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Arsenic is a toxicant commonly found in drinking water. Even though its main route of exposure is oral, little is known of the impact of in vivo arsenic exposure on small intestine. In vitro studies have shown that arsenic decreases differentiation of stem and progenitor cells in several different tissues. Thus, small intestinal organoids were used to assess if arsenic exposure would also impair intestinal stem cell differentiation. Unexpectedly, no changes in markers of differentiated epithelial cells were seen. However, exposing mice to 100 ppb arsenic in drinking water for 5 weeks impaired distinct populations of intestinal stromal cells. Arsenic reduced the width of the pericryptal lamina propria by 1.6-fold, and reduced Pdgfra mRNA expression, which is expressed in intestinal telocytes and trophocytes, by 4.2-fold. The height or extension of Pdgfra(+) telopodes into the villus tip was also significantly reduced. Transcript expression of several other stromal cell markers, such as Grem1, Gli, CD81, were reduced by 1.9-, 2.3-, and 1.4-fold, respectively. Further, significant correlations exist between levels of Pdgfra and Gli1, Grem1, and Bmp4. Our results suggest arsenic impairs intestinal trophocytes and telocytes, leading to alterations in the Bmp signaling pathway.

Arsenic is a contaminant found in many foods and drinking water. Exposure to arsenic during development can cause improper neuronal progenitor cell development, differentiation, and function, while in vitro studies have determined that acute arsenic exposure to stem and progenitor cells reduced their ability to differentiate. In the current study, P19 mouse embryonal stem cells were exposed continuously to 0.1-μM (7.5 ppb) arsenic for 32 weeks. A cell lineage array examining messenger RNA (mRNA) changes after 8 and 32 weeks of exposure showed that genes involved in pluripotency were increased, whereas those involved in differentiation were reduced. Therefore, temporal changes of select pluripotency and neuronal differentiation markers throughout the 32-week chronic arsenic exposure were investigated. Sox2 and Oct4 mRNA expression were increased by 1.9- to 2.5-fold in the arsenic-exposed cells, beginning at Week 12. Sox2 protein expression was similarly increased starting at Week 16 and remained elevated by 1.5-fold to sixfold. One target of Sox2 is N-cadherin, whose expression is a hallmark of epithelial-mesenchymal transitions (EMTs). Exposure to arsenic significantly increased N-cadherin protein levels beginning at Week 20, concurrent with increased grouping of N-cadherin positive cells at the perimeter of the embryoid body. Expression of Zeb1, which helps increase the expression of Sox2, was also increased started at Week 16. In contrast, Gdf3 mRNA expression was reduced by 3.4- to 7.2-fold beginning at Week 16, and expression of its target protein, phospho-Smad2/3, was also reduced. These results suggest that chronic, low-level arsenic exposure may delay neuronal differentiation and maintain pluripotency.

In the search for novel broad-spectrum therapeutics to fight chronic infections, inflammation, and cancer, host defense peptides (HDPs) have garnered increasing interest. Characterizing their biologically-active conformations and minimum motifs for function represents a requisite step to developing them into efficacious and safe therapeutics. Here, we demonstrate that metallating HDPs with Cu2+ is an effective chemical strategy to improve their cytotoxicity on cancer cells. Mechanistically, we find that prepared as Cu2+-complexes, the peptides not only physically but also chemically damage lipid membranes. Our testing ground features piscidins 1 and 3 (P1/3), two amphipathic, histidine-rich, membrane-interacting, and cell-penetrating HDPs that are α-helical bound to membranes. To investigate their membrane location, permeabilization effects, and lipid-oxidation capability, we employ neutron reflectometry, impedance spectroscopy, neutron diffraction, and UV spectroscopy. While P1-apo is more potent than P3-apo, metallation boosts their cytotoxicities by up to two- and seven-fold, respectively. Remarkably, P3-Cu2+ is particularly effective at inserting in bilayers, causing water crevices in the hydrocarbon region and placing Cu2+ near the double bonds of the acyl chains, as needed to oxidize them. This study points at a new paradigm where complexing HDPs with Cu2+ to expand their mechanistic reach could be explored to design more potent peptide-based anticancer therapeutics.

In the search for novel broad-spectrum therapeutics to fight chronic infections, inflammation, and cancer, host defense peptides (HDPs) have garnered increasing interest. Characterizing their biologically-active conformations and minimum motifs for function represents a requisite step to developing them into efficacious and safe therapeutics. Here, we demonstrate that metallating HDPs is an effective chemical strategy to improve their cytotoxicity on cancer cells. Mechanistically, we find that the metallated peptides not only physically but also chemically damage lipid membranes. Our testing ground features piscidins 1 and 3 (P1/3), two amphipathic, histidine-rich, membrane-interacting, and cell-penetrating HDPs that are α-helical bound to membranes. To investigate their membrane location, permeabilization effects, and lipid-oxidation capability, we employ neutron reflectometry, impedance spectroscopy, neutron diffraction, and UV spectroscopy. While P1-apo is more potent than P3-apo, metallation boosts their cytotoxicities by up to two-and seven-fold, respectively. Remarkably, P3 is particularly effective at inserting its metallated motif in bilayers, causing water crevices in the hydrocarbon region and placing Cu 2+ near the double bonds of the acyl chains, as needed to oxidize them. This study points at a new paradigm where metallating HDPs to expand their mechanistic reach could be explored to design more potent peptide-based anticancer therapeutics.

Arsenic is a global health concern that causes toxicity through ingestion of contaminated water and food. In vitro studies suggest that arsenic reduces stem and progenitor cell differentiation. Thus, this study determined if arsenic disrupted intestinal stem cell (ISC) differentiation, thereby altering the number, location, and/or function of intestinal epithelial cells. Adult male C57BL/6 mice were exposed to 0 or 100 ppb sodium arsenite (AsIII) through drinking water for 5 weeks. Duodenal sections were collected to assess changes in morphology, proliferation, and cell types. qPCR analysis revealed a 40% reduction in Lgr5 transcripts, an ISC marker, in the arsenic-exposed mice, although there were no changes in the protein expression of Olfm4. Secretory cell-specific transcript markers of Paneth (Defa1), Goblet (Tff3), and secretory transit amplifying (Math1) cells were reduced by 51%, 44%, and 30% respectively, in the arsenic-exposed mice, indicating significant impacts on the Wnt-dependent differentiation pathway. Further, protein levels of phosphorylated β-catenin were reduced in the arsenic-exposed mice, which increased the expression of Wnt-dependent transcripts CD44 and c-myc. PCA analysis, followed by MANOVA and regression analyses, revealed significant changes and correlations between Lgr5 and the transit amplifying (TA) cell markers Math1 and Hes1, which are in the secretory cell pathway. Similar comparisons between Math1 and Defa1 show that terminal differentiation into Paneth cells is also reduced in the arsenic-exposed mice. The data suggests that ISCs are not lost following arsenic exposure, but rather, specific Wnt-dependent progenitor cell formation and terminal differentiation in the small intestine is reduced.