Arachidonic acid alleviates the detrimental effects of acetylsalicylic acid on human granulosa cells performance in vitro

Here, we investigated the biological effects of arachidonic acid (AA) in human cumulus granulosa cells (CGCs) after exposure to ASA. Cells were isolated from the follicular fluid and incubated with 0.5 mM acetylsalicylic acid (ASA) and 50 µM AA. Cell viability was analyzed by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay. E₂ and P₄ levels were measured by chemiluminescence assay. Expression of genes including CYP19A1, FACN, and SCD1 was measured by real‐time polymerase chain reaction assay. Oxidative status was analyzed by monitoring glutathione peroxidase activity. The fatty acid profile was analyzed by the gas chromatography technique. Enzyme‐linked immunosorbent assay was used to measure prostaglandin E₂ (PGE₂) in CGCs after exposure to ASA and AA. Protein levels of the estrogen receptor were studied by immunofluorescence staining. Ultrastructural changes were evaluated by transmission electron microscopy imaging. ASA treatment reduced E₂ production, Cyp19a1 expression, glutathione peroxidase (GPx) activity, and estradiol receptor expression in CGCs. The addition of AA prevented the ASA‐induced E₂ reduction (p < .05) and expression of Cyp19a1. Moreover, AA increased the antioxidant capacity of CGCs exposed to ASA by promoting GPx activity (p < .05). AA increased monounsaturated fatty acid/saturated fatty acid ratio compared with the ASA group (p < .05). AA supplementation triggered the synthesis and secretion of PGE₂ in ASA‐treated CGCS (p < .05). Cytoplasmic vacuolation observed in the ASA group and treatment with AA intensified vacuolation rate. The expression of the estrogen receptor was increased after AA supplementation. Data demonstrated that AA decreased the detrimental effects of ASA on human CGCs after 72 hr.     

Storage of potato microplants in vitro in the presence of acetylsalicylic acid

Acetylsalicylic acid was investigated as an alternative medium supplement to mannitol for slow-growth in vitro storage of potato microplants. At 8°C, culture in the presence of either 100 μM acetylsalicylic acid or 4% mannitolretarded microplant stem growth, and required intervals between subcultures ranged from 8 to over 12 months, depending on the genotype. Several media were tested for recovery of clones from slow-growth storage, with similar efficiences of recovery achieved for microplants from either mannitol or acetylsalicylic acid media. Prolonged culture on acetylsalicylic acid had no adverse effects on the yield of minitubers on plantlets transferred to glasshouse cultivation. At 18°C also, intervals between subcultures could be prolonged to 5.5–6 months by culture on either mannitol or acetylsalicylic acid media. Microplants cultured on acetylsalicylic acid did not exhibit the frequent phenotypic abnormalities found in microplants on mannitol medium. This revised version was published online in June 2006 with corrections to the Cover Date.     

Superiority of zinc complex of acetylsalicylic acid to acetylsalicylic acid in preventing postischemic myocardial dysfunction

The pathophysiology of ischemic myocardial injury involves cellular events, reactive oxygen species, and an inflammatory reaction cascade. The zinc complex of acetylsalicylic acid (Zn(ASA)₂) has been found to possess higher anti-inflammatory and lower ulcerogenic activities than acetylsalicylic acid (ASA). Herein, we studied the effects of both ASA and Zn(ASA)₂ against acute myocardial ischemia. Rats were pretreated with ASA (75 mg/kg) or Zn(ASA)₂ (100 mg/kg) orally for five consecutive days. Isoproterenol (85 mg/kg, subcutaneously [s.c.]) was applied to produce myocardial infarction. After 17–22 h, animals were anesthetized with sodium pentobarbital (60 mg/kg, intraperitoneally [i.p.]) and both electrical and mechanical parameters of cardiac function were evaluated in vivo. Myocardial histological and gene expression analyses were performed. In isoproterenol-treated rats, Zn(ASA)₂ treatment normalized significantly impaired left-ventricular contractility index (E_max 2.6 ± 0.7 mmHg/µL vs. 4.6 ± 0.5 mmHg/µL, P < 0.05), increased stroke volume (30 ± 3 µL vs. 50 ± 6 µL, P < 0.05), decreased systemic vascular resistance (7.2 ± 0.7 mmHg/min/mL vs. 4.2 ± 0.5 mmHg/min/mL, P < 0.05) and reduced inflammatory infiltrate into the myocardial tissues. ECG revealed a restoration of elevated ST-segment (0.21 ± 0.03 mV vs. 0.09 ± 0.02 mV, P < 0.05) and prolonged QT-interval (79.2 ± 3.2 ms vs. 69.5 ± 2.5 ms, P < 0.05) by Zn(ASA)₂. ASA treatment did not result in an improvement of these parameters. Additionally, Zn(ASA)₂ significantly increased the mRNA-expression of superoxide dismutase 1 (+73 ± 15%), glutathione peroxidase 4 (+44 ± 12%), and transforming growth factor (TGF)-β₁ (+102 ± 22%). In conclusion, our data demonstrate that oral administration of zinc and ASA in the form of bis(aspirinato)zinc(II) complex is superior to ASA in preventing electrical, mechanical, and histological changes after acute myocardial ischemia. The induction of antioxidant enzymes and the anti-inflammatory cytokine TGF-β₁ may play a pivotal role in the mechanism of action of Zn(ASA)₂.     

Evaluation of clastogenicity of formic acid, acetic acid and lactic acid on cultured mammalian cells

Using Chinese hamster ovary K1 cells, chromosomal aberration tests were carried out with formic acid, acetic acid and lactic acid, and the relationship between the pH of the medium and the clastogenic activity was examined. The medium used was Ham's F12 supplemented with 17 mM NaHCO3 and 10% fetal calf serum. All of these acids induced chromosomal aberrations at the initial pH of ca. 6.0 or below (about 10-14 mM of each acid) both with and without S9 mix. Exposure of cells to about pH 5.7 or below (about 12-16 mM of each acid) was found to be toxic. When the culture medium was first acidified with each of these acids and then neutralized to pH 6.4 or pH 7.2 with NaOH, no clastogenic activity was observed. Using F12 medium supplemented with 34 mM NaHCO3 as a buffer, no clastogenic activity was observed at doses up to 25 mM of these acids (initial pH 5.8-6.0). However, it was found that about 10% of the cells had aberrations at pH 5.7 or below (27.5-32.5 mM of each acid). Furthermore, when 30 mM HEPES was used as a buffer, chromosomal aberrations were not induced at doses up to 20 mM formic acid and acetic acid (initial pH 7.0-7.1), and at doses up to 30 mM lactic acid (initial pH 6.6). In the initial pH range of 6.4-6.7 (25-32.5 mM of each acid), chromosomal aberrations were observed. The above results show that these acids themselves are non-clastogenic, and the pseudo-positive reactions attributable to non-physiological pH could be eliminated by either neutralization of the treatment medium or enhancement of the buffering ability.     

Further investigations on the polypeptides and reconstitution of prasinophycean ejectisomes

Ejectisome fragments were isolated from the prasinophyte Pyramimonas grossii and subjected to different treatments, i.e. Percoll density gradient centrifugation, incubation at pH 2.5 or at pH 10.8, or incubation in 6 M guanidine hydrochloride. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that Percoll density gradient centrifugation did not improve the purity of the ejectisome fragment-enriched fractions. The ejectisome fragments withstood pH 2.5 and pH 10.8 treatment, and no loosely bound polypeptides became detached. The disintegration of ejectisome fragments was achieved in 6 M guanidine hydrochloride, and reassembly into filamentous, ejectisome-like structures occurred after dialysis against distilled water. Fractions enriched either in ejectisome fragments or in reconstituted ejectisome-like structures were dominated by three polypeptides with relative molecular weights of approximately 12.5–19 kDa and two additional polypeptides of 23 and 26 kDa. A polyclonal antiserum directed against an ejectisome fragment-enriched fraction weakly cross-reacted with these polypeptides, and no significant immuno-labelling of ejectisome fragments was registered. A positive immuno-label was achieved using immunoglobulin (IgG) fractions which were gained by selectively incubating nitrocellulose stripes of these polypeptides with the antiserum.     

Metal chelates of 2-hydroxy-4-methylthiobutanoic acid in animal feeding. Part 2: Further characterizations, in vitro and in vivo investigations

The alpha-hydroxyacid 2-hydroxy-4-methylthiobutanoic acid (the so-called methionine hydroxy-analogue, MHA), largely used in animal nutrition as a source of methionine, forms stable metal chelates with divalent metals of formula [{CH(3)SCH(2) CH(2)CH(OH)COO}(2)M].nH(2)O. Protonation and iron(III) and copper(II) complex formation constants have been determined by potentiometry at 25 degrees C. Distribution diagrams show that no free Fe(3+) cations are present in solution at pH>2.5. ESI-MS (Electron-Spray Ionization Mass Spectrometry) investigations carried out both on iron and zinc complexes in solution have evidenced various species with different MHA/metal ratios. In vivo trials were carried out with rats. After receiving a zinc-deficient diet for 3 weeks, animals were fed the same diet added with zinc sulfate or zinc/MHA chelate; the zinc content of faeces was higher (+45%; P<0.05) in sulfate fed rats, whereas zinc retention was higher (+61%; P<0.05) in the Zn/MHA diet. Experiments in vitro with human intestinal Caco-2 cells indicated that the MHA/Fe chelate was taken up by the cells without any apparent toxic effect. The iron uptake was higher than that of iron nitrilotriacetate (Fe(3+)NTA), an effective chelate for delivering iron to milk diets. In conclusion, these data indicate that the use of MHA chelates could be a valuable tool to increase bioavailability of trace minerals and reduce the environmental impact of animal manure.     

Effect of acetylsalicylic acid on prostacyclin production in trophoblast

In attempt to elucidate whether acetylsalicylic acid (ASA) has an in vivo effect on prostacyclin (PGI2)-like activity released from trophoblast we have evaluated PGI2-like activity in pregnant women scheduled for pregnancy termination after ASA ingestion. Following subjects were studied: Group I: 7 healthy pregnant women who were treated with 1.5 g ASA for two days; Group II: 18 control pregnant women who received placebo for two days. Trophoblast specimens were obtained by legal abortions; PGI2-like activity in trophoblast was measured by the method of Moncada. In normal pregnant women (8-10 weeks gestation) treated with ASA the mean PGI2-like activity of trophoblast significantly decreased compared to the controls. These data indicate that treatment with ASA of early pregnant women might have a harmful effect on trophoblast and the problem should be further explored before allowing the administration of cyclooxygenase inhibiting drugs during early pregnancy.