Identification of Environmental Quaternary Ammonium Compounds as Direct Inhibitors of Cholesterol Biosynthesis

Abstract

In this study, we aim to identify environmental molecules that can inhibit cholesterol biosynthesis, potentially leading to the same biochemical defects as observed in cholesterol biosynthesis disorders, which are often characterized by congenital malformations and developmental delay. Using the Distributed Structure-Searchable Toxicity (DSSTox) Database Network developed by EPA, we first carried out in silico screening of environmental molecules that display structures similar to AY9944, a known potent inhibitor of 3β-hydroxysterol-Δ(7)-reductase (DHCR7)-the last step of cholesterol biosynthesis. Molecules that display high similarity to AY9944 were subjected to test in mouse and human neuroblastoma cells for their effectiveness in inhibiting cholesterol biosynthesis by analyzing cholesterol and its precursor using gas chromatography-mass spectrometry. We found that a common disinfectant mixture, benzalkonium chlorides (BACs), exhibits high potency in inhibiting DHCR7, as suggested by greatly elevated levels of the cholesterol precursor, 7-dehydrocholesterol (7-DHC). Subsequent structure-activity studies suggested that the potency of BACs as Dhcr7 inhibitors decrease with the length of their hydrocarbon chain: C10 > C12 ≫ C14 > C16. Real-time qPCR analysis revealed upregulation of the genes related to cholesterol biosynthesis and downregulation of the genes related to cholesterol efflux, suggesting a feedback response to the inhibition. Furthermore, an oxidative metabolite of 7-DHC that was previously identified as a biomarker in vivo was also found in cells exposed to BACs by liquid chromatography-mass spectrometry. Our findings suggest that certain environmental molecules could potently inhibit cholesterol biosynthesis, which could be a new link between environment and developmental disorders.

Assessment of altered lipid homeostasis by HILIC-ion mobility-mass spectrometry-based lipidomics

“…The lipidomic results on BAC-exposed Neuro2a cells are consistent with our previous studies on the biological activities of individual BACs (47). Because BAC-C10 acts as a potent inhibitor of DHCR7, we anticipated that the effects of AY9944 and BAC-C10 treatments on the Neuro2a lipidome would be similar.…”

“…In the context of elevated phospholipid levels, the decreased DG levels in BAC-C16 treatment suggest increased glycerophospholipid biosynthesis from DGs rather than decreased DG synthesis. In our previous study, gene expression analysis revealed that fatty acid synthase was significantly upregulated in BAC-C16-treated Neuro2a cells (47), which is consistent with the elevated phospholipid levels observed in BAC-C16 treatment.…”

“…Many of the lipid species affected, such as PEs and SMs, play significant roles in neurological development and function (62,63). These results in conjunction with our previous findings, suggest that BAC exposure could lead to or contribute to the pathogenesis of developmental disorders if exposure occurs during developmental stages (47). …”

“…AY9944 is a known inhibitor of 3-hydroxysterol- 7 -reductase (DHCR7), the last step of cholesterol biosynthesis (46). We recently found that BACs, a class of environmental toxins, also inhibit cholesterol biosynthesis, but their activities appear to vary with the length of the alkyl chain (47). Specifically, BAC-C10 acts as a potent inhibitor of DHCR7, whereas BAC-C16 does not, but BAC-C16 induced significantly more changes to genes associated with lipid homeostasis (47).…”

“…The prominent biochemical features of DHCR7 inhibition are decreased cholesterol levels and elevated levels of 7-DHC, 7-dehydrodesmosterol (7-DHD), as well as the 7-DHC-derived oxysterols 4-OH-7-DHC, 4-OH-7-DHC and DHCEO (46,53,54). We have previously shown that 7-DHC, 7-DHD, and DHCEO are significantly elevated in AY9944-and BAC-C10-treated Neuro2a cells (47). It is likely that the observed oxysterols are 7-DHC-or 7-DHD-derived oxysterols, given that the mass difference between the tentative oxysterols is the same as the mass difference between 7-DHC and 7-DHD (i.e., 384.3 Da and 382.3 Da, respectively).…”