Original Contributions
Salicylate Hydroxylation as an Indicator of Hydroxyl Radical Generation in Dextran Sulfate-Induced Colitis

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Abstract

Reactive oxygen and nitrogen species have been implicated as mediators of mucosal injury in inflammatory bowel disease. This study investigated hydroxyl radical (OH) generation in the inflamed colon of dextran sulfate sodium (DSS)-induced colitis by measuring the OH-specific product of salicylate hydroxylation, 2,3-dihydroxybenzoic acid (DHB). Colitis was induced in 6–7 week old CBA/H male mice by supplementing the drinking water with 5% DSS for 7 days. On the last day of dextran exposure, mice were injected with salicylate (SAL) (100 mg/kg i.p.) 60 min before sacrifice, and mucosal homogenates were assayed for SAL and 2,3-DHB by HPLC with fluorescence and electrochemical detection. Mucosal 2,3-DHB levels in mice exposed to 5% DSS were increased by 83% (p < .005); however, SAL levels were also elevated by 182% (p < .001). This translated to a 34% decrease in the ratio 2,3-DHB:SAL in inflamed mucosa, possibly indicating greater catabolism or decreased production of 2,3-DHB. In vitro investigation of the stability of DHBs and SAL in the presence of oxidants of inflammatory lesions revealed that 2,3-DHB and 2,5-DHB were rapidly degraded by hypochlorous acid (HOCl), with initial decomposition rates of 190 and 281 nmol/min, respectively (100μM DHB with 200μM HOCl). Methionine prevented decomposition of DHBs in vitro; however, in mice with 5% DSS-induced colitis, where mucosal myeloperoxidase activity was ten-fold control levels (p < .001), administration of methionine (up to 200 mg/kg i.p.) with SAL was ineffective at increasing the ratio 2,3-DHB:SAL. SAL was also degraded in vitro by HOCl (4.7 nmol/min) resulting in the formation of new fluorescent species which may be useful as indicators of HOCl-mediated injury. Salicylate hydroxylation was unable to provide conclusive evidence supporting a role for OH in the tissue injury of DSS-induced colitis, as metabolic disturbances in the diseased animals other than changes in OH generation may have altered 2,3-DHB levels. This problem is relevant to any study involving the in vivo use of trapping molecules. In particular, the susceptibility of 2,3-DHB to degradation by HOCl brings into question the usefulness of salicylate hydroxylation for measurement of OH-generation in any neutrophilic inflammatory lesion.

Introduction

Inflammatory bowel disease (IBD) is a chronic inflammatory disorder of unknown etiology where reactive oxygen and nitrogen species (RONS), produced by stimulated inflammatory cells such as macrophages and neutrophils, have been implicated as mediators of mucosal injury.1, 2, 3Superoxide (O2•−) and hydrogen peroxide (H2O2) generated by these inflammatory cells may interact with catalytic iron to generate hydroxyl radical (OH) according to the Haber-Weiss reaction.4, 5In the inflamed colon, OH may contribute to the tissue injury of colitis as iron capable of catalysing this reaction is potentially abundant, with unabsorbed dietary iron concentrated in the faecal material, especially in cases of oral iron therapy for chronic anemia, and characteristic mucosal bleeding supplying catalytic haem iron.[6]

Experimental evidence supporting a role for OH in colitis includes the detection of a metabolite of 5-aminosalicylic acid (5-ASA) in faecal extracts from inflammatory bowel disease patients that is also formed by irradiation of 5-ASA in vitro,[7]and the ability of 5-ASA to scavenge OH in vitro.8, 9These observations suggest that OH are generated in the inflamed colon and that scavenging of reactive oxygen species by 5-ASA may contribute to its therapeutic efficacy.[7]In animal models, iron chelation by desferrioxamine and OH scavenging by dimethylsulfoxide significantly attenuated formylmethionyl-leucyl-phenylalanine (fMLP)-induced mucosal damage.[10]These agents were ineffective, however, in decreasing the severity of inflammation in acetic acid-induced colitis.[11]

While OH has an extremely short half-life and cannot be measured directly, substantial evidence of OH formation in in vitro systems can be obtained by using salicylate (SAL) as an OH trap, which forms the hydroxylation products 2,3- and 2,5-dihydroxybenzoic acid (DHB).[12]The lack of toxicity of SAL, ease of administration, and stability of the extracted reaction products make SAL a useful OH trap for animal studies. 2,5-DHB is also produced by the microsomal cytochrome P-450 system[13]and by myeloperoxidase (MPO),[14]however, no enzymatic pathway of 2,3-DHB generation has been identified, thus the formation of 2,3-DHB is considered indicative of OH generation in vivo.12, 13, 152,3-DHB formation has been used to measure OH generation in several model systems including in the perfusate of isolated organs such as ischaemic/reperfused hearts,16, 17in intestinal ischaemia in rats,18, 19and in acute lung inflammation in the rat following instillation of silica.[20]

Dextran sulfate sodium (DSS)-induced colitis in mice, a disease similar to human ulcerative colitis, has been characterised histologically and inflammatory cells implicated in its pathology.21, 22The importance of neutrophils to the development of severe mucosal damage[23]and significant depletion of several mucosal antioxidants[24]suggest that RONS may be involved in tissue injury of DSS-induced colitis. The similarity of these changes to the human disease[24]suggests that DSS-induced colitis is a relevant model of the involvement of RONS in IBD. Although the possible involvement of OH in colitis has been hypothesised,[6]and OH scavengers have been used in models of colitis,10, 11no attempt has been made to measure OH production in vivo in colitis.

In this study, the technique of measuring 2,3-DHB levels within a tissue as an indicator of OH production was applied to mice with DSS-induced colitis. The results obtained were difficult to interpret, influenced by metabolic changes in the inflamed lesion and possibly by the high reactivity of DHBs with hypochlorous acid (HOCl) resulting in loss of 2,3-DHB. These findings highlight the complexities of using SAL for in vivo measurement of OH production in inflammatory lesions.

Section snippets

Induction of Colitis

Colitis was induced in 6–7 week old male CBA/H mice by administering distilled water supplemented with 5% dextran sulfate sodium (average mol wt 44,000, 15% sulfur content, TdB Consultancy AB, Uppsala, Sweden) for 7 days or 3% DSS for 15 days ad libitum. Mice were observed daily for fluid intake and for the major symptoms of diarrhoea and rectal bleeding.

Mice were sacrificed on the last day of DSS administration for the collection of colonic mucosa. Colons were removed and flushed with

Colitis and 2,3-dihydroxybenzoic Acid Levels

Mice exposed to 5% DSS developed symptoms of acute colitis, with diarrhoea being observed first, followed by rectal bleeding and severe weight loss, while administration of 3% DSS induced less severe diarrhoea and little rectal bleeding. Histologically, 5% DSS resulted in large areas of epithelial crypt loss, a predominantly neutrophilic infiltrate throughout the mucosa, ulceration and mucosal bleeding, while 3% DSS resulted in only small lesions of crypt loss without ulceration. The extent of

Discussion

The possibility that cell-generated oxidants may interact with iron sources in inflamed tissues to result in OH formation has long been discussed.4, 5, 31In intestinal inflammation, the potential abundance of iron suggests that the mucosa may be subject to OH attack.[6]This study examined the issue of OH production in colonic mucosa of mice with DSS-induced colitis by investigating salicylate hydroxylation, an in vivo measure of OH generation.

Significant changes in salicylate hydroxylation

Acknowledgements

We thank Prof. C. C. Winterbourn and Dr. A. J. Kettle, Free Radical Research Group, Christchurch School of Medicine, Christchurch, New Zealand, for constructive discussions and helpful comments during the preparation of this manuscript.

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