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Publication Detail

Title: Metabolism of Benzalkonium Chloride by CYP-Mediated ω-Oxidation and C-C Cleavage Followed by β-Oxidation in Humans.

Authors: Seguin, Ryan; Xu, Libin

Published In FASEB J, (2022 May)

Abstract: INTRODUCTION: Benzalkonium chlorides (BACs) are widely used quaternary ammonium disinfectants. A recent study found that median levels of BACs in human blood have increased 174% during COVID from pre-COVID levels, with BACs being detected in 98% of blood samples. Although the parent BACs are not readily excreted into urine, we have identified several BAC metabolites in anonymous human urine samples. Formation of these metabolites requires ω-oxidation and oxidative C-C cleavage of BAC alkyl chains followed by β-oxidation to produce a series of chain-shortened carboxylic acid (COOH) BAC metabolites. Here, we aim to elucidate the metabolic route(s) that can account for the observed urinary metabolites of BACs. Our approach includes LC-MS-based investigation of metabolite profiles in microsomes, hepatocytes, and recombinant CYPs in comparison with those observed in human urine. BACs of the alkyl chain lengths found in commercial mixtures (C12, C14, and C16) served as substrates. Authentic metabolite standards were synthesized and confirmed by NMR and LC-MS/MS. Selective CYP inhibitors were employed to assign specific CYP isoform contributions to metabolite formation. RESULTS: As an extension of our previous work, we confirmed that NADPH-dependent ω-oxidation in microsomes and recombinant CYPs produced ω-OH and ω-COOH metabolites, as well as ω-1 oxidized products, of all BAC chain lengths. We further found that the responsible CYPs (2D6, 4F2, 4F11, and 4F12) are all capable of performing C-C cleavage of BAC side chains' terminal carbons. The resulting products were chain-shortened ω-COOH BACs having lost 1 or 2 carbons (-CH2 or -C2H4). In hepatocytes, subsequent β-oxidation of the ω-COOH C10, C12, C14, and C16 BAC metabolites (having lost 0 or 2 carbons during CYP metabolism) produces a series of even-carbon ω-COOH products (C4, C6, and C8) given the successive 2 carbon removal (-C2H4) from the even-carbon starting chain lengths. Additionally, hepatocytes produce a series of odd-carbon ω-COOH products (C5, C7, and C9) that logically originate from β-oxidation of ω-COOH C11, C13, and C15 BAC metabolites (having lost 1 carbon during CYP metabolism). The ω-COOH BAC metabolites produced in hepatocytes were the same as those detected in human urine samples, and the relative abundance among the various chain lengths of ω-COOH products was similar between human hepatocytes and urine. In most urine samples, C8, C10, and C12 were the most abundant COOH metabolites of even-carbon length, and C9 was most abundant among the odd-carbon series. In hepatocytes, addition of HET0016 and quinidine to inhibit CYP4F/CYP2D6 abolished all metabolite formation, including the β-oxidation products. CONCLUSION: The above data confirm that CYP4F and/or CYP2D6 are required to initiate BAC metabolism. The relative abundance of ω- and β-oxidized products (ω-COOH BACs C4 - C16) observed in urine was consistent with the human cellular metabolism of BACs observed in hepatocytes. While parent BACs are commonly found in human blood, they are not readily excreted to urine. Thus, we propose to expand monitoring of human urine samples for chain-shortened ω-COOH BAC metabolites as a practical surrogate for assessing BAC exposure in the general population.

PubMed ID: 35567457 Exiting the NIEHS site

MeSH Terms: Benzalkonium Compounds*/chemistry; Benzalkonium Compounds*/metabolism; COVID-19*; Carbon/metabolism; Chromatography, Liquid; Cytochrome P-450 CYP2D6/metabolism; Cytochrome P-450 Enzyme System/metabolism; Humans; Microsomes, Liver; Tandem Mass Spectrometry

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