Effects of methotrexate on the intestinal epithelium are influenced by environmental changes

Abstract

The functions of the intestinal epithelium of patients receiving cytostatic drugs is severely impaired, leading to malabsorption and malnutrition. More insight in mechanisms that mediate mucosal damage and repair is needed for the development of prophylactic regimes. Aim: To further investigate the effects of the cytostatic drug methotrexate (MTX) on functional capacities of intestinal epithelial cells during damage and regeneration by studying epithelial morphology and gene expression. Methods: Rats were subjected to a single or double dose of intraveneously injected MTX ranging from 10 to 60 mg/kg. Rats were kept in cages either alone or grouped (n=4) and sacrificed at 2, 4, 6 or 8 days after the final injection. Jejunal segments were isolated and processed for histochemical analysis. Next to morphology, changes in epithelium-specific differentiation markers were studied at the level of mRNA (in situ hybridization) and protein (immunohistochemistry). Sucraseisomaltase (SI), intestinal (i-) and liver (1-) fatty acid binding protein (FABP) and sodium-glucosetransporter 1 (SGLT1) were used as markers for enterocytes. Mucin (Muc2) and the protease matrilysin were used for goblet cells and Paneth cells, respectively. Results: After a week of adaptation to solitary caging, a single dose of 30, 45 or 60 mg/kg MTX did not lead to any epithelial alterations. However, when two injections of 30 mg/kg MTX were given with a 24 hour interval, 100% mortality occurred at day 6 after the last injection. In rats adapted to group-caging, two-day treatment with 20 mg/kg and 10 mg/kg per day led to villus atrophy that was maximal at day 4 after the last dose and associated with severe cryptloss. In contrast to iand 1-FABP mRNA's, no expression of SI mRNA and SGLT1 mRNA were detected at day 4. At the level of protein, changes in gene expression parallelled the mRNA patterns. Between day 6 and 8, expression 'patterns of all markers returned to normal as the crypt villus architecture restored. Expression patterns of MUC2 and matrilysin mRNA and protein did not show major changes during the period analysed. When rats were caged alone when treatment with MTX started, a single dose of 30 mg/kg affected the epithelium comparable to treatment with 20 mg/kg + 10 mg/kg in rats adapted to group-caging. Conclusions: The observed differences in intestinal epithelial damage between rats adapted to solitary or group-caging versus rats suffering social loss at the moment of MTX treatment, indicate that environmental changes induce a condition of stress leading to increased epithelial sensitivity towards MTX. Still, epithelial damage and mortality also occur in rats kept in a socially known environment using longer period of treatment (2 days). During MTX-induced mucosal damage, enterocyte function is affected with respect to SI and SGLTI expression whereas iand 1-FABP expression is unaltered, suggesting changes in enterocyte metabolism from sugar to fatty acid absorption. Expression of luminal cytoprotective MUC2 and basolateral metalloprotease matrilysin are maintained during MTX-indnced epithelial damage, since these markers remained detectable throughout the study.