Metabolism of trinitrobenzene sulfonic acid by the rat colon produces reactive oxygen species.
Recent studies have shown that intrarectal administration of 2,4,6-trinitrobenzene sulfonic acid (TNBS) in ethanol or intramural injection of TNBS in saline produces an acute and possibly chronic colitis in rats. It has been assumed that interstitial TNBS initiates the inflammatory response via macrophage-mediated recognition and degradation of TNBS-modified mucosal cells and proteins. However, it is known that certain flavoproteins and/or reductants interact with compounds containing the nitro functional group to generate pro-inflammatory, nitrogen-centered free radicals and reactive oxygen metabolites. The objective of this study was to assess the ability of the rat colon, using either colon homogenates, isolated colonocytes, or intestinal interstitial fluid, to produce reactive oxygen species via enzymatic and/or nonenzymatic metabolism of TNBS. It was found that the addition of TNBS (1 mmol/L) to the 10,000 x g supernatant of rat colon homogenates increased the rate of superoxide production from normally undetectable levels to 2.6 +/- 0.23 nmol.min-1.mg protein-1. Addition of nicotinamide adenine dinucleotide, reduced form (NADH; 1 mmol/L) to colon homogenates containing TNBS significantly enhanced superoxide production to 10.4 +/- 0.9 nmol.min-1.mg-1. Similarly, addition of nicotinamide adenine dinucleotide phosphate, reduced form (NADPH; 1 mmol/L) to colon extracts containing TNBS produced an even further increase in the rate of superoxide formation to 25.2 +/- 1.1 nmol.min-1.mg-1. Addition of NADH or NADPH to the colon homogenate in the absence of TNBS produced no detectable superoxide formation, suggesting that TNBS was required for the enhanced oxidative metabolism. In a separate series of experiments, it was found that isolated colonocytes produced small but significant amounts of superoxide (3.15 +/- 0.6 nmol/2 x 10(6) cells) that were significantly increased in the presence of ethanol to 6.55 +/- 1.14 nmol/2 x 10(6) cells. Using purified preparations of two flavoproteins found in the rat colon, it was shown that the addition of TNBS (1 mmol/L) to purified NADH dehydrogenase or glutathione reductase increased the rate of superoxide formation by these enzymes from normally undetectable levels to 1.6 nmol/min and 1.2 nmol/min, respectively. In addition, it was found that intestinal interstitial fluid (lymph) initiated redox cycling of TNBS such that 28.1 +/- 1.6 nmol of oxygen was consumed per minute per milliliter of lymph. This increase in oxygen consumption was inhibited by the addition of superoxide dismutase and catalase. One possible metabolite involved in both mucosal and lymph-mediated metabolism of TNBS is ascorbic acid.(ABSTRACT TRUNCATED AT 400 WORDS)
Grisham
M B
MB
Department of Physiology and Biophysics, Louisiana State University Medical Center, Shreveport.
Volkmer
C
C
Tso
P
P
Yamada
T
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D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology; D003106:Colon / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D000431:Ethanol / Q000494:pharmacology; D066298:In Vitro Techniques; D008196:Lymph / Q000378:metabolism; D008297:Male; D009243:NAD / Q000494:pharmacology; D009249:NADP / Q000494:pharmacology; D010084:Oxidation-Reduction; D010101:Oxygen Consumption / Q000187:drug effects; D051381:Rats; D013481:Superoxides / Q000378:metabolism*; D014302:Trinitrobenzenesulfonic Acid / Q000378:metabolism*
D009243:NAD; D013481:Superoxides; D000431:Ethanol; D009249:NADP; D014302:Trinitrobenzenesulfonic Acid; D001205:Ascorbic Acid