Assessment of Altered Cholesterol Homeostasis by Xenobiotics Using Ultra‐High Performance Liquid Chromatography–Tandem Mass Spectrometry

Herron, Josi; Hines, Kelly M.; Xu, Libin

doi:10.1002/cptx.65

Cited by 16 publications

(26 citation statements)

References 23 publications

(30 reference statements)

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“…Analysis of Cholesterol and synthesis precursors: 7-Dehydrocholesterol (7-DHC), 8-Dehydrocholesterol (8-DHC), 7-Dehydrodesmosterol (7-DHD), Lanosterol, Zymosterol, Lathosterol, and Desmosterol, were determined by mass spectrometry. Lipids were extracted from the whole blood and the sterols analyzed by reverse phase LC–MS/MS assays as described previously [ 21 ].…”

Section: Methodsmentioning

confidence: 99%

Altered toxicological endpoints in humans from common quaternary ammonium compound disinfectant exposure

Hrubec

Seguin

et al. 2021

Toxicology Reports

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Section: Methodsmentioning

confidence: 99%

Altered toxicological endpoints in humans from common quaternary ammonium compound disinfectant exposure

Hrubec

Seguin

et al. 2021

Toxicology Reports

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“…Sterol and oxysterol quantitation was performed by ultrahigh-performance liquid chromatography–tandem mass spectrometry using stable isotope–labeled internal standards (d 7 -cholesterol, d 7 -7-dehydrocholesterol, 13 C 3 -desmosterol, and 13 C 3 -lanosterol for sterols and d 7 -7-ketocholesterol for oxysterols) as described previously ( Herron et al, 2018 ; Raleigh et al, 2018 ). Each sterol or oxysterol level was normalized to the corresponding protein weight of the sample.…”

Section: Methodsmentioning

confidence: 99%

Sterol and oxysterol synthases near the ciliary base activate the Hedgehog pathway

Findakly

Daggubati

García

et al. 2020

Journal of Cell Biology

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Vertebrate Hedgehog signals are transduced through the primary cilium, a specialized lipid microdomain that is required for Smoothened activation. Cilia-associated sterol and oxysterol lipids bind to Smoothened to activate the Hedgehog pathway, but how ciliary lipids are regulated is incompletely understood. Here we identified DHCR7, an enzyme that produces cholesterol, activates the Hedgehog pathway, and localizes near the ciliary base. We found that Hedgehog stimulation negatively regulates DHCR7 activity and removes DHCR7 from the ciliary microenvironment, suggesting that DHCR7 primes cilia for Hedgehog pathway activation. In contrast, we found that Hedgehog stimulation positively regulates the oxysterol synthase CYP7A1, which accumulates near the ciliary base and produces oxysterols that promote Hedgehog signaling in response to pathway activation. Our results reveal that enzymes involved in lipid biosynthesis in the ciliary microenvironment promote Hedgehog signaling, shedding light on how ciliary lipids are established and regulated to transduce Hedgehog signals.

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“…Lipids were then extracted using the Folch method and analyzed by HLPC-MS/MS, according to our previously described method. 36,37 LC-MS Methods.…”

Section: Cell Culture Treatment and Sterol Analysismentioning

confidence: 99%

Metabolism of Benzalkonium Chlorides by Human Hepatic Cytochromes P450

Seguin

Herron

Lopez

et al. 2019

Chem. Res. Toxicol.

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Benzalkonium chlorides (BACs) are widely used as disinfectants in cleaning products, medical products, and the food processing industry. Despite a wide range of reported toxicities, limited studies have been conducted on the metabolism of these compounds in animal models and none in human-derived cells or tissues. In this work, we report on the metabolism of BACs in human liver microsomes (HLM) and by recombinant human hepatic cytochrome P450 (CYP) enzymes. BAC metabolism in HLM was NADPH-dependent and displayed apparent half-lives that increased with BAC alkyl chain length (C 10 < C 12 < C 14 < C 16), suggesting enhanced metabolic stability of the more lipophilic, longer chain BACs. Metabolites of d 7-benzyl labeled BAC substrates retained all deuteriums and there was no evidence of N-dealkylation. MS/MS fragmentation of BAC metabolites confirmed oxidation occurs on the alkyl chain region. Major metabolites of C 10-BAC were identified as ω-hydroxy-, (ω−1)-hydroxy-, (ω, ω−1)-diol-, (ω−1)-ketone-, and ω-carboxylic acid-C 10-BAC by LC-MS comparison with synthetic standards. In a screen of hepatic CYP isoforms, recombinant CYP2D6, CYP4F2, and CYP4F12 consumed substantial quantities of BAC substrates and produced the major microsomal metabolites. The use of potent pan-CYP4 inhibitor HET0016, the specific CYP2D6 inhibitor quinidine, or both confirmed major contributions of CYP4-and CYP2D6-mediated metabolism in the microsomal disappearance of BACs. Kinetic characterization of C 10-BAC metabolite formation in HLM demonstrated robust Michaelis-Menten kinetic parameters for ω-hydroxylation (V max = 380 pmol/min/mg, K m = 0.69 μM) and (ω −1)-hydroxylation (V max = 126 pmol/min/mg, K m = 0.13 μM) reactions. This work illustrates important roles for CYP4-mediated ω-hydroxylation and CYP2D6/CYP4-mediated (ω−1)hydroxylation during the hepatic elimination of BACs, an environmental contaminant of emerging concern. Furthermore, we demonstrate that CYP-mediated oxidation of C 10-BAC mitigates the potent inhibition of cholesterol biosynthesis exhibited by this short-chain BAC.

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Assessment of Altered Cholesterol Homeostasis by Xenobiotics Using Ultra‐High Performance Liquid Chromatography–Tandem Mass Spectrometry

Cited by 16 publications

References 23 publications

Altered toxicological endpoints in humans from common quaternary ammonium compound disinfectant exposure

Altered toxicological endpoints in humans from common quaternary ammonium compound disinfectant exposure

Sterol and oxysterol synthases near the ciliary base activate the Hedgehog pathway

Metabolism of Benzalkonium Chlorides by Human Hepatic Cytochromes P450

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