Jayasree Basivireddy

Oxidative stress in the development of liver cirrhosis: a comparison of two different experimental models.

Background/Aims:  Oxidative stress has been implicated in liver cirrhosis. Carbon tetrachloride and thioacetamide are the most widely used models to develop cirrhosis in rats and the present study compares oxidative stress in the liver induced by these compounds at different stages of cirrhosis development.

Indomethacin-induced mitochondrial dysfunction and oxidative stress in villus enterocytes

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.

Indomethacin-induced free radical-mediated changes in the intestinal brush border membranes.

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.

Intestinal mucosal alterations in rats with carbon tetrachloride-induced cirrhosis: changes in glycosylation and luminal bacteria.

Spontaneous bacterial peritonitis is a major cause of mortality after liver cirrhosis. Altered permeability of the mucosa and deficiencies in host immune defenses through bacterial translocation from the intestine due to intestinal bacterial overgrowth have been implicated in the development of this complication. Molecular mechanisms underlying the process are not well known. In order to understand mechanisms involved in translocation of bacteria, this study explored the role of oxidative stress in mediating changes in intestinal mucosal glycosylation and luminal bacterial content during cirrhosis. CCl4‐induced cirrhosis in rats led to prolonged oxidative stress in the intestine, accompanied by increased sugar content of both intestinal brush border and surfactant layers. This was accompanied by changes in bacterial flora in the gut, which showed increased hydrophobicity and adherence to the mucosa. Inhibition of xanthine oxidase using sodium tungstate or antioxidant supplementation using vitamin E reversed the oxidative stress, changes in brush border membrane sugar content, and bacterial adherence. In conclusion, oxidative stress in the intestine during cirrhosis alters mucosal glycosylation, accompanied by an increased hydrophobicity of luminal bacteria, enabling increased bacterial adherence onto epithelial cells. This might facilitate translocation across the mucosa, resulting in complications such as spontaneous bacterial peritonitis. (HEPATOLOGY 2006;43:837–846.)

Renal damage in experimentally‐induced cirrhosis in rats: Role of oxygen free radicals

Cirrhosis with ascites is associated with impaired renal function accompanied by sodium and water retention. Although it has been suggested that mediators such as nitric oxide play a role in the development of renal failure in this situation, other mechanisms underlying the process are not well understood. This study examined the role of oxidative stress in mediating renal damage during the development of cirrhosis in order to understand mechanisms involved in the process. It was shown that carbon tetrachloride– or thioacetamide‐induced cirrhosis in rats results in oxidative stress in the kidney as seen by increased lipid peroxidation and protein oxidation, accompanied by altered antioxidant status. Cirrhosis was also found to affect renal mitochondrial function, as assessed by measurement of the respiratory control ratio, the swelling of mitochondria, and calcium flux across mitochondrial membranes. Increased lipid peroxidation and changes in lipid composition were evident in the renal brush border membranes, with compromised transport of 14C glucose across these membranes. In conclusion, renal alterations produced as a result of cirrhosis in the rat are possibly mediated by oxidative stress. (HEPATOLOGY 2006;43: 1248–1256.)

Oral glutamine attenuates indomethacin-induced small intestinal damage

The use of NSAIDs (non-steroidal anti-inflammatory drugs), although of great therapeutic value clinically, is limited by their tendency to cause mucosal damage in the gastrointestinal tract. In the small intestine, the effects these drugs have been shown to produce include inhibition of cyclo-oxygenase, mitochondrial dysfunction and free radical-induced oxidative changes, all of which contribute to the mucosal damage seen. Glutamine is a fuel preferentially used by enterocytes and is known to contribute to maintaining the integrity of these cells. In the present study, we investigated the effect of glutamine on indomethacin-induced changes in the small intestinal mucosa. Rats were given 2% glutamine or glutamic acid or isonitrogenous amino acids, glycine or alanine, in the diet for 7 days. Indomethacin was then administered orally at a dose of 40 mg/kg of body weight. After 1 h, the small intestine was removed and used for the measurement of parameters of oxidative stress and mitochondrial and BBM (brush border membrane) function. Evidence of oxidative stress was found in the mucosa of the small intestine of drug-treated rats, as indicated by significantly increased activity of xanthine oxidase (P < 0.001) and myeloperoxidase (P < 0.001), with corresponding decreases in the levels of several free radical scavenging enzymes and alpha-tocopherol (P < 0.001 in all cases). Levels of products of peroxidation were also significantly elevated (P < 0.001 for all the parameters measured). In addition, oxidative stress was evident in isolated intestinal mitochondria and BBMs (P < 0.001 for all the parameters measured), with associated alterations in function of these organelles (P < 0.001 for all the parameters measured). Supplementation of the diet with glutamine or glutamic acid prior to treatment with indomethacin produced significant amelioration in all the effects produced by the drug in the small intestine (P < 0.001 for all the parameters measured). Glycine and alanine were found to be much less effective in these respects.

A simple method of rat renal brush border membrane preparation using polyethylene glycol precipitation

A simple method for preparation of brush border membranes (BBM) from rat kidney using polyethylene glycol (PEG) precipitation has been described. This method avoids the use of cations for the preparation, which might alter membrane lipid composition. These preparations were assessed for enrichment of marker enzymes, contamination by subcellular structures, lipid composition and transport function. An enrichment of 11.8910-fold of alkaline phosphatase, 13.9500-fold of amino peptidase and 13.6500-fold of gamma-glutamyl transpeptidase and an approximate yield of 60% were seen in the final membrane preparation as compared to the homogenate. There was very little contamination of basolateral membranes, peroxisomes, microsomes or lysosomes in the final membrane preparation. Analysis of sugars indicated high content of fucose and sailic acid as compared to hexoses. Isolated membranes appeared as vesicles as seen by electron microscopy. Lipid analysis indicated the presence of various neutral and phospholipids with a high content of sphingomyelin along with a cholesterol/phospholipid ratio of 0.4850. The isolated membrane vesicles were able to transport glucose. This study has shown a simple method of renal brush border membrane preparation, which is comparatively pure and functionally active.

Activation of phospholipase A2 is involved in indomethacin-induced damage in Caco-2 cells.

Nonsteroidal anti-inflammatory drugs (NSAIDs), widely used in clinical practice, cause adverse effects in the gastrointestinal tract. These effects have been attributed to mechanisms such as drug-induced cyclooxygenase inhibition, oxidative stress, mitochondrial dysfunction and changes in cell membrane lipids. Our previous study showed that indomethacin (an NSAID commonly used in toxicity studies) caused activation of cytosolic phospholipase A(2) (cPLA(2)) in the rat small intestine. We hypothesized that activation of cPLA(2) is an important event in the pathogenesis of indomethacin-induced damage in enterocytes. To test this, we incubated enterocyte-like Caco-2 cells with indomethacin, with and without pretreatment with methyl arachidonyl fluorophosphonate (MAFP), an inhibitor of cPLA(2). Cells treated with indomethacin showed decreased viability and evidence of oxidative stress and morphological cell damage. Phospholipids were degraded in these cells, with increases in the levels of lysophospholipids and arachidonic acid. There was no evidence of apoptosis in the cells in response to the drug. Pretreatment of the cells with MAFP attenuated the drug-induced effects seen. This shows that activation of phospholipase A(2) appears to be an important event in the pathogenesis of indomethacin-induced damage in Caco-2 cells. To our knowledge, this is the first report that implicates the involvement of this enzyme in NSAID-induced enteropathy.