The Effects of Benzalkonium Chloride Disinfectants on Lipid Homeostasis and Neurodevelopment

Thesis (Ph.D.)--University of Washington, 2019Developmental neurotoxicity (DNT) is one of the least tested health effects of the more than 80,000 chemicals registered for use today. In order to more efficiently test chemicals for DNT, more knowledge is needed on mechanisms that trigger adverse neurodevelopmental outcomes. Lipids are critical for neurodevelopment; therefore, disruption of lipid homeostasis by chemicals is expected to have detrimental effects on this process. However, few have investigated this as a possible mechanism of DNT. In preliminary studies, we demonstrated that benzalkonium chloride compounds (BACs) alter cholesterol biosynthesis and lipid homeostasis in neuronal cells. BACs are the most commonly used quaternary ammonium compound (QAC) disinfectants. They are applied in food processing lines, health care facilities, residential settings, and are common ingredients in over-the-counter cosmetics, hand sanitizers, and pharmaceutical products. Therefore, exposure to BACs is prevalent given the diversity of applications and may occur through dermal/eye contact, inhalation, and ingestion. Recent studies demonstrate that BAC exposure leads to an increased incidence of neural tube defects in utero and increased apoptosis of neural progenitor cells (NPCs). However, the effects of BACs on neurodevelopment as a result of altered lipid homeostasis has not been investigated. In my dissertation work, I characterized the effects of BACs on lipid homeostasis and neurodevelopmental processes. First, I showed that BACs potently inhibit cholesterol biosynthesis in mouse and human neuronal cells. Building on this work, I showed that BACs can cross the blood-placental barrier and enter the developing mouse brain following in utero exposure via maternal diet. Further transciptomic analyses of the developing brain elucidated key signaling pathways affected by BACs, including 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. Finally, I investigated the effects of BACs on neurospheres, free floating structures of NPCs, which are used as a three-dimensional (3-D) in vitro model of neurodevelopment. I found that BACs depleted the pool of NPCs and increased apoptosis, which contributed to a reduction in neurosphere growth. Transcriptome analysis revealed that BACs activated the integrated stress response, a mechanism used by cells to adapt to a variety of stressors including oxidative stress, mitochondrial dysfunction, nutrient deficiency, or hypoxia. Altogether, the findings of this dissertation demonstrate that a class of commonly used disinfectants alter lipid homeostasis and impact neurodevelopmental processes. These data add to a growing body of literature reporting the adverse effects of BACs on neurodevelopment. Importantly, this work supports a novel mechanism by which environmental chemicals may target neurodevelopment

Data from: Multi-omics 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

Neonatal brain transcriptome profiles

C57BL/6J neonates were exposed in utero, through maternal dietary exposure to control, BAC C12 (120 mg/kg/day), or BAC C16 (120 mg/kg/day). At postnatal day 0, neonates were sacrificed and brains were collected. This excel sheet contains 14 columns. Four biological replicates per treatment were used for transcriptomics analyses. Rows contain FPKM values of each transcript, signified by Ensembl IDs and Gene Names

Prevention of Retinal Degeneration in a Rat Model of Smith-Lemli-Opitz Syndrome

Abstract Smith-Lemli-Opitz Syndrome (SLOS) is a recessive human disease caused by defective cholesterol (CHOL) synthesis at the level of DHCR7 (7-dehydrocholesterol reductase), which normally catalyzes the conversion of 7-dehydrocholesterol (7DHC) to CHOL. Formation and abnormal accumulation of 7DHC and 7DHC-derived oxysterols occur in SLOS patients and in rats treated with the DHCR7 inhibitor AY9944. The rat SLOS model exhibits progressive and irreversible retinal dysfunction and degeneration, which is only partially ameliorated by dietary CHOL supplementation. We hypothesized that 7DHC-derived oxysterols are causally involved in this retinal degeneration, and that blocking or reducing their formation should minimize the phenotype. Here, using the SLOS rat model, we demonstrate that combined dietary supplementation with CHOL plus antioxidants (vitamins E and C, plus sodium selenite) provides better outcomes than dietary CHOL supplementation alone with regard to preservation of retinal structure and function and lowering 7DHC-derived oxysterol formation. These proof-of-principle findings provide a translational, pre-clinical framework for designing clinical trials using CHOL-antioxidant combination therapy as an improved therapeutic intervention over the current standard of care for the treatment of SLOS

Transcriptome profiling of equine vitamin E deficient neuroaxonal dystrophy identifies upregulation of liver X receptor target genes.

Specific spontaneous heritable neurodegenerative diseases have been associated with lower serum and cerebrospinal fluid α-tocopherol (α-TOH) concentrations. Equine neuroaxonal dystrophy (eNAD) has similar histologic lesions to human ataxia with vitamin E deficiency caused by mutations in the α-TOH transfer protein gene (TTPA). Mutations in TTPA are not present with eNAD and the molecular basis remains unknown. Given the neuropathologic phenotypic similarity of the conditions, we assessed the molecular basis of eNAD by global transcriptome sequencing of the cervical spinal cord. Differential gene expression analysis identified 157 significantly (FDR<0.05) dysregulated transcripts within the spinal cord of eNAD-affected horses. Statistical enrichment analysis identified significant downregulation of the ionotropic and metabotropic group III glutamate receptor, synaptic vesicle trafficking and cholesterol biosynthesis pathways. Gene co-expression analysis identified one module of upregulated genes significantly associated with the eNAD phenotype that included the liver X receptor (LXR) targets CYP7A1, APOE, PLTP and ABCA1. Validation of CYP7A1 and APOE dysregulation was performed in an independent biologic group and CYP7A1 was found to be additionally upregulated in the medulla oblongata of eNAD horses. Evidence of LXR activation supports a role for modulation of oxysterol-dependent LXR transcription factor activity by tocopherols. We hypothesize that the protective role of α-TOH in eNAD may reside in its ability to prevent oxysterol accumulation and subsequent activation of the LXR in order to decrease lipid peroxidation associated neurodegeneration

Transcriptome profiling of equine vitamin E deficient neuroaxonal dystrophy identifies upregulation of liver X receptor target genes.

Specific spontaneous heritable neurodegenerative diseases have been associated with lower serum and cerebrospinal fluid α-tocopherol (α-TOH) concentrations. Equine neuroaxonal dystrophy (eNAD) has similar histologic lesions to human ataxia with vitamin E deficiency caused by mutations in the α-TOH transfer protein gene (TTPA). Mutations in TTPA are not present with eNAD and the molecular basis remains unknown. Given the neuropathologic phenotypic similarity of the conditions, we assessed the molecular basis of eNAD by global transcriptome sequencing of the cervical spinal cord. Differential gene expression analysis identified 157 significantly (FDR<0.05) dysregulated transcripts within the spinal cord of eNAD-affected horses. Statistical enrichment analysis identified significant downregulation of the ionotropic and metabotropic group III glutamate receptor, synaptic vesicle trafficking and cholesterol biosynthesis pathways. Gene co-expression analysis identified one module of upregulated genes significantly associated with the eNAD phenotype that included the liver X receptor (LXR) targets CYP7A1, APOE, PLTP and ABCA1. Validation of CYP7A1 and APOE dysregulation was performed in an independent biologic group and CYP7A1 was found to be additionally upregulated in the medulla oblongata of eNAD horses. Evidence of LXR activation supports a role for modulation of oxysterol-dependent LXR transcription factor activity by tocopherols. We hypothesize that the protective role of α-TOH in eNAD may reside in its ability to prevent oxysterol accumulation and subsequent activation of the LXR in order to decrease lipid peroxidation associated neurodegeneration