Xenobiotic-metabolizing Cytochrome P450 Enzymes Research Articles

Circulating Extracellular Vesicles Containing Xenobiotic Metabolizing CYP Enzymes and Their Potential Roles in Extrahepatic Cells Via Cell-Cell Interactions.

The cytochrome P450 (CYP) family of enzymes is known to metabolize the majority of xenobiotics. Hepatocytes, powerhouses of CYP enzymes, are where most drugs are metabolized into non-toxic metabolites. Additional tissues/cells such as gut, kidneys, lungs, blood, and brain cells express selective CYP enzymes. Extrahepatic CYP enzymes, especially in kidneys, also metabolize drugs into excretable forms. However, extrahepatic cells express a much lower level of CYPs than hepatocytes. It is possible that the liver secretes CYP enzymes, which circulate via plasma and are eventually delivered to extrahepatic cells (e.g., brain cells). CYP circulation likely occurs via extracellular vesicles (EVs), which carry important biomolecules for delivery to distant cells. Recent studies have revealed an abundance of several CYPs in plasma EVs and other cell-derived EVs, and have demonstrated the role of CYP-containing EVs in xenobiotic-induced toxicity via cell–cell interactions. Thus, it is important to study the mechanism for packaging CYP into EVs, their circulation via plasma, and their role in extrahepatic cells. Future studies could help to find novel EV biomarkers and help to utilize EVs in novel interventions via CYP-containing EV drug delivery. This review mainly covers the abundance of CYPs in plasma EVs and EVs derived from CYP-expressing cells, as well as the potential role of EV CYPs in cell–cell communication and their application with respect to novel biomarkers and therapeutic interventions.

Impact of Panax ginseng and Ginkgo biloba extracts on expression level of transcriptional factors and xenobiotic-metabolizing cytochrome P450 enzymes

Summary Introduction: Despite widespread use of Panax ginseng and Ginkgo biloba, the data on the safety as well as herb-drug interactions are very limited. Therefore, we postulate that P. ginseng and G. biloba may modulate the activity and content of cytochrome P450 isozymes involved in the biotransformation of diverse xenobiotic substances. Objective: The aim of our study was to determine the influence of herbal remedies on the expression level of CYP enzymes and transcriptional factors. Methods: Male Wistar rats were given standardized Panax ginseng (30 mg/kg p.o.) or standardized Ginkgo biloba (200 mg/kg p.o.) for 3 and 10 days. The expression in liver tissue was analyzed by realtime PCR method. Results: Our results showed a decrease of CYP3A1 (homologue to human CYP3A4) mRNA level after P. ginseng extract treatment. The CYP2C6 (homologue to human CYP2C9) expression was also reduced. Additionally, after 10 days of the treatment with P. ginseng an increase of CYP1A1 (homologue to human CYP1A1) and CYP1A2 (homologue to human CYP1A2) expression was observed. Moreover, G. biloba extract also caused an increase of expression level for CYP1A1, CYP2C6, CYP3A1 and CYP3A2. Conclusion: Our findings suggest that herbal extracts can modulate the expression of transcriptional factors and CYP enzymes involved in xenobiotic metabolism and chemical carcinogenesis.

Benzo[a]pyrene-mediated toxicity in primary pig bladder epithelial cells: A proteomic approach

The studies described in this paper deal with a sequence of cellular events induced by the environmental toxicant benzo[a]pyrene (B[a]P) that were investigated in primary urinary bladder epithelia cells (PUBEC) from pigs by using a proteomic approach. Two-dimensional (2DE) gel electrophoresis unveiled the differences in protein expression between cells exposed to 0.5 μM B[a]P for 24 h and control cells. Twenty-five differentially expressed proteins involved in DNA repair, mitochondrial dysfunction, and apoptosis were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). These findings were supported by the concentration-dependent increase in olive tail moments as determined by the comet assay and by a time-dependent increase in histone H2A.x (H2AX) phosphorylation upon B[a]P exposure. On the other hand, the expression of voltage-dependent anion channel 2 (VDAC2), cathepsin D (CTSD), heat shock protein 27 (HSP27), and heat shock protein 70 (HSP70) hinted to apoptosis occurring through the intrinsic apoptotic mitochondrial pathway. Taken together, these data suggest that B[a]P is capable of inducing DNA damage in urinary bladder epithelial cells at low concentrations during a short exposure period, thus eventually leading to cell death by apoptosis. Epidemiological studies have indicated PAHs as potential candidates for initiating bladder cancer development, although the precise risk is still unknown (Kaufman et al. (2009)). In recent years, the understanding of the metabolic capacity of urothelial cells has broadened continuously; i.e. a wide range of xenobiotic metabolizing cytochrome P450 enzymes (CYP) were detected in urothelial cells from humans and animals (Roos et al., 2006; Guhe et al., 1996), thus indicating that urothelial cells are not only passively exposed to reactive metabolites but also actively by intracellularly producing reactive intermediates that can induce cancer. Moreover, small quantities of non-metabolized B[a]P and its hydroxylated derivatives have been identified in blood and urine (Rossella et al. (2009)). Thus, it appears plausible that B[a]P, a highly lipophilic compound, is taken up by the urothelium and metabolically activated to carcinogenic intermediates in these cells. In our previous studies with primary uroepithelial cells isolated from freshly slaughtered pigs we demonstrated the ability of these cells for a strong uptake of B[a]P and its conversion to the oxidative metabolite (3-OH-B[a]P) (Verma et al. (2012)). The present study is a continuation of this previous work exhibiting the effects of B[a]P exposure on cellular functions of PUBEC. The results indicated caspase-dependent apoptosis induced by B[a]P due to DNA damage (possibly lethal double-strand breaks as indicated by H2AX phosphorylation). Taken together, these studies provide strong evidence for the ability of B[a]P to act as a bladder carcinogen.

Molecular probes of the mechanism of cytochrome P450. Oxygen traps a substrate radical intermediate

The diagnostic substrate tetramethylcyclopropane (TMCP) has been reexamined as a substrate with three drug- and xenobiotic-metabolizing cytochrome P450 enzymes, human CYP2E1, CYP3A4 and rat CYP2B1. The major hydroxylation product in all cases was the unrearranged primary alcohol along with smaller amounts of a rearranged tertiary alcohol. Significantly, another ring-opened product, diacetone alcohol, was also observed. With CYP2E1 this product accounted for 20% of the total turnover. Diacetone alcohol also was detected as a product from TMCP with a biomimetic model catalyst, FeTMPyP, but not with a ruthenium porphyrin catalyst. Lifetimes of the intermediate radicals were determined from the ratios of rearranged and unrearranged products to be 120, 13 and 1 ps for CYP2E1, CYP3A4 and CYP2B1, respectively, corresponding to rebound rates of 0.9 × 10 10 s −1, 7.2 × 10 10 s −1 and 1.0 × 10 12 s −1. For the model iron porphyrin, FeTMPyP, a radical lifetime of 81 ps and a rebound rate of 1.2 × 10 10 s −1 were determined. These apparent radical lifetimes are consistent with earlier reports with a variety of CYP enzymes and radical clock substrates, however, the large amounts of diacetone alcohol with CYP2E1 and the iron porphyrin suggest that for these systems a considerable amount of the intermediate carbon radical is trapped by molecular oxygen. These results add to the view that cage escape of the intermediate carbon radical in [Fe IV–OH R] can compete with cage collapse to form a C–O bond. The results could be significant with regard to our understanding of iron-catalyzed C–H hydroxylation, the observation of P450-dependent peroxidation and the development of oxidative stress, especially for CYP2E1.

(Far) Outside the box: Genomic approach to acute porphyria

If I were living in Caucasus I would be writing fairy tales there Chekov, 1888 The question of the reasons for the extreme variation in morbidity among the gene carriers of acute porphyria and the great diversity of the precipitating factors are approached by the aid of a model of interacting genomic circuits. It is based on the current paradigm of the acute porphyric attack as a result of a toxic proximal overload of the enzyme-deficient heme-biosynthetic patway. Porphyrogenic influx of precursors is seen as a consequence of uncontrolled induction of its gate-keeping enzyme, ubiquitous 5-aminolevulinate synthase (ALAS1), due to attenuated post-translational control of the enzyme combined with activated gene transcription. Focus is directed on the genomic control of the master-regulator of ALAS1-transcription, the nuclear receptor pair constitutively active receptor (CAR) and pregnane xenobiotic receptor (PXR). On activation by their ligands, i.e. lipophilic drugs, solvents, alcohols, hormonal steroids and biocides, these DNA-binding proteins transform xenobiotic or steroid stimuli to coordinated activations of gene transcription-programs for ALAS1 and apo-cytochromes P450 (apo-CYPs), thus effecting the formation of xenobiotic-metabolizing cytochrome P450 enzymes. The potency of the CAR/PXR-transduction axis is enhanced by co-activators generated in at least four other genomic circuits, each triggered by different external and internal stimuli clinically experienced to be porphyrogenic, and each controlled by co-activating and co-repressing modulators. The expressions of the genes for CAR and PXR are thus augmented by binding glucocorticoid receptor (GR) activated by a steroid hormone, e.g, cortisol generated in fasting, infection or different forms of stress. The promotor regions of ALAS1 and apoCYPs contain binding sites for at least three co-activating transcription factors enhancing CAR/PXR transduction: i.e. the ligand-independent growth hormone (GH)-pulse controlled hepatocyte nuclear factor 4 (HNF4), the insulin-responsive forkhead box class O-(FOXO) protein pathway activated in stress and infection, and the proliferator-activated receptor gamma co-activator 1 alpha (PGC-1alpha) circuit responding to glucagon liberated in fasting. Many interactions and cross-talk take place within the tangle of genomic circuits that control ALAS1-transcription, which may explain the extreme inter- and intra-individual variability in morbidity in acute porphyria. Reasons for gender-differences are found in sex-dependent control of HPA- and GH-activity as well as in direct, or GR-mediated effects on CAR/PCR activation. Constitutional differences in individual porphyric morbidity may be discussed along lines of mutations or duplications of genes for co-activating or co-repressing nuclear proteins active at different levels within the circuits.

Dehydroepiandrosterone sulfotransferase is a target for transcriptional induction by the vitamin D receptor.

Dehydroepiandrosterone sulfotransferase (SULT2A1) is a cytosolic enzyme that mediates sulfo-conjugation of endogenous hydroxysteroids (dehydroepiandrosterone, testosterone, bile acids), and diverse xenobiotic compounds. Upon sulfonation, SULT2A1 substrates become polar and water-soluble and thus suitable for rapid excretion. SULT2A1 is abundantly expressed in the liver and intestine. Recent evidence has shown that the ligand-activated vitamin D receptor (VDR) can transcriptionally induce the xenobiotic-metabolizing cytochrome P450 enzymes. Herein, we report that VDR also targets SULT2A1 for transcriptional activation. Vitamin D stimulated endogenous SULT2A1 expression and induced transfected human, mouse, and rat SULT2A1 promoters in liver and intestinal cells upon cotransfection with VDR. An inverted repeat DNA element (IR0), located within -191 to -168 positions of mouse and rat Sult2A1, mediates VDR induction of Sult2A1. DNase1 footprinting, competition EMSA, and antibody supershift assay showed that the IR0 is a binding site for the RXR-alpha/VDR heterodimer. Point mutations within the IR0 prevented RXR/VDR binding and abolished VDR-mediated Sult2A1 induction. The IR0 element conferred VDR responsiveness on a thymidine kinase promoter. Thus, VDR-mediated nuclear signaling may be important in the phase II metabolism involving Sult2A1. The rodent Sult2A1 gene is also induced by the farnesoid X receptor (FXR) and pregnane X receptor (PXR) through the same IR0. In competition transfections, FXR or PXR inhibited VDR induction of the IR0. Competitive functional interactions among VDR, PXR, and FXR suggest that the intracellular hormonal and metabolic milieu may determine the extent to which a specific nuclear receptor pathway would influence steroid/xenobiotic metabolism using dehydroepiandrosterone sulfotransferase.

Expression and Regulation of Xenobiotic-Metabolizing Cytochrome P450 (CYP) Enzymes in Human Lung

Pathogenesis of lung diseases, such as lung cancer and chronic obstructive pulmonary disease, is tightly linked to exposure to environmental chemicals, most notably tobacco smoke. Many of the compounds associated with these diseases require an enzymatic activation to exert their deleterious effects on pulmonary cells. These activation reactions are mostly catalyzed by cytochrome P450 (CYP) enzymes. Interindividual differences in the in situ activation and inactivation of chemical toxicants may contribute to the risk of developing lung diseases associated with these compounds. This review summarizes in detail the expression of individual CYP forms in human pulmonary tissue and gives a view on the significance of the pulmonary expression of CYP enzymes. The localization of individual CYP enzymes in various cell types of human lung and the emerging field of regulation of human pulmonary CYP enzymes are discussed. At least CYP1A1 (in smokers), CYP1B1, CYP2B6, CYP2E1, CYP2J2, and CYP3A5 proteins are expressed in human lung, and also other CYP forms are likely to be expressed. Xenobiotic-metabolizing CYP enzymes are mostly expressed in bronchial and bronchiolar epithelium, Clara cells, type II pneumocytes, and alveolar macrophages in human lung, although individual CYP forms have different patterns of localization in pulmonary tissues. Problems in animal to human lung toxicity extrapolation and several specific aspects requiring more detailed assessment are identified.

Discovery of a N′-hydroxyphenylformamidine derivative HET0016 as a potent and selective 20-HETE synthase inhibitor

N-(4-Butyl-2-methylphenyl)- N′-hydroxyformamidine (HET0016) was evaluated as the first potent and selective inhibitor of 20-hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE) synthase. The IC 50 value of HET0016 for the production of 20-HETE from arachidonic acid (AA) by human renal microsomes was 8.9±2.7 nM, with over 200 times the selectivity of xenobiotic-metabolizing cytochrome P450 enzymes. An examination of the structure–activity relationship revealed that the unsubstituted hydroxyformamidine moiety and the substituent at the para-position of the N-hydroxyformamidine moiety are necessary for the potent activity of HET0016.

Liver Injury and Expression of Cytochromes P450: Evidence That Regulation of CYP2A5 Is Different from That of Other Major Xenobiotic Metabolizing CYP Enzymes

The purpose of this study was to find out how liver injury caused by two well-known hepatotoxins, chloroform and thioacetamide, alters the expression of hepatic xenobiotic metabolizing cytochrome P450 (CYP) enzymes of DBA/2N mice. Dose-dependent toxic effects of the two hepatotoxins were verified by histological examination. Along with the toxicity, intense staining of immunoreactive material was detected in the centrilobular zone, with anti-CYP2A5 antibody in hepatic tissue. This apparent increase in the expression ofCyp2a-5was verified by Northern blot and Western blot analyses and by determining the enzymatic activity, coumarin 7-hydroxylase, in hepatic tissue. The results suggest that liver injury due to these hepatotoxins increases the expression ofCyp2a-5and that the expression is pretranslationally regulated. The increased expression ofCyp2a-5is in contrast with that of other xenobiotic metabolizing CYPs because a dose-dependent decrease of the total hepatic P450 content and either a decrease or no change in the levels of CYP1A, 2B, 2C, 2E1, and 3A4 were observed. The results suggest that essential differences exist in the regulation of CYP2A5 and other major xenobiotic metabolizing CYP enzymes and that in a damaged liver CYP2A5 may be a major catalyst of xenobiotic metabolism.