Cytochrome P450 (P450) 3A4 is the most abundant human P450 and oxidizes a diversity of substrates, including various drugs, steroids, carcinogens, and macrolide natural products. In some reactions, positive cooperativity has been reported in microsomal studies. Flavonoids, e.g., 7,8-benzoflavone (alpha-naphthoflavone, alpha NF), have been shown to stimulate some reactions but not others. In systems containing purified recombinant bacterial P450 3A4, positive cooperativity was seen in oxidations of several substrates, including testosterone, 17 beta-estradiol, amitriptyline, and most notably aflatoxin (AF) B1. With these and other reactions, alpha NF typically reduced cooperativity (i.e., the n value in a Hill plot) while either stimulating or inhibiting reactions. With the substrate AFB1, alpha NF both stimulated 8,9-epoxidation and inhibited 3 alpha-hydroxylation. The same patterns were seen with AFB1 in a fused P450 3A4-NADPH-P450 reductase protein. alpha NF did not alter patterns of activity plotted as a function of NADPH-P450 reductase concentration in systems containing the individual proteins. The patterns of AFB1 oxidation to the two products were modified considerably in systems in which NADPH-P450 reductase was replaced with a flavodoxin or ferredoxin system, iodosylbenzene, or cumene hydroperoxide. AFB2, which differs from AFB1 only in the presence of a saturated 8,9-bond, was not oxidized by P450 3A4 but could inhibit AFB1 oxidation. These and other results are considered in the context of several possible models. The results support a model in which an allosteric site is involved, although the proximity of this putative site to the catalytic site cannot be ascertained as of yet. In order to explain the differential effects of alpha NF and reduction systems on the two oxidations of AFB1, a model is presented in which binding of substrate in a particular conformation can facilitate oxygen activation to enhance catalysis.
Background New drugs are routinely screened for acute IKr blocking properties thought to predict QT prolonging and arrhythmogenic liability. However, recent data suggest that chronic (hours) drug exposure to PI3 kinase (PI3K) inhibitors used in cancer can prolong QT by inhibiting potassium currents and increasing late sodium current (INa-L) in cardiomyocytes. We tested the extent to which IKr blockers with known QT liability generate arrhythmias through this pathway. Methods and Results Acute exposure to dofetilide, an IKr blocker without other recognized electropharmacologic actions, produced no change in ion currents or action potentials in adult mouse cardiomyocytes, which lack IKr. By contrast, 2–48 hours’ exposure to the drug generated arrhythmogenic afterdepolarizations and up to 15-fold increases in INa-L. Including PIP3, a downstream effector for the PI3K pathway, in the pipette inhibited these effects. INa-L was also increased, and inhibitable by PIP3, with hours of dofetilide exposure in human iPSC-derived cardiomyocytes and in CHO cells transfected with SCN5A, encoding INa. Cardiomyocytes from dofetilide-treated mice similarly demonstrated increased INa-L and afterdepolarizations. Other agents with variable IKr blocking potencies and arrhythmia liability produced a range of effects on INa-L, from marked increases (E-4031, d-sotalol, thioridazine, erythromycin) to little or no effect (haloperidol, moxifloxacin, verapamil). Conclusions Some but not all drugs designated as arrhythmogenic IKr blockers can generate arrhythmias by augmenting INa-L through the PI3K pathway. These data identify a potential mechanism for individual susceptibility to proarrhythmia and highlight the need for a new paradigm to screen drugs for QT prolonging and arrhythmogenic liability.
Resveratrol (3,5,4'-trihydroxystilbene), a naturally occurring phytoalexin present in grapes and other foods, has been reported to possess chemopreventive effects as revealed by its striking inhibition of diverse cellular events associated with tumor initiation, promotion and progression. In our present study, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), when treated with the cultured human mammary epithelial (MCF-10A) cells, induced the expression of cytochrome P450 1A1 (CYP1A1) and 1B1 (CYP1B1) that are responsible for the oxidation of 17beta-estradiol to produce catechol estrogens. Resveratrol strongly inhibited the TCDD-induced aryl hydrocarbon receptor (AhR) DNA binding activity, the expression of CYP1A1 and CYP1B1 and their catalytic activities in MCF-10A cells. It also reduced the formation of 2-hydroxyestradiol and 4-hydroxyestradiol from 17beta-estradiol by recombinant human CYP1A1 and CYP1B1, respectively. Furthermore, resveratrol significantly attenuated the intracellular reactive oxygen species (ROS) formation and oxidative DNA damage as well as the cytotoxicity induced by the catechol estrogens. Our data suggest that CYP1A1- and CYP1B1-catalyzed catechol estrogen formation might play a key role in TCDD-induced oxidative damage, and resveratrol can act as a potential chemopreventive against dioxin-induced human mammary carcinogenesis by blocking the metabolic formation of the catechol estrogens and scavenging the ROS generated during their redox cycling.
Rationale The lack of measurable single cell contractility of human induced pluripotent stem cell-derived cardiac myocytes (hiPSC-CMs) currently limits the utility of hiPSC-CMs for evaluating contractile performance for both basic research and drug discovery. Objective To develop a culture method that rapidly generates contracting single hiPSC-CMs and allows quantification of cell shortening with standard equipment used for studying adult cardiac myocytes (CMs). Methods and Results Single hiPSC-CMs were cultured for 5 – 7 days on a 0.4 – 0.8 mm thick mattress of undiluted Matrigel (“mattress hiPSC-CM”) and compared to hiPSC-CMs maintained on control substrate (<0.1 mm thick 1:60 diluted matrigel, “control hiPSC-CM”). Compared to control hiPSC-CM, mattress hiPSC-CMs had more rod-shape morphology and significantly increased sarcomere length. Contractile parameters of mattress hiPSC-CMs measured with video-based edge detection was comparable to that of freshly isolated adult rabbit ventricular CMs. Morphological and contractile properties of mattress hiPSC-CM were consistent across cryopreserved hiPSC-CMs generated independently at another institution. Unlike control hiPSC-CM, mattress hiPSC-CMs display robust contractile responses to positive inotropic agents such as myofilament calcium sensitizers. Mattress hiPSC-CMs exhibit molecular changes that include increased expression of the maturation marker cardiac troponin I and significantly increased action potential upstroke velocity due to a 2-fold increase in sodium current (INa). Conclusions The Matrigel mattress method enables the rapid generation of robustly contracting hiPSC-CMs and enhances maturation. This new method allows quantification of contractile performance at the single cell level, which should be valuable to disease modeling, drug discovery and preclinical cardiotoxicity testing.
This study conducted comprehensive and comparative metabolic and lipidomic profiling of a human epithelial breast cell line (MCF-10A), a slightly metastatic (MCF-7), and a highly metastatic (MDA-MB-231) breast cancer cell line using gas chromatography mass spectrometry (GC-MS) and direct infusion mass spectrometry (DI-MS). Among 39 metabolites identified by GC-MS analysis, xanthine, glucose-6-phosphate, mannose-6-phosphate, guanine, and adenine were selected as prognostic markers of breast cancer metastasis. Major metabolic pathways involved in differentiation of the cell lines were alanine, aspartate, and glutamate metabolism, purine metabolism and glycine, serine, and threonine metabolism. Among 44 intact lipid species identified by DI-MS analysis, the levels of most phospholipids were higher in both metastatic groups than in normal cells. Specifically, the levels of phosphatidylserine (PS) 18:0/20:4, phosphatidylinositol (PI) 18:0/20:4, and phosphatidylcholine (PC) 18:0/20:4 were markedly higher while those of phosphatidylethanolamine (PE) 18:1/18:1 and PI 18:0/18:1 were lower in MDA-MB-231 cells than in MCF-7 cells. A partial-least-squares regression model was developed and validated for predicting the metastatic potential of breast cancer cells. The information obtained in this study will be useful when developing diagnostic tools and for identifying potential therapeutic targets for metastatic breast cancer.
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