Gene-Environment Interactions in Developmental Neurotoxicity: a Case Study of Synergy between Chlorpyrifos and CHD8 Knockout in Human BrainSpheres

Abstract

Background: Autism spectrum disorder (ASD) is a major public health concern caused by complex genetic and environmental components. Mechanisms of gene-environment ($G\times E$) interactions and reliable biomarkers associated with ASD are mostly unknown or controversial. Induced pluripotent stem cells (iPSCs) from patients or with clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR/Cas9)-introduced mutations in candidate ASD genes provide an opportunity to study ($G\times E$) interactions.

Objectives: In this study, we aimed to identify a potential synergy between mutation in the high-risk autism gene encoding chromodomain helicase DNA binding protein 8 (CHD8) and environmental exposure to an organophosphate pesticide (chlorpyrifos; CPF) in an iPSC-derived human three-dimensional (3D) brain model.

Methods: This study employed human iPSC-derived 3D brain organoids (BrainSpheres) carrying a heterozygote CRISPR/Cas9-introduced inactivating mutation in CHD8 and exposed to CPF or its oxon-metabolite (CPO). Neural differentiation, viability, oxidative stress, and neurite outgrowth were assessed, and levels of main neurotransmitters and selected metabolites were validated against human data on ASD metabolic derangements.

Results: Expression of CHD8 protein was significantly lower in CHD8 heterozygous knockout ($CHD{8}^{+/-}$) BrainSpheres compared with $CHD{8}^{+/+}$ ones. Exposure to CPF/CPO treatment further reduced CHD8 protein levels, showing the potential ($G\times E$) interaction synergy. A novel approach for validation of the model was chosen: from the literature, we identified a panel of metabolic biomarkers in patients and assessed them by targeted metabolomics in vitro. A synergistic effect was observed on the cholinergic system, S-adenosylmethionine, S-adenosylhomocysteine, lactic acid, tryptophan, kynurenic acid, and $\alpha \text{-hydroxyglutaric}$ acid levels. Neurite outgrowth was perturbed by CPF/CPO exposure. Heterozygous knockout of CHD8 in BrainSpheres led to an imbalance of excitatory/inhibitory neurotransmitters and lower levels of dopamine.

Discussion: This study pioneered ($G\times E$) interaction in iPSC-derived organoids. The experimental strategy enables biomonitoring and environmental risk assessment for ASD. Our findings reflected some metabolic perturbations and disruption of neurotransmitter systems involved in ASD. The increased susceptibility of $\text{CHD}{8}^{+/-}$ BrainSpheres to chemical insult establishes a possibly broader role of ($G\times E$) interaction in ASD. https://doi.org/10.1289/EHP8580.

Figure 1.

$CHD{\mathit{8}}^{+/+}$ and $CHD{\mathit{8}}^{+/-}$ NPC characterization. Scheme of BrainSphere differentiation. Prior differentiation to BrainSpheres, NPCs were expanded in monolayers and characterized. (A) CHD8 protein levels in $CHD{\mathit{8}}^{+/-}$ vs. $CHD{\mathit{8}}^{+/+}$ NPCs analyzed by western blot. (B) Representative images showing expression of the NPC markers Sox2 (green) and Nestin (red) in $CHD{\mathit{8}}^{+/+}$ and $CHD{\mathit{8}}^{+/-}$ NPC cultures prior to induction of differentiation into BrainSpheres. The nuclei were visualized with Hoechst 33342. Scale bar: $50\mathrm{\mu }\mathrm{m}$. (C) Differentiation and toxicant treatment scheme. Note: CPF, chlorpyrifos; CPO, oxon-metabolite of chlorpyrifos; EB, embryoid bodies; NPC, neural progenitor cell; 3D, three dimensional.

Figure 2.

Cell viability and AChE activity after exposure to CPF or CPO. Resazurin reduction assay in 47 and $100\mathrm{\mu }\mathrm{M}$ (A) CPF- and (B) CPO-treated $CHD{\mathit{8}}^{+/+}$ (gray) and $CHD{\mathit{8}}^{+/-}$ (white) BrainSpheres for 24 h at 4 and 8 wk of differentiation. The data represents three independent experiments with three technical replicates each (9 replicates in total, and 6 for 8-wk CPO treatment) normalized to DMSO vehicle-treated controls. Corresponding summary data are shown in Table S7. Asterisks after E, G, and I denote discovery of two-way ANOVA with posttest multiple comparisons for exposure effect between $\text{CHD}{8}^{+/+}$ and $\text{CHD}{8}^{+/-}$ BrainSpheres by the two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli and a desired false discovery rate (FDR) of 0.05. *$p<0.05$, **$p<0.01$, ***$p<0.001$. (C) AChE activity: the data represent the AChE activity normalized to the protein amount in each sample ($\text{mean}\pm \text{SEM}$, three independent experiments, *$p<0.01$, ****$p<0.0001$); (D) mRNA expression (five independent experiments, with 7 technical replicates in total, *$p<0.05$, **$p<0.01$); intracellular levels of (E) acetylcholine and (F) choline measured at 4 wk of differentiation in $CHD{\mathit{8}}^{+/+}$ and $CHD{\mathit{8}}^{+/-}$ spheroids after exposure to $100\mathrm{\mu }\mathrm{M}$ CPF (dark gray) and $100\mathrm{\mu }\mathrm{M}$ CPO (light gray). DMSO vehicle-treated controls are depicted in white. Acetylcholine and choline were measured by LC-MS/MS in three independent experiments (12 technical replicates in total). Data were normalized to DMSO vehicle-treated $CHD{\mathit{8}}^{+/+}$ controls. Discovery of effect by genetics or by exposure or by ($\mathrm{G}\times \mathrm{E}$) interaction on the metabolite levels was based on a two-way ANOVA with posttest multiple comparisons to *$CHD{\mathit{8}}^{+/+}$ DMSO or ^#$CHD{\mathit{8}}^{+/-}$ DMSO by the two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli and a desired FDR of 0.01. *$p<0.01$, ****$p<0.0001$. Corresponding summary data are shown in Tables S8 and S10. Note: AChE, acetylcholinesterase; ANOVA, analysis of variance; CPF, chlorpyrifos; CPO, oxon-metabolite of chlorpyrifos; DMSO, dimethyl sulfoxide; E, discovery of effect by exposure; G, discovery of effect by genetics; $\mathrm{G}\times \mathrm{E}$, gene–environment; I, discovery of effects by $\mathrm{G}\times \mathrm{E}$; n.s., not significant; SEM, standard error of the mean.

Figure 3.

Neurite outgrowth upon $100\mathrm{\mu }\mathrm{M}$ CPF treatment and co-treatment with $100\mathrm{\mu }\mathrm{M}$ tocopherol. (A) Sholl analysis of neurite outgrowth in $CHD{\mathit{8}}^{+/+}$ and $CHD{\mathit{8}}^{+/-}$ showing number of intersections (neurite density) starting from the edge of the spheroid. Each curve represents the $\text{mean}\pm \text{SEM}$ from 8 to 12 spheroids. (B) Area under the curve (AUC), calculated for each condition shown in (A). **$p<0.01$ and *** $p<0.001$, one-way ANOVA with Holm-Sidak’s posttest. (C) Representative images for each treatment. Scale bar: $200\mathrm{\mu }\mathrm{m}$. Note: ANOVA, analysis of variance; CPF, chlorpyrifos; DMSO, dimethyl sulfoxide; SD, standard deviation; SEM, standard error of the mean; Vit. E, tocopherol.

Figure 4.

CHD8 expression upon CPF/CPO treatment. (A) CHD8 gene expression in course of differentiation (four independent experiments). (B) CHD8 gene expression in $CHD{\mathit{8}}^{+/+}$ and $CHD{\mathit{8}}^{+/-}$ BrainSpheres exposed to CPF/CPO (four independent experiments). (C) Western Blot quantification of CHD8 protein in $CHD{\mathit{8}}^{+/+}$ and $CHD{\mathit{8}}^{+/-}$ BrainSpheres treated with CPF. CHD8 protein level was normalized to GAPDH and is expressed as a percentage of $CHD{\mathit{8}}^{+/+}$ DMSO vehicle-treated control BrainSpheres. Dendrogram is shown as $\text{means}\pm \text{SEMs}$ (five independent experiments). Statistical significance was calculated by two-way ANOVA with posttest multiple comparisons to *$CHD{\mathit{8}}^{+/+}$ DMSO or ^#$CHD{\mathit{8}}^{+/-}$ DMSO by the two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli and a desired false discovery rate of 0.05. *$p<0.05$, **$p<0.01$, ***$p<0.001$, ****$p<0.0001$. A representative blot is shown on the right side of (C). (D) Immunostaining of $CHD{\mathit{8}}^{+/+}$ and $CHD{\mathit{8}}^{+/-}$ BrainSpheres with CHD8 antibody (red) after CPF or CPO exposure. Representative images from three experiments are shown. Nuclei are stained with Hoechst 33342 (blue). Scale bar: $100\mathrm{\mu }\mathrm{m}$. Summary data (mean, SEM, N) are shown in Table S9. Note: ANOVA, analysis of variance; CPF, chlorpyrifos; CPO, oxon-metabolite of chlorpyrifos; DMSO, dimethyl sulfoxide; E, discovery of effect by exposure; G, discovery of effect by genetics; $\mathrm{G}\times \mathrm{E}$, gene–environment; GADPH, glyceraldehyde-3-phosphate dehydrogenase; I, discovery of effect by ($\mathrm{G}\times \mathrm{E}$) interaction; MS, mass spectrometry; NPC, neural progenitor cell; ns, not significant; SEM, standard error of the mean.

Figure 5.

Effects of CHD8 mutation and CPF/CPO exposure on key adverse outcome pathways of ASD. (A) Methyl donor system: SAM, SAH, SAM/SAH ratio, and folic acid. (B) l-alanine and lactic acid, (C) tryptophan and KA, and (D) $\mathrm{\alpha }\text{-hydroxyglutaric}$ acid levels were measured by LC-MS/MS. DMSO vehicle-treated controls are depicted in white, CPF-treated in dark gray, and CPO in light gray. Data are normalized to DMSO vehicle-treated $CHD{\mathit{8}}^{+/+}$ control and represent results from three independent experiments (12 technical replicates in total, 8 for KA). Discovery of effect by genetics or by exposure was based on a two-way ANOVA with posttest multiple comparisons to *$CHD{\mathit{8}}^{+/+}$ DMSO or ^#$CHD{\mathit{8}}^{+/-}$ DMSO by the two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli and a desired false discovery rate of 0.01. *^,#$p<0.01$, ***$p<0.001$, ****$p<0.0001$. Summary data (mean, SD, SEM, N) are shown in Table S10. Note: ANOVA, analysis of variance; ASD, autism spectrum disorder; CPF, chlorpyrifos; CPO, oxon-metabolite of chlorpyrifos; DMSO, dimethyl sulfoxide; E, discovery of effect by exposure; G, discovery of effect by genetics; KA, kynurenic acid; LC, liquid chromatography; MS/MS, tandem mass spectrometry; SAM, S-adenosylmethionine; SAH, S-adenosylhomocysteine; SD, standard deviation; SEM, standard error of the mean.

Figure 6.

Excitatory/inhibitory neurotransmitters detection in $CHD{\mathit{8}}^{+/+}$ and $CHD{\mathit{8}}^{+/-}$ BrainSpheres after CPF/CPO exposure. (A) Glutamate and (B) GABA levels and (C) their ratio in $CHD{\mathit{8}}^{+/-}$ vs. $CHD{\mathit{8}}^{+/+}$ BrainSpheres treated with CPF (dark gray), CPO (light gray), or vehicle (white) were assessed by LC-MS/MS. Data from three independent experiments (12 technical replicates in total) normalized to DMSO vehicle-treated $CHD{\mathit{8}}^{+/+}$ controls is shown. Discovery of effect by genetics or by exposure was based on a two-way ANOVA with posttest multiple comparisons to *$CHD{\mathit{8}}^{+/+}$ DMSO or ^#$CHD{\mathit{8}}^{+/-}$ DMSO by the two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli and a desired false discovery rate of 0.01. *^,#$p<0.01$, ****$p<0.0001$. Summary data (mean, SD, SEM, N) are shown in Table S10. Note: ANOVA, analysis of variance; CPF, chlorpyrifos; CPO, oxon-metabolite of chlorpyrifos; DMSO, dimethyl sulfoxide; E, discovery of effect by exposure; G, discovery of effect by genetics; GABA, gamma-aminobutyric acid; Glu, Glutamate; Gly, glycine; LC, liquid chromatography; MS/MS, tandem mass spectrometry; SD, standard deviation; SEM, standard error of the mean.

Figure 7.

Perturbations of the dopaminergic system in ${\text{CHD}8}^{+/+}$ vs. ${\text{CHD}8}^{+/–}$ BrainSpheres after CPF/CPO treatment. (A) Levels of phenylalanine, tyrosine, l-DOPA, and dopamine were measured by LC-MS/MS. Data from three independent experiments (12 technical replicates in total) normalized to DMSO vehicle-treated $\text{CHD}{8}^{+/+}$ controls is shown. Discovery of effect by genetics or by exposure was based on a two-way ANOVA with posttest multiple comparisons to $CHD{\mathit{8}}^{+/+}$ DMSO by the two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli and a desired false discovery rate (FDR) of 0.01. *$p<0.01$, ****$p<0.0001$. (B) RT-PCR of tyrosine hydroxylase (TH) and catechol-O-methyltransferase (COMT) in both cell lines treated with CPF. Data represent ${\log }_2\left(2^{-\mathrm{\Delta }\mathrm{\Delta }\text{Ct}}\right)$ of at least three independent experiments with 11 to 16 technical replicates in total. Discovery of effect by genetics or by exposure was based on a two-way ANOVA with posttest multiple comparisons to $CHD{\mathit{8}}^{+/+}$ DMSO by the two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli and a desired FDR of 0.05. *$p<0.05$, ***$p<0.001$, ****$p<0.0001$. (C) Immunohistochemistry with anti-TH–specific antibody (green) of $\text{CHD}{8}^{+/+}$ and $\text{CHD}{8}^{+/-}$ BrainSpheres treated with CPF/CPO. White arrowheads indicate neuronal-shaped cells, blue arrows indicate flat clusters of cells. Nuclei were stained with Hoechst 33342 (blue). Scale bar: $100\mathrm{\mu }\mathrm{m}$. Summary data (mean, SD, SEM, N) are shown in Tables S9 and S10. Note: ANOVA, analysis of variance; CPF, chlorpyrifos; CPO, oxon-metabolite of chlorpyrifos; DMSO, dimethyl sulfoxide; E, discovery of effect by exposure; G, discovery of effect by genetics; LC, liquid chromatography; l-DOPA, 1-3,4-dihydroxypyhenylalanine (or levodopa); MS/MS, tandem mass spectroscopy; ns, not significant; RT-PCR, real-time polymerase chain reaction; SD, standard deviation; SEM, standard error of the mean.