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Ulcerative colitis is a condition characterised by the infiltration of leukocytes into the gastrointestinal wall. Leukocyte-MPO catalyses hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN) formation from chloride (Cl−) and thiocyanous (SCN−) anions, respectively. While HOCl indiscriminately oxidises biomolecules, HOSCN primarily targets low-molecular weight protein thiols. Oxidative damage mediated by HOSCN may be reversible, potentially decreasing MPO-associated host tissue destruction. This study investigated the effect of SCN− supplementation in a model of acute colitis. Female mice were supplemented dextran sodium sulphate (DSS, 3% w/v) in the presence of 10 mM Cl− or SCN− in drinking water ad libitum, or with salts (NaCl and NaSCN only) or water only (controls). Behavioural studies showed mice tolerated NaSCN and NaCl-treated water with water-seeking frequency. Ion-exchange chromatography showed increased fecal and plasma SCN− levels in thiocyanate supplemented mice; plasma SCN− reached similar fold-increase for smokers. Overall there was no difference in weight loss and clinical score, mucin levels, crypt integrity and extent of cellular infiltration between DSS/SCN− and DSS/Cl− groups. Neutrophil recruitment remained unchanged in DSS-treated mice, as assessed by fecal calprotectin levels. Total thiol and tyrosine phosphatase activity remained unchanged between DSS/Cl− and DSS/SCN− groups, however, colonic tissue showed a trend in decreased 3-chlorotyrosine (1.5-fold reduction, p