RESPONSE OF THE INTESTINAL EPITHELIUM TO METHOTREXATE INDUCED DAMAGE

Abstract

One of the major side effects of chemotherapy is severe damage to the intestinal epithelium, leading to impairment of the intestinal function. Especially in pediatric patients, clinical symptoms like malabsorption, malnutrition and anorexia are of great concern. Aim: To gain insight in the regulation of epithelial proliferation, differentiation and cell death during damage and regeneration in order to develop prophylactic/therapeutic regimes to avoid or repair intestinal damage. Methods: Since morphological alterations after chemotherapy are similar in rat and human small intestine, we focussed on rats treated with methotrexate (MTX). Duodenal and jejunal segments were excised on various days after a single i.v. dose of MTX. Next to standard histological evaluation, differentiation of the epithelial cells was assessed by the expression of cell-type specific differentiation markers at mRNA (in situ hybridization) and protein (immunohistochemistry) levels. Sucrase-isomaltase (SI) and intestinal fatty acid binding protein (iFABP) were used as differentiation markers for enterocytes, mucin (MUC2) for goblet cells, and lysozyme for Paneth cells. In addition, proliferating and apoptotic epithelial cells were assessed by immunodetection of proliferating cell nuclear antigen (PCNA) and the terminal uridine deoxynucleotide nick-end labeling (TUNEL) method, respectively. Results: Morphologically, intestinal damage at 2 days after MTX injection is characterized by disturbance of the crypt architecture and shortening of the villi. On day 4 the villus atrophy is even more pronounced, while the crypts are elongated. Both, the crypt- and villus architecture has been restored completely on day 6. Expression of the enterocyte specific differentiation markers showed a decrease of SI while iFABP remained stable at both mRNA- and protein levels. Moreover, expression of the Paneth cell marker lysozyme (on day 2-4) and the goblet cell marker MUC2 (on day 4) are increased at both levels investigated. There is an increase in TUNEL positive cells in the crypts on day 2, which is followed by an increase in PCNA-positive cells on day 4. Conclusions: During MTX-induced damage, lysozyme and MUC2 expression are increased, which implies an increased production of protective molecules. The MTX-induced decrease in SI expression and the unaltered iFABP expression suggest a differential effect on expression of these genes in enterocytes. This might indicate a switch in enterocyte metabolism from sugar absorption to fatty acid absorption. Since alterations in mRNA and corresponding protein levels correlate well for each differentiation marker, the effects of MTX occur mainly at the mRNA level. Furthermore, single dose MTX treatment leads to increased apoptosis in crypts followed by an increase of proliferative activity in crypts as demonstrated by the histological changes, TUNEL method and PCNA expression patterns.