DUOX2 and DUOXA2 form the predominant enzyme system capable of producing the reactive oxygen species H2O2 in active ulcerative colitis and are modulated by 5-aminosalicylic acid.
BACKGROUND
NADPH oxidase-derived reactive oxygen species, such as H2O2, are part of the intestinal innate immune system but may drive carcinogenesis through DNA damage. We sought to identify the predominant enzyme system capable of producing H2O2 in active ulcerative colitis and assess whether it is affected by 5-aminosalicylic acid (5-ASA).


METHODS
We studied human mucosal biopsies by expression arrays, quantitative real-time polymerase chain reaction for NADPH oxidase family members, in situ hybridization (DUOX2 and DUOXA2) and immunofluorescence for DUOX, 8-OHdG (DNA damage), and γH2AX (DNA damage response) and sought effects of 5-ASA on ex vivo cultured biopsies and cultured rectal cancer cells.


RESULTS
DUOX2 with maturation partner DUOXA2 forms the predominant system for H2O2 production in human colon and is upregulated in active colitis. DUOX2 in situ is exclusively epithelial, varies between and within individual crypts, and increases near inflammation. 8-OHdG and γH2AX were observed in damaged crypt epithelium. 5-ASA upregulated DUOX2 and DUOXA2 levels in the setting of active versus quiescent disease and altered DUOX2 expression in cultured biopsies. Ingenuity pathway analysis confirmed that inflammation status and 5-ASA increase expression of DUOX2 and DUOXA2. An epithelial cell model confirmed that cultured cancer cells expressed DUOX protein and produced H2O2 in response to hypoxia and 5-ASA exposure.


CONCLUSIONS
Both DUOX2 and DUOXA2 expression are involved specifically in inflammation and are regulated on a crypt-by-crypt basis in ulcerative colitis tissues. Synergy between inflammation, hypoxia, and 5-ASA to increase H2O2 production could explain how 5-ASA supports innate defense, although potentially increasing the burden of DNA damage.
*Centre for Digestive Diseases, Blizard Institute, Barts and The London School of Medicine and Dentistry, Whitechapel, London, United Kingdom; †National Centre for Bowel Research and Surgical Innovation, Barts and The London School of Medicine and Dentistry, Whitechapel, London, United Kingdom; ‡Flow Cytometry Core Facility, Blizard Institute, Barts and The London School of Medicine and Dentistry, Whitechapel, London, United Kingdom; §Department of Pathology, Medical Faculty, Otto-von-Guericke University Magdeburg, Magdeburg, Germany; ‖Department of Cellular Pathology, Royal London Hospital, London, United Kingdom; ¶Division of Pulmonary and Critical Care Medicine, Genome and Biomedical Sciences Facility, University of California at Davis, Davis, California; and **Department of Pathology, Genentech, Inc., South San Francisco, California.
Tammie S
TS
MacFie
Richard
R
Poulsom
Alexandra
A
Parker
Gary
G
Warnes
Tatjana
T
Boitsova
Anke
A
Nijhuis
Nirosha
N
Suraweera
Angela
A
Poehlmann
Jaroslaw
J
Szary
Roger
R
Feakins
Rosemary
R
Jeffery
Richart W
RW
Harper
Adrian M
AM
Jubb
James O
JO
Lindsay
Andrew
A
Silver
D000236:Adenoma / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology; D015153:Blotting, Western; D002478:Cells, Cultured; D003093:Colitis, Ulcerative / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology*; D003110:Colonic Neoplasms / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology*; D000074623:Dual Oxidases; D005455:Fluorescent Antibody Technique; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism*; D000860:Hypoxia / Q000378:metabolism / Q000473:pathology; D017403:In Situ Hybridization; D007249:Inflammation / Q000378:metabolism / Q000473:pathology; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D019804:Mesalamine / Q000494:pharmacology*; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism*; D016877:Oxidants / Q000378:metabolism; D010084:Oxidation-Reduction; D012333:RNA, Messenger / Q000235:genetics; D017382:Reactive Oxygen Species / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction
D000894:Anti-Inflammatory Agents, Non-Steroidal; C513411:DUOXA2 protein, human; D008565:Membrane Proteins; D016877:Oxidants; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D019804:Mesalamine; D006861:Hydrogen Peroxide; D000074623:Dual Oxidases; D019255:NADPH Oxidases; C431515:DUOX2 protein, human