Graphical abstract

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.

Conventional strategies for drug metabolite identification employ a combination of liquid chromatography-mass spectrometry (LC-MS), which offers higher throughput but provides limited structural information, and nuclear magnetic resonance spectroscopy, which can achieve the most definitive identification but lacks throughput. Ion mobility-mass spectrometry (IM-MS) is a rapid, two-dimensional analysis that separates ions on the basis of their gas-phase size and shape (reflected by collision cross section, CCS) and their mass-to-charge (m/z) ratios, respectively. The rapid nature of IM separation combined with the structural information provided by CCS make IM-MS a promising technique for obtaining more structural information of drug metabolites without sacrificing analytical throughput. Here, we present an in vitro-biosynthesis coupled with IM-MS strategy for rapid generation and analysis of drug metabolites. Drug metabolites were generated in vitro using pooled subcellular fractions derived from human liver and analyzed using a rapid flow injection-IM-MS method. We measured CCS values for 19 parent drugs and their 37 metabolites generated in vitro (78 values in total), representing a wide variety of metabolic modifications. Post-IM fragmentation and computational modeling were used to support metabolite identifications and explore the structural characteristics driving behaviors observed in IM separation. Overall, we found the effects of metabolic modifications on the gas-phase structures of the metabolites to be highly dependent upon the structural characteristics of the parent compounds and the specific position of the modification. This in vitro-biosynthesis coupled with rapid IM-MS analysis workflow represents a promising platform for rapid and highconfidence identification of drug metabolites, applicable at a large scale.

Lipids are critical for neurodevelopment; therefore, disruption of lipid homeostasis by environmental chemicals is expected to have detrimental effects on this process. Previously, we demonstrated that the benzalkonium chlorides (BACs), a class of commonly used disinfectants, alter cholesterol biosynthesis and lipid homeostasis in neuronal cell cultures in a manner dependent on their alkyl chain length. However, the ability of BACs to reach the neonatal brain and alter sterol and lipid homeostasis during neurodevelopment in vivo has not been characterized. Therefore, the goal of this study was to use targeted and untargeted mass spectrometry and transcriptomics to investigate the effect of BACs on sterol and lipid homeostasis and to predict the mechanism of toxicity of BACs on neurodevelopmental processes. After maternal dietary exposure to 120 mg BAC/kg body weight/day, we quantified BAC levels in the mouse neonatal brain, demonstrating for the first time that BACs can cross the blood-placental barrier and enter the developing brain. Transcriptomic analysis of neonatal brains using RNA sequencing revealed alterations in canonical pathways related to cholesterol biosynthesis, liver X receptor-retinoid X receptor (LXR/RXR) signaling, and glutamate receptor signaling. Mass spectrometry analysis revealed decreases in total sterol levels and downregulation of triglycerides and diglycerides, which were consistent with the upregulation of genes involved in sterol biosynthesis and uptake as well as inhibition of LXR signaling. In conclusion, these findings demonstrate that BACs target sterol and lipid homeostasis and provide new insights for the possible mechanisms of action of BACs as developmental neurotoxicants.

The rate-determining step in free radical lipid peroxidation is the propagation of the peroxyl radical, where generally two types of reactions occur: (a) hydrogen-atom transfer (HAT) from a donor to the peroxyl radical; (b) peroxyl radical addition (PRA) to a "CC" double bond. Peroxyl radical clocks have been used to determine the rate constants of HAT reactions (k H ), but no radical clock is available to measure the rate constants of PRA reactions (k add ). In this work, we modified the analytical approach on the linoleate-based peroxyl radical clock to enable the simultaneous measurement of both k H and k add . Compared to the original approach, this new approach involves the use of a strong reducing agent, LiAlH 4 , to completely reduce both HAT and PRAderived products and the relative quantitation of total linoleate oxidation products with or without reduction. The new approach was then applied to measuring the k H and k add values for several series of organic substrates, including para-and meta-substituted styrenes, substituted conjugated dienes, and cyclic alkenes. Furthermore, the k H and k add values for a variety of biologically important lipids were determined for the first time, including conjugated fatty acids, sterols, coenzyme Q10, and lipophilic vitamins, such as vitamins D 3 and A.