Vancamp P et al. (2021), The pyriproxyfen metabolite, 4'-OH-PPF, disrupt...

XB-ART-58340

Environ Pollut 2021 Sep 15;285:117654. doi: 10.1016/j.envpol.2021.117654.

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The pyriproxyfen metabolite, 4'-OH-PPF, disrupts thyroid hormone signaling in neural stem cells, modifying neurodevelopmental genes affected by ZIKA virus infection.

Vancamp P , Spirhanzlova P , Sébillot A , Butruille L , Gothié JD , Le Mével S , Leemans M , Wejaphikul K , Meima M , Mughal BB , Roques P , Remaud S , Fini JB , Demeneix BA .

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North-Eastern Brazil saw intensive application of the insecticide pyriproxyfen (PPF) during the microcephaly outbreak caused by the Zika virus (ZIKV). ZIKV requires the neural RNA-binding protein Musashi-1 to replicate. Thyroid hormone (TH) represses MSI1. PPF is a suspected TH disruptor. We hypothesized that co-exposure to the main metabolite of PPF, 4'-OH-PPF, could exacerbate ZIKV effects through increased MSI1 expression. Exposing an in vivo reporter model, Xenopus laevis, to 4'-OH-PPF decreased TH signaling and increased msi1 mRNA and protein, confirming TH-antagonistic properties. Next, we investigated the metabolite's effects on mouse subventricular zone-derived neural stem cells (NSCs). Exposure to 4'-OH-PPF dose-dependently reduced neuroprogenitor proliferation and dysregulated genes implicated in neurogliogenesis. The highest dose induced Msi1 mRNA and protein, increasing cell apoptosis and the ratio of neurons to glial cells. Given these effects of the metabolite alone, we considered if combined infection with ZIKV worsened neurogenic events. Only at the fourth and last day of incubation did co-exposure of 4'-OH-PPF and ZIKV decrease viral replication, but viral RNA copies stayed within the same order of magnitude. Intracellular RNA content of NSCs was decreased in the combined presence of 4'-OH-PPF and ZIKV, suggesting a synergistic block of transcriptional machinery. Seven out of 12 tested key genes in TH signaling and neuroglial commitment were dysregulated by co-exposure, of which four were unaltered when exposed to 4'-OH-PPF alone. We conclude that 4'-OH-PPF is an active TH-antagonist, altering NSC processes known to underlie correct cortical development. A combination of the TH-disrupting metabolite and ZIKV could aggravate the microcephaly phenotype.

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Species referenced: Xenopus laevis
Genes referenced: casp2 dlx2 fasn msi1 sox10
GO keywords: brain development [+]

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	Graphical Abstract
	Fig. 1. Structural similarity of 3,3′,5′-triiodothyronine (T3), the larvicide pyriproxyfen (PPF), and its hydroxylated metabolite 4′–OH–PPF.
	Fig. 2. Exposure to 4′–OH–PPF affects thyroid hormone signaling, msi1 gene expression and its encoded protein in Xenopus laevis. (A) Tg(thibz:eGFP) tadpoles exposed for 72 h to (from left to right) vehicle control, 5 nM T3 and 5 nM T3 + 3 × 10−1 mg/L 4‘–OH–PPF. Scale bar: 500 μm. (B) 72 h exposure of NF45 Tg(thibz:eGFP) tadpoles to 4’–OH–PPF with (left graph) or without 5 nM T3 (right graph). Values normalized to control group or T3-treated group, respectively (3 independent experiments; n = 15 per experiment). (C) Representative pictures of dorsal views of NF45 Xenopus tadpoles exposed to 5 nM T3 or T3 + 10−2 mg/L 4′–OH–PPF and stained immunohistochemistically for Msi1. Msi1 levels are visualized as a heat map representing integrated density with arbitrary values from 0 to 256. Dark blue areas – no or low expression of Msi1; yellow/white – highest expression of Msi1; fb: forebrain, mb: midbrain, hb: hindbrain. (D) msi1 gene (upper graph) and Msi1 protein (lower graph) expression in dissected brains from NF45 tadpoles exposed for 24 h to 4′–OH–PPF (10−2; 10−1 mg/L) together with 5 nM T3 (3 independent experiments, n = 5 per experiment). Protein levels were quantified by measuring fluorescence intensity from dorsal views (2 independent experiments with 4–10 tadpoles per experiment). Data were analyzed by a one-way ANOVA with Dunnet's post-hoc test (Mean ± SEMs, p < 0.05, p < 0.01, **p < 0.001). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
	Fig. 3. Exposure to 4′–OH–PPF decreases NSC proliferation and disrupts gene expression dose-dependently. (A) Schematic timeline illustrating the experimental design for the neurosphere assay. (B) Two representative pictures of neurospheres in control and 3 × 10−1 mg/L 4′–OH–PPF conditions. The graphs show the neurosphere diameter (left) and average neurosphere number (right) in presence of 10 nM T3 ± different doses of 4′–OH–PPF versus controls without added T3 (n = 12 wells/group, Kruskal-Wallis ANOVAs: p < 0.001, followed by Dunn's post-tests: p < 0.001). (C) Graphs showing Msi1, Fasn, Dlx2, Ng2, and Sox10 mRNA expression levels in presence of 10 nM T3 ± different doses of 4′–OH–PPF (n = 3–6 per group, Kruskal-Wallis ANOVAs: Msi1, p = 0.0009, Fasn, p = 0.0019, Dlx2, p = 0.0088, Ng2, p = 0.0159, Sox10, p = 0.0032, followed by Dunn's post-tests). The panel at the lower right indicates how the genes are implicated in mouse SVZ-NSC fate. All graphs depict means ± SDs. DIV: days in vitro, EGF: epidermal growth factor, FGF: fibroblast growth factor, NSC: neural stem cell, OL: oligodendrocyte, OPC: oligodendrocyte progenitor cell, SVZ: subventricular zone. p < 0.05, p < 0.01, **p < 0.001.
	Fig. 4. Exposure to 4′–OH–PPF increases cellular MSI1 protein, cell apoptosis and the neuro/glia ratio. (A) Schematic timeline illustrating the experimental design for the neurosphere assay. (B) Representative images of immunostained MSI1 protein (green, upper images) and activated caspase-3 (green, lower images) in cultured adult mouse NSCs (blue) following exposure to 0.01% DMSO (CTL) or 3 × 10−1 mg/L 4′–OH–PPF for 1 day. Inserts show the proteins within individual cells. Scale bars: 20 μm. (C) Left upper graph showing a borderline-significantly increased proportion of MSI1-positive cells (two-tailed t-test, t56 = 1.923, p = 0.0596). The right upper graph shows an increased integrative density of the fluorescent signal per cell, indicating elevated MSI1 protein levels (Mann-Whitney test, p < 0.001). The left lower graph shows an increase in activated caspase-3-positive cells under 4′–OH–PPF exposure (Mann-Whitney test, p < 0.001). (D) Representative images of immunostained DCX (green, neuroblasts) and OLIG2 (red, oligodendrocytes) in cultured mouse SVZ-NSCs (blue) following exposure to 0.01% DMSO (CTL) or 3 × 10−1 mg/L 4′–OH–PPF for 5 days. (E) Graph showing the proportion of DCX- (Mann-Whitney test, p < 0.001) and OLIG2-positive cells (two-tailed t-test, t58 = 3.294, p = 0.0017). (F) Graph showing the ratio of DCX- vs OLIG2-positive cells changed significantly (Mann-Whitney test, p < 0.001). For all experiments, n = 3 replicates/group, n = 9–10 images/replicate. All graphs depict means ± SDs. CTL: control, DIV: day in vitro, EGF: epidermal growth factor, FGF: fibroblast growth factor, NSC: neural stem cell, OPC: oligodendrocyte progenitor cells, SVZ: subventricular zone. ***p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
	Fig. 5. Exposure to 4′–OH–PPF does not alter ZIKV replication in NSCs, but decreases intracellular RNA content and dysregulates mRNA expression. (A) Schematic timeline of 4'-OH-PPF exposure and viral infection. (B) Left graph showing an exponential increase in ZIKV virus RNA copies/mL during 4 days post-infection. ZIKV virus RNA copies/mL were reduced at day 4 under 4'-OH-PPF exposure as compared to controls, but no other day (2 independent experiments, n = 6/group, two-way ANOVA: p < 0.001, followed by Bonferroni's multiple comparisons test: day 0–3: p > 0.05, day 4: p < 0.001). The right graph shows neuroprogenitor intracellular RNA content (n = 6–12, one-way ANOVA: p < 0.001, followed by Tukey's post-test: p < 0.01). (C) Heatmap showing relative mRNA expression of genes implicated in thyroid hormone signaling, neuroprogenitors and neuroglial commitment. While the expression of only 3 genes was altered with 4′–OH–PPF exposure alone, ZIKV infection with or without 4′–OH–PPF dysregulated respectively 7 to 8 of the 12 tested genes (n = 6–12, one-way ANOVAs followed by Tukey's post-test in case of significance). Graphs depict means ± SDs. CTL: control, DIV: days in vitro, EGF: epidermal growth factor, FGF: fibroblast growth factor, NB: neuroblast, OL: oligodendrocyte, SVZ: subventricular zone, ZIKV: Zika virus. p < 0.05, p < 0.01, **p < 0.001.

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