Date of Award
Master of Science (MS)
Lisa J Bain
Charles D Rice
Matthew W Turnbull
Arsenic (As) is a global contaminant listed as the highest priority pollutant on the Substance Priority List by the ATSDR. The primary route of exposure is by ingestion of water and food. The World Health Organization (WHO) describes environmental exposure to arsenic as the largest mass poisoning of a population in history and designated a provisional guideline value of 10ppb in drinking water. Though the primary route of exposure to As is by ingestion, there is relatively scarce research investigating the effects of As on the small intestines. Arsenic has been shown to impair cellular differentiation and proliferation in other stem and progenitor cells, and the intestines are considered the most proliferative tissue in the body. A previous study conducted in killifish (Fundulus heteroclitus) exposed embryonically to arsenic showed significant reduction in intestinal villus height and PCNA+ cells at 8-, 16-, and 40-weeks post-exposure. The goal of this study was to determine if arsenic altered intestinal epithelial morphology, stem cell homeostasis, and the numbers and subtypes of differentiated intestinal cells
Twelve adult male mice were exposed to either 0ppb or 100ppb As in drinking water for five weeks. On the last day of the exposure, a permeability assay using a FITC-labeled dextran was conducted. Sections of the duodenal tissue was resected and used for histochemistry, protein expression, and transcript expression.
The results indicate that arsenic reduced mRNA expression of Lgr5, a marker of intestinal stem cells, by 40%. Likewise, the secretory pathway transcript markers Math1, Defa1, and TFF3 were reduced by 30%, 50%, and 42%, respectively. Lgr5 protein levels are too lowly expressed to quantify using immunoblotting or IHC, therefore we examined protein expression and localization of Olfm4, another robust marker of intestinal stem cells. However, its expression was not changed. To maintain homeostasis after an insult, differentiated cells can dedifferentiate and assume the role of the intestinal stem cell and express markers of proliferation. Though not significant, the percentage of Ki67+ cells in the crypt was an average of 7% higher in the treated mice compared to the controls. H&E staining was used to determine alterations in intestinal epithelium morphology given the changes seen at the molecular level. Though reductions in villi height were not seen, the treated villi were on average nearly 30% wider compared to the controls. No changes were seen in crypt depth.
Linear correlations and MANOVA analyses were used to compare gene expression of individual cell types within a differentiation pathway. Significant correlations and similar changes in means suggests related impacts by an arsenic exposure. These analyses reveal significant reductions in related Wnt-dependent secretory differentiation pathways, between Lgr5,Math1, and Defa1 in treated mice when compared to control mice. Interestingly, comparisons between intestinal villi width, Lgr5, and Defa1 show significant correlations by increased villi width and a decrease in Lgr5 and Defa1 expression in the treated mice. These changes in villi width appear to be mesenchymal tissue related, although this was not investigated.
In conclusion, this study shows that Wnt-dependent intestinal cell differentiation pathways are negatively impacted by arsenic in drinking water, though specific mechanisms still remain elusive. Hypothesized reductions in proliferation and villi height were not seen, though villi width was significantly increased in treated mice. This study in part supports previous research using in vitro models that show significant reduction of Wnt signals and pathway markers.
Jatko, Jordan Thomas, "Effects of a 5-week 100ppb Arsenic Exposure in Drinking Water on Mouse Intestinal Epithelium" (2020). All Theses. 3400.