7-Dehydrocholesterol-derived oxysterols cause neurogenic defects in Smith-Lemli-Opitz syndrome.

Defective 3ß-hydroxysterol-Δ7 -reductase (DHCR7) in the developmental disorder, Smith-Lemli-Opitz syndrome (SLOS), results in a deficiency in cholesterol and accumulation of its precursor, 7-dehydrocholesterol (7-DHC). Here, we show that loss of DHCR7 causes accumulation of 7-DHC-derived oxysterol metabolites, premature neurogenesis from murine or human cortical neural precursors, and depletion of the cortical precursor pool, both in vitro and in vivo. We found that a major oxysterol, 3ß,5α-dihydroxycholest-7-en-6-one (DHCEO), mediates these effects by initiating crosstalk between glucocorticoid receptor (GR) and neurotrophin receptor kinase TrkB. Either loss of DHCR7 or direct exposure to DHCEO causes hyperactivation of GR and TrkB and their downstream MEK-ERK-C/EBP signaling pathway in cortical neural precursors. Moreover, direct inhibition of GR activation with an antagonist or inhibition of DHCEO accumulation with antioxidants rescues the premature neurogenesis phenotype caused by the loss of DHCR7. These results suggest that GR could be a new therapeutic target against the neurological defects observed in SLOS.

Benzalkonium Chloride Disinfectants Induce Apoptosis, Inhibit Proliferation, and Activate the Integrated Stress Response in a 3-D in Vitro Model of Neurodevelopment.

We previously found that the widely used disinfectants, benzalkonium chlorides (BACs), alter cholesterol and lipid homeostasis in neuronal cell lines and in neonatal mouse brains. Here, we investigate the effects of BACs on neurospheres, an in vitro three-dimensional model of neurodevelopment. Neurospheres cultured from mouse embryonic neural progenitor cells (NPCs) were exposed to increasing concentrations (from 1 to 100 nM) of a short-chain BAC (BAC C12), a long-chain BAC (BAC C16), and AY9944 (a known DHCR7 inhibitor). We found that the sizes of neurospheres were decreased by both BACs but not by AY9944. Furthermore, we observed potent inhibition of cholesterol biosynthesis at the step of DHCR7 by BAC C12 but not by BAC C16, suggesting that cholesterol biosynthesis inhibition is not responsible for the observed reduction in neurosphere growth. By using immunostaining and cell cycle analysis, we found that both BACs induced apoptosis and decreased proliferation of NPCs. To explore the mechanisms underlying their effect on neurosphere growth, we carried out RNA sequencing on neurospheres exposed to each BAC at 50 nM for 24 h, which revealed the activation of the integrated stress response by both BACs. Overall, these results suggest that BACs affect neurodevelopment by inducing the integrated stress response in a manner independent of their effects on cholesterol biosynthesis.

Metabolism of Benzalkonium Chlorides by Human Hepatic Cytochromes P450.

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 (C10 < C12 < C14 < C16), suggesting enhanced metabolic stability of the more lipophilic, longer chain BACs. Metabolites of d7-benzyl labeled BAC substrates retained all deuteriums and there was no evidence of N-dealkylation. Tandem mass spectrometry fragmentation of BAC metabolites confirmed that oxidation occurs on the alkyl chain region. Major metabolites of C10-BAC were identified as ω-hydroxy-, (ω-1)-hydroxy-, (ω, ω-1)-diol-, (ω-1)-ketone-, and ω-carboxylic acid-C10-BAC by liquid chromatography-mass spectrometry 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 C10-BAC metabolite formation in HLM demonstrated robust Michaelis-Menten kinetic parameters for ω-hydroxylation (Vmax = 380 pmol/min/mg, Km = 0.69 µM) and (ω-1)-hydroxylation (Vmax = 126 pmol/min/mg, Km = 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 C10-BAC mitigates the potent inhibition of cholesterol biosynthesis exhibited by this short-chain BAC.

Multiomics Investigation Reveals Benzalkonium Chloride Disinfectants Alter Sterol and Lipid Homeostasis in the Mouse Neonatal Brain.

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.