Expression Of Cytochrome P450 Enzymes Research Articles

AHR and GPER mediate the stimulatory effects induced by 3-methylcholanthrene in breast cancer cells and cancer-associated fibroblasts (CAFs)

BackgroundThe chemical carcinogen 3-methylcholanthrene (3MC) binds to the aryl hydrocarbon receptor (AHR) that regulates the expression of cytochrome P450 (CYP) enzymes as CYP1B1, which is involved in the oncogenic activation of environmental pollutants as well as in the estrogen biosynthesis and metabolism. 3MC was shown to induce estrogenic responses binding to the estrogen receptor (ER) α and stimulating a functional interaction between AHR and ERα. Recently, the G protein estrogen receptor (GPER) has been reported to mediate certain biological responses induced by endogenous estrogens and environmental compounds eliciting an estrogen-like activity.MethodsMolecular dynamics and docking simulations were performed to evaluate the potential of 3MC to interact with GPER. SkBr3 breast cancer cells and cancer-associated fibroblasts (CAFs) derived from breast tumor patients were used as model system. Real-time PCR and western blotting analysis were performed in order to evaluate the activation of transduction mediators as well as the mRNA and protein levels of CYP1B1 and cyclin D1. Co-immunoprecipitation studies were performed in order to explore the potential of 3MC to trigger the association of GPER with AHR and EGFR. Luciferase assays were carried out to determine the activity of CYP1B1 promoter deletion constructs upon 3MC exposure, while the nuclear shuttle of AHR induced by 3MC was assessed through confocal microscopy. Cell proliferation stimulated by 3MC was determined as biological counterpart of the aforementioned experimental assays. The statistical analysis was performed by ANOVA.ResultsWe first ascertained by docking simulations the ability of 3MC to interact with GPER. Thereafter, we established that 3MC activates the EGFR/ERK/c-Fos transduction signaling through both AHR and GPER in SkBr3 cells and CAFs. Then, we found that these receptors are involved in the up-regulation of CYP1B1 and cyclin D1 as well as in the stimulation of growth responses induced by 3MC.ConclusionsIn the present study we have provided novel insights regarding the molecular mechanisms by which 3MC may trigger a physical and functional interaction between AHR and GPER, leading to the stimulation of both SkBr3 breast cancer cells and CAFs. Altogether, our results indicate that 3MC may engage both GPER and AHR transduction pathways toward breast cancer progression.

Functional expression and activity screening of all human cytochrome P450 enzymes in fission yeast.

Here, a complete set of recombinant fission yeast strains that coexpress each of the 57 human cytochrome P450 (CYP) enzymes together with their natural human electron transfer partner(s) was cloned. This strain collection was tested with two luminogenic probe substrates, and 31 human CYPs (including the orphan enzymes CYP2A7, CYP4A22 and CYP20A1) were found to metabolize at least one of these. Since other substrates are known for the remaining enzymes, all human CYPs are now shown to be active. Interestingly, CYP5A1 was found for the first time to work on a substrate other than prostaglandin H2 , and, moreover, to catalyze an aliphatic hydroxylation reaction that consumes molecular oxygen. Also, the ability of CYP11A1 to catalyze an aryl hydroxylation is another unexpected result.

Effect of "Jiaji" (EX-B2)-electroacupuncture preconditioning on expression of myocardial cytochrome P450 enzyme in rats with acute myocardial ischemia-reperfusion injury

To investigate the effect of "Jiaji"(EX-B2)-electroacupuncture(EA) preconditioning on structural changes of myocardium and expression of cytochrome P450 (CYP450) enzymes in acute myocardial ischemia-reperfusion injury (MIRI) rats. Sixty male Wistar rats were randomly divided into sham operation, model, EX-B2, Neiguan (PC6), Yanglingquan(GB34) and Quchi (LI11) groups (n＝10 in each group). The MIRI model was established by occlusion of the descending anterior branch of the left coronary artery for 30 min, followed by reperfusion for 30 min. EA preconditioning (2 Hz/100 Hz, 1 mA) was respectively applied to EX-B2, PC6, GB34, and LI11 for 30 min, once daily for 7 days. Morphological changes of myocardium were observed by H．E. staining. The ultrastructure of myocardial cells was observed by transmission electron microscope (TEM). The protein expression levels of myocardial CYP450 (CYP1A1, CYP2B2, CYP2C11 and CYP4A2) were detected by Western blot. In MIRI rats, myocardial pathological changes as swollen, disorganized arrangement, and partially ruptured myocardial fibers with unclear limit and necrosis and infiltration of lots of inflammatory cells under microscope, and focal myofilament degeneration, dissolution and necrosis with interstitial edema, vague mitochondria with swelling and vacuolation, ruptured and disorganized arrangement of crests under TEM were found in the ischemic area, which was obviously milder in rats of both EX-B2 and PC6 groups. After modeling, the expression of myocardial CYP1A1 and CYP2B2 protein were significantly up-regulated in the model group relevant to the sham operation group (P<0.05). In EA preconditioning groups, the expression levels of CYP1A1 and CYP4A2 proteins in the EX-B2, PC6 and GB34 groups were obviously down-regulated (P<0.01), and those of CYP2B2 and CYP2C11 proteins in both EX-B2 and PC6 groups were markedly up-regulated relevant to the model group (P<0.01). Compared with the EX-B2 group, the expression levels of CYP1A1, CYP4A2 proteins in the LI11 group were significantly up-regulated (P<0.05), and CYP2B2, CYP2C11 proteins were significantly down-regulated (P<0.05). No significant differences were found between the model and sham operation groups in the expression levels of myocardial CYP2C11 and CYP4A2 proteins, between the LI11 and model groups in the expression of CYP1A1, CYP2B2, CYP2C11 and CYP4A2, between the GB34 and model groups in the expression of CYP2B2, CYP2C11 (all P>0.05)．. EA preconditioning at EX-B2 and PC6 can suppress MIRI induced up-regulation of myocardial CYP450 ω-hydroxylase CYP1A1 and CYP4A2 expression and further up-regulate the expression of myocardial epoxygenase CYP2B2 and CYP2C11 proteins in acute MIRI rats, thus playing a role in myocardial protection.

Cold storage of porcine hepatocyte spheroids for spheroid bioartificial liver.

Cell-based therapies for liver disease such as bioartificial liver rely on a large quantity and high quality of hepatocytes. Cold storage was previously shown to be a better way to preserve the viability and functionality of hepatocytes during transportation rather than freezing, but this was only proved at a lower density of rat hepatocytes spheroids. The purpose of this study was to optimize conditions for cold storage of high density of primary porcine hepatocyte spheroids. Porcine hepatocytes were isolated by a three-step perfusion method; hepatocyte spheroids were formed by a 24hours rocked culture technique. Hepatocyte cell density was 5×106 /mL in 1000mL spheroid forming medium. Spheroids were then maintained in rocked culture at 37°C (control condition) or cold stored at 4°C for 24, 48 or 72hours in four different cold storage solutions: histidine-tryptophan-ketoglutarate (HTK) alone; HTK+1mM deferoxamine (DEF); HTK+5mM N-acetyl-L-cysteine (NAC); and HTK+1mM DEF+5mM NAC. The viability, ammonia clearance, albumin production, gene expression, and functional activity of cytochrome P450 enzymes were measured after recovery from the cold storage. In this study, we observed that cold-induced injury was reduced by the addition of the iron chelator. Viability of HTK+DEF group hepatocyte spheroids was increased compared with other cold storage groups (P<0.05). Performance metrics of porcine hepatocyte spheroids cold stored for 24hours were similar to those in control conditions. The hepatocyte spheroids in control conditions started to lose their ability to clear ammonia while production of albumin was still active at 48 and 72hours (P<0.05). In contrast, the viability and functionality of hepatocyte spheroids including ammonia clearance and albumin secretion were preserved in HTK+DEF group at both 48- and 72-hour time points (P<0.05). The beneficial effects of HTK supplemented with DEF were more obvious after cold storage of high density of porcine hepatocyte spheroids for 72hours. The porcine hepatocyte spheroids were above the cutoff criteria for use in a spheroid-based bioartificial liver.

Role of CYP3A4 in bone marrow microenvironment–mediated protection of FLT3/ITD AML from tyrosine kinase inhibitors

AbstractJones et al address a critical issue in the field of FMS-like tyrosine kinase 3/internal tandem duplication–positive (FLT3/ITD+) leukemias, the fact that FLT3 inhibitors demonstrate superior activity against peripheral compared to bone marrow disease. They demonstrate that expression of cytochrome P450 enzymes (CYPs), in particular CYP3A4, play a major role in bone marrow stromal cell–mediated inhibition in the activity of several FLT3 inhibitors. Their findings suggest combining CYP inhibitors with FLT3 inhibitors may enhance clearance of bone marrow disease. These findings may have major implications for the treatment of FLT3+ acute myeloid leukemia.

Epigenetic Modifications of the Liver Tumor Cell Line HepG2 Increase Their Drug Metabolic Capacity.

Although human liver tumor cells have reduced metabolic functions as compared to primary human hepatocytes (PHH) they are widely used for pre-screening tests of drug metabolism and toxicity. The aim of the present study was to modify liver cancer cell lines in order to improve their drug-metabolizing activities towards PHH. It is well-known that epigenetics is strongly modified in tumor cells and that epigenetic regulators influence the expression and function of Cytochrome P450 (CYP) enzymes through altering crucial transcription factors responsible for drug-metabolizing enzymes. Therefore, we screened the epigenetic status of four different liver cancer cell lines (Huh7, HLE, HepG2 and AKN-1) which were reported to have metabolizing drug activities. Our results showed that HepG2 cells demonstrated the highest similarity compared to PHH. Thus, we modified the epigenetic status of HepG2 cells towards ‘normal’ liver cells by 5-Azacytidine (5-AZA) and Vitamin C exposure. Then, mRNA expression of Epithelial-mesenchymal transition (EMT) marker SNAIL and CYP enzymes were measured by PCR and determinate specific drug metabolites, associated with CYP enzymes by LC/MS. Our results demonstrated an epigenetic shift in HepG2 cells towards PHH after exposure to 5-AZA and Vitamin C which resulted in a higher expression and activity of specific drug metabolizing CYP enzymes. Finally, we observed that 5-AZA and Vitamin C led to an increased expression of Hepatocyte nuclear factor 4α (HNF4α) and E-Cadherin and a significant down regulation of Snail1 (SNAIL), the key transcriptional repressor of E-Cadherin. Our study shows, that certain phase I genes and their enzyme activities are increased by epigenetic modification in HepG2 cells with a concomitant reduction of EMT marker gene SNAIL. The enhancing of liver specific functions in hepatoma cells using epigenetic modifiers opens new opportunities for the usage of cell lines as a potential liver in vitro model for drug testing and development.

Postmortem protein stability investigations of the human hepatic drug-metabolizing cytochrome P450 enzymes CYP1A2 and CYP3A4 using mass spectrometry

Variability in expression and activity of hepatic drug-metabolizing cytochrome P450 (CYP) enzymes can play a causal role in fatal intoxication cases and is thus of forensic interest. We investigated the feasibility of LC-MS/MS based quantification and in vitro enzyme activity measurements of two major drug-metabolizing enzymes CYP1A2 and CYP3A4 in postmortem human liver microsomes (HLM). In autopsy cases (postmortem interval 24–36 h) we found CYP1A2 and CYP3A4 protein levels similar to that measured in a non-decayed reference HLM pool, whereas CYP1A2 and CYP3A4 enzyme activities were absent or severely decreased. Stability studies showed that CYP1A2 and CYP3A4 protein abundances were relatively stable in tissue stored in vitro for up to seven days at 4 °C. When tissue was stored for more than one day at 21 °C variable and case-specific decay patterns were observed, and CYP abundances declined especially after 3–4 days storage. Investigations of 50 autopsy cases revealed mean CYP1A2 and CYP3A4 levels of 49 and 47 pmol per mg HLM protein and inter-individual variabilities similar to those reported in other studies. This study supports postmortem quantification of CYP proteins in autopsy hepatic tissue by mass spectrometry. SignificanceThis study indicates that MS-based detection of drug-metabolizing cytochrome P450 (CYP) proteins is achievable in postmortem hepatic tissue and that acceptable quantification data are obtainable but dependent on the storage conditions and postmortem sampling time. CYP abundance data could contribute to a conceivable way of assessing individual CYP activity phenotypes in a postmortem context.

Reliable and easy-to-use LC–MS/MS-method for simultaneous determination of the antihypertensives metoprolol, amlodipine, canrenone and hydrochlorothiazide in patients with therapy-refractory arterial hypertension

BackgroundTherapy-refractory arterial hypertension is defined as a blood pressure (BP) in a subset of patients who fail to achieve BP control despite a three-drug regimen (including a diuretic). Various factors have impact on loss of therapy response. Drug-drug-interactions (DDIs) may cause altered pharmacokinetics (PK) of antihypertensive drugs. Upregulation of activity and expression of cytochrome P450 (CYP) enzymes can result in decreased plasma drug levels. Besides these PK considerations a significant problem could be nonadherence to drug therapy. In this regard Therapeutic Drug Monitoring (TDM) is a useful tool for detecting nonadherence. Therefore a LC–MS/MS-method for determination of Metoprolol (MET), Amlodipine (AML), Canrenone (CAN) and Hydrochlorothiazide (HCT) was developed. MethodsAn UHPLC-MS/MS method was developed and validated for simultaneous determination of MET, AML, CAN and HCT in plasma matrix. Extraction of serum samples consisted of simple protein precipitation using acetonitrile. Stable isotope labeled analogues for each antihypertensive were obtained for internal standardization and quantitative analysis ([2H7]-MET, ([13C6]-AML, [2H4]-CAN, [13C6]-HCT). Calibrators and quality controls were prepared in plasma matrix of normal individuals. Sample preparation: protein precipitation with acetonitrile and addition of internal standard-mix. ResultsAll analytes were eluted within a runtime of 2.5 min. Linearity experiments were demonstrated in plasma over following concentration ranges: MET: 5–750 μg/l, AML: 1–50 μg/l, CAN: 10–500 μg/l, HCT: 5–500 μg/l (R2 > 0.993).Chromatographic separation was achieved using a C18 column (50 × 2.1 mm, 1.9 μm particle size) and an isocratic elution. LC–MS/MS analyses were performed on a triple quadrupole mass spectrometer using positive and negative electrospray ionization in selected reaction monitoring (SRM) mode. Ion transitions monitored for quantitation were m/z 268.2 → 74.1 for MET, m/z 409.1 → 238.0 for AML, m/z 341.2 → 91.0 for CAN and m/z 296.0 → 205.1 for HCT. For all analytes, inter- and intra-day precision (CV, %) varied between 1.7 and 14.0 and inter- and intra-day accuracy values ranged from −2.5 to 7.1%. The lower limits of detection and quantification were: 0.08 and 0.23; 0.05 and 0.15; 2.82 and 8.54; and 0.02 and 0.05 μg/l for MET, AML, CAN and HCT, respectively. Results of stability experiments were within the required range of +/− 15%. ConclusionsAlthough the level of recommendation of TDM of antihypertensive drugs in patients with refractory hypertension is not yet established, the present LC–MS/MS-method can serve as an effective tool for detection of PK-alterations/nonadherence and may help to monitor antihypertensive pharmacotherapy.

Primary and secondary pyrrolic metabolites of pyrrolizidine alkaloids form DNA adducts in human A549 cells

Humans and animals can be exposed to carcinogenic pyrrolizidine alkaloids (PAs) through consumption of plants commonly found in many parts of the world. Although the liver is the primary target organ for carcinogenic PAs, they have also induced lung tumors in rodents. Hepatic cytochrome P450 activity converts PAs into dehydro-PAs that can be hydrolyzed to dehydropyrrolizidine (DHP); these reactive pyrrolic metabolites can produce four characteristic DNA adducts associated with PA-induced liver tumor initiation in laboratory animals. We reported recently that these four DNA adducts are also formed when 7-glutathione-DHP (7-GS-DHP) or 7-cysteine-DHP is incubated with calf thymus DNA. Here we showed that the four characteristic DNA adducts were formed when human A549 brochoalveolar carcinoma cells were treated with three dehydro-PAs (dehydroriddelliine, dehydromonocrotaline, or dehydroretronecine) or with 7-GS-DHP or 7-cysteine-DHP. For comparison, two parent PAs (riddelliine and monocrotaline) and 7,9-di-glutathionine-DHP were studied. No DHP-DNA adducts were detected with these incubations, confirming that A549 lung carcinoma cells do not express cytochrome P450 enzymes required for metabolic activation of PAs. Our results show that primary and secondary pyrrolic metabolites of carcinogenic PAs produce characteristic DHP-containing DNA adducts in A549 lung cancer cells, suggesting that they are DNA reactive metabolites.

Glutathione S-Transferases and Cytochrome P450 Enzyme Expression in Patients with Intracranial Tumors: Preliminary Report of 55 Patients

Objective: Intracranial tumors are one of the most frightening and difficult-to-treat tumor types. In addition to surgery, protocols such as chemotherapy and radiotherapy also take place in the treatment. Glutathione S-transferase (GST) and cytochrome P450 (CYP) enzymes are prominent drug-metabolizing enzymes in the human body. The aim of this study is to show the expression of GSTP1, GSTM1, CYP1A1, and CYP1B1 in different types of brain tumors and compare our results with those in the literature. Subjects and Methods: The expression of GSTP1, GSTM1, CYP1A1, and CYP1B1 was analyzed using immunostaining in 55 patients with intracranial tumors in 2016–2017. For GST and CYP expression in normal brain tissue, samples of a portion of surrounding normal brain tissue as well as a matched far neighbor of tumor tissue were used. The demographic features of the patients were documented and the expression results compared. Results: The mean age of the patients was 46.72 years; 29 patients were female and 26 were male. Fifty-seven specimens were obtained from 55 patients. Among them, meningioma was diagnosed in 12, metastases in 12, glioblastoma in 9, and pituitary adenoma in 5. The highest GSTP1, GSTM1, and CYP­1A1 expressions were observed in pituitary adenomas. The lowest GSTP1 expression was detected in glioblastomas and the lowest CYP1B1 expression in pituitary adenomas. Conclusion: GSTP1 and CYP expression is increased in intracranial tumors. These results should be confirmed with a larger series and different enzyme subtypes.

In vitro safety pharmacology evaluation of 2-hydroxybenzylamine acetate

2-hydroxybenzylamine (2-HOBA), a compound found in buckwheat, is a potent scavenger of reactive γ-ketoaldehydes, which are increased in diseases associated with inflammation and oxidative stress. While the potential of 2-HOBA is promising, studies were needed to characterize the safety of the compound before clinical trials. In a series of experiments, the risks of 2-HOBA-mediated mutagenicity and cardio-toxicity were assessed in vitro. The effects of 2-HOBA on the mRNA expression of select cytochrome P450 (CYP) enzymes were also assessed in cryopreserved human hepatocytes. Further, the distribution and metabolism of 2-HOBA in blood were determined. Our results indicate that 2-HOBA is not cytotoxic or mutagenic in vitro and does not induce the expression of CYP1A2, CYP2B6, or CYP3A4 in human hepatocytes. The results of the hERG testing showed a low risk of cardiac QT wave prolongation. Plasma protein binding and red blood cell distribution characteristics indicate low protein binding and no preferential distribution into erythrocytes. The major metabolites identified were salicylic acid and the glycoside conjugate of 2-HOBA. Together, these findings support development of 2-HOBA as a nutritional supplement and provide important information for the design of further preclinical safety studies in animals as well as for human clinical trials with 2-HOBA.

Sensitive and Comprehensive LC-MS/MS Analyses of Chiral Pharmaceuticals and Their Hepatic Metabolites Using Ovomucoid Column.

A sensitive analytical method was developed for the simultaneous detection of 11 chiral pharmaceuticals and their hepatic metabolites by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using an ovomucoid chiral column. After optimization of the LC conditions, all pharmaceuticals examined were enantio-separated with Rs of >0.82 in LC-MS/MS analysis. The limit of detections of all pharmaceuticals by MS/MS detection ranged from 1.2 to 92.3 nM, which is approximately 1000 - 25000 times lower than those obtained by UV detection. From hepatic metabolite analyses in P450-expressing cells, metabolites of three pharmaceuticals were detected and enantio-separated. By using the proposed method, changes in the optical isomer ratio of the hepatic metabolites chlorpheniramine and verapamil caused by differential cytochrome P450 enzyme expression for each isomer, could be successfully traced.

Task Force on Gene Testing for Antidepressant Efficacy Concludes Tests Not Yet Ready for Widespread Use

Task Force on Gene Testing for Antidepressant Efficacy Concludes Tests Not Yet Ready for Widespread Use

Posttranslational regulation of CYP2J2 by nitric oxide.

Nitric oxide (NO) is an essential signaling molecule in the body, regulating numerous biological processes. Beside its physiological roles, NO affects drug metabolism by modulating the activity and/or expression of cytochrome P450 enzymes. Previously, our lab showed that NO generation caused by inflammatory stimuli results in CYP2B6 degradation via the ubiquitin-proteasome pathway. In the current study, we tested the NO-mediated regulation of CYP2J2 that metabolizes arachidonic acids to bioactive epoxyeicosatrienoic acids, as well as therapeutic drugs such as astemizole and ebastine. To investigate the effects of NO on CYP2J2 expression and activity, Huh7 cells stably transduced with CYP2J2 with a C-terminal V5 tag were treated with dipropylenetriamine-NONOate (DPTA), a NO donor. The level of CYP2J2 proteins were decreased in a time- and concentration-dependent manner, and the activity was also rapidly inhibited. However, mRNA expression was not altered and the protein synthesis inhibitor cycloheximide did not attenuate DPTA-mediated downregulation of CYP2J2. Removal of DPTA from the culture media quickly restored the activity of remaining CYP2J2, and no further CYP2J2 degradation occurred. To determine the mechanism of CYP2J2 down-regulation by NO, cells were treated with DPTA in the presence or absence of protease inhibitors including proteasomal, lysosomal and calpain inhibitors. Remarkably, the down-regulation of CYP2J2 by NO was attenuated by calpeptin, a calpain inhibitor. However, other calpain inhibitors or calcium chelator show no inhibitory effects on the degradation. The proteasome inhibitor bortezomib showed small but significant restoration of CYP2J2 levels although stimulated ubiquitination of CYP2J2 was not detected. In conclusion, these data suggest that NO regulates CYP2J2 posttranslationally and NO-evoked CYP2J2 degradation undergoes ubiquitin-independent proteasomal degradation pathway unlike CYP2B6.

Progression of conventional hepatic cell culture models to bioengineered HepG2 cells for evaluation of herbal bioactivities.

Cancer cell lines of human tissue origin have been extensively used to investigate antiproliferative activity and toxicity of herbal extracts, isolated compounds, and anticancer drugs. These cell lines are genetically and/or epigenetically well characterized to determine the altered expression of proteins within given cellular pathways and critical genes in cancer. Human derived hepatoma (HepG2) cell line has been extensively exploited to examine cytoprotective, antioxidative, hepatoprotective, anti-hepatoma, hypocholesterolemic, anti-steatosis, bioenergetic homeostatic and anti-insulin resistant properties. Moreover, mechanism of action of various botanicals and bioactive constituents has been reported using these cells. HepG2 cells have significant differences as compared to primary hepatocytes with respect to expression of cytochrome P450 enzymes and xenobiotic receptors in conventional in vitro culture conditions. Therefore, strategies have been employed to overcome limitations of two dimensional (2D) in vitro HepG2 cell culture in order to recognize functional biomarkers more accurately and to boost its predictive value in clinical research. In consequence, three dimensional (3D) human hepatoma cell culture models are being developed as a resource to achieve these goals of simulating the in vivo tumor microenvironment. It is assumed that bioengineered 3D hepatoma cell culture models can provide significant assistance in scrutinizing the molecular response of herbal natural products to recognize novel prognostic targets and crucial biomarkers in treatment strategies for cancer patients in near future.

Manipulating Cytochrome P450 Enzymes: New Perspectives for Cancer Treatment

Increasing drug efficiency and reducing drug overall toxicity are two of the main goals of today`s drug chemotherapy of cancer. In general this could be achieved by searching new ways for selective active drug accumulation in cancer cells by manipulating local drug metabolism or delivery. In this short review, on the basis of the main directions described by McFadyen et al. (Mol Cancer Ther 2004; 3(3): 363-371), new data is reported for localization and expression of cytochrome P450 enzymes in human tumors, development of cytochrome P450-based and gene-directed enzyme activated prodrugs, antisense-based P450 and immune-based therapy, cytochrome P450 polymorphism in development of anticancer drugs. New discoveries of molecular biology of cancer give us hope for more successful development of modern cancer chemotherapy. Biomed Rev 2017; 28: 120-124. Keywords: cytochrome P450 isoforms, cancer cells, drug metabolism, drug delivery

What Pharmacogenetic Testing Can, Can’t Tell You About Your Patient

What Pharmacogenetic Testing Can, Can’t Tell You About Your Patient

Effect of citral on mouse hepatic cytochrome P450 enzymes

Context: Citral is used as a potential natural treatment for various infectious diseases.Objective: To examine the effect of citral on the mRNA expression and activities of cytochrome P450 (CYP450) enzymes and establish the relationship between citral-induced liver injury and oxidative stress.Materials and methods: ICR mice were randomly divided into citral (20, 200, and 2000 mg/kglow), Tween-80, and control groups (0.9% saline), 10 mice in each group. The citral-treated groups were intragastrically administered citral for 3 d, control groups treated with 0.5% Tween-80 and 0.9% saline in the same way. Liver injury and CYP450 enzymes were analyzed by analyzing the histopathological changes and the changes of related enzymes.Results: Citral treatment (2000 mg/kg) for 3 d increased serum glutamic pyruvic transaminase and glutamic oxaloacetic transaminase levels, as well as glutathione, gydroxyl radicals, malonaldehyde and total superoxide dismutase contents, but decreased the content of total antioxidant capacity. In doses of 20 and 200 mg/kg groups mice, the contents of NO were decreased significantly and other changes were similar to the 2000 mg/kg group mice, but the liver damage was most severe in the 2000 mg/kg group. Citral induced the mRNA expression and activities of CYP450 1A2, 2D22, and 2E1 in the liver of mice at doses of 20 and 200 mg/kg. There were no changes in testing indexes in Tween-80 treated group mice. Due to its toxic effects, the CYP induction effect of citral negatively correlated with its dose. Although the mRNA expression of CYP450 3A11 was induced by citral, its activity was not affected by low and moderate doses of citral. CYP450 3A11 activity was significantly decreased by high-dose citral.Conclusions: Citral is hepatotoxic and induced oxidative stress in higher dose, which has a negative effect on CYP450 enzymes. These data suggest caution needs to be taken in order to avoid citral-drug interactions in human beings.

2,3,4\u2032,5-tetrahydroxystilbene-2-O-\u03b2-D-glucoside exacerbates acetaminophen-induced hepatotoxicity by inducing hepatic expression of CYP2E1, CYP3A4 and CYP1A2

Hepatotoxicity induced by medicinal herb Polygonum multiflorum Thunb. attracts wide attention in the world recently. 2,3,4′,5-tetrahydroxystilbene-2-O-β-D-glucoside (TSG) is a main active compound in Polygonum multiflorum Thunb. This study aims to observe TSG-provided the aggravation on acetaminophen (APAP)-induced hepatotoxicity in mice by inducing hepatic expression of cytochrome P450 (CYP450) enzymes. Serum alanine/aspartate aminotransferase (ALT/AST) analysis and liver histological evaluation showed that TSG (200, 400, 800 mg/kg) exacerbated the hepatotoxicity induced by sub-toxic dose of APAP (200 mg/kg) in mice, but TSG alone had no hepatotoxicity. TSG aggravated hepatic reduced glutathione (GSH) depletion and APAP-cysteine adducts (APAP-CYS) formation induced by APAP in mice. TSG increased the expression of CYP2E1, CYP3A4 and CYP1A2 both in mice and in human normal liver L-02 hepatocytes. TSG also enhanced liver catalytic activity of CYP2E1, CYP3A4 and CYP1A2 in mice. TSG induced the nuclear translocation of aryl hydrocarbon receptor (AHR) and pregnane X receptor (PXR), and TSG-provided the aggravation on APAP-induced hepatotoxicity in mice was reversed by PXR or AHR inhibitors. In summary, our results demonstrate that TSG enhances hepatic expression of CYP3A4, CYP2E1 and CYP1A2, and thus exacerbates the hepatotoxicity induced by APAP in mice. PXR and AHR both play some important roles in this process.

Osthole prevents acetaminophen-induced liver injury in mice.

Acetaminophen (APAP) overdose leads to severe hepatotoxicity. Osthole, a natural coumarin found in traditional Chinese medicinal herbs, has therapeutic potential in the treatment of various diseases. In this study, we investigated the effects of osthole against APAP-induced hepatotoxicity in mice. Mice were administered osthole (100 mg·kg-1·d-1, ip) for 3 d, then on the fourth day APAP (300 mg/kg, ip) was co-administered with osthole. The mice were euthanized post-APAP, their serum and livers were collected for analysis. Pretreatment with osthole significantly attenuated APAP-induced hepatocyte necrosis and the increases in ALT and AST activities. Compared with the mice treated with APAP alone, osthole pretreatment significantly reduced serum MDA levels and hepatic H2O2 levels, and improved liver GSH levels and the GSSG-to-GSH ratio. Meanwhile, osthole pretreatment markedly alleviated the APAP-induced up-regulation of inflammatory cytokines in the livers, and inhibited the expression of hepatic cytochrome P450 enzymes, but it increased the expression of hepatic UDP-glucuronosyltransferases (UGTs) and sulfotransferases (SULTs). Furthermore, osthole pretreatment reversed APAP-induced reduction of hepatic cAMP levels, but pretreatment with H89, a potent selective PKA inhibitor, failed to abolish the beneficial effect of osthole, whereas pretreatment with L-buthionine sulfoximine, a GSH synthesis inhibitor, abrogated the protective effects of osthole on APAP-induced liver injury, and abolished osthole-caused alterations in APAP-metabolizing enzymes. In cultured murine primary hepatocytes and Raw264.7 cells, however, osthole (40 μmol/L) did not alleviate APAP-induced cell death, but it significantly suppressed APAP-caused elevation of inflammatory cytokines. Collectively, we have demonstrated that osthole exerts a preventive effect against APAP-induced hepatotoxicity by inhibiting the metabolic activation of APAP and enhancing its clearance through an antioxidation mechanism.