Lethal Effect of Neutral Mannan Fraction of Bakers' Yeast in Mice

A simple polysaccharide, the neutral mannan from Saccharomyces cerevisiae wild type strain (WNM) was found to kill ddY strain mice by intravenous administration, showing a LD₅₀ value of 12.2 mg/kg. On the other hand, the acidic mannan fraction from the same yeast containing phosphate (WAM025), and chemically phosphorylated WNM (WNM-P) were practically non-toxic. Concerning the relationship between chemical structure and lethal effect of these mannans, it was demonstrated that a mannan possessing a highly branched structure exhibited stronger lethality than those with less branched structures. Against C3H/HeJ strain mice with no responsiveness to lipopolysaccharide, the LD₅₀ value of WNM was as high as 75 mg/kg. Pretreatment with 500 mg/kg of D-mannose, N-acetyl-D-glucosamine, D-galactose, and L-fucose prevented mice from the lethal effects of WNM. However, WNM (LD₁₀₀) did not show any lethal effect in mice for 2 to 12 hr after treatment with dexamethasone, an anti-inflammatory steroid.     

Comparison of the effectiveness of 2,3-dimercaptopropanol (BAL) andmeso-2,3-dimercaptosuccinic acid (DMSA) as protective agents against mercuric chloride-induced nephrotoxicity in rats

The effectiveness of 2,3-dimercaptopropanol (BAL) andmeso-2,3-dimercaptosuccinic acid (DMSA) on HgCl₂-induced nephrotoxicity was studied in the rat. Seven groups of adult male rats were given a single sc toxic dose of HgCl₂ (0.68 mg/kg) followed by 0.9% saline (positive control group), BAL (15, 30, and 60 mg/kg) or DMSA (50, 100, and 200 mg/kg) administered ip at 0, 24, 48, and 72 h thereafter. Although the renal function of HgCl₂-exposed rats was slightly improved after BAL administration, Hg concentrations in the kidney were only reduced at 60 mg/kg. In addition, the protective effect of BAL was not dose-related. In contrast to BAL, DMSA was effective in increasing the urinary excretion of Hg and in reducing the renal Hg content. These results show that DMSA would be more effective than BAL in preventing or in protecting against inorganic Hg-induced nephrotoxicity.     

FACTORS INFLUENCING THE TOXICITY AND ANIMAL SUSCEPTIBILITY OF ANABAENA FLOS-AQUAE (CYANOPHYTA) BLOOMS1

Biological factors have been found which can cause variable toxicity of colony and clonal isolates of Anabaena flos-aquae (Lyngb.) de Bréb. when cultured in the laboratory. These factors help to explain some of the variable toxicity of and animal susceptibility to A. flos-aquae blooms in nature. Two bacteria in the Enterobacteriaceae isolated from a toxic waterbloom, depressed toxin production in selected bacteria-free toxic clones of A. flos-aquae. These toxin-depressing bacteria decreased culture toxicity 3-fold from 80 to 240 mg/kg (intra-peritoneal in male mice). Many colony isolates from a toxic bloom had minimum lethal dosages (LD_min) greater than 240 mg/kg. This was because they were composed of mixtures of toxic and nontoxic filaments. The oral LD_min of the toxin from A. flos-aquae clonal isolate NRC-44-1 varied significantly for 6 different animal species. Using these oral LD_min it is estimated that a surface-concentrated bloom of toxic A. flos-aquae, having a biomass density of 20 mg/ml dry weight, would cause death of ducks or calves when 20 ml/kg was consumed whereas a monogastric animal such as a rat would require 80 ml/kg.     

Mechanism of enhanced bioavailability and diuretic effect of azosemide by ascorbic acid in rats

To increase the extent of comparative oral bioavailability (F) value and the diuretic and natriuretic effects of orally administered azosemide, ascorbic acid was coadministered to rats. The rationales for this study are that ascorbic acid might inhibit intestinal first-pass effect of azosemide and might increase the unionized fraction of azosemide at the receptor sites. After oral administration of azosemide (20 mg/kg) with 100 mg of ascorbic acid, the F value (138% vs. 100%), 8-h urinary excretion of azosemide (5.18% vs. 1.32% of oral dose), 8-h urine output (41.3 vs. 23.0 ml), and 8-h urinary excretion of sodium (24.6 vs. 15.3 mmol/kg) were greater than controls (without ascorbic acid). The amount of spiked azosemide remaining after 30 min incubation of 50 mug of azosemide with the 9000 g supernatant fraction of rat small intestine was significantly greater by 100 microg of ascorbic acid (45.3 vs. 40.9 microg) than controls (without ascorbic acid). After oral administration of azosemide with NH4Cl, the urine pH decreased by 0.5 U, and 8-h urine output (25.8 vs. 11.0 ml) and 8-h urinary excretion of sodium (13.3 vs. 6.89 mmol/kg) were significantly greater than controls (without NH4Cl). The increase in F value and diuretic and natriuretic effects of azosemide with coadministration of ascorbic acid seemed to be due to reduced intestinal first-pass metabolism of azosemide, increased urinary excretion of azosemide, and increased unionized fraction of azosemide at the renal tubular receptor sites.     

Fatty Acid Metabolism in Microorganisms

During the investigation on the metabolism of azelaic acid by Micrococcus sp., it was found that the bacterium produced a large amount of keto acid (α-ketoglutaric acid) under the restricted condition for nitrogen source. The acid was identified as α-ketoglutaric acid by physico-chemical and biological methods. The mechanism of the production of α-ketoglutaric acid from azelaic acid was investigated. From the result, it was suggested that α-ketoglutaric acid production proceeded thrpugh the further oxidation of acetic acid produced from azelaic acid and that the production might be functioned by TCA cycle enzymes of the bacterium. Similarly, α-ketoglutaric acid was found to be produced remarkably from other various fatty acids.     

Inhibition of prostaglandin synthesis in man

7α-Hydroxy-5,11-diketotetranor-prostane-1,16-dioic acid, the major urinary metabolite of prostaglandins E₁ and E₂ in man, was determined in human urine by a method based on the use of the bis (O-²H₃-methyloxime) derivative of dimethyl 7α-hydroxy-5,11-diketotetranor-prostane-1,16-dioate as internal standard and determination of the ratio between unlabeled and deuterium-labeled molecules by multiple-ion analysis. Male subjects excreted larger amounts of the metabolite (6.5–46.7 μg/24 hours, n=10) than did female subjects (2.5–5.3 μg/24 hours, n=10). The excretion rate was strongly suppressed following oral administration of therapeutic doses of indomethacin, aspirin and sodium salicylate.     

Genetic Effects Induced by Nickel Sulfate in Germline and Somatic Cells of WR Mice

We examined the effects of nickel sulfate at doses 0.5 to 5.0 mg/kg (1/200–1/20 LD₅₀) on the frequency of dominant lethal mutations and double-strand DNA breaks (DSBs) in germline cells and on an increase in frequency in gene mutations W ^y in pigment cells of first-generation mice. The results indicated that spermatogenesis stages most sensitive to nickel sulfate (at a dose of 1.0 mg/kg) are spermatozoids, early spermatids, late spermatocytes, and stem spermatogonia. No statistically significant increase in the total TSB level was detected in spermatozoids 4 weeks after exposure. At the same time, a significant (P < 0.05) increase in percentage of cells with an extremely high level of DNA fragmentation (supposedly apoptotic cells) was observed upon exposure at a dose of 0.5 mg/kg. Nickel sulfate at doses of 5.0 and 1.0 mg/kg induced a marked increase in the c-kit gene expression in pigment cells of heterozygous first-generation WR mice as compared to control (P < 0.001). It was shown that the nonobservable adverse effect level (NOAEL) of nickel sulfate on the dominant lethal mutation frequency and gene mutations was 1/200 LD₅₀, while the lowest observable adverse effect level (LOAEL) was 1/100 LD₅₀.__________Translated from Genetika, Vol. 41, No. 7, 2005, pp. 894–901.Original Russian Text Copyright © 2005 by Domshlak, Elakov, Osipov.     

Biliary, fecal and uninary excretion of [H]-prostaglandins in the rat

The profiles of biliary, fecal and urinary excretion of tritium labeled prostaglandins (PG's) of differing biological activity were investigated in the rat. The PG's (10 μg/kg: 2 to 50 μCi/rat, in 1 ml polyethylene glycol-400) were administered intragastrically. Excretion data were expressed as a percentage of the total administered radioactivity. for the orally administered PG's 11R-methyl-16R-fluoro-15R-hydroxy-9-oxoprosta- -5- -13-dienoic acid and its methyl ester, excretion was equally divided between urine and feces. The fecal and urinary profile of excretion of ³H after prostacyclin (PGl₂) was similar to that following administration of 11R, 16, 16-trimethyl-15R-hydroxy-9-oxoprosta- -5- -13-dienoic acid (trimoprostil), a PG with antisecretory-antiulcer potential. However, PGl₂ was very poorly absorbed from the intestine, while the absorption of trimoprostil was very efficient. Biliary excretion, with little entero-porto-hepatic biliary circulation, was the main route of elimination of trimoprostil, thereby resulting in rapid elimination of drug-related products and diminishing the potential for systemic liability in the rat.     

Effect of triethylenepentaminehexaacetic acid on the renal damage in cadmium-treated Syrian hamsters

Cadmium (Cd)-induced nephropathy was treated by triethylene-pentaminehexaacetic acid (TTHA) in male Syrian hamsters. Hamsters injected three times a week with 3 mg/kg body wt CdCl₂ showed proteinuria, urinaryN-acetyl-β-d-inglucosaminidase (NAG), and fractional excretion of sodium (FENa) when compared to saline-injected control. Cd-treated hamsters injected ip with TTHA 10 mg/kg body wt five times a week showed reduction of renal damage, including reductions in urinary protein (from 6.7±2.2 to 4.3±0.5 mg/d) and NAG (0.17±0.06 to 0.04±0.02 U/d). Urinary excretion of Cd was significantly increased (from 87±51.3 to 3052±1485 mg/L) by TTHA administration. Cd concentration in renal cortical tissue was slightly reduced (26.4±3.0 to 21.8±2.7 mg/g. protein). Excretion of malondialdehyde (MDA) was increased only in Cd-injected hamsters (to 2.1±1.6 nM/L), and elevated MDA in renal cortical tissue was not reduced by the administration of TTHA (1041±105 vs 1104±358 nM/g protein). Glutathione (GSH) concentration in the renal cortex was significantly elevated after Cd administration and further increased after TTHA administration (5.5±2.1 to 9.8±2.0 μg/50 mg protein). There were no marked effects on creatinine clearance (Ccr) and hematocrit. Moreover, renal morphological changes were improved significantly by treatment with TTHA. We demonstrated the efficacy of TTHA in the treatment of Cd-induced nephropathy in hamsters. Although the precise mechanism of the TTHA effects on Cd-induced nephropathy has not been elucidated, it might involve GSH reducing the elevated MDA concentration in renal tissue.     

Inhibition of δ-aminolevulinic acid synthetase and δ-aminolevulinic acid dehydrase by L-2-amino-4-methoxy-trans-3-butenoic acid in the rat

The rate limiting enzyme of heme biosynthesis, δ-aminolevulinic acid synthetase (ALA synthetase), and the second enzyme in the heme biosynthetic pathway, δ-aminolevulinic acid dehydrase (ALA dehydrase), were inhibited by the olefinic amino acid L-2-amino-4-methoxy - $\text{trans}$-3-butenoic acid (AMTB). Administration of AMTB (20 mg/kg; i.p.) to rats inhibited ALA synthetase and ALA dehydrase in control animals and in animals with markedly elevated activity of ALA synthetase which resulted from the administration of 3,5-dicarbethoxy-1,4-dimethyl-collidine (DDC, 200 mg/kg, i.p.) or allylisopropylacetamide (200 mg/kg, s.c.). AMTB also blocked the synthesis of rat hepatic porphyrins and inhibited the increase in the urinary excretion of δ-aminolevulinic acid and porphobilinogen following DDC (150 mg/kg, p.o.) administration. Preincubation of AMTB with liver mitochondria or a soluble fraction of liver decreased the activity of mitochondrial ALA synthetase and soluble ALA dehydrase, respectively.     

A comparative study of the effects of nicotinamide and diethylnicotinamid on hepatic monooxygenase, glucuronyl and glutathione conjugating systems

The nicotinamide administration to rats (50 mg/kg, subcutaneously, over 5 days) increased the concentration of liver cytochrome b5, the activities of cytosol and microsomal glutathione S-transferase, UDP-glucuronosyltransferase and urinary excretion of bound glucuronic acid by 26.7, 33.1, 33.3, 53.0 and 31.0%, respectively. The chloral hydrate-induced sleep time in mice was reduced by 65%. Under similar experimental conditions the administration of equimolar amounts of diethylamide of nicotinic acid (75 mg/kg) exerted a more pronounced enzyme-stimulating effect. The cytochrome P-450 concentration, the activities of cytosol and microsomal glutathione S-transferase, UDP-glucuronosyltransferase as well as the sulphobromophthalein elimination from blood plasma and urinary excretion of bound glucuronic acid were increased by 37.0, 33.1, 54.6, 80.5, 24.5 and 49.0%, whereas the chloral hydrate-induced sleep time decreased by 75%. The nicotinamide and diethylamide of nicotinic acid stimulating effects on xenobiotic biotransformation in rat liver are assumed to be due to enhanced NADPH, glutathione and UDP-glucuronic acid biosynthesis as well as their antioxidant properties.     

Amino acid sequence of piratoxin-I, a myotoxin fromBothrops pirajai snake venom, and its biological activity after alkylation withp-bromophenacyl bromide

The complete sequence of the 121 amino acid residues of piratoxin-I (PrTX-I), a phospholipase A₂ (PLA₂)-like myotoxin fromBothrops pirajai snake (Bahia jararacussu) venom, is reported. From the sequence, anM _r of 13,825 and an approximatepI of 8.3 were calculated. PrTX-I shows a high sequence homology with Lys-49 myotoxins from other bothropic (∼95%) and nonbothropic (∼80%) venoms, but only 70–75% homology w hen aligned with the catalytically active Asp-49 PLA₂s. When compared with bothropstoxin-I fromBothrops jararacussu, which is morphologically almost identical toB. pirajai, only two changes out of 121 total amino acid residues have been observed. The approximate minimal lethal doseLD ₅₀ (mice, i.p., 24 hr) of PrTX-I was 8 (6.8–9.1) mg/kg, and the minimal edematogenic dose (MED) in a rat paw model was 39.5±1.8 ug. After alkylation of His-48 withp-bromophenacyl bromide, the MED was 40.1±1.9 ug, but up to 4LD ₅₀ were unable to cause death in any of a group of eight mice after 72 hr. Therefore the edematogenic activity was retained and apparently did not involve His-48, suggesting that at least two biologically active sites are present in PrTX-I.     

Amino acid sequence of piratoxin-I, a myotoxin fromBothrops pirajai snake venom, and its biological activity after alkylation withp-bromophenacyl bromide

The complete sequence of the 121 amino acid residues of piratoxin-I (PrTX-I), a phospholipase A₂ (PLA₂)-like myotoxin fromBothrops pirajai snake (Bahia jararacussu) venom, is reported. From the sequence, anM _r of 13,825 and an approximatepI of 8.3 were calculated. PrTX-I shows a high sequence homology with Lys-49 myotoxins from other bothropic (∼95%) and nonbothropic (∼80%) venoms, but only 70–75% homology w hen aligned with the catalytically active Asp-49 PLA₂s. When compared with bothropstoxin-I fromBothrops jararacussu, which is morphologically almost identical toB. pirajai, only two changes out of 121 total amino acid residues have been observed. The approximate minimal lethal doseLD ₅₀ (mice, i.p., 24 hr) of PrTX-I was 8 (6.8–9.1) mg/kg, and the minimal edematogenic dose (MED) in a rat paw model was 39.5±1.8 ug. After alkylation of His-48 withp-bromophenacyl bromide, the MED was 40.1±1.9 ug, but up to 4LD ₅₀ were unable to cause death in any of a group of eight mice after 72 hr. Therefore the edematogenic activity was retained and apparently did not involve His-48, suggesting that at least two biologically active sites are present in PrTX-I.     

Primary poisoning risk for encapsulated sodium nitrite,a new tool for pest control

ABSTRACT

Acute toxicity of sodium nitrite (NaNO₂) was assessed in chickens (Gallus gallus domesticus) and domestic mallard ducks (Anas platyrhynchos domestica) by oral gavage and in free-feeding trials with chickens, domestic mallard ducks, pigeons (Columba livia f. domestica), budgerigars (Melopsittacus undulates) and wētā (Family: Rhaphidophoridae). Free-feeding trials involved the presentation of toxic paste and pellet baits containing encapsulated NaNO₂ developed for the control of common brushtail possums (Trichosurus vulpecula) and feral pigs (Sus scrofa). The oral gavage LD₅₀ value for NaNO₂ in solution was approximately 68.50?mg/kg (95% CI 55.00–80.00?mg/kg) for both chickens and ducks. In feeding trials, six out of 12 chickens consumed toxic paste bait and four of these birds consumed a lethal dose. When chickens consumed toxic paste bait, the LD₅₀ value was approximately 254.6?mg/kg (95% CI 249.1–260.2?mg/kg). Of the other three species of birds presented with toxic baits only one duck consumed a lethal dose of paste bait. There was no evidence of wētā feeding on toxic baits.     

Isolation and Characterization of Antitumor Lipopolysaccharide from Proteus mirabilis

Systematic isolation of the cell constituents of Proteus mirabilis RMS–203 was performed to find out localization of antitumor principle only in the lipopolysaccharide (LPS) layer of the cell wall fraction.

LPS with strong antitumor activity was extracted from P. mirabilis RMS–203 by phenol-water method followed by purification on DEAE-Sephadex A–50 column chromatography.

The main components of purified LPS were galactose, hexosamine, 2-keto-deoxy-octonic acid (KDO), myristic acid, β-hydroxymyristic acid and α,ε-diaminopimelic acid.

The minimal effective dose of LPS against Ehrlich solid carcinoma in mice was 0.1~1.0 μg/mouse. LD₅₀ in mice and pyrogenicity in rabbits were 28 mg/kg and 10^?3–10^?5 μg/rabbit, respectively.     

Involvement of heparan sulfate in the renoprotective effects of imidapril,an angiotensin-converting enzyme inhibitor,in diabetic db/db mice

Abstract

We investigated the renoprotective effects of imidapril hydrochloride ((-)-(4?S)-3-[(2?S)-2-[[(1?S)-1-ethoxycarbonyl-3-phenylpropyl] amino] propionyl]-1-methyl-2-oxoimidazolidine-4-carboxylic acid hydrochloride, imidapril), an angiotensin-converting enzyme inhibitor, in a diabetic animal model. We used BKS.Cg-+Lepr^db/+Lepr^db (db/db) mice, a genetic animal model of obese type 2 diabetes. Diabetic db/db mice suffered from glomerular hyperfiltration, albuminuria and hypoalbuminemia. Oral administration of 5?mg/kg/day of imidapril for 3 weeks suppressed renal hyperfiltration, reduced albuminuria and normalized hypoalbuminemia. Imidapril did not influence body weights, blood pressure or blood glucose concentrations in db/db mice. Urinary excretion of heparan sulfate (HS) in non-treated 11-week-old db/db mice was significantly lower than that in age-matched non-diabetic db/+m mice. HS is a component of HS proteoglycans, which are present in glomerular basement membranes and glycocalyx of cell surfaces. Reduced urinary HS excretion indicated glomerular HS loss in db/db mice. Imidapril increased urinary excretion of HS to concentrations observed in db/+m mice, indicating that imidapril prevented the loss of renal HS. These results suggest that imidapril ameliorates renal hyperfiltration and loss of renal contents of HS. Improvement of filtration function and maintenance of HS, which is an important structural component of glomeruli, may contribute to renoprotective effects of imidapril.     

Acute toxicity of long-circulating and pH-sensitive liposomes containing cisplatin in mice after intraperitoneal administration

AimsThe objective of this work was to evaluate the acute toxicity of long-circulating and pH-sensitive liposomes containing cisplatin (SpHL-CDDP), after their intraperitoneal administration in male and female mice.Main methodsAfter single administration of free CDDP (5,10,and 20 mg/kg) or SpHL-CDDP (7,12,30,45 and 80 mg/kg), the body weight was recorded and the LD₅₀ was calculated. Blood samples were collected for biochemical and hematological analysis. Kidneys, liver, spleen and bone marrow were removed to histopathological examination.Key findingsMice treated with high doses of free CDDP showed a greater loss of body weight and more delayed recovery time than those treated with SpHL-CDDP. The LD₅₀ values for SpHL-CDDP treatment for male and female mice groups were 2.7 and 3.2 fold higher, respectively, than that obtained for free CDDP. The red and white blood cells counts and quantification of hemoglobin and hematocrit presented no change upon administration of SpHL-CDDP treatment. Free CDDP treatment, however, did lead to an appearance of mild anemia and a reduction in total white blood cell counts. As regards nephrotoxicity, it was observed that free CDDP treatment caused pronounced alterations in the blood urea and creatinine levels of mice. In contrast, these parameters were slightly altered only after SpHL-CDDP treatment at a dose of 30 mg/kg. Microscopic analysis of kidneys from mice treated with SpHL-CDDP showed no morphological alteration. Concerning hepatotoxicity, no histopathological alteration was observed after both treatments.SignificanceThese findings reveal that SpHL-CDDP can eliminate CDDP-induced toxicity and is thus a promising candidate for intraperitoneal chemotherapy.     

Quantitative studies on prostaglandin synthesis in man. 3. Excretion of the major urinary metabolite of prostaglandins F 1 alpha and F 2 alpha during pregnancy

The mean urinary excretion of 5α, 7α-dihydroxy-11-ketotetranor-prostane-1,16-dioic acid, the major urinary metabolite of prostaglandins F_1α and F_2α, in eight women in pregnancy weeks 37–40 was 32.5 ± 12.2 μg/24 hours, representing a 2–5 fold increase compared to non-pregnant women. Determination of the metabolite throughout pregnancy in three subjects showed that there was a gradual increase in the urinary excretion as pregnancy progressed with maximum excretion (4–5 times the individual pre-pregnant value) at the end of pregnancy. After pregnancy there was a rapid normalization back to the pre-pregnant excretion value.     

Cadmium-induced excretion of urinary lipid metabolites,DNA damage,glutathione depletion,and hepatic lipid peroxidation in sprague-dawley rats

Recent studies have described lipid peroxidation to be an early and sensitive consequence of cadmium exposure, and free radical scavengers and antioxidants have been reported to attenuate cadmium-induced toxicity. These observations suggest that cadmium produces reactive oxygen species that may mediate many of the untoward effects of cadmium. Therefore, the effects of cadmium (II) chloride on reactive oxygen species production were examined following a single oral exposure (0.50 LD₅₀) by assessing hepatic mitochondrial and microsomal lipid peroxidation, glutathione content in the liver, excretion of urinary lipid metabolites, and the incidence of hepatic nuclear DNA damage. Increases in lipid peroxidation of 4.0- and 4.2-fold occurred in hepatic mitochondria and microsomes, respectively, 48 h after the oral administration of 44 mg cadmium (II) chloride/kg, while a 65% decrease in glutathione content was observed in the liver. The urinary excretion of malondialdehyde (MDA), formaldehyde (FA), acetaldehyde (ACT), and acetone (ACON) were determined at 0–96 h after Cd administration. Between 48 and 72 h posttreatment maximal excretion of the four urinary lipid metabolites was observed with increases of 2.2- to 3.6-fold in cadmium (II) chloride-treated rats. Increases in DNA single-strand breaks of 1.7-fold were observed 48 h after administration of cadmium. These results support the hypothesis that cadmium induces production of reactive oxygen species, which may contribute to the tissue-damaging effects of this metal ion.     

Removal of thallium by combining desferrioxamine and deferiprone chelators in rats

The hypothesis that two known chelators deferiprone (1,2-dimethy1-3-hydroxypyrid-4-one, L₁) and desferrioxamine (DFO) might be more efficient as combined treatment than as monotherapies in removing thallium from the body was tested in rats. Six-week-old male Wistar rats received chelators: L₁ (p.o.), DFO (i.p.) or L₁ + DFO as 110 or 220 mg/kg dose half an hour after a single i.p. administration of 8 mg Tl/kg body weight in the form of chloride. Serum thallium concentration, urinary thallium and iron excretions were determined by graphite furnace atomic absorption spectrometry. Both chelators were effective only at the higher dose level, while DFO was more effective than L₁ in enhancing urinary thallium excretion, L₁ was more effective than DFO in enhancing urinary iron excretion. In the combined treatment group, L₁ did not increase the DFO effect on thallium and DFO did not increase the effect of L₁ on iron elimination. Our results support the usefulness of this animal model for preliminary in vivo testing of thallium chelators. Urinary values were more useful because of the high variability of serum results. Result of combined chelators treatment should be confirmed in a different experimental model before extrapolation to other systems.