Title: Intestinal Bacteria are Necessary for Doxorubicin-induced Intestinal Damage but not

25 Doxorubicin (DOXO) induces significant, but transient, increases in apoptosis in the stem cell 26 zone of the jejunum, followed by mucosal damage involving a decrease in crypt proliferation, 27 crypt number, and villus height. The gastrointestinal tract is home to a vast population of 28 commensal bacteria and numerous studies have demonstrated a symbiotic relationship 29 between intestinal bacteria and intestinal epithelial cells (IEC) in maintaining homeostatic 30 functions of the intestine. However, whether enteric bacteria play a role in DOXO-induced 31 damage is not well understood. We hypothesized that enteric bacteria are necessary for 32 induction of apoptosis and damage associated with DOXO treatment. Conventionally raised 33 (CONV) and germ free (GF) mice were given a single injection of DOXO, and intestinal tissue 34 was collected at 6, 72, and 120 h after treatment and from no treatment (0 h) controls. 35 Histology and morphometric analyses quantified apoptosis, mitosis, crypt depth, villus height, 36 and crypt density. Immunostaining for muc2 and lysozyme evaluated Paneth cells, goblet 37 cells or dual stained intermediate cells. DOXO administration induced significant increases 38 in apoptosis in jejunal epithelium regardless of the presence of enteric bacteria; however, the 39 resulting injury, as demonstrated by statistically significant changes in crypt depth, crypt 40 number, and proliferative cell number, was dependent upon the presence of enteric bacteria. 41 Furthermore, we observed expansion of Paneth and goblet cells and presence of 42 intermediate cells only in CONV and not GF mice. These findings provide evidence that 43 manipulation and/or depletion of the enteric microbiota may have clinical significance in 44 limiting chemotherapy-induced mucositis. 45