Oral administration of Nigella sativa oil attenuates arsenic-induced redox imbalance,DNA damage,metabolic distress,and histopathological alterations in rat intestine

BackgroundExposure to arsenic, a widespread environmental toxin, produces multiple organ toxicity, including gastrointestinal toxicity. Nigella sativa (NS) has long been revered for its numerous health benefits under normal and pathological states. In view of this, the present study attempts to evaluate the protective efficacy of orally administered Nigella sativa oil (NSO) against arsenic-induced cytotoxic and genotoxic alterations in rat intestine and elucidate the underlying mechanism of its action.MethodsRats were categorized into the control, NaAs, NSO, and NaAs+NSO groups. After pre-treatment of rats in the NaAs+NSO and NSO groups daily with NSO (2 ml/kg bwt, orally) for 14 days, NSO treatment was further continued for 30 days, with and without NaAs treatment (5 mg/kg bwt, orally), respectively. Various biochemical parameters, such as enzymatic and non-enzymatic antioxidants, carbohydrate metabolic and brush border membrane marker enzyme activities were evaluated in the mucosal homogenates of all the groups. Intestinal brush border membrane vesicles (BBMV) were isolated, and the activities of membrane marker enzyme viz. ALP, GGTase, LAP, and sucrase were determined. Further, the effect on kinetic parameters viz K_M (Michaelis-Menten constant) and V_max of these enzymes was assessed. Integrity of enterocyte DNA was examined using the comet assay. Histopathology of the intestines was performed to evaluate the histoarchitectural alterations induced by chronic arsenic exposure and/or NSO supplementation. Arsenic accumulation in the intestine was studied by inductively coupled plasma-mass spectroscopy (ICP-MS).ResultsNaAs treatment caused substantial changes in the activities of brush border membrane (BBM), carbohydrate metabolism, and antioxidant defense enzymes in the intestinal mucosal homogenates. The isolated BBM vesicles (BBMV) also showed marked suppression in the marker enzyme activities. Severe DNA damage and mucosal arsenic accumulation were observed in rats treated with NaAs alone. In contrast, oral NSO supplementation significantly alleviated all the adverse alterations induced by NaAs treatment. Histopathological examination supported the biochemical findings.ConclusionNSO, by improving the antioxidant status and energy metabolism, could significantly alter the ability of the intestine to protect against free radical-mediated arsenic toxicity in intestine. Thus, NSO may have an excellent scope in managing gastrointestinal distress in arsenic intoxication.     

Regional variations in intestinal brush border membrane fluidity and function during diabetes and the role of oxidative stress and non-enzymatic glycation

The physical state (fluidity) of lipids modulates the activities of several membrane bound enzymes and transport proteins. Alteration of brush border membrane (BBM) fluidity is one of the several changes exhibited by the small intestine during diabetes. In the present study, an investigation of the diabetes induced regional changes in fluidity, oxidative damage, non-enzymatic glycation as well as the activities and the kinetic parameters of the enzymes alkaline phosphatase and -glutamyl transpeptidase was carried out on the intestinal BBM. At the end of 6 weeks of diabetes, significant increases in the extent of both oxidative damage and non-enzymatic glycation were observed along the length of the intestine along with a simultaneous decrease in membrane fluidity. A significant correlation between the decrease in BBM fluidity and increase in non-enzymatic glycation was observed in the duodenum and jejunum. Additionally regional variations in the activities and kinetic parameters of both the enzymes were observed.     

Changes in leucine transport activity in Chironomus riparius larvae after short-term exposure to potassium dichromate and fenitrothion

The effect of sublethal concentrations of potassium dichromate and fenitrothion on sodium-leucine cotransport in brush border membrane vesicles from Chironomus riparius larvae has been investigated. Exposure to potassium dichromate and fenitrothion caused a dose- and time-dependent inhibition of leucine uptake. Transport inhibition is easily detectable at doses 100-fold lower than LD50. Kinetic experiments showed that inhibition was mainly caused by a decrease of the Vmax (680 +/- 53 vs. 382 +/- 23 and 555 +/- 27 nmol/15s/mg protein in control and exposed larvae to K2Cr2O7 and fenitrothion, respectively). Inhibition is possibly related to a variation of sodium ions permeability as evidenced by increased membrane lipid peroxidation. Appropriate control experiments ruled out that the observed differences could be due to changes in general features of membrane preparations. Transport inhibition observed in larvae exposed to potassium dichromate was accompanied by changes in ascorbate peroxidase and dehydroascorbate reductase activities, whereas those exposed to fenitrothion displayed an increase in transaminase activity. The possible value of leucine uptake as biochemical biomarker is briefly discussed. Arch. Insect Biochem. Physiol. 55:90-101, 2004.     

Acute dieldrin toxicity: effect on the uptake of glucose and leucine and on brush border enzymes in monkey intestine

Administration of a single oral dose of dieldrin (20 mg/kg body wt.) to rhesus monkeys considerably elevated the uptake of glucose and the activities of brush border sucrase, lactase, maltase and alkaline phosphatase in intestine compared to control animals. Leucine uptake and leucine amino peptidase activity was significantly depressed in pesticide-treated animals. Kinetic studies with brush border sucrase revealed that augmentation of enzyme activity in pesticide-fed animals was due to an increase in the disaccharidase content.     

Modification of biochemical parameters of gentamicin nephrotoxicity by coenzyme Q10 and green tea in rats

The present study was designed to investigate the possible potential protective role of coenzymeQ10 (CoQ10; 10 mg/kg/day, ip) and/or green tea (GT; 25mg/kg/day, po) against gentamicin (GM) nephrotoxicity. Marked increase in the level of serum urea. creatinine and lipid peroxidation (LPO) content was found after administration of gentamicin (80 mg/kg/day, ip) for eight days along with significant decrease in the antioxidant enzymes, superoxide dismutase (SOD), reduced glutathione (GSH), catalase (CAT) as well as brush border enzymes (Na+/K+ ATPase, Mg(+2)ATPase and Ca2+ ATPase).Treatment with CoQ10 or green tea alone with GM showed significant decrease in serum urea, creatinine and tissue LPO content and significant increase in antioxidant and membrane bound enzymes. Combined treatment with CoQ10 and green tea was more effective in mitigating adverse effect of GM nephrotoxicity. The present work indicated that CoQ10 and green tea due to their antioxidant activity modified the biochemical changes occurred during gentamicin nephrotoxicity and thus had a potential protective effect.     

Effect of cisplatin on brush border membrane enzymes and anti-oxidant system of rat intestine

Cisplatin (CP) is a widely used antineoplastic agent which exhibits gastrointestinal toxicity. The present work was done to study the effect of administration of CP on brush border membrane (BBM) enzymes and anti-oxidant system of rat intestine. Male Wistar rats were given a single intraperitoneal dose of CP (6 mg/kg body weight) and then sacrificed 1, 3, 5 and 7 days after this treatment. Control animals were given saline only. The administration of CP led to significant decline in the specific activities of BBM enzymes both in the mucosal homogenates and isolated membrane vesicles. Kinetic studies showed that the V(max) of the enzymes was decreased in BBM vesicles from CP treated rats while the K(m) remained unchanged. The activities of catalase, Cu-Zn superoxide dismutase, glucose 6-phosphate dehydrogenase and glutathione reductase decreased while the activities of glutathione S-transferase and thioredoxin reductase increased in CP treated animals compared to the control group. Lipid peroxidation and total sulfhydryl groups were also altered upon CP treatment indicating the generation of oxidative stress. The maximum changes in all the parameters studied above were 3 days after administration of CP and then recovery took place on days 5 and 7. Thus, the administration of CP leads to significant alterations in the activities of BBM enzymes and the anti-oxidant status of rat intestine.     

Digestive and absorptive functions along dog small intestine: comparative distributions in relation to biochemical and morphological parameters

The digestive (hydrolytic enzymes) and absorptive (sugar and amino acid transport) functions of dog small intestine have been evaluated in different segments and analysed in relation to morphometric and biochemical parameters. The dog small intestine is a cylinder of decreasing diameter in which the underlying mucosa thins down from duodenum to ileum, though maintaining its cellular homogeneity as revealed by measuring the mucosal weight, the total DNA and protein content and the protein content of the brush border membrane. Sucrase, gamma-glutamyltranspeptidase, leucylnaphthylamidase and alkaline phosphatase specific activities, measured both in homogenates of the mucosa and purified brush border membrane fractions, were found distributed along proximo-distal gradients of activity. However, different patterns were obtained which are specific for the enzyme considered. Kinetic parameters, Vmax and Km, were estimated for sucrase and alkaline phosphatase in purified brush border membrane fractions. It appeared that Vmax correlated well with the observed distribution of catalytic sites along the small intestine. Sugar (glucose) and amino acid (alanine and leucine) transport capacities were also distributed according to specific proximo-distal gradients but passive and facilitated diffusions were not affected. Only the active, Na+ -dependent component of transport was sensitive to position along the small intestine and we postulated that this adaptation should involve variations in carrier densities. It is therefore concluded that absorbo-digestive functions are intrinsic characteristics of the brush border membrane which are regulated according to the position along the small intestine.     

Differential sensitivity of intestinal brush border enzymes to pancreatic and lysosomal proteases.

Isolated human intestinal brush border membranes were used as sources of enzyme to study their degradation by proteolytic enzymes. Human intestinal brush border hydrolases undergo degradation by two separate proteolytic systems. Sucrase and alkaline phosphatase are degraded by pancreatic proteases (e.g. chymotrypsin) at neutral pH, whereas trehalase is degraded by lysosomal extracts at acid pH. Both the membrane bound and membrane free isolated enzymes had similar sensitivity to proteolytic enzymes. Thus, initial removal from the membrane is not essential as a prerequisite to proteolysis. It is postulated that the brush border membrane of the intestine is subject to proteolysis by pancreatic enzymes from the external cell surface and by lysosomal proteases within the cell.     

Distribution and biosynthesis of aminopeptidase N and dipeptidyl aminopeptidase IV in rat small intestine

The regional, cellular and subcellular distribution patterns of aminopeptidase N and dipeptidyl aminopeptidase IV were examined in rat small intestine. Aminopeptidase N of brush border membrane had maximal activity in the upper and middle intestine, while dipeptidyl aminopeptidase IV had a more uniform distribution profile with relatively high activity in the ileum. Along the villus and crypt cell gradient, the activity of both enzymes was maximally expressed in the mid-villus cells. However there was substantial dipeptidyl aminopeptidase IV activity in the crypt cells. Both enzymes were primarily associated with brush border membranes in all segments, however, in the proximal intestine, a significant amount of dipeptidyl aminopeptidase IV activity was associated with the cytosol fraction. The cytosol and brush border membrane forms of dipeptidyl aminopeptidase IV were immunologically identical and had the same electrophoretic mobility on disc gels. In contrast, the soluble and brush border membrane-bound forms of aminopeptidase N were immunologically distinct. When the total amount of aminopeptidase N and dipeptidyl aminopeptidase IV was determined by competitive radioimmunoassay, there were no regional or cellular differences in specific activity (enzyme activity/mg of enzyme protein) of either enzyme in brush border membrane and homogenate. The specific activity of both enzymes in a purified Golgi membrane fraction as measured by radioimmunoassay was about half that of the brush border membrane fraction. These results suggest that (1) aminopeptidase N and dipeptidyl aminopeptidase IV have different regional, cellular and subcellular distribution patterns; (2) there are enzymatically inactive forms of both enzymes present in a constant proportion to active molecules and that (3) a two-fold activation of precursor enzyme forms occurs during transfer from the Golgi membranes to the brush border membranes.     

Expression of sodium-glucose co-transporter and brush border disaccharidases in Giardia lamblia infected rat intestine

The absorption of D-glucose and brush border membrane disaccharidases in the intestine of rat during infection by Giardia lamblia has been studied. The level of mRNA encoding Na+/glucose co-transporter (SGLT1) and brush border sucrase and lactase activities were also analyzed. At the peak of infection, i.e, day 7, 11 and 15 post-infection, there was a marked decrease in the signal of 4.5 kb and 2.8 kb mRNAs encoding SGTL1 compared to the controls. A similar decrease in sucrase and lactase mRNA's (6.5 kb and 6.8 kb respectively) was also observed under these conditions. This corresponds to observed decrease in the rate of Na(+)-dependent D-glucose uptake and low activities of brush border sucrase and lactase under these conditions. There was no change in Na(+)-independent D-glucose uptake in giardia infected rat intestine. These findings suggest that the down regulation of the expression of SGLT1 and brush border sucrase and lactase activities may be responsible for the observed malabsorption in G. lamblia infection.     

Demonstration of hydrolase-related glucose transport in brush border membrane vesicles prepared from guinea pig small intestine.

Osmotically active brush border-membrane vesicles were prepared from guinea pig small intestine. Transport into these vesicles of glucose released by the action of the membrane enzymes, sucrase and alkaline phosphatase, was measured. At comparable hydrolytic rates, several fold more glucose was transferred into the intravesicular space from sucrose than from G-l-P, thus confirming the existence of hydrolase-related transport in the brush border membrane.     

Organ culture of adult mouse intestine. III. Behavior of the proteins, DNA content and brush border membrane enzymatic activities.

The proteins, the DNA content and the brush border membrane enzyme activities of adult mouse intestinal explants have been measured during a 24-hr organ culture. These activities were not modified in comparison with the controls at the beginning of the culture. The activity of the enzymes secreted in the medium during the 24-hr culture was equal to the activity present in the explants at the start of the culture. These results show that several metabolic functions of the intestine are fully preserved in organ culture.     

Age related effects of organochlorine insecticide lindane on intestinal brush border membrane in rats.

The effect of oral administration of lindane (gamma-HCH) has been studied on the intestine in 10-day, 20-day and 100-day old rats. In 10 day-old suckling pups exposed to lindane, there was a significant decrease in the activities of sucrase (29%), lactase (20%) and that of alkaline phosphatase (24%) compared to control. Sialic acid content of the brush borders was significantly decreased (29%) in 10-day old as well as in 20- and 100-day old rats (20 and 25% respectively), while fucose content of the membranes was significantly enhanced in all the age groups upon pesticide treatment. Among the brush border lipids, cholesterol content was significantly increased in all the age groups studied, the maximum increase of 35% being observed in 10-day-old rats. Membrane phospholipids were also increased in 20- and 100-day old animals (22% each) on lindane exposure. The present studies indicated that brush border membranes of suckling rat intestine were more susceptible to pesticide induced changes compared to older animals.     

Chemoprotective Effect of Taurine on Potassium Bromate-Induced DNA Damage,DNA-Protein Cross-Linking and Oxidative Stress in Rat Intestine

Potassium bromate (KBrO₃) is widely used as a food additive and is a major water disinfection by-product. It induces multiple organ toxicity in humans and experimental animals and is a probable human carcinogen. The present study reports the protective effect of dietary antioxidant taurine on KBrO₃-induced damage to the rat intestine. Animals were randomly divided into four groups: control, KBrO₃ alone, taurine alone and taurine+ KBrO₃. Administration of KBrO₃ alone led to decrease in the activities of intestinal brush border membrane enzymes while those of antioxidant defence and carbohydrate metabolism were also severely altered. There was increase in DNA damage and DNA-protein cross-linking. Treatment with taurine, prior to administration of KBrO₃, resulted in significant attenuation in all these parameters but the administration of taurine alone had no effect. Histological studies supported these biochemical results showing extensive intestinal damage in KBrO₃-treated animals and greatly reduced tissue injury in the taurine+ KBrO₃ group. These results show that taurine ameliorates bromate induced tissue toxicity and oxidative damage by improving the antioxidant defence, tissue integrity and energy metabolism. Taurine can, therefore, be potentially used as a therapeutic/protective agent against toxicity of KBrO₃ and related compounds.     

Evaluation of the fluidity and functionality of the renal cortical brush border membrane in experimental diabetes in rats

The present study was aimed at addressing the effect of hyperglycemia on the renal cortical brush border membrane. The fluidity and the functionality of the renal cortical brush border membrane have been evaluated after 6 weeks of streptozotocin-induced diabetes in rats. Lipid peroxidation and protein oxidation were first performed to confirm a state of oxidative stress. The fluidity of the brush border membrane of diabetic rats decreased significantly by 15.76%. There was an increase in the amount of early (19.39%) and advanced (42.23%) glycation end-products suggesting the accumulation of significant amount of non-enzymic glycation products at 6 weeks of diabetes. Although, the activities of both gamma-glutamyl transpeptidase and alkaline phosphatase of the brush border membrane decreased, that of the latter decreased to a significant extent with an increase in K(m) (81%) and no change in the V(max). A study of the activities of glutathione-dependent antioxidant enzymes in the renal cortical homogenates showed that the activities of glutathione peroxidase and glyoxalase II were altered significantly. Our study seems to suggest that increased free radical generation accompanied by non-enzymic glycation may be responsible for oxidative stress and an increased rigidity of the diabetic brush border membrane. Alkaline phosphatase may thus serve as a potentially useful marker of free radical induced damage to the renal cortical brush border membrane. The results also suggest that enhanced susceptibility to oxidative stress during early stages may be an important factor in the development of secondary complications of diabetes.     

Comparison of brush border membrane glycoproteins and glycoenzymes in the proximal and distal rat small intestine

Brush border membranes isolated from the proximal and distal portions of the rat small intestine were examined to see whether qualitative differences exist in their glycoprotein constituents. After SDS-polyacrylamide gel electrophoresis distinct differences were observed, indicating that the protein and glycoprotein profiles of the distal intestine are less complex. A competitive radioassay of lectin receptors revealed that there are significantly more wheat germ agglutinin and succinylated wheat germ agglutinin receptors present on brush border membranes from proximal intestine as compared to distal intestine. However, binding of Ricinus communis agglutinin I to brush border membranes of distal intestine was 2-times higher than that of proximal intestine. These segmental differences were also reflected in the binding patterns of individual brush border membrane hydrolases to wheat germ agglutinin and R. communis agglutinin I. Carbohydrate analysis demonstrated that the overall sugar content of brush border membranes is higher in distal intestine, with more galactose and sialic acid residues. No difference was found in the content of N-acetylglucosamine between the two segments. When brush border membranes from both segments were used as acceptors for galactosyltransferase, those from proximal intestine were better acceptors. Neuraminidase treatment significantly enhanced galactose oxidase/sodium borotritide labeling of brush border membranes from distal intestine and altered the electrophoretic mobility of dipeptidyl aminopeptidase IV and aminopeptidase N. No significant changes in labeling or enzyme electrophoretic mobility were noted in brush border membranes from proximal intestine after neuraminidase treatment. These studies indicate that the glycoproteins from brush border membranes of proximal and distal intestine are qualitatively different and that the glycoproteins from distal intestine may have more completed oligosaccharide side chains.     

Alterations in brush border membrane enzymes,carbohydrate metabolism and oxidative damage to rat intestine by potassium bromate

The acute toxicity of potassium bromate (KBrO₃) on rat small intestine was studied in this work. Animals were given a single oral dose of KBrO₃ (100 mg/kg body weight) and sacrificed 12, 24, 48, 96 and 168 h after the treatment; control animals were not given KBrO₃. The administration of KBrO₃ resulted in a reversible decline in the specific activities of several BBM enzymes. Lipid peroxidation, protein oxidation and hydrogen peroxide levels increased while total sulfhydryl groups and reduced glutathione decreased in KBrO₃-treated rats indicating induction of oxidative stress in the intestinal mucosa. The activities of anti-oxidant and carbohydrate metabolic enzymes were also altered upon KBrO₃ treatment. The maximum changes in all the parameters were 48 h after administration of KBrO₃ after which recovery took place, in many cases almost to control values after 168 h. Histopathological studies supported the biochemical findings showing extensive damage to the intestine at 48 h and recovery at 168 h. These results show that a single oral dose of KBrO₃ causes reversible oxidative damage to the intestine.     

Studies on the protective effect of dietary fish oil on gentamicin-induced nephrotoxicity and oxidative damage in rat kidney

Gentamicin (GM)-induced nephrotoxicity limits its long-term clinical use. Several agents/strategies were attempted to prevent GM nephrotoxicity but were not found suitable for clinical practice. Dietary fish oil (FO) retard the progression of certain types of cancers, cardiovascular and renal disorders. We aimed to evaluate protective effect of FO on GM-induced renal proximal tubular damage. The rats were pre-fed experimental diets for 10 days and then received GM (80 mg/kg body weight/day) treatment for 10 days while still on diet. Serum/urine parameters, enzymes of carbohydrate metabolism, brush border membrane (BBM), oxidative stress and phosphate transport in rat kidney were analyzed. GM nephrotoxicity was recorded by increased serum creatinine and blood urea nitrogen. GM increased the activities of lactate and glucose-6-phosphate dehydrogenases whereas decreased malate, isocitrate dehydrogenases; glucose-6 and fructose-1,6-bisphosphatases; superoxide dismutase, catalase, glutathione peroxidase and BBM enzymes. In contrast, FO alone increased enzyme activities of carbohydrate metabolism, BBM and oxidative stress. FO feeding to GM treated rats markedly enhanced resistance to GM elicited deleterious effects and prevented GM-induced decrease in 32Pi uptake across BBM. Dietary FO supplementation ameliorated GM-induced specific metabolic alterations and oxidative damage due to its intrinsic biochemical/antioxidant properties.     

Osmotic water permeability of rat intestinal brush border membrane vesicles: involvement of aquaporin-7 and aquaporin-8 and effect of metal ions.

Water channels AQP7 and AQP8 may be involved in transcellular water movement in the small intestine. We show that both AQP7 and AQP8 mRNA are expressed in rat small intestine. Immunoblot and immunohistochemistry experiments demonstrate that AQP7 and AQP8 proteins are present in the apical brush border membrane of intestinal epithelial cells. We investigated the effect of several metals and pH on the osmotic water permeability (Pf) of brush border membrane vesicles (BBMVs) and of AQP7 and AQP8 expressed in a cell line. Hg2+, Cu2+, and Zn2+ caused a significant decrease in the BBMV Pf, whereas Ni2+ and Li+ had no effect. AQP8-transfected cells showed a reduction in Pf in the presence of Hg2+ and Cu2+, whereas AQP7-transfected cells were insensitive to all tested metals. The Pf of both BBMVs and cells transfected with AQP7 and AQP8 was not affected by pH changes within the physiological range, and the Pf of BBMVs alone was not affected by phlorizin or amiloride. Our results indicate that AQP7 and AQP8 may play a role in water movement via the apical domain of small intestine epithelial cells. AQP8 may contribute to the water-imbalance-related clinical symptoms apparent after ingestion of high doses of Hg2+ and Cu2+.     

Studies on the protective effect of dietary fish oil on uranyl-nitrate-induced nephrotoxicity and oxidative damage in rat kidney

Human and animal exposure demonstrates that uranium is nephrotoxic. However, attempts to reduce it were not found suitable for clinical use. Dietary fish oil (FO) enriched in ω-3 fatty acids reduces the severity of cardiovascular and renal diseases. Present study investigates the protective effect of FO on uranyl nitrate (UN)-induced renal damage. Rats prefed with experimental diets for 15 days, given single nephrotoxic dose of UN (0.5 mg/kg body weight) intraperitoneally. After 5 d of UN treatment, serum/urine parameters, enzymes of carbohydrate metabolism, brush border membrane (BBM), oxidative stress and phosphate transport were analyzed in rat kidney. UN nephrotoxicity was characterized by increased serum creatinine and blood urea nitrogen. UN increased the activity of lactate dehydrogenase and NADP-malic enzyme whereas decreased malate, isocitrate and glucose-6-phophate dehydrogenases; glucose-6-phophatase, fructose-1, 6-bisphosphatase and BBM enzyme activities. UN caused oxidant/antioxidant imbalances as reflected by increased lipid peroxidation, activities of superoxide dismutase, glutathione peroxidase and decreased catalase activity. Feeding FO alone increased activities of enzymes of glucose metabolism, BBM, oxidative stress and Pi transport. UN-elicited alterations were prevented by FO feeding. However, corn oil had no such effects and was not similarly effective. In conclusion, FO appears to protect against UN-induced nephrotoxicity by improving energy metabolism and antioxidant defense mechanism.