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Nonsteroidal anti-inflammatory drugs are used extensively in clinical medicine. In spite of their therapeutic utility, however, they are known to cause significant gastrointestinal and renal toxicities, circumstances that limit their use. The side effects produced in these organs have been attributed mainly to the inhibitory effect of these drugs on the activity of cyclooxygenase, a key enzyme in prostaglandin synthesis. In addition to this, in the small intestine it is known that reactive oxygen species also contribute to the enteropathy seen in response to these drugs. In the kidney, however, there is little information whether other mechanisms contribute to the renal toxicity. This study was designed to look at the possible biochemical mechanisms involved in indomethacin-induced renal damage. Rats fasted overnight were dosed with indomethacin (20 mg/kg) by gavage and sacrificed 24 hr later. Histology of the kidney showed abnormalities in the mitochondria in the proximal tubules. Evidence of oxidative stress was found in the kidney associated with mitochondrial dysfunction and neutrophil infiltration. The lipid composition in the mitochondria was also altered. Such effects were abolished by the prior administration of arginine, a donor of nitric oxide. This study, thus, suggests that one of the mechanisms by which nonsteroidal anti-inflammatory drugs induce renal damage is through oxygen free radicals possibly generated by activated neutrophils and mitochondrial dysfunction.

Nonsteroidal anti-inflammatory drugs (NSAIDs) cause small intestinal damage but the pathogenesis of this toxicity is not well established. Our earlier work has shown that villus enterocytes are most susceptible to the effects of indomethacin, a commonly used NSAID. This study looked at the acute effect of indomethacin on brush border membranes (BBM), which are present mainly in the villus cells and are in immediate contact with the contents of the small intestinal lumen. Evidence of oxidative stress was found in the mucosa of the small intestine of rats dosed with indomethacin, as indicated by increased activity of xanthine oxidase with corresponding decrease in the levels of several free radical scavenging enzymes. These changes were associated with an increase in peroxidation parameters in the BBM and a fall in the level of alpha-tocopherol. These BBM also exhibited impairment in glucose transport. Significant changes were seen in the lipid composition of these membranes, with upregulation of an 85kDa isoform of phospholipase A(2). Pretreatment of animals with allopurinol, arginine or zinc protected against these effects of indomethacin. Thus this study suggests that in an acute model of indomethacin dosing there is impairment in structure and function of the BBM in enterocytes, with the effects possibly mediated by free radicals and phospholipases.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are known to cause small intestinal damage but the pathogenesis of this toxicity is not well established. Intestinal epithelial cells are thought to be affected by these drugs in the course of their absorption. These cells are of different types, viz. villus, middle and crypt cells. There is little information on which of these cells, if any, are particularly vulnerable to the effects of NSAIDs. This paper aimed to study the effects of indomethacin, an NSAID commonly used in toxicity studies, on different populations of enterocytes. Effects of the drug were assessed in terms of oxidative damage, mitotic activity, mitochondrial function and lipid composition in enterocytes isolated from the small intestine of rats that had been orally administered indomethacin. In addition, the effects of arginine and zinc in protecting against such changes were assessed. Cell viability, tetrazolium dye (MTT) reduction and oxygen uptake were significantly reduced in villus tip cells from rats dosed with the drug. Thymidine uptake was higher in the crypt cell fraction from these rats. Similarly, products of lipid peroxidation were elevated in the villus tip cells with a corresponding decrease in the level of the anti-oxidant, alpha-tocopherol. In isolated mitochondrial preparations from various enterocyte fractions, significant functional impairment and altered lipid composition were seen mainly in mitochondria from villus cells. Arginine and zinc pre-treatment were found to protect against these effects. These results suggest for the first time that the villus tip cells are more vulnerable to the damaging effects of indomethacin and that oxidative stress is possibly involved in this damage.

Inflammation is associated with oxidative stress and altered cellular calcium homeostasis. Our earlier studies have shown that, increased phosphatidic acid (PA) formation occurred in enterocyte mitochondria when exposed to superoxide, divalent metal ions or polyamines resulting in altered lipid composition. Since aminosalicylates are the drug of choice for gut inflammation, we have tested the effect of aminosalicylates on PA formation by enterocyte mitochondria. When stimulated by superoxide, Ca2+ or spermine, phosphatidyleth-anolamine (PE) degradation and PA formation occurred in enterocyte mitochondria which can be inhibited by aminosalicylates. The inhibition was 50-60% at 0.5-mM concentration and at 1- or 2-mM final concentration, complete inhibition was observed. Both 5-aminosalicylate (5-ASA) and 4-aminosalicylate (4-ASA) showed similar effects. The stimulation of PA formation by calcium or spermine was not due to increased generation of superoxide by mitochondria which was confirmed by measurement of superoxide production by the mitochondria. These studies suggest that in addition to other cellular effects, aminosalicylates may prevent the enterocyte mitochondrial damage by inhibition of PA formation and PE degradation and alteration of mitochondrial lipid composition.

The intestinal epithelium is likely to be exposed to oxidants derived from diet, bacterial metabolic products or endogenously formed cellular metabolites. Glutathione, being the major intracellular thiol, provides protection against oxidative injury. The present study looks at the effect of luminal exposure to oxidants on intestinal thiol status. Different oxidants were placed in the lumen of anesthetized rats for 30 min and the mucosal non-protein and protein-bound thiols were quantitated using HPLC. Oxidant exposure altered the thiol redox status and increased the oxidized glutathione (GSSG) level 6-7-fold and protein-bound reduced glutathione (GSH) 2-fold, whereas the non-protein GSH level remained the same or increased slightly. This could be due to either increased mucosal GSH synthesis or transport from circulation. After incubation with buthionine sulfoximine, a specific inhibitor of GSH synthesis, prior to oxidant exposure, the same increase in GSSG and GSH in the mucosa was seen. Oxidant exposure also decreased the plasma GSH level by 50%. The other cellular non-protein thiols, cysteine and cystine, did not change after luminal exposure to oxidants. These results suggest that oxidant exposure stimulates uptake of GSH from the circulation to maintain mucosal GSH.