Metabolic heterogeneity of isolated cortical and juxtamedullary glomeruli in adriamycin nephrotoxicity.

The anticancer drug adriamycin (ADR) is selectively toxic to glomerular cells when administered intravenously (5 mg/kg b.w.) to female MWF/Ztm rats. Recent data have shown that the proteinuria associated with the lesion does not occur in cortical glomeruli, suggesting the selective injury of juxtamedullary glomeruli. In the present study, the effect of ADR on glomerular metabolism was studied with special reference to possible differences between cortical and juxtamedullary glomeruli. On day 7 after ADR treatment, cortical and juxtamedullary glomeruli were separately isolated by the sieving method and 14C glucose oxidation to 14CO2 and the incorporation of 3H proline into macromolecules were measured in vitro and used to study target selective injury in ADR-treated rats compared to control rats. The investigations revealed differences in the response of cortical and juxtamedullary glomeruli to ADR. ADR treatment increased proline incorporation over a 4-hour incubation period in both glomerular populations compared to controls, but the effect was significantly (p less than 0.05) more pronounced in juxtamedullary glomeruli (juxtamedullary: 187 +/- 8% of control; cortical: 167 +/- 4% of control). Glucose oxidation was enhanced after 4 h only in juxtamedullary glomeruli (juxtamedullary: 132 +/- 3% of control; cortical: 82 +/- 10% of control). These data show that glomerular damage caused by ADR is associated with a stimulating effect on glomerular metabolism which is more marked in juxtamedullary than in cortical glomeruli, thus indicating a heterogenous response of different glomerular populations and supporting the concept that the selective damage of juxtamedullary glomeruli accounts for the proteinuria.     

Effects of hydrogen peroxide on relaxation through the NO/sGC/cGMP pathway in isolated rat iliac arteries

Abstract

The production of reactive oxygen species, including hydrogen peroxide (H₂O₂), is increased in diseased blood vessels. Although H₂O₂ leads to impairment of the nitric oxide (NO)/soluble guanylate cyclase (sGC)/cGMP signaling pathway, it is not clear whether this reactive molecule affects the redox state of sGC, a key determinant of NO bioavailability. To clarify this issue, mechanical responses of endothelium-denuded rat external iliac arteries to BAY 41-2272 (sGC stimulator), BAY 60-2770 (sGC activator), nitroglycerin (NO donor), acidified NaNO₂ (exogenous NO) and 8-Br-cGMP (cGMP analog) were studied under exposure to H₂O₂. The relaxant response to BAY 41-2272 (pD₂: 6.79?±?0.10 and 6.62?±?0.17), BAY 60-2770 (pD₂: 9.57?±?0.06 and 9.34?±?0.15) or 8-Br-cGMP (pD₂: 5.19?±?0.06 and 5.24?±?0.08) was not apparently affected by exposure to H₂O₂. In addition, vascular cGMP production stimulated with BAY 41-2272 or BAY 60-2770 in the presence of H₂O₂ was identical to that in its absence. On the other hand, nitroglycerin-induced relaxation was markedly attenuated by exposing the arteries to H₂O₂ (pD₂: 8.73?±?0.05 and 8.30?±?0.05), which was normalized in the presence of catalase (pD₂: 8.59?±?0.05). Likewise, H₂O₂ exposure impaired the relaxant response to acidified NaNO₂ (pD₂: 6.52?±?0.17 and 6.09?±?0.16). These findings suggest that H₂O₂ interferes with the NO-mediated action, but the sGC redox equilibrium and the downstream target(s) of cGMP are unlikely to be affected in the vasculature.     

Elevations of intracellular cAMP result in a change in cell shape that resembles dome formation in cultured rat glomerular epithelial cells

Summary Cultured glomerular epithelial cells form a continuous monolayer of polyhedral-shaped cells. PGE₂ (1 μg/ml) in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine (MIX) markedly raises intracellular and medium cyclic AMP (cAMP) levels at 20 min (intracellular: MIX alone, 112 ± 6.6 pmol cAMP/mg protein, MIX plus PGE₂, 2252±63 pmol cAMP/mg protein; medium: MIX, 20.6±2.1 pmol cAMP/mg protein; MIX plus PGE₂, 117±3.8 pmol cAMP/mg protein). By 2 h, when cellular and medium cAMP levels were still elevated, the cells underwent a change in shape that was similar to dome formation (15 to 20% of the monolayer changing shape). Derivatives of cAMP [i. e. dibutyryl and 8-(4-chlorophenylthio)-cAMP], when added to the incubation medium also caused shape change in glomerular epithelial cells at 2 h; cAMP itself did not. The formation of domes has been used as a morphological indicator of the vertorial transport of salt and water in other cultured epithelial cells. This work was supported by grant AM 29787 from the National Institutes of Health, Bethesda, MD.     

Cardiac Function in the Hagfish,Myxine (Myxinoidea:Cyclostomata)

The electrocardiogram of Myxine glutinosa is described and its events related to systole in the sinus venosus, atrium and ventricle. Intraatrial, ventricular, ventral aortic and dorsal aortic pressures are presented. Ventricular systolic pressure was 10.3 ± 2.0 cm H₂O; average dorsal aortic pressure was 7.0 ± 1.7 cm H₂O. The resistance of the gill vessels caused a loss of 28% of the ventral aortic pressure. The conclusion is drawn that the basic mechanism of the heart of Myxine is like that of fish and higher vertebrates, and that the low pressure it develops is to be ascribed to structural and functional features of the myocardium.     

Elevated exhalation of hydrogen peroxide in patients with non-small cell lung cancer is not affected by chemotherapy

Objectives: Reactive oxygen species, which are implicated in the process of carcinogenesis, are also responsible for cell death during chemotherapy (CHT). Therefore, the aim of the study was to evaluate exhaled H₂O₂ levels in non-small cell lung cancer (NSCLC) patients before and after CHT.

Methods: Thirty patients (age 61.3?±?9.3 years) with advanced NSCLC (stage IIIB–IV) and 15 age-matched healthy cigarette smokers were enrolled into the study. Patients received four cycles of cisplatin or carboplatin with vinorelbine every three weeks. Before and after the first, second, and fourth cycle, the concentration of H₂O₂ in exhaled breath condensate was measured with respect to treatment response.

Results: At the baseline, NSCLC patients exhaled 3.8 times more H₂O₂ than the control group (0.49?±?0.14 vs. 0.13?±?0.03?µmol/L, P?2O₂ levels independent of the treatment response (partial remission vs. progressive disease). Pre- and post-CHT cycles of H₂O₂ levels generally correlated positively.

Discussion: The study demonstrated the occurrence of oxidative stress in the airways of advanced NSCLC patients. Exhaled H₂O₂ level was not affected by CHT and independent of treatment results and changes in the number of circulating neutrophils.     

Acute effects of Cu on oxygen consumption and 96 hr-LC50 values in the freshwater fish Tilapia sparrmani (Teleostei: Cichlidae) in Mooi River hard water,South Africa

The median lethal copper (Cu) concentration (96 hr-LC₅₀) values for acute Cu toxicity for Tilapia sparrmanii (live mass: 30 ± 8g) in Mooi River hard water of dolomitic origin at 20 °C, pH 7.9, was 68.1 μmol l^?1. At this 96 hr-LC₅₀ value the specific oxygen consumption rate (∈ O₂) decreased by 44.2 (± 2.1) % from a non-exposed value of 6.6 (±0.32) mmol O₂ kg^?1 hr^?1 to 3.63 (±0.23) mmol O₂ kg ^?1 hr^?1. At 46.4 μmol Cu l^?1, 100% of the exposed T. sparrmanii were still alive after 96 hours, but the ∈ O₂ decreased by a mean value of 1.65 (± 0.16) mmol O₂ kg^?1 fish hr^?1 or 25% (± 2.4). Contrary to Pb and Cd, Cu as CuCl₂ 2H₂O was not precipitated in hard water four days after it was dissolved. Thus T. sparrmanii and other cichlids are shown to be more than an order of magnitude more resistant to Cu as a toxicant than most salmonids.     

Resting metabolic rate and lung function in fasted and fed rough-toothed dolphins,Steno bredanensis

We measured resting metabolic rate (RMR), tidal volume (V_T), breathing frequency (f_R), respiratory flow, and end-expired gases in rough-toothed dolphins (Steno bredanensis) housed in managed care after an overnight fast and 1–2 hr following a meal. The measured average (± standard deviation) V_T (4.0 ± 1.3 L) and f_R (1.9 ± 1.0 breaths/min) were higher and lower, respectively, as compared with estimated values from both terrestrial and aquatic mammals, and the average V_T was 43% of the estimated total lung capacity. The end-expired gas levels suggested that this species keep alveolar O₂ (10.6% or 80 mmHg) and CO₂ (7.6% or 57 mmHg), and likely arterial gas tensions, low and high, respectively, to maximize efficiency of gas exchange. We show that following an overnight fast, the RMR (566 ± 158 ml O₂/min) was 1.8 times the estimated value predicted by Kleiber for terrestrial mammals of the same size. We also show that between 1 and 2 hr after ingestion of a meal, the metabolic rate increases an average of 29% (709 ± 126 ml O₂/min). Both body mass (M_b) and f_R significantly altered the measured RMR and we propose that both these variables should be measured when estimating energy use in cetaceans.     

Free-radical scavenging by Ouratea parviflora in experimentally-induced liver injuries

Abstract

The antioxidant potential of crude extracts and fractions from leaves of Ouratea parviflora, a Brazilian medicinal plant used for the treatment of inflammatory diseases, was investigated in vitro through the scavenging of radicals 2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH), hydroxyl radical (HO^?), superoxide anion (O₂^??), and lipid peroxidation in rat liver homogenate. The crude extract (CEOP) and hydro-alcoholic fraction (OP4) showed strong inhibitory activity toward lipid peroxidation induced by tert-butyl peroxide (IC₅₀ = 2.3 ± 0.2 and 1.9 ± 0.1 μg/ml, respectively). The same products exhibited a strong concentration-dependent inhibition of deoxyribose oxidation (14.9 ± 0.2 and 0.2 ± 0.1 μg/ml, respectively), and also showed a considerable antioxidant activity against O₂^??(87.3 ± 0.1 and 73.1 ± 0.4 μg/ml, respectively) and DPPH radicals (55.4 ± 0.3 and 38.3 ± 0.4 μg/ml, respectively). The protective effects of CEOP and OP4 were also studied in mouse liver. CCl₄ significantly increased (by 90%) levels of lipid hydroperoxides, carbonyl protein content (64%), DNA damage index (133%), aspartate aminotransferase (261%), alanine aminotransferase (212%), catalase activity (23%), and also caused a decrease of 60% in GSH content. The results showed that CEOP and OP4 exerted cytoprotective effects against oxidative injury caused by CCl₄ in rat liver, probably related to the antioxidant activity showed by the in vitro free radical scavenging property.     

Glomerular compromise in mercuric chloride-induced nephrotoxicity

We have examined the effects of mercuric chloride on renal glomerular structure. Isolated glomeruli from mercury-treated rats (HgCl₂, 5 mg/kg body wt, s.c.) 1 hour post injection presented a diminished cross-sectional area as compared with control glomeruli [control (μm²) = 26,310 ± 2,545, HgCl₂ (μm²) = 18,474 ± 1,828] and higher glomerular calcium content (control = 23 ± 6 nmoles/mg prot, HgCl₂ = 43 ± 7 nmoles/mg prot). Renal sections prepared for immunohistochemical and histochemical analysis showed larger deposits of fibronectin and lipids and enhanced cellularity in glomerular structures from HgCl₂-treated rats. Moreover, mieloperoxidase activity measured in isolated glomeruli were also increased as compared with control preparations [MPO (U/mg prot): control = 59 ± 7, HgCl₂ = 134 ± 10]. When the animals were studied 24 hours post HgCl₂ injection, glomerular cross-sectional area values were not different from control values (25,276 ± 1,983 μm²), while calcium contents were higher than values observed 1 hour after treatment (92 ± 9 nmoles/mg prot). A similar pattern was observed in fibronectin deposits. Hypercellularity in glomerular structures and the higher mieloperoxidase levels were maintained at this time (MPO HgCl₂-rats 24 h = 148 ± 31 U/mg prot). The effects observed in this study are consistent with an inflammatory response in the glomerular structure of HgCl₂-treated rats that could explain the altered renal function described in previous reports in our laboratory. © 1997 John Wiley & Sons, Inc.     

Similarity of PO2 and PCO2 values in the air cell of eggs of birds and in the alveolar gas of humans at sea level and at high altitude

At the end of incubation, the partial pressures of oxygen and carbon dioxide in the air cell of sea-level avian eggs are similar to those in the expiratory air of adult birds. At high altitude, changes in the permeability of the shell and probably in the embryo metabolism partially compensates the increase in the gas diffusion constant resulting from the low barometric pressure. The aim of this study was to test whether-despite of the adaptive responses of the high altitude avian embryo-the air cell values would be similar to those of the alveolar air of high altitude human natives. Air cell O₂ (48.3±1.6 torr) and CO₂ (20.9±0.85 torr) pressure values were obtained by studying naturally incubated eggs of the Andean gull (Larus serranus)_at 4650m. Sea-level chicken (Gallus gallus) air cell pressure values of O₂ (102.3±2.7 torr) and of CO₂ (43.3±1.3 torr) were obtained from the literature for comparison. Both these values were similar to those found in the alveolar air of humans at sea level (O₂: 104.4±0.4 torr, CO₂:40.1±0.25 torr) and at high altitude (4540 m) (O₂:50.5±0.53 torr, CO₂: 29.1±0.37 torr). Despite very large evolutionary changes in morphology and physiology of the respiratory organs, the head pressure of O₂ that oxygenates the blood keeps a constant value in the pre-pipping avian embryo and in the alveolar air of adult mammals. This constancy holds valid at high altitude.     

Decreased paraoxonase 2 enzymatic activity in monocyte/macrophages cells. A comparative in vivo and in vitro study for diabetes

Aim: To investigate peripheral blood monocytes/macrophages (Mo/M?) paraoxonase 2 (PON2) in diabetes and the factors modulating its activity.

Methods: One hundred and eighteen patients with newly diagnosed uncomplicated type 2 diabetes mellitus were compared regarding clinical, biochemical and oxidative stress parameters with 80 healthy subjects. The capacity of the peripheral blood mononuclear cells (PBMNC) to release pro-oxidants and to neutralise them was determined by measuring the respiratory burst (RB) and the intracellular antioxidant enzyme PON2. In vitro experiments were conducted on a differentiated monocytes cell line (dU937) that was exposed to serum deprivation followed by addition of isolated lipoproteins (VLDL or LDL).

Results: Paraoxonase 2 activity in Mo/M? was significantly lower in type 2 diabetes patients (0.042?±?0.044 vs 0.165?±?0.133U lactonase activity/mg protein in controls, p?1c) and insulin resistance (HOMA-IR). In multivariate regression models, 15–34% of the PON2 variance was explained by diabetes. The in vitro addition of VLDL normalised the RB of serum deprived dU937 cells, S? (to 82?±?18% of the cells incubated with serum, S+) and PON2 activity (from 0.524?±?0.061 in S???to 0.298?±?0.048?U/mg protein). In contrast, when LDL was added, the RB remained lower (61?±?12% of S+, p?=?.03) and PON2 higher (0.580?±?0.030?U/mg protein, p?=?.003).

Conclusions: The decrease in monocyte/macrophage PON2 enzymatic activity observed in type 2 diabetes cannot be totally explained by abdominal obesity and insulin resistance. The underlying molecular mechanisms need to be identified.     

A three‐phase excess post‐exercise oxygen consumption in Atlantic salmon Salmo salar and its response to exercise training

The recovery of oxygen uptake to the standard metabolic rate (SMR) following exhaustive chasing exercise in Atlantic salmon Salmo salar parr occurred in three phases (rapid, plateau and slow). The initial recovery phase lasted 0·7 h and contributed 16% to the total excess post‐exercise oxygen consumption (EPOC). It was followed by a longer plateau phase that contributed 53% to the total EPOC. The slow recovery phase that completed recovery of SMR, which has not been reported previously, made a 31% contribution to the total EPOC. The plasticity of EPOC was demonstrated in exercise‐trained fish. Exercise training increased EPOC by 39% when compared with control fish (mean ± S.E., 877·7 ± 73·1 v . 629·2 ± 53·4 mg O₂ kg^?1, d.f. = 9, P <  0·05), with the duration of the plateau phase increasing by 38% (4·7 ± 0·58 v . 3·4 ± 0·16 h, d.f. = 9, P <  0·05) and the contribution of the slow phase to the total EPOC increasing by 80% (173·9 ± 23·9 v . 312·5 ± 50·4 mg O₂ kg^?1, d.f. = 9, P  < 0·05). As a result, the combination of the plateau and slow phases of exercise‐trained fish increased by 47% compared with control fish (756·6 ± 71·4 v . 513·6 ± 43·1 mg O₂ kg^?1; d.f. = 9, P  = 0·01). To substantiate the hypothesis that the plateau and slow recovery phase of EPOC was related to general metabolic recovery following exhaustive exercise, the time‐course for recovery of SMR was compared with previously published metabolite recovery profiles. The final phase of metabolic recovery was temporally associated with the final phases of gluconeogenesis, lactate oxidation and muscle intracellular pH regulation. Therefore, the plasticity of the latter phase of EPOC agreed with the known effects of exercise training in fishes.     

Comparison of two co-culture systems to assess the survival of in vitro produced bovine blastocysts after vitrification

The ability of bovine blastocysts to recover after cryopreservation and thawing procedures is often assessed by evaluating their re-expansion during in vitro co-culture. However, the influence of factors such as feeder cell type and gas atmosphere on blastocyst survival and evolution have never been considered. This study therefore compared two cell co-culture systems and two different gas atmospheres to assess survival of in vitro produced bovine blastocysts after vitrification. Day-7 blastocysts (n=181) were vitrified in a mixture of 25% glycerol/25% ethylene glycol. After warming and dilution, they were co-cultured either on Buffalo rat liver cells (BRL CC cell line) or on granulosa cells (GR CC primary culture) in TCM 199 supplemented with 10% FCS and under an atmosphere of 5% or 20% O₂. Surviving and hatching rates were recorded at 24 h intervals for 3 days. After 72 h of culture, surviving blastocysts were treated for differential counting of inner cell mass (ICM) and trophectoderm cells. Blastocyst survival rates were higher when BRL and granulosa co-culture were performed under 20% oxygen as compared to 5% oxygen (20% O₂: 62% vs. 5% O₂: 25%, P<0.0001). However, the quality of blastocysts surviving in the granulosa co-culture condition was lower under 20% O₂ than under 5% O₂ as indicated by lower total and trophectoderm cell numbers (respectively 79±6 and 56±6 at 20% O₂ vs. 100±10 and 74±10 at 5% O₂, P<0.05), by an altered ICM/trophectoderm ratio (20% O₂: 28% vs. 5% O₂: 23%, P<0.05), by a higher total nuclear fragmentation (20% O₂: 3.7% vs. 5% O₂: 1.5%, P<0.05) and a trend to decreased hatching (20% O₂: 32% vs. 5% O₂: 81%, P=0.07). Whereas, for BRL co-culture, 20% O₂ yielded higher quality blastocysts than 5% O₂ as evaluated by higher ICM and trophectoderm cell numbers (19±1 and 71±5 at 20% O₂ vs. 15±2 and 48±9 at 5% O₂, respectively, P<0.05), by lower nuclear fragmentation in the ICM (20% O₂: 2.2% vs. 5% O₂: 6.7%, P<0.05). In conclusion, co-culture conditions may influence blastocysts survival and quality after cryopreservation. In our conditions, co-culture with BRL cells under 20% O₂ seems to be the best combination to evaluate blastocyst survival and quality after vitrification.     

Inactivation of human pathogens and spoilage bacteria on the surface and internalized within fresh produce by using a combination of ultraviolet light and hydrogen peroxide

Aims: To evaluate the efficacy of ultraviolet (UV) light (254 nm) combined with hydrogen peroxide (H₂O₂) to inactivate bacteria on and within fresh produce. Methods and Results: The produce was steep inoculated in bacterial cell suspension followed by vacuum infiltration. The inoculated samples were sprayed with H₂O₂ under constant UV illumination. The log count reduction (LCR) of Salmonella on and within lettuce was dependent on the H₂O₂ concentration, temperature and treatment time with UV intensity being less significant. By using the optimized parameters (1·5% H₂O₂ at 50°C, UV dose of 37·8 mJ cm^?2), the surface Salmonella were reduced by 4·12 ± 0·45 and internal counts by 2·84 ± 0·34 log CFU, which was significantly higher compared with H₂O₂ or UV alone. Higher LCR of Escherichia coli O157:H7, Pectobacterium carotovora, Pseudomonas fluorescens and Salmonella were achieved on leafy vegetables compared with produce, such as cauliflower. In all cases, the surface LCR were significantly higher compared with the samples treated with 200 ppm hypochlorite. UV–H₂O₂‐treated lettuce did not develop brown discolouration during storage but growth of residual survivors occurred with samples held at 25°C. Conclusions: UV–H₂O₂ reduce the bacterial populations on and within fresh produce without affecting the shelf‐life stability. Significance of the Study: UV–H₂O₂ represent an alternative to hypochlorite washes to decontaminate fresh produce.     

Temperature response of carbon isotope discrimination and mesophyll conductance in tobacco

The partial pressure of CO₂ at the sites of carboxylation within chloroplasts depends on the conductance to CO₂ diffusion from intercellular airspace to the sites of carboxylation, termed mesophyll conductance (g_m). We investigated the temperature response of g_m in tobacco (Nicotiana tabacum) by combining gas exchange in high light, ambient CO₂ in either 2 or 21% O₂ with carbon isotope measurements using tuneable diode laser spectroscopy. The g_m increased linearly with temperature in 2 or 21% O₂. In 21% O₂, isotope discrimination associated with g_m decreased from 5.0 ± 0.2 to 1.8 ± 0.2‰ as temperature increased from 15 to 40 °C, but the photorespiratory contribution to the isotopic signal is significant. While the fractionation factor for photorespiration (f = 16.2 ± 0.7‰) was independent of temperature between 20 and 35 °C, discrimination associated with photorespiration increased from 1.1 ± 0.01 to 2.7 ± 0.02‰ from 15 to 40 °C. Other mitochondrial respiration contributed around 0.2 ± 0.03‰. The drawdown in CO₂ partial pressure from ambient air to intercellular airspaces was nearly independent of leaf temperature. By contrast, the increase in g_m with increasing leaf temperature resulted in the drawdown in CO₂ partial pressure between intercellular airspaces and the sites of carboxylation decreasing substantially at high temperature.     

Cytotoxic effect of adriamycin and agarose-coupled adriamycin on glomerular epithelial cells: Role of free radicals

Summary It has been suggested that the generation of toxic radicals plays an important role in toxicity by Adriamycin (ADR) on cancer cell lines and in vivo. We have examined the role of free radicals in determining toxicity and resistance to ADR of rat glomerular epithelial cells in culture; this method provides a good model for analyzing the mechanisms responsible for ADR experimental nephrosis in rats. Three points were established: a) the intra- or extracellular site of ADR toxicity; b) the role of the superoxide anion and of the hydroxyl radical in determining intra- and-extracellular cytotoxicity; and c) the implication of oxido-reduction cycling as a potential route for ADR semiquinone transformation. Free ADR was found to induce the same inhibition of [³H]thymidine incorporation into DNA as ADR bound to an agarose macroporous bed which prevents the intracellular incorporation of the drug. Specific scavenging of free radical activity by the enzymes catalase and superoxide dismutase, the hydroxyl radical inhibitors dimethyl sulfoxide and dimethylthiourea (DMTU) and by chelation of intracellular free iron with deferoxamine produced only a partial restoration of [³H]thymidine incorporation into DNA, which was maximal for DMTU (30% of normal incorporation). DMTU treatment was unsuccessful in preventing the extracellular cytostatic effect of ADR. Finally, glomerular epithelial cell killing (⁵¹Cr-release method) by 5-iminodaunorubicin, an ADR analogue with a modified quinone function that prohibits oxido-reduction cycling, was higher than unmodified ADR. These results indicate that ADR may exert its cytotoxic effects on glomerular epithelial cells by interaction at the cell surface, whereas the intracellular compartment, principally DNA, does not seem to be the target of ADR effects. They also suggest that the free radicals are in part responsible for ADR intracellular cytotoxicity, but other mechanisms should also be hypothesized. Finally, the participation of the ADR semiquinone radical in oxido-reduction cycling seems not important for the induction of the cellular damage.     

Prostaglandin Endoperoxide H Synthase-2 (PGHS-2) Variants and Risk of Obesity and Microvascular Dysfunction Among Tunisians: Relevance of rs5277 (306G/C) and rs5275 (8473T/C) Genetic Markers

The purpose of this study was to determine the impact of six PGHS-2 genetic variants on obesity development and microvascular dysfunction. The study included 305 Tunisian subjects (186 normal weights, 35 overweights and 84 obeses). PCR analyses were used for allelic discrimination between polymorphisms. Prostaglandin (PGE2, PGI2), leptin, and matrix metalloproteinase (MMP1, 2, 3, 9) levels were evaluated by ELISA. Fatty acid composition was performed by gas chromatography–mass spectrometry. Our results revealed that subjects carrying the PGHS-2 306CC (rs5277) and 8473CC (rs5275) genotypes present higher anthropometric values compared to wild-type genotypes (306GG, BMI (Kg/m²): 27.11?±?0.58; WC (cm): 93.09?±?1.58; 306CC, BMI: 33.83?±?2.46; WC: 109.93?±?5.41; 8473TT, BMI: 27.75?±?0.68; WC: 93.96?±?1.75; 8473CC, BMI: 33.72?±?2.2; WC: 117.89?±?2.94). A reduced microvascular reactivity and a higher PGE2 level were also found in individuals with the 306CC and 8473CC genotypes in comparison to 306GG and 8473TT carriers (306GG, Peak Ach-CVC (PU/mmHg): 0.46?±?0.03; PGE2 (pg/ml): 7933.1?±?702; 306CC, Peak Ach-CVC: 0.24?±?0.01; PGE2: 13,380.3?±?966.2; 8473TT, Peak Ach-CVC: 0.48?±?0.05; PGE2: 7086.41?±?700.31; 8473CC, Peak Ach-CVC: 0.23?±?0.01; PGE2: 13,175.7?±?1165.8). Fatty acid analysis showed a significant increase of palmitic acid (PA) (34.2?±?2.09 vs. 16.82%?±?1.76, P?<?0.001), stearic acid (SA) (25.76?±?3.29 vs. 9.05%?±?2.53, P?<?0.001), and linoleic acid (LA) (5.25?±?1.18 vs. 0.5%?±?0.09, P?<?0.001) levels in individuals carrying the PGHS-2 306CC genotype when compared to GG genotype individuals. Subjects with the 8473CC genotype showed also a significant increase of PA, SA ,and LA levels when compared to TT genotype carriers (PA: 38.02?±?1.51 vs. 12.65%?±?1.54, P?<?0.001; SA: 32.96?±?1.87 vs. 1.38%?±?0.56, P?<?0.001; LA: 26.84?±?2.09 vs. 3.7%?±?1.54, P?<?0.001). Logistic regression analysis revealed that PGHS-2 306CC and 8473CC variants are significantly associated with obesity status (OR 6.25, CI (1.8–21.6), P?=?0.004; OR 3.01, CI (1.13–8.52), P?=?0.03, respectively). Haplotypes containing the C₃₀₆:T₈₄₇₃ (OR 2.91; P?=?0.01) and G_306:C₈₄₇₃ (OR 5.25; P?=?0.002) combinations were associated with an enhanced risk for obesity development in the studied population. In conclusion, our results highlight that PGHS-2 306G/C and 8473T/C variants could be useful indicators of obesity development, inflammation, and microvascular dysfunction among Tunisians.

     

Nitric oxide protection against adriamycin-induced tubulointerstitial injury

It is well known that oxidative stress is related to the pathogenesis of adriamycin (ADR) nephropathy. However, it is unclear how nitric oxide (NO) is associated with the pathophysiological process after ADR administration. The NO level in a kidney homogenate was assayed by electron paramagnetic resonance (EPR) spectrometry using a direct in vivo NO trapping technique after ADR administration. N-(3-(aminomethyl)benzyl)acetamidine (1400W) was used as a specific, inducible nitric oxide synthase (iNOS) inhibitor. The levels of NO after ADR administration gradually increased for 6 h and then decreased until 24 h after ADR administration. The fractional excretion of Na (FE_Na) in the urine was elevated in the ADR group on day 1. Pre-treatment of the animals with 1400W attenuated the increase in NO levels despite further elevation of FE_Na. These findings suggest that iNOS-derived NO does not produce a harmful effect but rather protects the ADR-treated kidney against sodium excretion.     

Prothrombin and fibrinogen carbonylation: How that can affect the blood clotting

Objectives: The aim of the work was the development of a simple method for measuring the plasma prothrombin carbonylation and the study the impact of prothrombin and fibrinogen oxidation on the rate of plasma clotting.

Methods: A new method was based on the ability of prothrombin to be adsorbed by the barium sulfate. It consists of four steps: prothrombin mixing with the water suspension of BaSO₄; reaction of 2,4-dinitrophenylhydrazine with the BaSO₄-bound prothrombin; desorption of prothrombin-2,4-dinitrophenylhydrazone complex from BaSO₄ in an alkaline medium; neutralization and reading of the optical absorbance of the complex (λ?=?370?nm). The prothrombin/fibrinogen carbonylation and plasma clotting rate in vitro in the presence of reactive oxygen species (ROS)-generating agents (0.05–0.8?mM Fe²⁺/H₂O₂) were monitored.

Results: The plasma volume required for measurement of carbonylated prothrombin was 0.4?ml. High level of linearity and reproducibility was observed (r?=?0.9995, P?=?0.0005 – for the protein; r?=?0.9971, P?=?0.0029 – for carbonyls). In the intact rats, the concentration of blood plasma prothrombin was 0.355?±?0.009?mg/ml, and that of carbonyls was 4.94?±?0.09?nmol/mg.

Discussion: Prothrombin and plasma clotting rate was not affected by low concentrations of ROS (0.05–0.2?mM Fe²⁺/H₂O₂). The fibrinogen was susceptible to ROS-related effect over all the used range of concentration (0.05–0.8?mM Fe²⁺/H₂O₂). Carbonylation of fibrinogen did not affect the plasma clotting activity at low ROS concentration (0.05–0.2?mM Fe²⁺/H₂O₂), however it retarded the clotting at higher ROS (0.2–0.8?mM Fe²⁺/H₂O₂).