Ram K. Sindhu

Antioxidant-Rich Diet Relieves Hypertension and Reduces Renal Immune Infiltration in Spontaneously Hypertensive Rats

Abstract—Previous studies have demonstrated that oxidative stress contributes to hypertension and treatments with either antioxidant or immunosuppressive/anti-inflammatory agents improve hypertension in spontaneously hypertensive rats (SHR). The present study was performed to determine if the antihypertensive effects of an antioxidant-rich diet are associated with reduction in the renal immune infiltration. Rats were divided into experimental groups (n=5 each) that were followed 7 months after birth, during which they were fed either a regular or antioxidant-enriched (test) diet as follows: SHR-R group=regular diet; SHR-T group=test diet throughout the experiment; SHR-S group=test diet for 4 months switched to regular diet thereafter; WKY group=control rats given regular diet. The SHR-T rats showed a significant reduction in systolic blood pressure (mm Hg): SHR-T=179.6±12.9 versus SHR-R=207.5±9.6 (P <0.001) and plasma hydrogen peroxide concentration (SHR-T=15±4 &mgr;mol/L versus 34±9 in SHR-R rats). This was accompanied by significant reductions of renal tissue nitrotyrosine abundance, tubulointerstitial infiltration (cells/mm2) of lymphocytes (SHR-T=18±3 versus SHR-R=30±4, P <0.001), macrophages (SHR-T= 17±3 versus SHR-R=22±3), and angiotensin II–positive cells (SHR-T= 17±2 versus SHR-R=25±5, P <0.01). Results in the SHR-S group were intermediate between the SHR-R and SHR-T groups. The intensity of the infiltration of lymphocytes, macrophages, and angiotensin II–positive cells significantly correlated with systolic blood pressure. Thus, the present study demonstrates that an antioxidant-enriched diet reduces the renal interstitial inflammation and improves hypertension in SHR. These findings point to interrelation between oxidative stress and inflammatory reactivity in the pathogenesis of hypertension.

Dysregulation of hepatic superoxide dismutase, catalase and glutathione peroxidase in diabetes: Response to insulin and antioxidant therapies

Recent evidence suggests that impaired antioxidant status is involved in oxidative stress associated with diabetes. The main antioxidant enzymes include superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX). The aim of the present investigation was to evaluate the activities and protein expression of these antioxidant enzymes in streptozotocin‐induced diabetes. Furthermore, the effects of insulin and antioxidant therapy alone and in combination were studied. Male Sprague‐Dawley rats were rendered diabetic by streptozotocin administration and randomly assigned to untreated, insulin‐treated, antioxidant (vitamin E and C)‐treated and insulin plus antioxidant‐treated groups. Normal rats fed either a regular diet or the antioxidant (vitamin E and C)‐rich diet served as controls. The animals were observed for 4 weeks. Diabetic animals showed marked weight loss, decreased activities of Cu Zn SOD and CAT and normal GPX activity. Additionally, the expression of all antioxidant enzyme proteins was decreased in the diabetic rats compared to the untreated controls. Insulin therapy prevented weight loss and normalized the activities and protein expression of all antioxidant enzymes. Antioxidant therapy in the diabetic rats normalized Cu Zn SOD and GPX protein expression. Combined therapy with insulin and antioxidants normalized all measured antioxidant enzyme protein expression and activities. Thus diabetes‐associated reductions in antioxidant enzymes can be ameliorated by insulin and/or antioxidant therapy.

Superoxide dismutase, catalase and glutathione peroxidase in the spontaneously hypertensive rat kidney: effect of antioxidant-rich diet.

Background and objective Earlier studies have shown increased production of reactive oxygen species (ROS) and upregulation of ROS-generating enzyme, nicotinamide adenine dinucleotide (phosphate) oxidase, in the kidney of spontaneously hypertensive rats (SHR). This study aimed to examine the activities and protein abundance of the main antioxidant enzymes [i.e. superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX)] in the kidney of SHR fed a regular or an antioxidant-rich diet. Methods Pregnant SHR and their offspring were fed either a regular diet or an antioxidant-rich diet (α-tocopherol, ascorbic acid, zinc and selenium) and observed for 6 months. Wistar–Kyoto (WKY) rats fed a regular or antioxidant-fortified diet served as controls. Results The untreated SHR showed severe hypertension and significant increases in plasma hydrogen peroxide and renal tissue nitrotyrosine abundance, indicating the presence of oxidative/nitrosative stress. Despite oxidative stress, Cu Zn SOD, CAT and GPX activities were unchanged in the cortex and medulla of untreated SHR. Immunodetectable Mn SOD was reduced in the medulla and elevated in the cortex, whereas, Cu Zn SOD protein was unchanged in the cortex and reduced in the medulla. By contrast, CAT protein abundance was increased in both cortex and medulla while GPX protein was elevated in the cortex and unchanged in the medulla. Comparison of protein abundance and activities of the antioxidant enzymes revealed significant discordance in the untreated SHR. Lifelong antioxidant therapy diminished the severity of hypertension, improved oxidative stress and ameliorated or reversed abnormalities of antioxidant enzyme expressions and activities. By contrast, antioxidant therapy had no effect on the measured parameters in the WKY rat controls. Conclusions Oxidative stress in SHR was associated with a lack of coordinate upregulation of the antioxidant enzymes and discordance between their protein abundance and enzymatic activity. These findings suggest an impaired antioxidant defense system and the presence of functionally abnormal enzymes in the SHR kidney. Lifelong antioxidant therapy improved expression, activity and activity-to-mass relationship of the measured enzymes. The latter suggests oxidative and nitrosative modification of these molecules in the SHR kidney.

NAD(P)H oxidase, superoxide dismutase, catalase, glutathione peroxidase and nitric oxide synthase expression in subacute spinal cord injury

Primary trauma to the spinal cord triggers a cascade of cellular and molecular events that promote continued tissue damage and expansion of the lesion for extended periods following the initial injury. Oxidative and nitrosative stresses play an important role in progression of spinal cord injury (SCI). In an attempt to explore the biochemical origin of oxidative/nitrosative stress associated with secondary SCI, we studied expression of the superoxide (O2*-)-generating enzyme, NAD(P)H oxidase, antioxidant enzymes [superoxide dismutase (CuZn SOD, Mn SOD), catalase, glutathione peroxidase (GPX)], nitric oxide synthases (NOS) and a byproduct of NO-O2*- interaction (nitrotyrosine) in the spinal cord tissues of rats 16 h and 14 days after surgical resections of a 5-mm segment of the cord below T8 or sham-operation. Immunodetectable NAD(P)H oxidase subunits (gp91phox and P67phox), Mn SOD, inducible NOS (iNOS), endothelial NOS (eNOS), and nitrotyrosine were elevated in the transected cords on day 1 and day 14. Neuronal NOS (nNOS) was unchanged on day 1 and significantly depressed on day 14. GPX was unchanged on day 1 and significantly elevated on day 14. Catalase was unchanged in the cord tissue surrounding the transection site at both points. Thus, concurrent upregulations of NAD(P)H oxidase, eNOS and iNOS (but not nNOS), work in concert to maintain oxidative and nitrosative stress in the injured cord tissue.

Regional expression of NO synthase, NAD(P)H oxidase and superoxide dismutase in the rat brain.

Nitric oxide (NO) derived from the endothelial NO synthase (eNOS) contributes to regulation of cerebral circulation, whereas that produced by neuronal NOS (nNOS) participates in the regulation of brain function. In particular, NO plays an important role in modulation of sympathetic activity and hence central regulation of arterial pressure. Superoxide derived from NAD(P)H oxidase avidly reacts with and inactivates NO and, thereby, modulates its bioavailability. Calmodulin (CM) is required for activation of NOS and soluble guanylate cyclase (sGC) serves as a NO receptor. Superoxide is dismutated to H2O2 by superoxide dismutase (SOD) and H2O2 is converted to H2O by catalase or glutathione peroxidase (GPX). Given the importance of NO in the regulation of brain perfusion and function, we undertook the present study to determine the relative expressions of immunodetectable nNOS, eNOS, CM, sGC, NAD(P)H oxidase and SOD by Western analysis in different regions of the normal rat brain. nNOS was abundantly expressed in the pons cerebellum and hypothalamus and less so in the cortex and medulla. sGC abundance was highest in the hypothalamus and pons, and lowest in the cerebellum and medulla. eNOS and calmodulin were equally abundant in all regions. NAD(P)H oxide was most abundant in the pons compared to other regions. Cytoplasmic SOD was equally distributed among different regions but catalase and GPX were more abundant in pons, hypothalamus and medulla and less so in the cortex and cerebellum. Thus, the study documented regional distributions of NOS, NAD(P)H oxidase, antioxidant enzymes, sGC and calmodulin which collectively regulate production and biological activities of NO and superoxide, the two important small molecular size signaling molecules.

Differential regulation of hepatic cytochrome P450 monooxygenases in streptozotocin-induced diabetic rats

The present investigation was carried out to study the expression of major cytochrome P450 (CYP) isozymes in streptozotocin-induced diabetes with concomitant insulin therapy. Male Sprague-Dawley rats were randomly assigned to untreated control, streptozotocin-induced diabetic, insulin-treated groups and monitored for 4 weeks. Uncontrolled hyperglycemia in the early phase of diabetes resulted in differential regulation of cytochrome P450 isozymes. CYP1B1, CYP1A2, heme oxygenase (HO)-2 proteins and CYP1A2-dependent 7-ethoxyresorufin O-deethylase (EROD) activity were upregulated in the hepatic microsomes of diabetic rats. Insulin therapy ameliorated EROD activity and the expression of CYP1A2, CYP1B1 and HO-2 proteins. In addition, CYP2B1 and 2E1 proteins were markedly induced in the diabetic group. Insulin therapy resulted in complete amelioration of CYP2E1 whereas CYP2B1 protein was partially ameliorated. By contrast, CYP2C11 protein was decreased over 99% in the diabetic group and was partially ameliorated by insulin therapy. These results demonstrate widespread alterations in the expression of CYP isozymes in diabetic rats that are ameliorated by insulin therapy.

Induction of cytochrome P4501A1 by photooxidized tryptophan in Hepa lclc7 cells.

Mouse hepatoma Hepa-lclc7 (Hepa-1) cells were cultivated in the presence of UV-irradiated amino acids. The results demonstrated that all of the amino acids tested, UV-oxidized tryptophan caused the highest induction of 7-ethoxyresorufin O-deethylase (EROD) activity compared with the controls (P < 0.01). The induction of EROD activity by oxidized tryptophan was dose dependent, and maximal induction was obtained at 12 hr after administration. Studies with various Hepa-1 mutants, which are defective in either the aryl hydrocarbon (Ah) receptor or Ah receptor nuclear translocator protein, indicated that the induction of EROD activity by oxidized tryptophan occurs through the Ah receptor. Gel mobility shift assays using nuclear extracts of Hepa-1 cells revealed that oxidized products of tryptophan can induce both Ah receptor transformation and binding of the liganded Ah receptor complex to its specific DNA recognition site. CYP1A1 mRNA, quantified by reverse transcription-polymerase chain reaction, and CYP1A1 protein were induced markedly in the oxidized tryptophan group compared with the controls. Injection of isolated oxidized tryptophan products into adult male rats caused significant induction of EROD activity in the pulmonary and hepatic microsomes compared with the controls (P < 0.01). These results demonstrated that oxidized tryptophan induces Ah receptor activation and binding of the liganded Ah receptor complex to its specific DNA recognition site, thereby initiating transcription and translation of the CYP1A1 gene with concomitant increase of EROD activity in Hepa-1 cells. Induction of EROD activity in the liver and lungs after injection of isolated oxidized tryptophan products into rats suggests that a similar mechanism may be operative in vivo.

Hepatic cytochrome P450 enzyme imprinting in adult rat by neonatal benzo[a]pyrene administration

ABSTRACT: The effect of neonatal exposure to benzo[a]pyrene (BaP) on the hepatic cytochrome P450 of male and female adult rats has been examined. Newborn rats (<24 h old) were injected with a single dose of BaP (1 mg/rat, s.c.) and killed after 110 days. In both sexes, body and liver weight, microsomal protein content, and total cytochrome P450 were unchanged. Cytochrome P450 1A2 protein content and 7-ethoxyresorufin O-deethylase activity were significantly decreased (p < 0.05) in males, whereas these were unaltered in females. Male-specific cytochrome P450 2C11 of male rats was significantly increased as shown by Western blot and increased testosterone 2α- and 16α-hydroxylase activities by 29% (p < 0.01) and 22% (p < 0.05), respectively. Female-specific cytochrome P450 2C12 protein content was unaltered in females. In addition, the level of free hepatic glucocorticoid receptor in adult males was elevated by 35% after BaP exposure, whereas it was unchanged in adult females. These results indicate, for the first time, that neonatal BaP exposure results in gender-specific lasting effects on hepatic cytochrome P450 1A2, cytochrome P450 2C11, and glucocorticoid receptors in adult male rats, whereas these parameters are unchanged in adult female rats.,

Exposure to environmental tobacco smoke results in an increased production of (+)-anti-benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide in juvenile ferret lung homogenates

Six-week-old ferrets were exposed head-only to clean air or environmental tobacco smoke (ETS) at an average particulate concentration of 38 +/- 13 mg/m3 for 2 h/d, 5 d/wk for up to 15 wk. Twenty four hours after last exposure, the ferrets were sacrificed and the metabolism of benzo[a]pyrene and (-)-7R-trans-benzo[a]pyrene-7,8-dihydrodiol was studied in lung homogenates. The results show that after ETS exposure total metabolism of benzo[a]pyrene, measured by the accumulation of hexane nonextractable radioactivity, was increased by 35% in the males and 66% in the females (p < .05), respectively, of that observed with air-exposed controls. With (-)-7R-trans-benzo[a]pyrene-7,8-dihydrodiol as substrate, the formation of both benzo[a]pyrene-r-7,t-8,9,c-10-tetrahydrotetraol and (+)-anti-benzo[a]-pyrene-7,8-dihydrodiol-9,10-epoxide-derived tetraols by lung homogenates of ETS-exposed male and female ferrets was significantly increased compared to the air-exposed controls (p < .01). DNA-bound radioactivity was significantly increased in both the males (p < .01) and females (p < .05) compared to the air-exposed ferrets.

Effect of hyperoxia on rat pulmonary and hepatic cytochrome P450 monooxygenases

Abstract Exposure of adult male rats to hyperoxia (O2 > 95%) resulted in a tendency for all of the components of the pulmonary cytochrome P450 (P450) system to increase at 48 h after the exposure. However, the most pronounced effect of hyperoxia was observed on pulmonary ethoxycoumarin O-deethylase and ethoxyresorufin O-deethylase activities which were induced 4- and 25-fold respectively after 48 h. In the liver, P450 and NADH b5 reductase were increased after 48 h, while other components of the monooxygenase system remained unchanged. In the hepatic microsomes, contrary to the lungs, aminopyrine N-demethylase activity was decreased after 24 h of hyperoxic exposure (P < 0.05) and returned to the control level by 48 h. Similar changes were observed in benzphetamine N-demethylase activity. Aniline hydroxylase activity was decreased after 8 h of hyperoxic exposure (P < 0.01) and remained decreased at 24 h (P < 0.01) and 48 h (P < 0.05). The level of induction of ethoxycoumarin O-deethylase and ethoxyresorufin O-deethylase activities, however, was almost similar in the liver to that observed in the lungs.