are absorbed in the intestine and metabolized in the intestine and liver. In the early stages of drug development, it is important to predict first-pass metabolism accurately to select candidate drugs with high bioavailability. The Caco-2 cell line derived from colorectal cancer is widely used as an intestinal model to assess drug membrane permeability. However, because the expression of major drug-metabolizing enzymes, such as cytochrome P450 (CYP), is extremely low in Caco-2 cells, it is difficult to predict intestinal metabolism, which is a significant factor in predicting oral drug bioavailability. Previously, we constructed a mouse artificial chromosome vector carrying the CYP (CYP2C9, CYP2C19, CYP2D6, and CYP3A4) and P450 oxidoreductase (POR) (4CYPs-MAC) genes and increased CYP expression and metabolic activity in HepG2 cells via transfer of this vector.ResultsIn the current study, to improve the Caco-2 cell assay model by taking metabolism into account, we attempted to increase CYP expression by transferring the 4CYPs-MAC into Caco-2 cells. The Caco-2 cells carrying the 4CYPs-MAC showed higher CYP mRNA expression and activity. In addition, high metabolic activity, availability for permeation test, and the potential to assess drug–drug interactions were confirmed.ConclusionsThe established Caco-2 cells with the 4CYPs-MAC are expected to enable more accurate prediction of the absorption and metabolism in the human intestine than parental Caco-2 cells. The mammalian artificial chromosome vector system would provide useful models for drug development.

The role of the microbiome and psychosocial stress in the expression and activity of drug metabolizing enzymes in mice

The gut microbiota is involved in a number of different metabolic processes of the host organism, including the metabolism of xenobiotics. In our study, we focused on liver cytochromes P450 (CYPs), which can metabolize a wide range of exo- and endogenous molecules. We studied changes in mRNA expression and CYP enzyme activities, as well as the mRNA expression of transcription factors that have an important role in CYP expression, all in stressed germ-free (GF) and stressed specific-pathogen-free (SPF) mice. Besides the presence of the gut microbiota, we looked at the difference between acute and chronic stress. Our results show that stress has an impact on CYP mRNA expression, but it is mainly chronic stress that has a significant effect on enzyme activities along with the gut microbiome. In acutely stressed mice, we observed significant changes at the mRNA level, however, the corresponding enzyme activities were not influenced. Our study suggests an important role of the gut microbiota along with chronic psychosocial stress in the expression and activity of CYPs, which can potentially lead to less effective drug metabolism and, as a result, a harmful impact on the organism.

Mechanism of Chronic Kidney Disease Progression and Novel Biomarkers: A Metabolomic Analysis of Experimental Glomerulonephritis.

While a complex network of cellular and molecular events is known to be involved in the pathophysiological mechanism of chronic kidney disease (CKD), the divergence point between reversal and progression and the event that triggers CKD progression are still unknown. To understand the different mechanisms between reversible and irreversible kidney disease and to search for urinary biomarkers that can predict prognosis, a metabolomic analysis was applied to compare acute and chronic experimental glomerulonephritis (GN) models. Four metabolites, namely, epoxyoctadecenoic acid (EpOME), epoxyeicosatetraenoic acid (EpETE), α-linolenic acid (ALA), and hydroxyretinoic acid, were identified as predictive markers after comparing the chronic nephritis model with acute nephritis and control groups (false discovery rate adjusted p-value (q-value) < 0.05). Renal mRNA expression of cytochrome P450 and epoxide hydrolase was also identified as being involved in the production of epoxide metabolites from these polyunsaturated fatty acids (p < 0.05). These results suggested that the progression of chronic kidney disease is associated with abnormally activated epoxide hydrolase, leading to an increase in EpOME and EpETE as pro-inflammatory eicosanoids.

Janus kinase-dependent regulation of drug detoxifying protein expression by interleukin-22 in human hepatic cells

Interleukin (IL)-22 is a cytokine up-regulated in inflammatory situations and known to exert various hepatic effects. The potential impact of IL-22 towards liver drug detoxifying proteins remains nevertheless unknown, but may be important to determine owing to the well-established alterations of liver detoxification occuring during inflammation. The present study was therefore designed to analyze the effects of IL-22 towards drug metabolizing enzyme and drug transporter expression and activity in cultured human hepatic cells. Exposure of differentiated hepatoma HepaRG cells or primary human hepatocytes to 10 ng/mL IL-22 was found to repress mRNA expression of cytochrome P-450 (CYP) 1A2, CYP3A4, CYP2B6 and CYP2C9 and of the sinusoidal sodium-taurocholate co-transporting polypeptide (NTCP); such IL-22 effects were concentration-dependent for CYP3A4 (IC50 = 1.7 ng/mL), CYP2B6 (IC50 = 0.9 ng/mL) and NTCP (IC50 = 1.8 ng/mL). Activity of CYP1A2 (phenacetin O-deethylation), CYP3A4 (midazolam hydroxylation) and CYP2B6 (bupropion hydroxylation), as well as that of NTCP (taurocholate uptake) were concomitantly decreased in IL-22-treated HepaRG cells; by contrast, activity of organic anion transporter polypeptides (OATPs) (estrone-3-sulfate uptake) and of organic cation transporter (OCT) 1 (tetra-ethylammonium uptake) remained unchanged. IL-22 was next found to activate the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) 3 pathway, whose inhibition by the JAK inhibitor ruxolitinib fully prevented the IL-22-mediated CYP3A4, CYP2B6 and NTCP repression in HepaRG cells. This JAK-dependent down-regulation of hepatic drug detoxifying proteins, notably of CYPs, by IL-22 may contribute to alteration of pharmacokinetics in patients suffering from acute and chronic inflammatory diseases and may be the source of drug-drug interactions.

P6412The AKR1D1*36 (rs1872930) allelic variant is independently associated with adverse cardiac events during Clopidogrel treatment

Abstract Background The treatment of acute coronary syndrome (ACS) includes dual antiplatelet therapy (DAPT) comprising of aspirin and a P2Y12 inhibitor; clopidogrel is usually the P2Y12 inhibitor of choice in patients with ACS. However, its efficacy has been questioned because of the relatively high incidence of Major Adverse Cardiovascular and Cerebrovascular Events (MACCE) among ACS patients despite adherence to clopidogrel-DAPT. Several risk factors for MACCE following intervention in ACS patients have been described, among others, mutations in the CYP2C19 gene, a member of the cytochrome P450 (CYP450) network. The CYP2C19 enzyme is involved in the metabolism of clopidogrel Interindividual response variability to clopidogrel-DAPT and the increased risk for MACCE have been strongly linked to the CYP2C19*2 and CYP2C19*3 loss-of-function (LOF) allelic variants. Nonetheless, the known CYP2C19 polymorphisms fail to account for all adverse events related to clopidogrel insensitivity. Recent studies point to a putative role of the AKR1D1 gene as a trans-genetic regulator of the CYP450 network. The AKR1D1*36 (rs1872930) minor allelic variant was shown to augment hepatic CYP450 mRNA expression, consequently, increasing the CYP2C19 enzyme activity. To this end, we hypothesise that the AKR1D1*36 polymorphism contributes to the observed incidence of MACCE during clopidogrel antiplatelet therapy. Purpose To determine the association between the AKR1D1*36 (rs1872930) allele and MACCE in Acute Coronary Syndrome (ACS) patients on clopidogrel-DAPT Methods We evaluated 198 consecutive ACS patients indicated for coronary angiography from October to November 2010. We performed a 5-year retrospective medical record review and screened for AKR1D1 and CYP2C19 polymorphisms; 118 patients had a complete medical history and were included in the study. The study participants were prospectively followed for five years or until the first incidence of MACCE. The Median follow up time was 38.5 months (IQR 24–48 months) Results The median age of the cohort was 62.5 years (IQR 57–66), and 55% were females. Follow-up was complete, there were no mortality cases. ACS patients carrying the AKR1D1*36 had a significantly shorter event-free-survival compared to wildtype patients, Hazard Ratio = 2.193 [CI95% 1.091 to 4.406], p=0.0155 (Figure 1). Median time-to-event was 36 months. Evaluation of additional candidate risk predictors in a Cox Proportional Hazards Model confirmed the AKR1D1*36 allele as an independent risk factor (HR = 2.36; 95% CI, 1.34 to 4.18) and identified 3 additional risk factors for MACCE; previous percutaneous interventions (PCI), HR = 2.78; (95% CI, 1.34 to 5.78), a history of Myocardial Infarction, HR = 2.62; (95% CI, 1.48 to 4.64), at baseline and presence of the CYPC19*2 allele (HR = 2.33; 95% CI, 1.33 to 4.06). Figure 1. Time-to-Event curve of MACCE Conclusions The AKR1D1*36 (rs1872930) minor variant allele is independently associated with a higher risk for MACCE. Acknowledgement/Funding None

Tissue distribution and characterization of feline cytochrome P450 genes related to polychlorinated biphenyl exposure

Cats have been known to be extremely sensitive to chemical exposures. To understand these model species' sensitivity to chemicals and their toxicities, the expression profiles of xenobiotic-metabolizing enzymes should be studied. Unfortunately, the characterization of cytochrome P450 (CYP), the dominant enzyme in phase I metabolism, in cats has not extensively been studied. Polychlorinated biphenyls (PCBs) are known as CYP inducers in animals, but the information regarding the PCB-induced CYP expression in cats is limited. Therefore, in the present study, we aimed to elucidate the mRNA expression of the CYP1–CYP3 families in the cat tissues and to investigate the CYP mRNA expression related to PCB exposure. In cats, the greatest abundance of CYP1–CYP3 (CYP1A2, CYP2A13, CYP2C41, CYP2D6, CYP2E1, CYP2E2, CYP2F2, CYP2F5, CYP2J2, CYP2U1, and CYP3A132) was expressed in the liver, but some extrahepatic isozymes were found in the kidney (CYP1A1), heart (CYP1B1), lung (CYP2B11 and CYP2S1) and small intestine (CYP3A131). In cats, CYP1A1, CYP1A2 and CYP1B1 were significantly upregulated in the liver as well as in several tissues exposed to PCBs, indicating that these CYPs were distinctly induced by PCBs. The strong correlations between 3,3′,4,4′-tetrachlorobiphenyl (CB77) and CYP1A1 and CYP1B1 mRNA expressions were noted, demonstrating that CB77 could be a potent CYP1 inducer. In addition, these CYP isoforms could play an essential role in the PCBs biotransformation, particularly 3–4 Cl-PCBs, because a high hydroxylated metabolite level of 3–4 Cl-OH-PCBs was observed in the liver.

Alteration of avian hepatic cytochrome P450 gene expression and activity by certain feed additives.

We investigated the effect of four feed additives, namely β-glucan, a drinking water acidifier (DWA), a sanguinarine-containing product (SN) and fulvic acid, on hepatic cytochrome P450 (CYP) mRNA expression and CYP enzyme activity in chickens. The test substances were given to the chickens in the recommended dose or in tenfold dose. The administration of 5 mg/kg body weight (bw) β-glucan and 0.1 ml/kg bw DWA for five days decreased the relative gene expression of CYP1A4 and CYP2C23a. The dosing of 50 mg/kg bw β-glucan, 5 and 50 mg/kg bw SN, 1 ml/kg bw DWA and 250 mg/kg bw fulvic acid doubled the hepatic CYP1A4 activity. The activity of CYP2C and CYP3A remained unchanged. Avoidance of CYP1A-mediated feed-drug interactions requires accurate dosing of β-glucan, DWA and fulvic acid. According to our results, no treatment resulted in excessive or less CYP2C and CYP3A protein formation, which reduces the risk of potential feed additive-drug interactions in chickens. However, the administration of feed additive SN containing a plant alkaloid should be avoided concomitantly with CYP1A-metabolised medicines.

Chronic intake of high-dose of blueberry leaf extract does not augment the harmful effects of ethanol in rats.

Excessive alcohol consumption is a risk factor for liver diseases. Enhancement of alcohol metabolism could be an effective strategy to prevent these adverse effects since it promotes the clearance of ethanol and acetaldehyde from the serum. Polyphenol-rich products have shown to protect against alcohol-related liver damage. Blueberry leaves have attracted attention as they are rich polyphenols such as proantocyanidins and chlorogenic acid. In this study, we investigated the effects of a high dose of blueberry leaf extract (BLEx) on alcohol metabolism during chronic intake of ethanol. Seven-week old Sprague-Dawley (SD) rats were divided into four groups: normal liquid diet group (NLD), normal liquid diet + BLEx group (NLD + BLEx), alcohol liquid diet group (ALD), and alcohol liquid diet + BLEx (ALD + BLEx). Then, rats were fed experimental diet for 5 weeks and at the end of feeding period, body weight, food intake, liver weight, indices of liver injury, expression and activity of alcohol metabolism-related and anti-oxidative enzymes, and levels of carbonyl protein, triglyceride (TG), and total cholesterol (T-Chol) were measured. Body weight and food intake decreased, whereas liver aldehyde dehydrogenase (ALDH) activity, liver microsomal cytochrome P450 2E1 (CYP2E1) protein and mRNA expression, and heme oxygenase 1 (HO-1) mRNA expression were upregulated by ethanol intake. Dietary BLEx, however, did not affect any of these ethanol-related changes. Indices of liver injury, expression and activity of other alcohol metabolism-related enzymes, liver carbonyl protein, TG, and T-Chol levels were not altered by ethanol and BLEx. Thus, chronic BLEx intake does not ameliorate the harmful effects of ethanol.