Cyp1a1

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CYP1A1 is a gene that encodes a member of the cytochrome P450 superfamily of enzymes. The encoded protein plays a role in the metabolism of various endogenous and exogenous substances.

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Gentest human CYP1A1 (2 citations)

5 protocols using cyp1a1

Recombinant Human Cytochrome P450 Enzymes

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Baculovirus-insect
cell-expressed human CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2C8,
CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5, and CYP3A7 were purchased
from BD Biosciences Discovery Labware (Woburn, MA, USA) and used according
to the manufacturer’s instructions.

Juvonen R.O., Ahinko M., Jokinen E.M., Huuskonen J., Raunio H, & Pentikäinen O.T. (2021). Substrate Selectivity of Coumarin Derivatives by Human CYP1 Enzymes: In Vitro Enzyme Kinetics and In Silico Modeling. ACS Omega, 6(17), 11286-11296.

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In vitro Liver Microsome and Cytosol Preparation

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Fluorochloridone, NADPH, and glutathione (GSH) were purchased from Sigma-Aldrich (St. Louis, MO). Human liver microsomes (HLM) from a pool of 33 donors (Catalog. 452161) were purchased from BD Gentest Corp (Woburn, MA). Human liver cytosols (HLC) from a pool of 46 donors (Catalog. HMCYPL) were purchased from GIBCO (ThermoFisher Scientific, MA). The recombinant enzymes CYP1A1, CYP1A2, CYP1B1, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, and CYP3A5, produced in baculovirus were purchased from BD Gentest. Pooled mouse liver microsomes (MLM) and cytosols (MLC) were prepared from male C57BL/6 mouse liver homogenates by differential ultracentrifugation.^{15 (link)}

Shi J., Xie C., Liu H., Krausz K.W., Bewley C.A., Zhang S., Tang L., Zhou Z, & Gonzalez F.J. (2016). Metabolism and Bioactivation of Fluorochloridone, a Novel Selective Herbicide, in Vivo and in Vitro. Environmental science & technology, 50(17), 9652-9660.

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Human Liver Microsome Characterization

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BFT and BF were purchased from Shanghai Winherb Medical Technology Company (Shanghai, China). ABT, furafylline, sulfaphenazole, clomethiazole, omeprazole, 8-methoxypsoralen, ticlopidine, CYP3cide, glucose-6-phosphate dehydrogenase, D-glucose-6-phosphate, and NADP⁺ were obtained from Sigma (St. Louis, MO, United States). Montelukast, quinidine, ketoconazole was purchased from Jianglai Biotechnology Co., Ltd. (Shanghai, China). The pooled HLMs (from 50 donors, lot no. X008067) were obtained from BioreclamationIVT (Baltimore, MD, United States). A panel of baculovirus expressed human P450s (CYP1A1, 1A2, 2A6, 2A13, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4, 3A5, 4F2, and 4F3), co-expressing NADPH-CYP reductase and cytochrome b5 were obtained from BD Gentest Corp (Woburn, MA, United States). All chemicals and solvents were of analytical grade.

Dai Z.R., Ning J., Sun G.B., Wang P., Zhang F., Ma H.Y., Zou L.W., Hou J., Wu J.J., Ge G.B., Sun X.B, & Yang L. (2019). Cytochrome P450 3A Enzymes Are Key Contributors for Hepatic Metabolism of Bufotalin, a Natural Constitute in Chinese Medicine Chansu. Frontiers in Pharmacology, 10, 52.

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Synthesis and Characterization of Novel Compounds

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IMM-H004 (purity >99%), M1, and M2 (purity >90%) (Li et al., 2010 (link)) were synthesized by Laboratory of Chemical Synthesis, and IMM-H004G (M3) (purity >99%) for animal study was provided by Prof. Dai (Laboratory of Biosynthesis of Natural Products, Institute of Materia Medica, Chinese Academy of Medical Science and Peking Union Medical College, Beijing, China). Propranolol (internal standard, IS), β-glucuronidase, sulfatase, 3′-phosphoadenosine-5′-phosphosulfate (PAPS), uridine 5′-diphosphoglucuronic acid (UDPGA), and alamethicin were obtained from Sigma-Aldrich (St. Louis, MO). Rat liver microsomes (RLMs) and cytosols were prepared by differential ultracentrifugation, and the protein concentration was determined by bicinchoninic acid assay (Beyotime Institute of Biotechnology, Jiangsu, China). Pooled mixed-gender human liver microsomes (HLMs), human liver cytosols, recombinant human cytochrome P450 enzymes (CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP2J2, CYP3A4, CYP4A11, CYP4F2, and CYP4F3), recombinant human UDP-glucuronosyltransferases (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT12B4, UGT12B7, UGT12B10, UGT12B15, and UGT12B17), and recombinant human sulfotransferase (SULT1A1, SULT1A2, SULT1A3, SULT1B1, SULT1C2, SULT1C4, SULT1E1, and SULT2A1) were purchased from BD Gentest (Woburn, MA).

Zhang Z., Liu D., Jiang J., Song X., Zou X., Chu S., Xie K., Dai J., Chen N., Sheng L, & Li Y. (2019). Metabolism of IMM-H004 and Its Pharmacokinetic-Pharmacodynamic Analysis in Cerebral Ischemia/Reperfusion Injured Rats. Frontiers in Pharmacology, 10, 631.

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Measuring CYP1A1/1B1 Enzyme Activity

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Ethoxyresorufin-O-deethylation (EROD) activity was measured in both MCF-10A cells and with recombinant proteins as previously described for enzyme modulation and inhibition.^{42 (link),43 (link)} For enzyme modulation, MCF-10A cells were plated in 96-well plates for 24 h. Compounds were added 1 h prior to TCDD (10 nM) for 48 h. Cells were washed twice with PBS and incubated with ethoxyresorufin (2.5 μM) and salicylamide (1.5 mM) at 37°C. For enzyme inhibition, MCF-10A cells pretreated with TCDD for two days were washed with PBS and pre-incubated with test compounds for 5 min. Subsequently, ethoxyresorufin (2.5 μM) and salicylamide (1.5 mM) were added at 37 °C. For the recombinant protein inhibition assay, 0.15 pmole of CYP1A1 or 0.8 pmole of CYP1B1 (BD Biosciences, Woburn, MA) per well was pre-incubated with test compounds or 2,3′,4,5′-tetramethoxystilbene (TMS) for 5 min at 37 °C in 50 mM potassium phosphate buffer (pH = 7.4) with 1 mM NADPH before adding ethoxyresorufin (2.5 μM). EROD activity was measured as resorufin formation by fluorescence with excitation at 530 nm and emission at 590 nm every minute for 20 min at 37 °C using BioTek’s Synergy H4 Multi-Mode reader (Winooski, VT). A resorufin standard curve was used to calculate the P450 1A/1B activity.

Dunlap T.L., Wang S., Simmler C., Chen S.N., Pauli G.F., Dietz B.M, & Bolton J.L. (2015). Differential effects of Glycyrrhiza species on genotoxic estrogen metabolism: licochalcone A downregulates P450 1B1 whereas isoliquiritigenin stimulates. Chemical research in toxicology, 28(8), 1584-1594.

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