Wistar Rat Protocol for DMH-Induced Colorectal Cancer

Twenty-nine male Wistar rats, weighing 200–250 g and aged seven weeks, were acquired from Charles River (France). Sigma-Aldrich^® (Saint Louis, MO, USA) provided DMH, which was prepared immediately before its use in a saline solution containing 0.9% of NaCl (B. Braun, Melsungen, Germany) and 1 mM of ethylenediaminetetraacetic acid (EDTA; E5134, Sigma-Aldrich^®, Saint Louis, Missouri, USA) [10 (link)]. During the study, the rats were kept in controlled conditions of temperature (20 ± 2 °C), relative humidity (50 ± 10%), and light-dark cycle (12:12-h). The rats were housed in groups of three to five animals per cage (1500UEurostandard Type IV S cages, Tecniplast, Buguggiate, Italy). Food (Diet Standard 4RF21^®, Mucedola, Italy) and tap water were provided ad libitum throughout the study. Corncob was used as bedding and was changed weekly. Polyvinyl chloride tubes were placed into the cages as environmental enrichment for the animals. The viruses and bacteria specified in the Supplementary Materials (Table S1), as well as endo and ectoparasites, were not found in the rats.