Volume 119, Issue 6, Pages 1537-1547 (December 2000) Drug enterocyte adducts: Possible causal factor for diclofenac enteropathy in rats  Chessley R. Atchison, *, A.Brian West, ‡, Arun Balakumaran, *, Sally J. Hargus, §, Lance R. Pohl, ∥, Davis H. Daiker, *, Judith F. Aronson, *, Walter E. Hoffmann, ¶, Bryan K. Shipp, #, Mary Treinen–Moslen, *  Gastroenterology  Volume 119, Issue 6, Pages 1537-1547 (December 2000) DOI: 10.1053/gast.2000.20186 Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 1 (A) Mucosal aspect of segment of small intestine showing numerous diclofenac-induced ulcers distributed along either side of the mesenteric attachment. (B) Histologic section of small intestine showing part of an ulcer. Note abrupt loss of mucosa overlying the edge of the mesenteric attachment (H&E stain; original magnification 60×). (C) In a sequential section of the ulcer shown in B, immunoreactivity for diclofenac adduct (brown staining) is evident on the brush border of the remaining villi and on the floor of the ulcer (immunoperoxidase with hematoxylin counterstain; original magnification 60×). Gastroenterology 2000 119, 1537-1547DOI: (10.1053/gast.2000.20186) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 2 Distribution of ulcers (by number) in quintiles of small intestine at 12 hours after treatment with 10, 50, or 100 mg/kg diclofenac. Results are expressed as mean ± SEM for 3–4 animals/group. Gastroenterology 2000 119, 1537-1547DOI: (10.1053/gast.2000.20186) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 3 Time course of small intestinal ulcer number and area and of changes in serum total protein and albumin after treatment with 50 mg/kg diclofenac. Results are expressed as mean ± SEM for 4 animals/group. *P < 0.05 compared with 0 hour time point. Gastroenterology 2000 119, 1537-1547DOI: (10.1053/gast.2000.20186) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 4 Correlation between percentage area of small intestinal ulceration and serum albumin of individual rats at 0, 6, 12, and 24 hours after treatment with 50 mg/kg diclofenac. Gastroenterology 2000 119, 1537-1547DOI: (10.1053/gast.2000.20186) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 5 (A) Diclofenac adducts are present in the brush border of enterocytes both lining the small intestinal villi and overlying a lymphoid nodule (Peyer's patch) in this representative ileum 12 hours after 50 mg/kg diclofenac. (B) High-power view of the epithelium overlying the lymphoid nodule showing intense staining of the brush border. (Immunoperoxidase with hematoxylin counterstain; original magnifications: A, 65×; B, 630×.) Gastroenterology 2000 119, 1537-1547DOI: (10.1053/gast.2000.20186) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 6 Time course of diclofenac adduct expression in villi from the 3rd quintile of the small intestine after administration of 50 mg/kg diclofenac. The villus on the left, from an untreated control animal, shows no adducts. One hour after treatment, brown adduct staining is detectable in enterocyte cytoplasm. By 3 hours, adducts are chiefly localized to the enterocyte brush border where staining is more intense by 6 hours. (All immunoperoxidase with hematoxylin counterstain; original magnification 510×.) Gastroenterology 2000 119, 1537-1547DOI: (10.1053/gast.2000.20186) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 7 (A) Paraffin block containing 15 rings of small intestine, color-coded on the mucosal surface with black, blue, or yellow dye as to quintile of origin (5 rings from each of 3 quintiles). (B–D) Immunoreactivity of the villus brush border for diclofenac adducts in the (B) 1st, (C) 3rd, and (D) 5th quintiles. The most intense reaction is seen in the 3rd quintile. (B–D, immunoperoxidase with hematoxylin counterstain; original magnification 300×). Gastroenterology 2000 119, 1537-1547DOI: (10.1053/gast.2000.20186) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 8 Correlation between diclofenac adduct intensity score and ulcer number in 1st, 3rd, and 5th quintiles of animals killed at 12 hours after treatment with 50 mg/kg diclofenac. Intensity of adduct staining was assessed by using rings of tissue mounted in a single paraffin block (see Figure 7A) for processing, sectioning, and staining under identical conditions. Results are expressed as mean ± SEM for 4 animals/group. Gastroenterology 2000 119, 1537-1547DOI: (10.1053/gast.2000.20186) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 9 Schematic summary of the proposed relationship between adduction of enterocyte proteins by reactive metabolites of diclofenac and known events in the biotransformation and enteropathy of this NSAID. Gastroenterology 2000 119, 1537-1547DOI: (10.1053/gast.2000.20186) Copyright © 2000 American Gastroenterological Association Terms and Conditions