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, 128 (1), 17014

Screening ToxCast™ for Chemicals That Affect Cholesterol Biosynthesis: Studies in Cell Culture and Human Induced Pluripotent Stem Cell-Derived Neuroprogenitors

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Screening ToxCast™ for Chemicals That Affect Cholesterol Biosynthesis: Studies in Cell Culture and Human Induced Pluripotent Stem Cell-Derived Neuroprogenitors

Phillip A Wages et al. Environ Health Perspect.

Abstract

Background: Changes in cholesterol metabolism are common hallmarks of neurodevelopmental pathologies. A diverse array of genetic disorders of cholesterol metabolism support this claim as do multiple lines of research that demonstrate chemical inhibition of cholesterol biosynthesis compromises neurodevelopment. Recent work has revealed that a number of commonly used pharmaceuticals induce changes in cholesterol metabolism that are similar to changes induced by genetic disorders with devastating neurodevelopmental deficiencies.

Objectives: We tested the hypothesis that common environmental toxicants may also impair cholesterol metabolism and thereby possibly contribute to neurodevelopmental toxicity.

Methods: Using high-throughput screening with a targeted lipidomic analysis and the mouse neuroblastoma cell line, Neuro-2a, the ToxCast™ chemical library was screened for compounds that impact sterol metabolism. Validation of chemical effects was conducted by assessing cholesterol biosynthesis in human induced pluripotent stem cell (hiPSC)-derived neuroprogenitors using an isotopically labeled cholesterol precursor and by monitoring product formation with UPLC-MS/MS.

Results: Twenty-nine compounds were identified as validated lead-hits, and four were prioritized for further study (endosulfan sulfate, tributyltin chloride, fenpropimorph, and spiroxamine). All four compounds were validated to cause hypocholesterolemia in Neuro-2a cells. The morpholine-like fungicides, fenpropimorph and spiroxamine, mirrored their Neuro-2a activity in four immortalized human cell lines and in a human neuroprogenitor model derived from hiPSCs, but endosulfan sulfate and tributyltin chloride did not.

Conclusions: These data reveal the existence of environmental compounds that interrupt cholesterol biosynthesis and that methodologically hiPSC neuroprogenitor cells provide a particularly sensitive system to monitor the effect of small molecules on de novo cholesterol formation. https://doi.org/10.1289/EHP5053.

Figures

Figure 1 is a schematic diagram of the following chemical compounds: (1) lanosterol, (2) 7-dehydrodesmosterol, (3) desmosterol, (4) dihydrolanosterol, (5) 7-dehydrocholesterol, (6) cholesterol, (7) 8-dehydrocholesterol, and (8) cholecalciferol (Vitamin D3). Compound 1 synthesises to compound 2 through CYP51 and SC5D. Compound 2 synthesises to compound 3 through DHCR7. Compounds 1, 2, and 3 also synthesize to compounds 4, 5 (through DHCR24), and 6 (through DHCR24), respectively. Compound 4 synthesises to compound 5 through CYP51 and SC5D. Compound 5 synthesises to compound 6 through DHCR7. Compound 5 also synthesizes to compound 7 through EBP and compound 8 through UV light. Compound 7 can synthesise to compound 5 through EBP.
Figure 1.
Schematic of cholesterol biosynthesis. Note: Selected enzymes involved in cholesterol biosynthesis are shown in dashed boxes.
Figure 2A is comprises a workflow with the following steps taking place one after the other: ToxCast chemical screen (1851 compounds) leads to lead hit validation (46 compounds), which leads to Lan (17 compounds) and 7DHC (21 compounds). Lan leads to endosulafan and tributyltin chloride, and 7DHC leads to frenpropimorph and spiroxamine. Figures 2B and 2C are horizontal bar graphs, plotting 7 dehydrocholesterol and lanosterol (y-axis), respectively, across z score. Those prioritized in elevating 7DHC are spiroxamine and fenpropimorph. Those prioritized in elevating lan are endosulfan sulfate and tributyltin chloride.
Figure 2.
Lead-hit determination of ToxCast™ Chemical Library for environmental cholesterol biosynthesis disruptors. (A) Workflow of high-throughput screen from entire library screened to the four selected lead-hit compounds, from bottom left, clockwise: tributyltin chloride, endosulfan sulfate, fenpropimorph, spiroxamine. Lead-hit compounds were identified through the results of two independent screens of the ToxCast™ library using Neuro-2a cells as an in vitro model at a screening exposure of 1μM for 24 h. Of the compounds screened, those determined as lead-hits for elevating 7-dehydrocholesterol (B) and lanosterol (C) are presented with their respective z-score values and those prioritized indicated with blue arrows.
Figures 3A, 3B, and 3C are chemical reactions. In 3A, 7DHC leads to 7DHC-PTAD through PTAD. In 3B, Des leads to Des-PTAD through PTAD. In 3C, 8DHC leads to Ene through PTAD, and Ene leads to 5, 7, 9 triene (which also leads to 5, 7, 9 PTAD) plus 5, 9, 14 triene (which also leads to 5, 9, 14 MeOH adduct).
Figure 3.
Proposed mechanism for the reaction of (A) 7-dehydrocholesterol, (B) desmosterol (or lanosterol), and (C) 8-dehydrocholesterol with PTAD. 7-DHC undergoes a Diels-Alder reaction, whereas Des and Lan undergo an ene reaction on the tail olefin. For 8-DHC, the Diels-Alder and MeOH adducts are the major products formed through a series of ene, elimination, and Diels-Alder reactions.
Figures 4A and 4B are three graphs each. 4A plots relative abundance (y-axis) for 5, 7, 9 PTAD (0.43), ene (0.42), and 5, 9, 14 MeOH (0.37), respectively, across time (x-axis). 4B plots relative abundance (y-axis) for 5, 7, 9 PTAD (0.43), ene (nill), and 5, 9, 14 MeOH (0.37), respectively, across time (x-axis).
Figure 4.
LC-MS analysis of (A) isolated and characterized products compared with (B) reaction of 8-dehydrocholesterol with PTAD under conditions used in sample analysis. The products were analyzed on an UPLC C18 column (Acquity UPLC BEH C18, 1.7μm, 2.1×50mm) with 100% MeOH (0.1% v/v acetic acid) mobile phase at a flow rate of 500μL/min and runtime of 1.2 min. The following SRMs were monitored: 5,7,9-PTAD 558363, ene 560365, and 5,9,14-MeOH 399363.
Figure 5 comprises the synthesis of chemical compounds. Compound 1 leads to compound 2 through a. Compound 2 leads to 5, 7, 9 triene through b. 5, 7, 9 triene leads to 5, 7, 9 PTAD through c. Compound 1 also leads to compound 3 below. Compound 3 leads to 8DHC through e. 8DHC leads to 5, 9, 14 triene through c. 5, 9, 14 triene leads to 5, 9, 14 MeOH through f. Reagents a, b, c, d, e, and f are Hg open parenthesis OAc close parenthesis sub 2; NaOH, MeOH virgule H sub 2 O; PTAD (1 eq), CH sub 2 CL sub 2; DEAD; Li super o, EtNH sub 2; and PTAD (10 eq), respectively.
Figure 5.
Synthesis of 5,7,9- and 5,9,14-trienes and their PTAD adducts.
Figure 6 comprises the synthesis of chemical compounds. Compound 4 leads to compound 5 through a, b. Compound 5 leads to compound 6 through c. Compound 6 leads to compound 7 through d. Compound 7 leads to compound 8 through e. Compound 8 leads to compound 9 through f. The reagents a, b, c, d, e, and f are TBDMSCL, im; phthalhydrazide, Pb open parenthesis OAc close parenthesis sub 4; O sub 3 and PPH sub 3; methyl (triphenylphosphoranylidene) acetate; H sub 2, Raney Ni; and LiAIH sub 4.
Figure 6.
Synthesis of 7-dehydrocholenol intermediate for d7-7-DHC and d7-8-DHC.
Figure 7 comprises the synthesis of chemical compounds. Compound 9 leads to compound 10 through a. Compound 10 leads to d sub 7 7 DHC. Compound 9 also leads to 11 major through d. 11 major leads to compound 12 through e, a. Compound 12 leads to d sub 7 8 DHC through b, c. The reagents a, b, c, d, and e are MsCl, pyr; d sub 7 2 bromopropane, Mg super o and Li sub 2 CuCl sub 4, and 6 or 9; TBAF; DEAD; and Li sub o, EtNH sub 2.
Figure 7.
Synthesis of d7-7-DHC and d7-8-DHC.
Figures 8A, 8B, 8C, and 8D are graphs plotting nanomoles Lan virgule 10 super 6 cells; nanomoles Des virgule 10 super 6 cells; nanomoles 7DHC virgule 10 super 6 cells; and nanomoles Des virgule 10 super 6 cells, respectively, (y-axis) across tributyltin chloride (micromolar); endosulfan sulfate (micromolar); fenpropimorph (micromolar); and spiroxamine (micromolar), respectively, (x-axis).
Figure 8.
Concentration-dependent response of lead-hit compounds on intracellular levels of lanosterol (A,B; solid squares), 7-dehydrocholesterol (C,D; open squares) and desmosterol (solid triangles) in Neuro-2a cells. Cells were exposed to tributyltin chloride (A), endosulfan sulfate (B), fenpropimorph (C), and spiroxamine (D) at the indicated concentration for 24 h. Data presented as nmol sterol/million cells (n=4, ±SEM). *p<0.05, **p<0.01, ***p<0.001 as determined by post hoc Dunnett’s test following one-way analysis of variance (ANOVA), using vehicle as the control comparison.
Figures 9A and 9B plot 7DHC nanomoles virgule 10 super 6 cells (y-axis) across fenpropimorph (SK N SH, A549, and Hep G2) (ranging from 0 nanomolar, 10 nanomolar, 100 nanomolar, and 1000 nanomolar) and spiroxamine (SK N SH, A549, and Hep G2) (ranging from 0 nanomolar, 10 nanomolar, 100 nanomolar, and 1000 nanomolar), respectively, (x-axis). Figures 9C and 9D plot 8DHC nanomoles virgule 10 super 6 cells (y-axis) across fenpropimorph (SK N SH, A549, and Hep G2) (ranging from 0 nanomolar, 10 nanomolar, 100 nanomolar, and 1000 nanomolar) and spiroxamine (SK N SH, A549, and Hep G2) (ranging from 0 nanomolar, 10 nanomolar, 100 nanomolar, and 1000 nanomolar), respectively, (x-axis). Figures 9E and 9F plot Des nanomoles virgule 10 super 6 cells (y-axis) across fenpropimorph (SK N SH, A549, and Hep G2) (ranging from 0 nanomolar, 10 nanomolar, 100 nanomolar, and 1000 nanomolar) and spiroxamine (SK N SH, A549, and Hep G2) (ranging from 0 nanomolar, 10 nanomolar, 100 nanomolar, and 1000 nanomolar), respectively, (x-axis). Figures 9G and 9H plot Chol nanomoles virgule 10 super 6 cells (y-axis) across fenpropimorph (SK N SH, A549, and Hep G2) (ranging from 0 nanomolar, 10 nanomolar, 100 nanomolar, and 1000 nanomolar) and spiroxamine (SK N SH, A549, and Hep G2) (ranging from 0 nanomolar, 10 nanomolar, 100 nanomolar, and 1000 nanomolar), respectively, (x-axis).
Figure 9.
Impact of fenpropimorph and spiroxamine on 7-dehydrocholesterol (7-DHC; A,B), 8-dehydrocholesterol (8-DHC; C,D), desmosterol (Des; E,F), and cholesterol levels (Chol; G,H) in three different human-derived cell lines: (from left to right) SK-N-SH, A549, and Hep-G2. Cells were exposed to compound (0, 10, 100, 1,000nM) for 24 h. Omitted bars reflect significant toxicity as determined by release of lactate dehydrogenase. Data presented as nmol sterol/million cells (n=4, ±SEM). *p<0.05, **p<0.01, ***p<0.001 as determined by post hoc Dunnett’s test following one-way analysis of variance (ANOVA), using vehicle as the control comparison.
Figure 10A comprises synthesis of chemical compounds, where 7dehydrocholesterol leads to cholesterol through DHCR7 and also to 8dehydrocholesterol through EBP. 8dehydrocholesterol also leads to 7dehydrocholesterol through EBP. Figures 10B and 10C plot 8DHC nanomoles virgule 10 super 6 cells (y-axis) across fenpropimorph (nanomolar) and spiroxamine (nanomolar), respectively, (x-axis).
Figure 10.
Biological synthesis of 8-dehydrocholesterol via isomerization of 7-dehydrocholesterol by the enzyme EBP is shown (A). SK-N-SH cells were exposed to fenpropimorph (B) and spiroxamine (C) at indicated concentrations for 24 h. Data presented as nmol 8-dehydrocholesterol/million cells (n=4, ±SEM). *p<0.05, ***p<0.001 as determined by post hoc Dunnett’s test following one-way analysis of variance (ANOVA), using vehicle as the control comparison.
Figure 11A is a differentiation of human induced pluripotent stem cells through a span of 10 days, where differentiated factors are LDN virgule SB431542 and media components are knockout serum and N2. The 8th day reveals replated NPs, and the 10th day reveals exposures. 11B and 11C plot nanomoles 7DHC virgule 10 super 6 cells and nanomoles Des virgule 10 super 6 cells, respectively, (y-axis) across time (hours) for fenpropimorph (ranging from 0 to 1000 nanomolar). 11D and 11D plot nanomoles 7DHC virgule 10 super 6 cells and nanomoles Des virgule 10 super 6 cells, respectively, (y-axis) across time (hours) for spiroxamine (ranging from 0 to 1000 nanomolar).
Figure 11.
Human induced pluripotent stem cells (hiPSCs) were differentiated towards a neuroectoderm lineage as shown (A). These hiPSC-derived neuroprogenitor cells were exposed on day 10 of differentiation to fenpropimorph (B,C) or spiroxamine (D,E) at 1,000nM (red triangle) for 4, 8, 12, and 24 h. Neuroprogenitor cells were then analyzed for 7-dehydrocholesterol (B,D) or desmosterol (C,E) and compared with vehicle control (black circle; 0.01% DMSO). Three distinct differentiations were conducted for each donor and averaged; shown is the average of the three donors ±SEM (n=3). *p<0.05, **p<0.01, ***p<0.001 as determined by Bonferroni posttests following two-way analysis of variance (ANOVA).
Figure 12A comprises synthesis of chemical compounds, where super 13 C sub 3 Lan leads to a combination of super 13 C sub 3 7DHC and super 13 C sub 3 Chol; 13 C sub 3 7DHC leads to super 13 C sub 3 Chol. Figure 12B is a grid with four cells. The cells in the clock-wise direction are labeled as follows: (1)8DHC, (2)desmosterol, (3)cholesterol, and (4)7DHC. 1 through EBP leads to 4 and 4 through EBP leads to 1. 2 through DCHR24 leads to 3. 4 through DHCR7 also leads to 3. Figures 12C, 12D, 12E, and 12F are graphs plotting 8DHC (picomoles virgule 10 super 6 cells), Des (picomoles virgule 10 super 6 cells), 7DHC (picomoles virgule 10 super 6 cells), and Chol (picomoles virgule 10 super 6 cells), respectively, (y-axis) across fenpropimorph, ranging from 0 to 1000 nanomolar. Figures 12G, 12H, 12I, and 12J are graphs plotting 8DHC (picomoles virgule 10 super 6 cells), Des (picomoles virgule 10 super 6 cells), 7DHC (picomoles virgule 10 super 6 cells), and Chol (picomoles virgule 10 super 6 cells), respectively, (y-axis) across spiroxamine, ranging from 0 to 1000 nanomolar.
Figure 12.
De novo synthesis of cholesterol and sterol precursors was accomplished by incubating hiPSC-derived neuroprogenitor cells with C313-lanosterol for 24 h and monitoring for C313-sterols including C313-7-dehydrocholesterol and C313-cholesterol (A). Simplified cholesterol biosynthetic pathway is shown (B). Neuroprogenitor cells were exposed to fenpropimorph (C–F) or spiroxamine (G–J) at 10nM and 1,000nM for the same duration as the C313-lanosterol incubation. Absolute values of C313-8-dehydrocholesterol (C,G), C313-desmosterol (D,H), C313-7-dehydrocholesterol (E,I), and C313-cholesterol (F,J) were detected, quantified and normalized to cell number. Three distinct differentiations were conducted for each donor and averaged; shown is the average of the three donors ±SEM (n=3). *p<0.05, **p<0.01, ***p<0.001 as determined by post hoc Dunnett’s test following one-way analysis of variance (ANOVA).
Figure 13 is a graph plotting nanomoles super 13 C sub 3 sterol virgule 10 super 6 cells (y-axis) for Lan, Des, 8DHC, 7DHC, and cholesterol across time (hours) (x-axis).
Figure 13.
De novo synthesis of cholesterol and sterol precursors was accomplished by incubating hiPSC-derived neuroprogenitor cells with C313-lanosterol for 24 h and monitoring for C313-sterols at indicated time points. Isotopically labeled sterols analyzed are lanosterol (solid line), desmosterol (solid square, dashed line), 8-DHC (open triangle, solid line), 7-DHC (solid circle, solid line), and cholesterol (open circle, dashed line). Three distinct differentiations were conducted for each donor and averaged; shown is the average of the three donors ±SEM (n=3). *p<0.05, **p<0.01, ***p<0.001 as determined by repeated measures one-way analysis of variance (ANOVA).

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