CYP1A1 Protein Expression Research Articles

Co-culture of JEG-3, BeWo and syncBeWo cell lines with adrenal H295R cell line: an alternative model for examining endocrine and metabolic properties of the fetoplacental unit

Monocultures of different placental cells are used for many physiological and toxicological studies; however, they are not a true reflection of the interaction between placenta and fetus. To develop the most appropriate model to study endocrine and metabolic properties of fetoplacental unit we used three co-culture models of placental cells nonfusogenic JEG-3, unsyncytialised BeWo (BeWo) and syncytialised BeWo (syncBeWo) cultured with adrenal (H295R) cells. As an end point of endocrine properties we investigated steroids receptors expression and steroid secretion, while as metabolic properties AhR, CYP1A1and COMT expression. Progesterone (P4), estradiol (E2) and human chorionic gonadotropin (hCG) secretion (ELISA) and 3βHSD, CYP19, estrogen (ERα/β), progesterone (PR) and aryl hydrocarbon (AhR) receptors, CYP1A1 and COMT protein expression (Western blot) were evaluated. Comparing three co-culture models we observed: (1) there were no differences between JEG-3 and BeWo in the PR expression, however it was higher in BeWo compared to syncBeWo; (2) there were no differences in ERα protein expression in all models, while profile of ERβ expression was the highest in syncBeWo; (3) high P4 secretion in JEG-3 and BeWo while low in syncBeWo; (4) high E2 levels in JEG-3 and syncBeWo, while low E2 secretion in BeWo; (5) the highest hCG secretion in the JEG-3 and syncBeWo than in BeWo (6) the highest AhR, CYP1A1 and COMT expression in syncBeWo. Based on the results showing higher hCG secretion in the JEG-3 than in BeWo, representing villous and extravillous phenotype we suggest that JEG-3 model could be used to study fetoplacental steroidogenesis at the 1st, while BeWo model at the 3rd. Results showing comparable profiles of AhR, CYP1A1 and COMT expression in JEG-3 and BeWo models and the significantly higher expression in synBeWo points to synBeWo as a good model for study the metabolic properties.

MicroRNA regulation of CYP 1A2, CYP3A4 and CYP2E1 expression in acetaminophen toxicity

MicroRNAs (miRNAs) that regulate the cytochrome P-450 isoforms involved in acetaminophen (APAP) toxicity were examined in HepaRG cells treated with APAP (20 mM). In-vitro studies found that APAP protein adducts were increased at 1 h, followed by ALT increases at 12 and 24 h. CYP1A2, CYP3A4 and CYP2E1 mRNA levels were decreased, while miRNAs were increased for miR-122-5p, miR-378a-5p, miR-27b-3p at 6 h and miR-125b-5p at 12 h and miR-27b-3p at 24 h. Putative miRNA binding sites on the 3′UTRs of the CYPs were identified in-silico. Overexpression of miR-122-5p and miR-378a-5p in cells suppressed protein expression of CYP1A2, CYP3A4 and CYP2E1. Luciferase reporter assays confirmed the interaction between miR-122 and the 3′UTR of the CYP1A2 and CYP3A4. Thus, the in-vitro experiments showed that miR-122-5p and miR-378a-5p upregulation were associated with translational repression of CYPs. Serum samples of children with APAP overdose had significant elevation of miR-122-5p, miR-378a-5p, miR-125b-5p and miR-27b-3p, compared to healthy controls and receiver operator curves of the miRNAs had AUCs of 91 to 100%. Collectively, the data suggest that miRNA elevations in APAP toxicity represent a regulatory response to modify CYP1A2, CYP3A4 and CYP2E1 translation due to cellular stress and injury.

Modulation of pregnane X receptor (PXR) and constitutive androstane receptor (CAR) activation by ursolic acid (UA) attenuates rifampin-isoniazid cytotoxicity

BackgroundInteractions between transcriptional inducers of cytochrome P450 (CYP450) enzymes and therapeutic drugs may be prevented by antagonizing the activation of a nuclear receptor (NR), pregnane X receptor (PXR, NR1I2), thus improving therapeutic efficacy. PurposeIn the present study, we aim to identify that ursolic acid (UA), a widely distributed pentacyclic triterpene, may act as an effective antagonist of PXR and its sister NR receptor, constitutive androstane receptor (CAR, NR1I3). MethodsThe hepatocellular carcinoma cell line, HepG2, was used to evaluate the promoter activity of PXR and CAR target genes, CYP3A4 and CYP2B6, respectively. Catalytic activities, mRNA, and protein expression of CYP3A4 and CYP2B6 were evaluated in a differentiated HepaRG cell line. Coregulation of PXR with coregulators on CYP3A4 promoter response elements was also been characterized. ResultsTransient transfection assays showed that UA effectively attenuated CYP3A4 and CYP2B6 promoter activities mediated by rifampin (RIF, human PXR agonist) and CITCO (human CAR agonist). These inhibitory effects were well correlated with the expression and catalytic activities of CYP3A4 and CYP2B6. Furthermore, the interaction of co-regulators with PXR and the transcriptional complexes in the CYP3A4 promoter activity and CYP3A4 promoter xenobiotic response element (everted repeat 6, ER6), respectively, were disrupted in the presence of UA. UA showed an antagonistic effect against PXR, and reversed the cytotoxic effects of isoniazid (INH) induced by RIF. Taken together, these results show that UA inhibits the transactivation effects of PXR and CAR, and reduces the expression and function of CYP3A4 and CYP2B6. ConclusionThe present study suggests that UA could be a powerful agent for reducing potentially dangerous interactions between transcriptional inducers of CYP enzymes and therapeutic drugs.

CYP4A/CYP2C modulation of the interaction of calcium channel blockers with cyclosporine on EDHF-mediated renal vasodilations in rats

The endothelium-derived hyperpolarizing factor (EDHF) serves as a back-up mechanism that compensates for reduced nitric oxide (NO)/prostanoids bioavailability. Here we investigated whether (i) under conditions of vascular endothelium dysfunction, the immunosuppressant drug cyclosporine (CSA) upregulates EDHF-dependent renal vasodilations through altering CYP4A/CYP2C signaling, and (ii) calcium channel blockers modulate the CSA/EDHF/CYP interaction. Rats were treated with CSA, verapamil, nifedipine, or their combinations for 7days. Blood pressure (BP) was measured by tail-cuff plethysmography. Kidneys were then isolated, perfused with physiological solution containing L-NAME (NOS inhibitor) and diclofenac (cyclooxygenase inhibitor, DIC), and preconstricted with phenylephrine. CSA (25mgkg−1day−1 for 7days) increased BP and augmented carbachol renal vasodilations. The co-treatment with verapamil (2mgkg−1day−1) or nifedipine (3mgkg−1day−1) abolished CSA hypertension and conversely affected carbachol vasodilations (increases vs. decreases). Infusion of MSPPOH (epoxyeicosatrienoic acids, EETs, inhibitor) reduced carbachol vasodilations in kidneys of all rat groups, suggesting the importance of EETs in these responses. By contrast, 20-Hydroxyeicosatetraenoic Acid (20-HETE) inhibition by HET0016 increased carbachol vasodilations in control rats, an effect that disappeared by CSA treatment, and reappeared in rats treated with CSA/verapamil or CSA/nifedipine. Renal protein expression of CYP2C and CYP4A as well as their vasoactive products (EETs/20-HETE) were increased in CSA-treated rats. Whereas the CYP2C/EETs effects of CSA were abolished by verapamil and intensified by nifedipine, the CYP4A/20-HETE effects were reduced by either CCB. Overall, nifedipine and verapamil blunts CSA hypertension but variably affected concomitantly enhanced EDHF-dependent renal vasodilations and alterations in CYP2C/CYP4A signaling.

P19 Intracerebral microdialysis studies on the role of Pinocembrin on CYP3A in rat

Propolis is one of the most heterogeneous natural product of plant origin. Research on the flavonoids in propolis has been prompted by their beneficial effects on health. In propolis, pinocembrin (5,7-dihydroxyflavanone) is the flavonoid that is present in the highest concentration. As flavonoids have a broad spectrum of pharmacological activities, more and more biological actions of pinocembrin have been reported, including anti-microbial, antioxidant, anti-inflammatory, vasorelaxant, and neuroprotective effects, etc. The current study focused on investigating the effects of pinocembrin on CYP3A in the rat brain using intracerebral microdialysis. To investigate the pharmacokinetic profiles of midazolam and 1 ′ -hydroxymidazolam in the rat brain before and after intravenous administration of pinocembrin, microdialysates from rat brains were collected and analysed. For microdialysis, a probe was inserted into rats’ paracele and then the animals were administrated either 5% of β-cyclodextrin (control group) or pinocembrin at 3.75, 7.5, 15 mg/kg (treatment groups) via the femoral vein. Rats were then administrated 5 mg/kg midazolam. In the presence of pinocembrin, the pharmacokinetic parameters of midazolam and 1 ′ -hydroxymidazolam varied significantly ( P t 1 / 2 , MRT and Vd were changed from 0.73 ± 0.09 h −1 , 0.96 ± 0.12 h, 1.63 ± 0.16 h and 52.02 ± 8.61 L · kg −1 to 0.55 ± 0.12 h −1 , 1.29 ± 0.19 h, 1.98 ± 0.30 h and 65.1 ± 10.7 L · kg −1 , respectively. The results indicated that pinocembrin induced alterations in the pharmacokinetic profiles of midazolam/ 1 ′ -hydroxymidazolam. Our other experimental data showed that both protein expression and mRNA level of CYP3A were also decreased. So pinocembrin could slow the midazolam metabolism process by inhibiting CYP3A in the rat brain.

Altered hepatic drug-metabolizing activity in rats suffering from hypoxemia with experimentally induced acute lung impairment

1. Hepatic drug-metabolizing activity was investigated in vitro with liver microsomes prepared from rats suffering from hypoxemia with experimentally induced acute lung impairment (ALI).2. Male Wistar rats received an intrabronchial administration of dilute hydrochloride solution for ALI induction. Pooled liver microsomes were prepared for the normal and ALI rats, and the hepatic drug metabolism mediated by cytochrome P450 (CYP) 3 A was examined in an incubation study with the microsomes.3. The NADPH-dependent metabolism of midazolam significantly increases in ALI rats as compared with that in normal rats. Testosterone 6β-hydroxylation was also observed to significantly increase in ALI rats.4. When the hepatic expression of CYP3A proteins was examined, the protein expression of CYP3A1 was shown to significantly increase and that of CYP3A2 remained unaltered in ALI rats. The hepatic expression of NADPH-cytochrome P450 reductase (POR), a protein mediating electron transfer in CYP-mediated drug metabolism, was also revealed to significantly increases in ALI rats.5. With the findings regarding the midazolam elimination, the hepatic drug-metabolizing activity seems to increase in response to acute hypoxemia, partly due to an altered expression of the CYP3A enzymes, and an augmented electron transfer with an increased POR expression is probably involved in the increase.

The fungicide difenoconazole alters mRNA expression levels of human CYP3A4 in HepG2 cells

The fungicide difenoconazole has been frequently detected in agricultural products, soils and surface waters, causing increasingly public concern due to toxicological properties. Although systemic triazole fungicides can inhibit the enzymatic activity of many CYP450 isozymes, how difenoconazole affects the human CYP3A4 still remains largely unknown. We thus investigated the effect of difenoconazole on normal mRNA expression and protein expression of human CYP3A4 by real-time quantitative PCR and western blot, respectively. Results show that the exposure to difenoconazole from 0.01 to 0.5 μM for 24 h down-regulates mRNA expression levels of human CYP3A4 in HepG2 cells. We also found that difenoconazole could inhibit the enzymatic activity of human CYP3A4 in a concentration-dependent mode. The IC50 of difenoconazole for inhibition of CYP3A4 activity is 0.41 µM, showing a stronger inhibitor in comparison with ketoconazole. Overall, our findings indicate the potential risk of difenoconazole for the disruption of human CYP3A4.

Activated thyroid hormone receptor modulates dioxin-inducible aryl hydrocarbon receptor-mediated CYP1A1 induction in human hepatocytes but not in human hepatocarcinoma HepG2 cells

Aryl hydrocarbon receptor (AhR) is a transcription factor, the activity of which is modulated by hormones including glucocorticoids and estrogens. In this study, we examined the effects of triiodothyronine (T3), a ligand and activator of thyroid hormone receptor (TR), on transcriptional activity of AhR and the expression of its target gene CYP1A1. Study was carried out in human hepatocellular carcinoma cells HepG2 and primary cultures of human hepatocytes (HH). Gene reporter assay in stably transfected AZ-AhR cells revealed that T3 dose-dependently augmented 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-inducible AhR-dependent luciferase activity. In contrast, T3 had no effect on TCDD-inducible expression of CYP1A1 mRNA, protein and catalytic activity. Incubation of human hepatocytes with T3 had modulatory and inter-individual (7 cell cultures from 7 different liver donors) effects on both basal and dioxin-inducible CYP1A1/2. Since there was no correlation between T3 effects on CYP1A expression and T3-dependent expression of Spot14 mRNA, the involvement of additional factors besides TR is supposed. Overall, the co-incubation of normal and cancer human hepatic cells with TCDD and T3 suggested transcriptional cross-talk between AhR and TR, which may have physiological and toxicological implications.

Effects of Paeonia emodi on hepatic cytochrome P450 (CYP3A2 and CYP2C11) expression and pharmacokinetics of carbamazepine in rats

Herbal medicines, dietary supplements, and other foods may pharmacokinetically and/or pharmacodynamically interact with carbamazepine (CBZ), which could lead to potential clinical consequences. Paeonia emodi (PE) is one of the herbs used as complementary therapy in the treatment of epileptic patients in some cultures, and may also be co-administered with CBZ. This study evaluates the effects of PE on the pharmacokinetics of CBZ and determines a possible mechanism of interaction. Rats were administered vehicle saline or PE (200mg/kg, p.o. daily for 7days), then administered a single CBZ dose (80mg/kg, p.o.) on day 7. Plasma samples were analyzed for CBZ concentrations using a sensitive reversed-phase high-performance liquid chromatography (RP-HPLC) assay. Pharmacokinetic parameters were calculated using non-compartmental analysis. The co-administration of PE with CBZ resulted in increased plasma maximum concentration (Cmax), area under the curve (AUC0-∞), and half-life (T½), by 14.61%, 48.12%, and 43.72%, respectively. The calculated oral clearance (CL/F) was reduced by 33.54%, while the volume of distribution (Vss) was unaffected. The PE extract also showed a significant potential to reduce CYP3A and CYP2C protein expression by approximately 50%. Therefore, a reduction in the metabolic capacity responsible for CBZ clearance appears to be the mechanism behind this herb-drug interaction. Consequently, the concomitant administration of PE and CBZ should be viewed cautiously. Further studies are needed to determine the clinical relevance of these observations.

The associations of genetic polymorphisms in CYP1A2 and CYP3A4 with clinical outcomes of breast cancer patients in northern China.

BackgroundCytochrome P450 (CYP) 1A2 and CYP3A4 may play a role in the differentiation of clinical outcomes among breast cancer women. This study aimed to analyze the association of genetic polymorphisms in the CYP1A2 and CYP3A4 genes with clinicopathological features, protein expression and prognosis of breast cancer in the northern Chinese population.ResultsFirstly, SNP rs11636419, rs17861162 and rs2470890 in the CYP1A2 were significantly associated with age and menstruation status. And SNP rs11636419 and rs17861162 were associated with the P53 status. Secondly, SNP rs2470890 was correlated with CYP1A2 protein expression under the co-dominant and dominant model (P = 0.017, P = 0.006, respectively). Thirdly, for SNP rs2470890, the Kaplan–Meier 5 year survival curves showed that patients carrying genotypes CT or TT had a worse OS compared with the genotype CC carriers under both codominant and dominant model (P < 0.001, P < 0.001, respectively).Materials and MethodsFour single nucleotide polymorphisms (SNPs) were successfully genotyped in 459 breast cancer patients using the SNaPshot method. The associations of four polymorphisms with protein expression and clinicopathological characteristics were evaluated by Pearson's chi-square test. The Cox hazard regression analysis and Kaplan–Meier survival analysis were performed to evaluate the relationship between the SNPs and overall survival (OS) of breast cancer.ConclusionsCYP1A2 rs2470890 was significantly associated with the prognosis of patients with breast cancer and could serve as an independent impact factor of prognosis of breast carcinoma.

Hepatic deletion of X-box binding protein 1 impairs bile acid metabolism in mice

The unfolded protein response (UPR) is an adaptive response to endoplasmic reticulum stress and the inositol-requiring enzyme 1α/X-box binding protein 1 (IRE1α/XBP1) pathway of the UPR is important in lipid metabolism. However, its role in bile acid metabolism remains unknown. We demonstrate that liver-specific Xbp1 knockout (LS-Xbp1-/-) mice had a 45% reduction in total bile acid pool. LS-Xbp1-/- mice had lower serum 7α-hydroxy-4-cholesten-3-one (C4) levels compared with Xbp1fl/fl mice, indicating reduced cholesterol 7α-hydroxylase (CYP7A1) synthetic activity. This occurred without reductions of hepatic CYP7A1 protein expression. Feeding LS-Xbp1-/- mice cholestyramine increased hepatic CYP7A1 protein expression to levels 2-fold and 8-fold greater than cholestyramine-fed and chow-fed Xbp1fl/fl mice, respectively. However, serum C4 levels remained unchanged and were lower than both groups of Xbp1fl/fl mice. In contrast, although feeding LS-Xbp1-/- mice cholesterol did not increase CYP7A1 expression, serum C4 levels increased significantly up to levels similar to chow-fed Xbp1fl/fl mice and the total bile acid pool normalized. In conclusion, loss of hepatic XBP1 decreased the bile acid pool and CYP7A1 synthetic activity. Cholesterol feeding, but not induction of CYP7A1 with cholestyramine, increased CYP7A1 synthetic activity and corrected the genotype-specific total bile acid pools. These data demonstrate a novel role of IRE1α/XBP1 regulating bile acid metabolism.

Effects of tetrahydroberberine and tetrahydropalmatine on hepatic cytochrome P450 expression and their toxicity in mice

The aim of this study was to investigate the effects of tetrahydroberberine (THB) and tetrahydropalmatine (THP) on the expression of mouse liver cytochrome P450s, and evaluate their liver toxicity in mice. Real-time polymerase chain reaction (PCR) and western blot analyses were used to analyze the expression of major P450 isoforms. Liver toxicity was evaluated by measuring serum biochemical parameters and performing histopathological analysis. The real-time PCR results showed that THB induced Cyp1a2 (1.66 ± 0.34 fold, P < 0.05), Cyp3a11 (1.57 ± 0.24 fold, P < 0.05), and Cyp2e1 (1.75 ± 0.97 fold, P < 0.05) mRNA expression, while THP inhibited Cyp1a2 (0.66 ± 0.12 fold, P < 0.05) mRNA expression. The western blot results confirmed that the expression of CYP1A2, CYP3A, and CYP2E1 proteins in the mouse liver was induced by THB, whereas that of CYP1A2 was inhibited by THP. Toxicological studies showed that THB (40 mg/kg, oral gavage) increased mouse serum aspartate transaminase and total bilirubin, and liver malondialdehyde levels, and induced liver edema. No obvious changes in serum and liver tissue biochemical parameters were found and no significant pathological changes were detected in liver tissues after THP administration. Our results provide more information on the toxicity of THB and THP, and their related drug-drug interactions.

Dimethylarsinic acid modulates the aryl hydrocarbon receptor-regulated genes in C57BL/6 mice: in vivo study

1. Dimethylarsinic acid (DMA(V)) is the major metabolite of inorganic arsenic in human body. Thus we investigated the effect of DMA(V) on the alteration of phase I (typified by Cyp1a) and phase II (typified by Nqo1) AhR-regulated genes in vivo. C57BL/6 mice received DMA(V) (13.3 mg/kg, i.p.) with or without TCDD (15 μg/kg, i.p.), thereafter the liver, lung, and kidney were harvested at 6 and 24 h post-treatment.2. Results demonstrated that DMA(V) has no significant effect on Cyp1a mRNA and protein expression or catalytic activity in the liver. On the other hand, DMA(V) significantly potentiated the TCDD-mediated induction of Cyp1a mRNA and protein expression, with a subsequent potentiation of catalytic activity in the lung. Moreover, DMA(V) significantly inhibited the TCDD-mediated induction of Cyp1a mRNA and protein expression with subsequent inhibition of catalytic activity in the kidney.3. Regarding to phase II AhR-regulated genes, DMA(V) has no significant effect on Nqo1 mRNA and protein expression, or activity neither in the liver, lung, or kidney.4. In conclusion, the present work demonstrates for the first time that DMA(V) modulates AhR-regulated genes in a tissue- and enzyme-specific manner. This modulation may play a crucial role in arsenic-induced toxicity and carcinogenicity.

A 3D fish liver model for aquatic toxicology: Morphological changes and Cyp1a induction in PLHC-1 microtissues after repeated benzo(a)pyrene exposures

To identify the potential environmental impacts of aquatic pollutants, rapid and sensitive screening tools are needed to assess adaptive and toxic responses. This study characterizes a novel fish liver microtissue model, produced with the cell line PLHC-1, as an in vitro aquatic toxicity testing platform. These 3D microtissues remain viable and stable throughout the 8-day testing period and relative to 2D monolayers, show increased basal expression of the xenobiotic metabolizing enzyme cytochrome P450 1A (Cyp1a). To evaluate pulsed, low-dose exposures at environmentally relevant concentrations, microtissue responsiveness to the model toxicant benzo(a)pyrene was assessed after single and repeated exposures for determination of both immediate and persistent effects. Significant induction of Cyp1a gene and protein expression was detected after a single 24h exposure to as little as 1nM benzo(a)pyrene, and after a 24h recovery period, Cyp1a expression declined in a dose-dependent manner. However, cell death continued to increase during the recovery period and alterations in microtissue architecture occurred at higher concentrations. To evaluate a pulsed or repeated exposure scenario, microtissues were exposed to benzo(a)pyrene, allowed to recover, then exposed a second time for 24h. Following pre-exposure to benzo(a)pyrene, cyp1a expression remained equally inducible and the pattern and level of Cyp1a protein response to a second exposure were comparable. However, pre-exposure to 1μM or 5μM of benzo(a)pyrene resulted in increased cell death, greater disruption of microtissue architecture, and alterations in cell morphology. Together, this study demonstrates the capabilities of this PLHC-1 microtissue model for sensitive assessment of liver toxicants over time and following single and repeated exposures.

Upregulation of 20-HETE Synthetic Cytochrome P450 Isoforms by Oxygen-Glucose Deprivation in Cortical Neurons.

20-Hydroxyeicosatetraenoic acid (20-HETE), a potent vasoconstrictor, is a cytochrome P450 (CYP) 4A/4F-derived metabolite of arachidonic acid. Inhibition of 20-HETE synthesis protects brain from ischemic injury. However, that protection is not associated with changes in cerebral blood flow. The present study examined whether CYP4A isoforms are expressed in neurons, whether they produce 20-HETE in neurons, and whether neuronally derived 20-HETE exerts direct neurotoxicity after oxygen-glucose deprivation (OGD). The expression of Cyp4a10 and Cyp4a12a mRNA in cultured mouse cortical neurons increased significantly at 1 and 3h after exposure to 1h of OGD. Reoxygenation also markedly augmented the expression of CYP4A protein in neurons and increased 20-HETE levels in the culture medium. Cell viability after OGD increased after treatment with a 20-HETE synthesis inhibitor or an antagonist. That effect was reversed by co-administration of a 20-HETE agonist. These results indicate that neurons express Cyp4a10 and 4a12a, that expression of these isoforms is upregulated by OGD stress, and that neuronally derived 20-HETE directly contributes to neuronal death after reoxygenation.

Donor Genotype and Intragraft Expression of CYP3A5 Reflect the Response to Steroid Treatment During Acute Renal Allograft Rejection.

Glucocorticoid (GC)-refractory acute rejection (AR) is a risk factor for inferior renal allograft outcome. We investigated genetic predisposition to the response to steroid treatment of acute allograft rejection. Single nucleotide polymorphisms of genes involved in GC signaling (GR, GLCCI1) and drug metabolism and transport (CYP3A5, ABCB1, and PXR) were analyzed in kidney transplant recipients (1995-2005, Leiden cohort, n = 153) treated with methylprednisolone. Significant associations were verified in a second cohort (Berlin cohort, n = 66). Patients who received a CYP3A5*1 allele expressing allograft had a lower risk of resistance to methylprednisolone during AR (odds ratio, 0.29; 95% confidence interval, 0.11-0.79; P = 0.016 in combined cohorts analysis). No differences were observed for GC signaling or other drug metabolism/transport-related genes. Both before transplantation (n = 69) and at time of AR (n = 88), tissue CYP3A5 mRNA expression was significantly higher in CYP3A5*1 allele expressing donor kidneys than in CYP3A5*3/*3 allografts (P < 0.00001). Moreover, steroid-responsive patients (n = 64) expressed significantly higher intragraft CYP3A5 mRNA levels compared to steroid-refractory patients (n = 42) in AR (P = 0.006). CYP3A5 protein expression was detected in tubular epithelial cells and inflammatory cells within the grafts. Our findings show that steroid resistance during AR is associated with donor genotype and intragraft expression levels of CYP3A5.

Cholesterol-lowering effects of piceatannol, a stilbene from wine, using untargeted metabolomics

This study aims at examining the hypolipidemic effect of piceatannol on high fat diet (HFD)-induced hypercholesterolemic Sprague-Dawley rats and serum metabolite changes. Piceatannol supplement significantly lowered the total cholesterols, low density lipoprotein cholesterol levels and the atherogenic index as compared to the HFD model which only have increased dietary cholesterol intake. Using untargeted massspectrometry-based metabolomic platforms, multivariate statistics revealed that HFD significantly perturbed fatty acids, lysophospholipids, bile acids and conjugated bile acids. Reduced CYP7A1 protein expression and increase in glycocholate and taurodeoxycholate after piceatannol treatment suggested the conjugated bile acid might contribute to the cholesterol-lowering effect. For lipid profiles, lysoPC (20:2) and lysoPC (20:0) were decreased while the ratio of esterified arachidonic acid to esterified dihomo-γ linoleic acid was up-regulated for rats after piceatannol supplement. These results indicated that the therapeutic effect of piceatannol is associated with bile acid and fatty acid metabolisms and reduced absorption of dietary cholesterols.

Ginsenoside Rb1 Inhibits Doxorubicin-Triggered H9C2 Cell Apoptosis via Aryl Hydrocarbon Receptor.

Doxorubicin (DOX) is a highly effective chemotherapeutic agent; however, the dose-dependent cardiotoxicity associated with DOX significantly limits its clinical application. In the present study, we investigated whether Rb1 could prevent DOX-induced apoptosis in H9C2 cells via aryl hydrocarbon receptor (AhR). H9C2 cells were treated with various concentrations (− μM) of Rb1. AhR, CYP1A protein and mRNA expression were quantified with Western blot and real-time PCR analyses. We also evaluated the expression levels of caspase-3 to assess the anti-apoptotic effects of Rb1. Our results showed that Rb1 attenuated DOX-induced cardiomyocytes injury and apoptosis and reduced caspase-3 and caspase-8, but not caspase-9 activity in DOX-treated H9C2 cells. Meanwhile, pre-treatment with Rb1 decreased the expression of caspase-3 and PARP in the protein levels, with no effects on cytochrome c, Bax, and Bcl-2 in DOX-stimulated cells. Rb1 markedly decreased the CYP1A1 and CYP1A2 expression induced by DOX. Furthermore, transfection with AhR siRNA or pre-treatment with AhR antagonist CH-223191 significantly inhibited the ability of Rb1 to decrease the induction of CYP1A, as well as caspase-3 protein levels following stimulation with DOX. In conclusion, these findings indicate that AhR plays an important role in the protection of Ginsenoside Rb1 against DOX-triggered apoptosis of H9C2 cells.

Protective Effects of Essential Oils as Natural Antioxidants against Hepatotoxicity Induced by Cyclophosphamide in Mice.

Clinical application of cyclophosphamide (CP) as an anticancer drug is often limited due to its toxicity. CP is metabolized mainly in the liver by cytochrome P450 system into acrolein which is the proximate toxic metabolite. Many different natural antioxidants were found to alleviate the toxic effects of various toxic agents via different mechanisms. Therefore, the present study aimed at investigating the role of essential oils extracted from fennel, cumin and clove as natural antioxidants in the alleviation of hepatotoxicity induced by CP through assessment of hepatotoxicity biomarkers (AST, ALT, ALP), histopathology of liver tissues as well as other biochemical parameters involved in the metabolism of CP. The data of the present study showed that treatment of male mice with cyclophosphamide (2.5 mg/Kg BW) as repeated dose for 28 consecutive days was found to induce hepatotoxicity through the elevation in the activities of AST, ALT, and ALP. Combined administration of any of these oils with CP to mice partially normalized the altered hepatic biochemical markers caused by CP, whereas administration of fennel, clove or cumin essential oils alone couldn’t change liver function indices. Moreover, CP caused histological changes in livers of mice including swelling and dilation in sinusoidal space, inflammation in portal tract and hepatocytes, as well as, hyperplasia in Kuppfer cells. However, co-administration of any of the essential oils with CP alleviated to some extent the changes caused by CP but not as the normal liver. CP was also found to induce free radical levels (measured as thiobarbituric acid reactive substances) and inhibited the activities of superoxide dismutase, glutathione reductase, and catalase as well as activities and protein expressions of both glutathione S-transferase (GSTπ) and glutathione peroxidase. Essential oils restored changes in activities of antioxidant enzymes (SOD, CAT, GR, GST, and GPx) caused by CP to their normal levels compared to control group. In addition, treatment of mice with CP was found to induce the protein expression of CYP 3A4, 2B1/2, 2C6, 2C23. Moreover, the present study showed that essential oils reduced the expression of CYPs 2E1, 3A4 but could not restore the expression of CYP 2C6 and 2C23 compared to CP-treated mice. Interestingly, pretreatment of mice with essential oil of clove was found to restore activities of DMN-dI, AHH, and ECOD which were induced by CP to their normal control levels. It is concluded that EOs showed a marked hepatoprotective effect against hepatotoxicity induced by CP. In addition, co-administration of CP with any of these oils might be used as a new strategy for cancer treatment to alleviate the hepatotoxicity induced by CP.

Role of vascular reactive oxygen species in regulating cytochrome P450-4A enzyme expression in Dahl salt-sensitive rats.

The potential contribution of CYP4A enzymes to endothelial dysfunction in Dahl salt-sensitive rats was determined by comparison to SS-5BN consomic rats having chromosome 5 carrying CYP4A alleles from the BN rat introgressed into the SS genetic background. The following experiments were performed in cerebral arteries from HS-fed SS and SS-5BN rats ± the SOD inhibitor DETC and/or the superoxide scavenger Tempol: (i) endothelial function was determined via video microscopy ± acute addition of the CYP4A inhibitor DDMS or Tempol; (ii) vascular oxidative stress was assessed with DHE fluorescence ± acute addition of DDMS, l-NAME, or PEG-SOD; and (iii) CYP4A protein levels were compared by western blotting. In DETC-treated SS-5BN and HS-fed SS rats, (i) DDMS or Tempol ameliorated vascular dysfunction, (ii) DDMS reduced vascular oxidative stress to control levels, (iii) chronic Tempol treatment reduced vascular CYP4A protein expression, and (iv) combined treatment with Tempol and l-NAME prevented the reduction in CYP4A protein expression in MCA of HS-fed SS rats. The CYP4A pathway plays a role in vascular dysfunction in SS rats and there appears to be a direct role of reduced NO availability due to salt-induced oxidant stress in upregulating CYP4A enzyme expression.