Gabor Bekesi

In vitro effects of different steroid hormones on superoxide anion production of human neutrophil granulocytes.

Neutrophil granulocytes play an important role in atherogenesis also through their free radical generation. According to recent studies, a point of action by which estrogens can provide protection against atherosclerosis is their inhibiting effect on superoxide anion production. The aim of our study was to test whether this means a common effect of steroids on superoxide production, or whether various steroid hormones have different action on superoxide generation of human granulocytes. Neutrophils were separated from the blood samples of twelve healthy volunteers. Isolated cells were incubated with different concentrations (10(-9), 10(-8), 10(-7) M) of hydrocortisone, aldosterone, cortexolone, 17-beta-estradiol, progesterone, and testosterone. Superoxide anion production was determined by photometry using the reduction of ferricytochrome-C. Compared to that of control cells neutrophils incubated with 17-beta-estradiol, progesterone, testosterone and hydrocortisone showed significantly reduced superoxide production. No significant alteration of superoxide anion production was found after the incubation of cells with aldosterone and cortexolone. It is concluded that similarly to estradiol other sex steroids and cortisol can inhibit the free radical production of human granulocytes, but mineralocorticoid aldosterone and Reichsteins substance S do not show such activity. Our results provide new evidence supporting the theory that certain types of steroid hormones have antioxidant capacity. This may give further reasons for investigating the molecular background of the existence or absence of this property and thus might lead to the development of new free radical scavengers.

Induced myeloperoxidase activity and related superoxide inhibition during hormone replacement therapy.

Objective To test whether the menopause entails any changes in the myeloperoxidase activity of neutrophil granulocytes. The effects of hormone replacement therapy on myeloperoxidase activity and related changes in free radical production were also investigated.

The effect of estrogens on superoxide anion generation by human neutrophil granulocytes: Possible consequences of the antioxidant defense

The present study aimed to test whether, beyond the known antioxidant effect of estradiol, such a property is also possessed by estrone and estriol. For this purpose, an in vitro investigation of the effect of estrone and estriol on superoxide anion production by human neutrophil granulocytes was carried out. Blood samples were obtained from healthy volunteers and neutrophil granulocytes were separated for measurement of superoxide anion generation after incubation with estrone, estriol (10−7, 10−6 and 10−5 M) and 17β-estradiol (10−7 M). Superoxide anion production of isolated neutrophil granulocytes was quantified by photometry and using the reduction of ferricytochrome-C. When adding estrone and estriol to neutrophil granulocyte suspensions, the production of superoxide anion fell (10−5 M: 84.17 ± 3.14% and 88.77 ± 1.98% of control production, p < 0.01 and p < 0.05, respectively). Estradiol produced an antioxidant effect at lower concentration (10−7 M: 72.91 ± 7.94% of control production, p < 0.001). The weak estrogens estrone and estriol, similarly to estradiol, are also able to reduce the superoxide anion release in our experimental model. This may have importance in the antioxidant defense of biological systems.

Plasma concentration of myeloperoxidase enzyme in pre- and post-climacterial people: Related superoxide anion generation

Neutrophil granulocytes are involved in the pathogenesis of atherosclerosis also through their free radical generation. The aim of the study was to test how extracellular levels of myeloperoxidase (MPO; a granulocyte enzyme playing role in free radical production) change by age and what effect this change has on the production of the free radical superoxide anion by neutrophils. We also wanted to examine whether the antioxidant effect of different steroid hormones is realized through the MPO. Plasma myeloperoxidase concentrations of healthy blood donors were quantified by ELISA. Superoxide anion production was measured by photometry. Myeloperoxidase concentration was significantly lower in plasmas obtained from older women and men than in those from younger subjects. Adding the MPO inhibitors 4-aminobenzoic acid hydrazide (ABAH) and indomethacin to the granulocytes, the generation of superoxide anion increased and the decreasing effect of the steroids on superoxide production was inhibited. Incubating the neutrophils with the product of the reaction catalyzed by MPO itself (hypochlorite anion), we found significant decrease in superoxide generation. According to our results MPO seems to diminish the production of superoxide anion and so probably has an antioxidant ability. Therefore, its lower plasma levels may contribute to the increasing incidence of atherosclerosis and other free radical mediated disorders in old people. Thus, after further studies MPO might become one of the indicators of cardiovascular risk and the scavenger capacity in general.

Effect of inhibitors of myeloperoxidase on the development of aortic atherosclerosis in an animal model

Our earlier studies have shown that some steroids increase myeloperoxidase enzyme (MPO) release from human granulocytes, and that MPO plasma levels are significantly lower in postclimacteric people. Moreover, we have proven that MPO inhibits production of atherogenic free radical superoxide anion and MPO-inhibitors increase superoxide release. The aim of the present study was to investigate the effect of MPO-inhibitors on the early phase of aortic atherosclerosis, namely the extent of intimal plaques and the thickening of the medial layer. Adult male rabbits were fed with lipid rich food (cholesterol: 1.3%, peanut oil: 8%) for 8 weeks. During this period MPO-inhibitors were also given (4-aminobenzoicacid-hydrazide/ABAH/-13.3 mg/kg/day or indometacin-5 mg/kg/day). All animals developed intimal lipid plaques (raised fatty streaks). The relative plaque-covered areas of the aortas were compared and the media thickness of the aorta was measured on plaque-free as well as plaque-containing areas. The medial smooth muscle density and peroxidase activity of the aortic media were also determined. The media thickness increased (p<0.05) in the cholesterol+ABAH as well as in the cholesterol+indometacin groups up to 375.7 (+/-60.5) and 442.5 (+/-123.4) microm, respectively, compared to the control group (cholesterol feeding alone) where it measured only 308.4 (+/-51.67) microm. The medial peroxidase activity decreased significantly in the indometacin treated group and showed a decreasing tendency using ABAH. In parallel to this there was a tendency of increase in the relative plaque covered areas. The smooth muscle density showed no significant modifications, while inhibitors of the MPO seemed to enhance aortic medial thickness, i.e. the grade of a pre-atherosclerotic lesion, in our animal model. Collectively, the anti-atherogenic effect of certain steroid hormones might be realized through the impact on MPO activity.

Endothelial relaxation mechanisms and nitrative stress are partly restored by Vitamin D3 therapy in a rat model of polycystic ovary syndrome

AIMS In polycystic ovary syndrome (PCOS), metabolic and cardiovascular dysfunction is related to hyperandrogenic status and insulin resistance, however, Vitamin D3 has a beneficial effect partly due to its anti-oxidant capacity. Nitrative stress is a major factor in the development of cardiovascular dysfunction and insulin resistance in various diseases. Our aim was to determine the effects of vitamin D3 in a rat model of PCOS, particularly the pathogenic role of nitrative stress. MAIN METHODS Female Wistar rats weighing 100-140g were administered vehicle (C), dihydrotestosterone (DHT) or dihydrotestosterone plus vitamin D3 (DHT+D) (n=10 per group). On the 10th week, acetylcholine (Ach) induced relaxation ability of the isolated thoracic aorta rings was determined. In order to examine the possible role of endothelial nitric oxide synthase (eNOS) and cyclooxygenase-2 (COX-2) pathways in the impaired endothelial function, immunohistochemical labeling of aortas with anti-eNOS and anti-COX-2 antibodies was performed. Leukocyte smears, aorta and ovary tissue sections were also immunostained with anti-nitrotyrosine antibody to determine nitrative stress. KEY FINDINGS Relaxation ability of aorta was reduced in group DHT, and vitamin D3 partly restored Ach induced relaxation. eNOS labeling was significantly lower in DHT rats compared to the other two groups, however COX-2 staining showed an increment. Nitrative stress showed a significant increase in response to dihydrotestosterone, while vitamin D3 treatment, in case of the ovaries, was able to reverse this effect. SIGNIFICANCE Nitrative stress may play a role in the pathogenesis of PCOS and in the development of the therapeutic effect of vitamin D3.

Increased total scavenger capacity and decreased liver fat content in rats fed dehydroepiandrosterone and its sulphate on a high-fat diet

Background: Weak androgens have an antioxidant effect in vitro which is represented as a beneficial change in the antioxidant status. Objective: Our aim was to clarify whether dehydroepiandrosterone (DHEA) and dehydroepiandrosterone-sulphate (DHEAS) oral administration results in beneficial antioxidant changes in Sprague-Dawley adult male rats in vivo. Methods: Groups of experimental animals were fed a high-fat or a normal-fat diet and treated with DHEA or DHEAS in the drinking fluid. The control group was fed a high-fat diet together with untreated drinking fluid. Total scavenger capacity (TSC) was measured before and after 4 weeks of treatment in blood samples using a chemiluminometric assay. Fat content, superoxide dismutase (SOD), catalase and glutathione S-transferase (GST) activity in the liver were determined by Sudan staining and spectrophotometric assessments, respectively, from the fresh frozen tissue. Results: DHEA and the DHEAS treatment showed significantly increased TSC in the groups fed a high-fat diet. The control group and the DHEA- or DHEAS-treated groups on normal diets showed no significant changes in TSC. The total score of liver fat content in the high-fat diet groups showed a marked positivity with Sudan staining, and the groups treated with DHEA or DHEAS had a markedly decreased amount of fat in the liver slides compared to the untreated group on the high-fat diet. Liver SOD activity was decreased in all high-fat diet groups and elevated only in the groups on a normal diet with DHEA or DHEAS treatment. Liver catalase and GST activities were decreased in the groups where TSC was significantly increased. Conclusion: Our results support the hypothesis that DHEA and DHEAS supplementation can improve the antioxidant status in lipid-rich dietary habits.

Antioxidant effect of the active metabolites of tibolone

Abstract Certain steroidal compounds have an antioxidant effect in humans. Our aim was to test whether the synthetic steroid tibolone and its metabolites are also able to display such a property. For this, granulocytes from healthy men and women were incubated for two hours with different concentrations (10−7, 10−8, 10−9 M) of either estradiol, tibolone, 3α-hydroxytibolone, 3β-hydroxytibolone, Δ4-tibolone, 3α-sulfated-tibolone, 3α-17β-disulfated-tibolone, 3β-sulfated-tibolone or 3β-17β-disulfated-tibolone. Superoxide anion generation of neutrophils was measured by photometry. Results of different steroids were given as percentages of their controls. A more simple superoxide generating system, the xanthine–xanthine oxidase reaction was also tested. We found that granulocyte superoxide production did not differ from the control using 10−9 M of steroids. Using 10−8 M concentration: estradiol (80.9 ± 2.5%); 3β-sulfated-tibolone (83.3 ± 4.7%); 3β-17β-disulfated-tibolone (81.0 ± 4.2%) caused a significant decrease in superoxide production, compared to the control. In addition at 10−7 M, 3β-hydroxytibolone and 3α-sulfated-tibolone also showed antioxidant effects. In the xanthine–xanthine oxidase system estradiol (67.4 ± 1.0%), 3α-sulfated-tibolone (85.8 ± 5.3%), 3α-17β-disulfated-tibolone (71.9 ± 2.5%), 3β-sulfated-tibolone (73.9 ± 5.0%), and 3β-17β-disulfated-tibolone (65.8 ± 3.4%) caused a significant decrease in superoxide production. Conclusively, although tibolone itself did not show significant antioxidant capacity, most of its active metabolites have antioxidant effects.

The effect of indomethacin, myeloperoxidase, and certain steroid hormones on bactericidal activity: an ex vivo and in vivo experimental study

BackgroundThe role of myeloperoxidase (MPO) is essential in the killing of phagocytosed bacteria. Certain steroid hormones increase MPO plasma concentration. Our aim was to test the effect of MPO, its inhibitor indomethacin, and certain steroid hormones on bactericidal activity.MethodsHuman polymorphonuclear leukocytes (PMN) were incubated with opsonised Escherichia coli and either MPO, indomethacin, estradiol, or hydrocortisone. Intracellular killing capacity was evaluated with UV microscopy after treatment with fluorescent dye. Next, an in vivo experiment was performed with nine groups of rats: in the first phase of the study indomethacin treatment and Pasteurella multocida infection (Ii), indomethacin treatment without infection (I0), untreated control with infection (Mi) and untreated control without infection (M0); in the second phase of the study rats with infection and testosterone treatment (NT), castration, infection and testosterone treatment (CT), castration, infection and estradiol treatment (CE), non-castrated infected control (N0), and castrated infected control (C0). After treatment bacteria were reisolated from the liver and heart blood on agar plates, and laboratory parameters were analyzed. For the comparison of laboratory results ANOVA or Kruskal-Wallis test and LSD post hoc test was used.ResultsIndomethacin did not have a remarkable effect on the bacterial killing of PMNs, while the other compounds increased bacterial killing to various degrees. In the animal model indomethacin and infection caused a poor clinical state, a great number of reisolated bacteria, elevated white blood cell (WBC) count, decreased C-reactive protein (CRP) and serum albumin levels. Testosterone treatment resulted in less bacterial colony numbers in group NT, but not in group CT compared to respective controls (N0, C0). Estradiol treatment (CE) decreased colony numbers compared to control (C0). Hormone administration resulted in lower WBC counts, and in group CE, a decreased CRP.ConclusionsMPO, estradiol, and hydrocortisone improve bacterial killing activity of PMNs. Indomethacin treatment and castration weaken immune responses and clinical state of infected rats, while testosterone and estradiol have a beneficial effect.

Biomechanics and vasoreactivity of female intramural coronaries in angiotensin II induced hypertension

Hypertension causes small vessel remodeling, vasomotor alterations. We investigated diameter, tone and mechanics of intramural small coronaries of female rats that received chronic angiotensin treatment to induce hypertension.Angiotensin II infusion (AII, 100 ng/bwkg/min, sc.) was used to establish hypertension in 10 female rats. Other 10 rats served as controls. Following 4 weeks of treatment, side branches of the left anterior descendant coronary (diameter approximately 200 microm) were isolated, cannulated and pressure-diameter curves were registered between 2-90 mmHg. Changes in vessel diameter were measured in Krebs solution, in the presence of thromboxane A2 receptor agonist (U46619, 10(-6) M), bradykinin (BK, 10(-6) M), and finally at complete relaxation (in Ca2+-free solution). Chronic AII treatment raised the mean arterial pressure (130+/-5 mmHg vs. 96+/-2 mmHg, average +/-SEM) significantly. Wall thickness of the AII group was significantly greater (40.2+/-4.2 microm vs. 31.4+/-2.7 microm at 50 mmHg in Ca2+ -free solution), but cross-section of the vessel wall did not differ. Tangentional wall stress and elastic modulus decreased significantly in hypertensive animals. Constrictions in the presence of U46619 were greater in the AII group (24.4+/- 5.6% vs. 14.5+/-3.3% at 50 mmHg). In hypertension, intramural small coronaries showed inward eutrophic remodeling, as a morphological adaptation following AII treatment enhanced thromboxane A2-induced tone.