Benzalkonium Chloride Disinfectants Induce Apoptosis, Inhibit Proliferation, and Activate the Integrated Stress Response in a 3-D in Vitro Model of Neurodevelopment

Abstract

We previously found that the widely used disinfectants, benzalkonium chlorides (BACs), alter cholesterol and lipid homeostasis in neuronal cell lines and in neonatal mouse brains. Here, we investigate the effects of BACs on neurospheres, an in vitro three-dimensional model of neurodevelopment. Neurospheres cultured from mouse embryonic neural progenitor cells (NPCs) were exposed to increasing concentrations (from 1 to 100 nM) of a short-chain BAC (BAC C12), a long-chain BAC (BAC C16), and AY9944 (a known DHCR7 inhibitor). We found that the sizes of neurospheres were decreased by both BACs but not by AY9944. Furthermore, we observed potent inhibition of cholesterol biosynthesis at the step of DHCR7 by BAC C12 but not by BAC C16, suggesting that cholesterol biosynthesis inhibition is not responsible for the observed reduction in neurosphere growth. By using immunostaining and cell cycle analysis, we found that both BACs induced apoptosis and decreased proliferation of NPCs. To explore the mechanisms underlying their effect on neurosphere growth, we carried out RNA sequencing on neurospheres exposed to each BAC at 50 nM for 24 h, which revealed the activation of the integrated stress response by both BACs. Overall, these results suggest that BACs affect neurodevelopment by inducing the integrated stress response in a manner independent of their effects on cholesterol biosynthesis.

Figure 1.. BACs reduce neurosphere growth.

(A) Size distribution of neurospheres exposed to vehicle control, AY9944 (positive control), BAC C12, or BAC C16 at a concentration of 1, 10, 50, and 100 nM from DIV 4 to DIV 7. (B) Representative brightfield images of neurospheres exposed to a concentration of 50 nM from DIV 4 to DIV 7 (scale bar: 400 μm). (C) Number of neural progenitor cells from dissociated neurospheres exposed to vehicle control (0 nM), AY9944, BAC C12, or BAC C16 at 10 and 50 nM from DIV 4 to DIV 7. N = 4 biological replicates per condition. Adjusted P value: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 2.. BACs alter cholesterol biosynthesis.

Neurospheres exposed to vehicle control (0 nM), BAC C12 or BAC C16 at 1, 10, 50, and 100 nM from DIV 4 to DIV 7 show alterations in levels of sterols in the post-squalene cholesterol biosynthetic pathway. N = 4 biological replicates per condition. Adjusted P value: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 3.. BACs decrease pool of proliferative NPCs.

(A) Immunocytochemistry for Ki67 (red) and EdU (green), counterstained with DAPI (blue) of NPCs from dissociated neurospheres exposed to vehicle control (0 nM) or 50 nM of BAC C12 or BAC C16 from DIV 4 to DIV 7. (B) Quantification of proliferation rate (Edu/Ki67) and percentage of proliferative cells (Ki67) cells. Adjusted P value: *, P < 0.05; **, P < 0.01; ***, P < 0.001. (C) Cell cycle analysis of nuclei isolated from dissociated neurospheres exposed to vehicle control or 50 nM of BAC C12 or BAC C16 from DIV 4 to DIV 5. (D) Quantitation of population of cells in G1 or S phase in (C); *, P < 0.05 relative to Control. N = 4 biological replicates per condition.

Figure 4.. BACs induce apoptosis of NPCs.

(A) Immunocytochemistry for TUNEL (green) counterstained with DAPI (blue) of NPCs exposed to vehicle control (0 nM) or 50 nM of BAC C12 or BAC C16 from DIV 4 to DIV 7. (B) Quantification of percentage of TUNEL-positive NPCs. (C) DAPI-stained nuclei for each exposure group. Enlarged image in bottom right corner shows condensed nuclei. (D) Quantification of percentage of condensed nuclei. N = 4 biological replicates per condition. Adjusted P value: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 5.. Transcriptome analysis indicates a stress response in BAC-exposed neurospheres.

(A) Venn diagram of DEGs from neurospheres exposed to 50 nM BAC C12 or BAC C16 from DIV 4 to DIV 5. (B) Venn diagram of GO biological processes affected by either or both BACs. (C) Top 5 significantly enriched GO biological processes. (D) Co-regulated DEGs involved in the “response to stress” biological process. (E) STRING analysis of DEGs co-regulated by both BACs. Red indicates genes involved in the integrated stress response. N = 3 biological replicates per condition. Adjusted P values for all DEGs are < 0.05.