Effects of cytochrome P450 inhibitors and of steroid hormones on the formation of 7-hydroxylated metabolites of pregnenolone in mouse brain microsomes

Hydroxylations of pregnenolone (PREG) at the 7 alpha- and 7 beta-positions have been reported in numerous murine tissues and organs and responsible cytochrome P450 (CYP) species await identification. Using thin layer chromatography and gas chromatography-mass spectrometry, we report identification of 7 alpha-hydroxy-PREG and 7 beta-hydroxy-PREG metabolites produced in mouse brain microsome digests and kinetic studies of their production with apparent KM values of 0.5 +/- 0.1 microM and 5.1 +/- 0.6 microM for 7 alpha- and 7 beta-hydroxylation respectively. Investigation of CYP inhibitors and of steroid hormone effects on both 7 alpha- and 7 beta-hydroxylations of PREG showed that: (i) different CYP were involved in 7 alpha- and 7 beta-hydroxylation of PREG because solely 7 alpha-hydroxylation was extensively inhibited by metyrapone, alpha-naphthoflavone, ketoconazole and 3 beta-hydroxysteroids, (ii) CYP 1A2, 2D6, 2B1 and 2B11 were not responsible for 7 alpha- and 7 beta-hydroxylation of PREG because respective specific inhibitors furafylline, quinidine and chloramphenicol triggered no inhibition, (iii) CYP 1A1 was responsible for only part of the 7 beta-hydroxylation of PREG because use of alpha-naphthoflavone, which inhibits specifically CYP 1A1, did not suppress entirely 7 beta-hydroxylation, while ketoconazole, metyrapone and antipyrine, which do not inhibit CYP 1A1, decreased part of the 7 beta-hydroxylation, (iv) 7 alpha-hydroxylation of PREG may be shared with other 3 beta-hydroxysteroids such as isoandrosterone and 5-androstene-3 beta,17 beta-diol which were strong inhibitors, but not with dehydroepiandrosterone which was a non-competitive inhibitor as weak as 3-oxosteroids, and (v) 7 beta-hydroxylation of PREG was not markedly changed by other steroids. Taken together, these findings will be of use for identification of the CYP species responsible for 7 alpha- and 7 beta-hydroxylation of PREG and for studies of their activities in brain.

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Dehydroepiandrosterone 7 α ‐ and 7 β ‐Hydroxylation in Mouse Brain Microsomes. Effects of Cytochrome P450 Inhibitors and Structure‐Specific Inhibition by Steroid Hormones

Journal of Neuroendocrinology ◽

10.1046/j.1365-2826.1997.00661.x ◽

1997 ◽

Vol 9 (12) ◽

pp. 923-928 ◽

Cited By ~ 29

Author(s):

J. Doostzadeh ◽

A. C. Cotillon ◽

R. Morfin

Keyword(s):

Cytochrome P450 ◽

Steroid Hormones ◽

Mouse Brain ◽

Specific Inhibition ◽

Brain Microsomes ◽

P450 Inhibitors

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In vitro Inhibitory Effects of Isofraxidin on Human Liver Cytochrome P450 Enzymes

Pharmacology ◽

10.1159/000495212 ◽

2018 ◽

Vol 103 (3-4) ◽

pp. 120-127 ◽

Cited By ~ 3

Author(s):

Xiaoli Song ◽

Gang Dong ◽

Yun Zhou

Keyword(s):

Cytochrome P450 ◽

Human Liver ◽

Liver Microsomes ◽

Kinetic Studies ◽

Competitive Inhibitor ◽

Cytochrome P450 Enzymes ◽

Inhibitory Effects ◽

Liver Cytochrome ◽

Cyp Isoforms

Isofraxidin is a Coumarin compound widely distributed in plants, such as the Umbelliferae or Chloranthaceae, and it possesses numerous pharmacological activities. However, whether isofraxidin affects the activity of human liver cytochrome P450 (CYP) enzymes remains unclear. In this study, the inhibitory effects of isofraxidin on the 8 human liver CYP isoforms (i.e., 1A2, 3A4, 2A6, 2E1, 2D6, 2C9, 2C19, and 2C8) were investigated in vitro using human liver microsomes. The results showed that isofraxidin inhibited the activity of CYP1A2, 3A4, and 2E1, with IC50 values of 23.01, 15.49, and 15.98 µmol/L, respectively, but that other CYP isoforms were not affected. Enzyme kinetic studies showed that isofraxidin was not only a noncompetitive inhibitor of CYP3A4 but also a competitive inhibitor of CYP1A2 and 2E1, with Ki values of 7.91, 10.14, and 9.30 µmol/L, respectively. In addition, isofraxidin is a time-dependent inhibitor for CYP3A4 with Kinact/KI value of 0.047/12.33 µmol/L–1min–1. The in vitro studies of isofraxidin with CYP isoforms indicate that isofraxidin has the potential to cause pharmacokinetic drug interactions with other coadministered drugs metabolized by CYP1A2, 3A4, and 2E1. Further clinical studies are needed to evaluate the significance of this interaction.

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In vitro Inhibitory Effects of Cynaroside on Human Liver Cytochrome P450 Enzymes

Pharmacology ◽

10.1159/000502172 ◽

2019 ◽

Vol 104 (5-6) ◽

pp. 296-302 ◽

Cited By ~ 2

Author(s):

Lei Wang ◽

Xiuju Ma ◽

Jing Wang ◽

Chang Li

Keyword(s):

Cytochrome P450 ◽

Human Liver ◽

Liver Microsomes ◽

Kinetic Studies ◽

Competitive Inhibitor ◽

Cytochrome P450 Enzymes ◽

Inhibitory Effects ◽

Liver Cytochrome ◽

Cyp Isoforms

Introduction: Cynaroside is a biological component isolated from Lonicera japonica Thunb, and it possesses numerous pharmacological activities. However, whether cynaroside affects the activity of human liver cytochrome P450 (CYP) enzymes remains unclear. The purpose of this study was to investigate the effects of cynaroside on 8 major CYP isoforms in human liver microsomes (HLMs). Methods: In this study, the inhibitory effects of cynaroside on the 8 human liver CYP isoforms (i.e., 1A2, 3A4, 2A6, 2E1, 2D6, 2C9, 2C19, and 2C8) were investigated in vitro using HLMs. Results: The results showed that cynaroside inhibited the activity of CYP1A2, 3A4, and 2C9, with IC50 values of 21.74, 15.88, and 16.58 μmol/L, respectively, but that other CYP isoforms were not affected. Enzyme kinetic studies showed that cynaroside was not only a noncompetitive inhibitor of CYP3A4 but also a competitive inhibitor of CYP1A2 and 2C9, with Ki values of 7.33, 11.60, and 8.09 μmol/L, respectively. In addition, cynaroside is a time-dependent inhibitor for CYP3A4 with Kinact/KI value of 0.049/11.62 μmol/L–1min–1. Conclusion: The in vitro studies of cynaroside with CYP isoforms indicate that cynaroside has the potential to cause pharmacokinetic drug interactions with other coadministered drugs metabolized by CYP1A2, 3A4, and 2C9. Further clinical studies are needed to evaluate the significance of this interaction.

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Fatty Liver and Impaired Hepatic Metabolism Alters the Congener-Specific Distribution of Polychlorinated Biphenyls (PCBs) in Mice with a Liver-Specific Deletion of Cytochrome P450 Reductase

10.26434/chemrxiv.12026904.v1 ◽

2020 ◽

Author(s):

Xueshu Li ◽

Chun-Yun Zhang ◽

Hans-Joachim Lehmler

Keyword(s):

Cytochrome P450 ◽

Polychlorinated Biphenyls ◽

Fatty Liver ◽

Partition Coefficients ◽

Hepatic Metabolism ◽

Gas Chromatography Mass Spectrometry ◽

Cytochrome P450 Reductase ◽

P450 Reductase ◽

Specific Distribution ◽

Specific Deletion

Polychlorinated biphenyls (PCBs) are persistent organic pollutants that are linked to adverse health outcomes. PCB tissue levels are determinants of PCB toxicity; however, it is unclear how factors, such as an altered metabolism and/or a fatty liver, affect PCB distribution in vivo. We determined the congener-specific disposition of PCBs in mice with a liver specific deletion of cytochrome P450 reductase (KO), a model of fatty liver with impaired hepatic metabolism, and wildtype (WT) mice. Male and female KO and WT mice were exposed orally to Aroclor 1254, a technical PCB mixture. PCBs were quantified in adipose, blood, brain and liver tissues by gas chromatography-mass spectrometry. PCB profiles and levels in tissues were genotype and sex dependent. PCB levels were higher in the liver from KO compared to WT mice. PCB profiles showed clear differences between tissues from the same exposure group. While experimental tissue : blood partition coefficients in KO and WT mice did not follow the trends predicted using a composition-based model, the agreement between experimental and calculated partition coefficients was still reasonable. Thus, a fatty liver and/or an impaired hepatic metabolism alter the distribution of PCBs in mice and the magnitude of the partitioning of PCBs from blood into tissues can be approximated using composition-based models.<br>

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Serum Cholinesterase Variants: Examination of Several Differential Inhibitors, Salts, and Buffers Used to Measure Enzyme Activity

Clinical Chemistry ◽

10.1093/clinchem/17.3.183 ◽

1971 ◽

Vol 17 (3) ◽

pp. 183-191 ◽

Cited By ~ 30

Author(s):

Philip J Garry

Keyword(s):

Enzyme Activity ◽

Sodium Fluoride ◽

Phosphate Buffer ◽

Divalent Cations ◽

Kinetic Studies ◽

Competitive Inhibitor ◽

Serum Cholinesterase ◽

Low Concentrations

Abstract Dibucaine, used as a differential inhibitor with acetyl-, propionyl-, and butyrylthiocholine as substrate, clearly identified the "usual" and "atypical" serum cholinesterases. Succinylcholine was also used successfully as a differential inhibitor with butyrylthiocholine as substrate. Sodium fluoride, used as a differential inhibitor, gave conflicting results, depending on whether Tris or phosphate buffer was used in the assay. Mono- and divalent cations (NaCl, KCl, MgCl2, CaCl2, and BaCl2) activated the "usual" and inhibited the "atypical" enzyme at low concentrations. The "usual" enzyme had the same activity in 0.05 mol of Tris or phosphate buffer per liter, while the heterozygous and "atypical" enzymes showed 12 and 42% inhibition, respectively, when assayed in the phosphate buffer. Kinetic studies showed the phosphate acted as a competitive inhibitor of "atypical" enzyme. Km values, determined for "usual" and "atypical" enzymes, were 0.057 and 0.226 mmol/liter, respectively, with butyrylthiocholine as substrate.

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Analytical and technical aspects of testing for drug abuse: confirmatory procedures.

Clinical Chemistry ◽

10.1093/clinchem/34.3.471 ◽

1988 ◽

Vol 34 (3) ◽

pp. 471-473 ◽

Cited By ~ 14

Author(s):

M A Peat

Keyword(s):

Drug Testing ◽

Analytical Procedure ◽

Gas Chromatography Mass Spectrometry ◽

Technical Aspects ◽

Governmental Agencies ◽

Accuracy And Precision ◽

Urine Drug Testing ◽

Urine Drug ◽

Chemical Technique ◽

Layer Chromatography

Abstract Many laboratories are now performing urine drug testing for employers, governmental agencies, and other institutions. It is now recognized that presumptive positive screening results have to be confirmed by an analytical procedure based on a different chemical technique with greater than or equal sensitivity to the screening test. Thin-layer chromatography has been widely used for this; however, it is relatively insensitive for certain drugs, and it cannot satisfy the accuracy and precision requirements needed to determine threshold concentrations reliably. Gas chromatography-mass spectrometry is able to satisfy these threshold requirements and has become the method of choice for confirming initial immunoassay results.

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