Structure-acute toxicity relationship of dinitriles in mice

Acute toxicity and metabolism of seven dinitriles in mice was studied in relation to the chemical structures. The oral LD₅₀ for each nitrile was determined under different conditions for mice pretreated with either carbon tetrachloride (CCl₄) or olive oil. All test nitriles were metabolized into cyanide in vivo and in vitro. The cyanide level was variable among the compounds (0.35–0.74 g CN/g brain) at death in the brains of mice, the level for malono- and adiponitrile being comparable to that found in mice killed by dosing with potassium cyanide. After receiving each nitrile, the mean survival time of mice pretreated with CCl₄ was prolonged and their brain cyanide level decreased when compared with the corresponding control. With malononitrile, the former did not significantly change and the latter decreased slightly. Brain cyanide levels of control mice at death showed a peak at the lower log P region, while those of CCl₄-pretreated animals remained lower independently of log P, with the exception of malononitrile. Microsomal metabolism of nitriles to cyanide was greatly inhibited when microsomes were prepared from livers of mice pretreated with CCl₄. The relationship between log (1/LD₅₀-CCl₄), LD₅₀ in mice pretreated with CCl₄, and log P fits a parabolic plot.Part of this work was presented at the Symposium QSAR in Toxicology and Xenobiochemistry, Praha, September 12–14, 1984.     

The effect of prior treatment with 4-dimethylaminophenol (DMAP) on animals experimentally poisoned with hydrogen cyanide

Six dogs were given sufficient oral 4-dimethylaminophenol (DMAP) to produce a peak methaemoglobin level of 12–15%. Five out of the six dogs then survived an intravenous injection of approximately 2 LD₅₀'s of hydrogen cyanide given when the methaemoglobin had reached 8–10%. The sixth dog died after 44 min. When the same dose of hydrogen cyanide was given to dogs, not previously given DMAP, all three died within 11/2 min. It was concluded that prior treatment with oral DMAP provided a large measure of protection against cyanide poisoning. Comparison of cyanide levels in whole blood and plasma in the two groups of dogs lent support to the hypothesis that methaemoglobin complexed with cyanide in the erythrocytes causing the plasma cyanide to remain lower than it did in unprotected animals.     

Bioavailability of iron and cyanide from oral potassium ferric hexacyanoferrate(II) in humans

After oral administration of 500 mg KFe[Fe(CN)₆] labelled with⁵⁹Fe either in the ferric or ferrous position and with¹⁴C in the cyanide group only 0.22% of the Fe^II and <0.04% of the Fe^III were absorbed in three male volunteers. Only 2 mg non-complex bound relabelled cyanide (0.03 mg CN-/kg body wt) were absorbed from 500 mg [¹⁴C]KFeHCF, which is about a factor of 20–100 below the lethal dose in humans (0.5–3.5 mg CN-/kg body wt). Therefore, iron(III) hexacyanoferrates(II) can be considered as safe antidotes, i.e. for inhibiting the intestinal absorption of radiocaesium or for accelerating the excretion of already absorbed^134/137Cs in the case of a severe nuclear accident.     

Pharmacokinetics of cyanide in poisoning of dogs,and the effect of 4-dimethylaminophenol or thiosulfate

Cyanide in blood, plasma, and urine of dogs after administration of K¹⁴CN was determined with the isotope dilution technique. The addition of large amounts of inactive KCN as soon as possible to a sample to be analyzed inhibited the decrease of the original cyanide concentration.After administration of several lethal doses of cyanide into the stomach or by slow intravenous infusion a concentration of about 40 M cyanide in plasma was found at the moment of respiratory arrest. Since 60% of the cyanide in plasma was bound to proteins the concentration of free cyanide which stopped respiration was about 16 M.Quick formation of ferrihemoglobin by i.v. injection of 4-dimethylaminophenol after plasma cyanide had risen to or above 40 M decreased the cyanide concentration in plasma and restored respiration, while cyanide was accumulated in red cells by formation of ferrihemoglobin cyanide.Equilibrium constants calculated for the reaction between ferrihemoglobin and cyanide in vivo indicated that the reaction approached equilibrium in a few minutes.Up to 60% of the radioactive cyanide absorbed was found as non-cyanide radioactivity in the urine.Abbreviations Used DMAP 4-Dimethylaminophenol hydrochloride - HbFe²⁺ Ferrohemoglobin - HbFe³⁺ Ferrihemoglobin - HbFe³⁺CN^– Ferrihemoglobin cyanide - C_a Molarity of all radioactive compounds calculated on the assumption that one mole cyanide yields one mole metabolite - NCR Molarity of non-cyanide radioactive compounds calculated on the assumption that one mole cyanide yields one mole of metabolite (C_a-[CN^–])     

Enzyme therapy in cyanide poisoning: Effect of rhodanese and sulfur compounds

Crystalline bovine liver rhodanese (thiosulfate: cyanide sulfurtransferase, EC 2.8.1.1) was evaluated as an antidote in combination with different sulfur compounds against cyanide poisoning in mice. The prophylactic antidote effect, when the antidote was injected i.v. 1 min prior to i.p. injection of cyanide, was dependent on both the dose of the enzyme and the dose of the sulfur compound. An optimal dose of the enzyme of about 2,000 U/kg (3 mg/kg of pure enzyme) was found. This enzyme dose combined with 2 mmol/kg of sodium thiosulfate raised the LD₅₀ for potassium cyanide 7.6 times. When thiosulfate was replaced with equimolar doses of ethanethiosulfonate and propanethiosulfonate, the corresponding values were 10.3 and 9.3 times, respectively. Maximum antidote effect was obtained when the doses of ethanethiosulfonate and propanethiosulfonate were raised to 4 mmol/kg, increasing the LD₅₀ for cyanide 20.8 and 15.4 times, respectively. On the other hand, when given without rhodanese, ethanethiosulfonate and propanethiosulfonate were no better antidotes than thiosulfate.Rhodanese and a sulfur compound given therapeutically to mice when symptoms of cyanide poisoning had occurred, also had a very good antidote effect. The prophylactic antidote effect of rhodanese plus thiosulfate rapidly decreased with increasing time interval between injection of the antidote and cyanide. Thus, when rhodanese and thiosulfate were given 20 min prior to cyanide, the antidote effect was of the same order as that of thiosulfate alone. The antidote effect of the latter did not decrease significantly within the same time interval.Enzyme activity in plasma decreased rapidly after i.v. injection of rhodanese, and enzyme activity in urine was detected following injection. No appreciable inactivation occurred when the enzyme was incubated with whole blood in vitro, but a strong and rapid inhibition, about 85%, of the enzyme occurred in fresh mouse urine in vitro.     

Acute toxicity of some synthetic cyanogens in rats and their response to oral treatment with alpha-ketoglutarate

Oral toxicity of several cyanogens and their reversal by alpha-ketoglutarate (A-KG; oral) were studied in rats. LD₅₀ of acetonitrile (ATCN), acrylonitrile (ACN), malononitrile (MCN), propionitrile (PCN), sodium nitroprusside (SNP), and succinonitrile (SCN) was 4891, 143.3, 69.8, 122.9, 69.8 and 488.7 mg/kg, respectively while the protection index of A-KG (ratio of LD₅₀ of cyanogens in the presence or absence of A-KG) was >2.0 against MCN (7.6), PCN (2.7) and SNP (3.6) only. We further studied the efficacy of A-KG against acute toxicity of these three cyanogens (0.75 LD₅₀) on various hematological and biochemical variables in blood and soft tissues 24 h post-exposure. We observed increase in white blood cells (SNP), plasma alanine (PCN, SNP) and aspartate (PCN) aminotransferase, lactate dehydrogenase (MCN, PCN, SNP), Na⁺ (MCN, PCN) and cyanide (PCN), and decrease in K⁺ (MCN, SNP) accompanied by an increase in brain, kidney and liver malondialdehyde (PCN), decrease in brain glutathione peroxidase, glutathione reductase (PCN, SNP), reduced glutathione (MCN, PCN, SNP) and cytochrome oxidase (PCN), liver rhodanese (PCN, SNP), and kidney cytochrome oxidase (PCN). The study indicates that (i) PCN was most toxic among all the cyanogens and (ii) beside cyanide, A-KG could be considered as an effective antidote for cyanogens.     

Metabolism of tetrachlorophenols in the rat

The three isomers of tetrachlorophenol were administrated intraperitoneally to rats and the urinary excretion products studied. Tetrachloro-p-hydroquinone was identified as a major metabolite of 2,3,5,6-tetrachlorophenol, constituing about 35% of the dose given. Trichloro-p-hydroquinone was identified as a minor metabolite of both 2,3,4,5- and 2,3,4,6-tetrachlorophenol. 2,3,5,6-tetrachlorophenol was eliminated within 24 h, 2,3,4,6-tetrachlorophenol within 48 h while only 60% of the given dose of 2,3,4,5-tetrachlorophenol could be recovered within 72 h.The acute toxicity of the tetrachlorophenols and tetrachloro-p-hydroquinone was studied in mice upon oral and intraperitoneal administration. 2,3,5,6-tetrachlorophenol (LD₅₀ p.o. 109 mg · kg^–1) was the most toxic compound followed y 2,3,4,6- and 2,3,4,5-tetrachlorophenol (LD₅₀ p.o. 131 and 400 mg · kg^–1, respectively). Tetrachloro-p-hydroquinone proved to have low oral toxicity (LD₅₀ p.o. 500 mg · kg^–1) but was more toxic than the tetrachlorophenols when administered intraperitoneally. The oral LD₅₀ for pentachlorophenol, under identical experimental conditions, was found to be 74 mg · kg^–1.

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Zusammenfassung Die drei Isomeren von Tetrachlorphenol wurden an Ratten intraperitoneal verabreicht und die Ausscheidungsprodukte in Harn untersucht. Tetrachlor-p-hydrochinon erwies sich als Hauptmetabolit von 2,3,5,6-Tetrachlorphenol, entsprechend etwa 35% der zugeführten Menge. Bei sowohl 2,3,4,5-als auch 2,3,4,6-Tetrachlorphenol wurde Trichlor-p-hydrochinon als Metabolit identifiziert, jedoch in geringer Menge. Das 2,3,5,6-Tetrachlorphenol wurde innerhalb von 24 Std und das 2,3,4,6-Tetrachlorphenol innerhalb von 48 Std eliminiert, während bei 2,3,4,5-Tetrachlorphenol innerhalb von 72 Std nur 60% der gegebenen Menge erfaßt werden konnte.Die akute Toxizität von Tetrachlorphenolen und Tetrachlor-p-hydrochinon wurden an Mäusen oral sowie intraperitoneal studiert. 2,3,5,6-Tetrachlorphenol erwies sich als die toxischste der Substanzen (LD₅₀ p.o. 109 mg · kg^–1), gefolgt von 2,3,4,6- und 2,3,4,5-Tetrachlorphenol (LD₅₀ p.o. 131 mg · kg^–1 und 400 mg · kg^–1). Tetrachlor-p-hydrochinon zeigte nur geringe Toxizität, wenn oral verabreicht (LD₅₀ 500 mg · kg^–1), war aber toxischer als die Tetrachlorphenole, wenn intraperitoneal gegeben.

     

Simultaneous determination of hydrogen cyanide and volatile aliphatic nitriles by headspace gas chromatography,and its application to an in vivo study of the metabolism of acrylonitrile in the rat

A method for the simultaneous determination of hydrogen cyanide (HCN) and aliphatic nitriles using manual headspace (HS) gas chromatography (GC) with a capillary porous polymer column GS-Q and a nitrogen–phosphorus detector is described. With a HS incubation at 50°C for 30 min and a GC temperature at 180°C, HCN and volatile nitriles [acetonitrile, acrylonitrile (VCN), propionitrile, isobutyronitrile] were well separated and could be detected within 7 min with a detection limit of 0.7–2.4 ng/ml in blood samples. The HS-GC method was used in an in vivo study of VCN metabolism. VCN was administered orally (at nearly one-half its LD₅₀) to rats, and heart blood and urine were sampled. Blood concentrations of HCN and VCN were measured by HS-GC, and plasma and urinary thiocyanate concentrations were measured by the König colorimetric method. Blood levels of HCN and VCN peaked 1.5 h after VCN administration, at which time the cyanide level (about 0.7 g/ml) is close to the fatal level. HCN levels were observed to be at almost background levels at 10 h, although 50 ng/ml VCN was still detectable. The plasma thiocyanate level increased, reaching a peak (about 30 g/ml) at 5 h. The cumulative urinary thiocyanate amount gradually increased, and at 10 h more than 1 mg thiocyanate was excreted into the urine. It is therefore possible to clarify the cause of cyanide poisoning using HS-GC analysis, when someone has taken volatile nitriles.     

Acute toxicity of some nerve agents and pesticides in rats

Objectives: Highly toxic organophosphorus compounds (V- and G-nerve agents) were originally synthesized for warfare or as agricultural pesticides. Data on their acute toxicity are rare and patchy. Therefore, there is a need for integrated summary comparing acute toxicity of organophosphates using different administration routes in the same animal model with the same methodology. Based on original data, a summary of in vivo acute toxicity of selected V- and G-nerve agents (tabun, sarin, soman, VX, Russian VX) and organophosphates paraoxon (POX) and diisopropyl fluorophosphate (DFP) in rats has been investigated. Materials and methods: Male Wistar rats were exposed to organophosphates in several administration routes (i.m., i.p., p.o, s.c., p.c.). The acute toxicity was evaluated by the assessment of median lethal dose (LD₅₀, mg?kg^?1) 2, 4, and 24 hours post exposure. Results: V-agents were the most toxic presented with LD₅₀ ranged from 0.0082?mg?kg^?1 (VX, i.m.) to 1.402?mg?kg^?1 (Russian VX, p.o.), followed by G-agents (LD₅₀?=?0.069?mg?kg^?1/soman, i.m./ – 117.9?mg?kg^?1/sarin, p.c./), organophosphate POX and DFP (LD₅₀?=?0.321?mg?kg^?1/POX, i.m./ – 420?mg?kg^?1/DFP, p.c./). Generally, i.m. administration was the most toxic throughout all tested agents and ways of administration (LD₅₀?=?0.0082?mg?kg^?1/VX/ – 1.399?mg?kg^?1/DFP/) whereas p.c. way was responsible for lowest acute toxicity (LD₅₀?=?0.085?mg?kg^?1/VX/ – 420?mg?kg^?1/DFP/). Conclusion: The acute toxicity of selected organophosphorus compounds is summarized throughout this study. Although the data assessed in rats are rather illustrative prediction for human, it presents a valuable contribution, indicating the toxic potential and harmfulness of organophosphates.     

The fate of 2-chlorobenzylidene malononitrile (CS) in rats

1. The fate of ³H-ring labelled, ¹⁴C-cyanide labelled and (¹⁴C=C) side chain labelled 2-chlorobenzylidene malononitrile (CS), 2-chlorobenzyl alcohol and 2-chlorobenzyl malononitrile (dihydro CS) in rats and isolated rat liver cells has been examined.

2. CS was administered both i.v. and i.g. to rats at doses from 0.08 to 159 ümol/kgand in most cases the greatest proportion of the dose was eliminated in the urine (44–100%). The principal urinary metabolites were 2-chlorohippuric acid, 1-O-(2-chlorobenzyl) glucuronic acid, 2-chlorobenzyl cysteine and 2-chlorobenzoic acid. Lesser amounts of 2-chlorophenyl acetyl glycine, 2-chlorobenzyl alcohol and 2-chlorophenyl 2-cyano propionate were identified.

3. The major urinary metabolite from 2-chlorobenzyl alcohol was 2-chlorohippuric acid (43%), 2-chlorobenzyl cysteine, 2-chlorobenzoic acid and 2-chlorobenzyl glucuronic acid were identified.

4. The products of dihydro-CS metabolism were 2-chlorophenyl acetyl glycine, 2-chlorophenyl 2-cyanopropionate and 2-chlorophenyl proprionamide.

5. Urinary thiocyanate levels increased with the dose of CS up to 159 ümol/kg. At 212ümol/kg there was a large increase in the amount of thiocyanate produced (molar conversion: 21.5–29.9%). Similarly malononitrile, the hydrolysis product of CS, gave a dose related increase in urinary thiocyanate levels. However at a higher dose (212 ümol/kg) the molar conversion was greater than 60%.

6. The metabolism of CS by isolated rat liver cells confirmed the results in vivo but demonstrated a marked limitation of this preparation to form conjugates.

7. It is concluded that CS in vivo is hydrolysed mainly to 2-chlorobenzaldehyde which is then either oxidized to 2-chlorobenzoic acid for subsequent glycine conjugation, or reduced to 2-chlorobenzyl alcohol for ultimate excretion as 2-chlorobenzyi acetyl cysteine or 1-O-(2-chlorobenzyl) glucuronic acid. Malononitrik is converted to thiocyanate via the formation of cyanide.     

Formation of cyanide after i. v. administration of the oxime HI 6 to dogs

HI6(pyridinium, 1-[[[4-(aminocarbonyl)pyridinio] methoxy]methyl]-2-[(hydroxyimino)methyl]-dichloride belongs to a series of bisquaternary pyridinium oximes that are effective against poisoning with extremely toxic organophosphates. Since HI6 has been shown to be unstable at pH 7.4 and to release significant amounts of cyanide, a study was undertaken to determine the degree of cyanide formation from HI 6 in vivo. When HI 6 (100 mol/kg) was administered i. v. to dogs, the animals showed no signs of cyanide toxicity but exhibited some cholinomimetic symptoms, including retching, hypersalivation and enhanced intestinal motility. Cyanide content in whole blood was monitored after production of methemoglobinemia (30%) by 4-dimethylaminophenol in order to sequester cyanide within red cells. Maximal cyanide contents of 20 mol/l were found in blood after 90 min. Calculation of the area under the concentration versus time curve for blood cyanide indicates that about 4% of HI 6 produced cyanide. Determination of the pharmacokinetic parameters of HI 6 (V_D=0.31 l/kg; k_el=0.76 h^–1) and of cyanide (V_D=0.086 l/kg; k_el=0.52 h^–1) together with the apparent first order rate constant of cyanide formation from HI 6 in vitro (0.174 h^–1, pH 7.4, 37°) allowed the simulation of a cyanide concentration curve that fitted with the experimental data points, indicating that cyanide formation in vivo was not bio-catalyzed. It is concluded that cyanide formation from HI 6 may not be regarded as a potential hazard, since cyanide elimination exceeded markedly its formation. Whether this conclusion also holds true for man has to be established.     

Efficacy of oral administration of sodium thiosulfate in a large,swine model of oral cyanide toxicity

IntroductionCyanide is a deadly poison, particularly with oral exposure where larger doses can occur before symptoms develop. Prior studies and multiple governmentagencies highlight oral cyanide as an agent with the potential for use in a terrorist attack. Currently, there are no FDA approved antidotes specific to oralcyanide. An oral countermeasure that can neutralize and prevent absorption of cyanide from the GI tract after oral exposure is needed. Our objective was toevaluate the efficacy of oral sodium thiosulfate on survival and clinical outcomes in a large, swine model of severe cyanide toxicity.MethodsSwine (45-55kg) were instrumented, sedated, and stabilized. Potassium cyanide (8 mg/kg KCN) in saline was delivered as a one-time bolus via an orogastric tube. Three minutes after cyanide, animals randomized to the treatment group received sodium thiosulfate (510 mg/kg, 3.25 M solution) via orogastric tube. Our primary outcome was survival at 60 minutes after exposure. We compared survival between groups by log-rank, Mantel-Cox analysis and trended labs and vital signs.ResultsAt baseline and time of treatment all animals had similar weights, vital signs, and laboratory values. Survival at 60 min was 100% in treated animals compared to 0% in the control group (p=0.0027). Animals in the control group became apneic and subsequently died by 35.0 min (20.2,48.5) after cyanide exposure. Mean arterial pressure was significantly higher in the treatment group compared to controls (p=0.008). Blood lactate (p=0.02) and oxygen saturation (p=0.02) were also significantly different between treatment and control groups at study end.ConclusionOral administration of sodium thiosulfate improved survival, blood pressure, respirations, and blood lactate concentrations in a large animal model of acute oral cyanide toxicity.     

Parenteral dosage form development and testing of dimethyl trisulfide,as an antidote candidate to combat cyanide intoxication

This study focused on the solubility enhancement and the in vivo antidotal efficacy testing of a new potential cyanide (CN) countermeasure, dimethyl trisulfide (DMTS). Various FDA approved cyclodextrins (HPβCD, RMβCD, HPγCD), cosolvents (ethanol, polyethylene glycols, propylene glycol), surfactants (cremophor EL, cremophor RH 40, sodium cholate, sodium deoxycholate, polysorbate 80) and their combinations were applied. Based on the solubility enhancing potential of the tested systems, polysorbate 80 was chosen for further in vivo efficacy studies. A composition comprising 15% polysorbate 80 and 50 mg/ml DMTS with the applied DMTS dose of 100 mg/kg provided a therapeutic antidotal protection of 3.4?×?LD₅₀. For comparison, the present therapy of sodium thiosulfate (TS) with the dose of 100 mg/kg provided only 1.1?×?LD₅₀ protection, and at the dose of 200 mg/kg, the LD₅₀ was enhanced by 1.3 times. No difference in the therapeutic protection by DMTS was detected when the concentration of polysorbate 80 was increased to 20% (3.2?×?LD₅₀ protection). These data demonstrate the potential importance of DMTS as a CN countermeasure, and the formulation comprising polysorbate 80 provides the base of an injectable intramuscular dosage form that can later serve as a CN antidotal kit suitable for mass scenario.     

Effect of pretreatment with CBDP on the toxicokinetics of soman stereoisomers in rats and guinea pigs

Pretreatment of rats and guinea pigs with the specific carboxylesterase inhibitor 2-(o-cresyl)-4H-1 3 2-benzodioxaphosphorin-2-oxide (CBDP) reduces the LD₅₀ of the nerve agent C(±)P(±)-soman in these species to the same range as in primates. This suggests that such CBDP-pretreated animals can be used in investigations that are relevant for prophylaxis and therapy of intoxication with C(±)P(±)-soman in primates including humans. In order to test this hypothesis we have studied the toxicokinetics of the toxic C(±)P(–)-isomers of soman in artificially respirated and CBDP-pretreated rats and guinea pigs at intravenous doses corresponding to 6 × LD₅₀. A comparison of the areas under the curve (AUCs) of the blood levels of C(±)P(–)-soman in pretreated and non-pretreated animals at the same absolute dose shows extreme nonlinearity with dose, indicating that CBDP occupies highly reactive binding sites which are no longer available for sequestration of the soman isomers. The AUCs of C(±)P(–)-soman at equitoxic doses of 6× LD₅₀ are reduced by pretreatment with CBDP from 1683 to 464 ng.min.ml^–1 in rats and from 978 to 176 ng.min.ml^–1 in guinea pigs, which is in the range of the AUC in non-pretreated marmosets at an equitoxic dose (419 ng.min.ml^–1). The blood levels of the C(±)P(–)-isomers in marmosets and CBDP rats are rather similar during the first 7 min, but persist in CBDP rats for 2 h longer at toxicologically relevant levels than in marmosets. The levels of C(±)P(–)-soman in CBDP-pretreated guinea pigs are substantially lower than in marmosets for an initial period of 80 min. Nevertheless, they drop below toxicologically relevant levels approximately 50 min later than in marmosets. Evidently, one should be cautious in considering CBDP, pretreated rats and guinea pigs as substitute primates.This research was supported in part by the US Army Medical Research and Development Command under grants DAMD17-87-G-7015 and DAMD17-90-Z-0034.     

The ophthalmic toxicology of o-chlorobenzylidene malononitrile (CS)

Rabbit eyes were contaminated with the riot control agent o-chlorobenzylidene malononitrile (CS) in solution (0.5 to 10 % in polyethylene glycol 300), as a solid (0.5 to 5 mg), and as a pyrotechnically generated smoke (15 min exposure to 6 g/m³). CS caused lachrymation, blepharitis and conjunctival irritation by all the methods of contamination, the severity and duration of which increased with the amount of material applied. Effects were most severe with CS in solution, less with solid, and least marked with smokes. Mild and transient keratitis and iritis occurred with solutions containing 1 % CS, being more severe and prolonged with the higher concentrations. Damage was due to CS, and not to its products of hydrolysis. Keratitis was not seen after exposure to smokes containing CS, and superficial corneal damage of short duration occurred only in a few animals treated with 5 mg solid CS. A concentration-dependent increase in intraocular tension, of less than 1 h duration, occurred with solutions of CS.     

Approximate lethal dose versus median lethal dose in acute toxicity testing of pharmaceuticals

The relation between approximate lethal doses (ALD, i. e. the lowest dose at which mortality occurs) and the corresponding median lethal doses (LD₅₀) was investigated in 231 acute toxicity studies in mice and rats. The ALD values were divided into four classes (<5 mg/kg, 5–50 mg/kg, 50–500 mg/kg, 500–2000 mg/kg) and the LD₅₀/ALD factors were calculated. In intravenous studies the LD₅₀ values were higher than the ALD values by mean factors of 1.27–1.61 in mice and 1.25–2.84 in rats. In oral studies the LD₅₀ values were higher by mean factors of 1.46–2.5 in mice and 1.59–2.1 in rats. Only in 20 cases (8.7%) did the LD₅₀ values differ by factors higher than 2.     

In Vitro and In Vivo Evaluation of Various Carbonyl Compounds against Cyanide Toxicity with Particular Reference to Alpha-Ketoglutaric Acid

Cyanide is a rapidly acting neurotoxin that necessitates immediate, vigorous therapy. The commonly used treatment regimen for cyanide includes the intravenous administration of sodium nitrite (SN) and sodium thiosulphate (STS). Due to many limitations of these antidotes, a search for more effective, safer molecules continues. Cyanide is known to react with carbonyl compounds to form the cyanohydrin complex. The present study addresses the efficacy of several carbonyl compounds and their metabolites or nutrients with alpha-ketoglutaric acid (A-KG), citric acid, succinic acid, maleic acid, malic acid, fumaric and oxaloacetic acid, glucose, sucrose, fructose, mannitol, sorbitol, dihydroxyacetone, and glyoxal (5 or 10 mM; ?10 min) against toxicity of potassium cyanide (KCN; 10 mM) in rat thymocytes in vitro. Six hours after KCN, cell viability measured by MTT assay and crystal violet dye exclusion revealed maximum cytoprotection by A-KG, followed by oxaloacetic acid. A-KG also resolved the leakage of intracellular lactate dehydrogenase, loss in nuclear integrity (propidium iodide staining), and altered mitochondrial membrane potential (rhodamine 123 assay) as a result of cyanide toxicity. Protection Index (ratio of LD₅₀ of KCN in protected and unprotected animals; PI) of all the compounds (oral; 1.0 g/kg; ?10 min) determined in male mice, revealed that maximum protection was afforded by A-KG (7.6 PI), followed by oxaloacetic acid (6.4 PI). Comparative evaluation of various salts of A-KG alone or with STS (intraperitoneal; 1.0 g/kg; ?15 min) showed that maximum protection was conferred by disodium anhydrous salt of A-KG, which also significantly prevented the inhibition of brain cytochrome oxidase caused by 0.75 LD₅₀ KCN. This study indicates the potential of A-KG as alternative cyanide antidote.     

Melatonin reduces oxidative damage induced by aluminium in rat kidney

We evaluated the effect of melatonin (Mel), in male Wistar rats which received aluminium (Al) lactate for 12 weeks (0.57 mg Al/100 g body weight (b.w.), i.p. three times per week). Moreover rats received Mel (10 mg/kg b.w. i.p. 5 days/weeks) for 12 weeks. At the end of the treatment water and sodium balances were studied, and nephrogenic cyclic adenosine monophosphate (cAMP) was also measured. Urinary osmolality was measured after the administration of desmopressin (vasopressin agonist) to assess concentrating capacity. Oxidative stress in renal tissue and Na⁺–K⁺ATPase and gamma-glutamyl transferase (GGT) activities in whole plasma membrane were determined. Sodium and water balances were impaired by Al. We found decreased urinary concentrating ability and nephrogenic cAMP excretion. Al increased the Na⁺–K⁺ATPase activity, and serum aldosterone concentration. Mel normalized serum aldosterone level, the Na⁺–K⁺ATPase activity and potassium urinary without improving water and sodium excretion. Mel treatment did not improve the impaired urinary concentrating ability. Al reduced the GGT activity, an effect that persists in Al⁺ Mel. Al exposure promoted oxidative stress with an increase in lipid peroxidation (LPO), and a decrease in glutathione (GSH) and glutathione peroxidase (GSH-Px) and catalase (CAT) activities. Mel markedly attenuated oxidative stress produced by Al. This may result from the higher efficacy of melatonin in scavenging various free radicals and also because of its ability in stimulating the antioxidant enzymes. However, it only reduced some alterations in the renal functions particularly related to the water and sodium excretion, which would be independent of the increased production of reactive oxygen substances.     

Structure-toxicity relationship of ethylene glycol ethers

The ultimate purpose of the present study was to evaluate correlations between acute in vivo and in vitro toxicity and log P (P is n-octanol-water partition coefficient). The in vitro toxicity to cloned cells (neuroblastoma N18TG-2 and glioma C6) in culture (ED₅₀) and the in vivo toxicity to mice (LD₅₀) of ethylene glycol ethers were studied in terms of the structure-activity relationship. The test ethers showed a wide range of ED₅₀ values in both cells. LD₅₀ was determined under two conditions: LD₅₀-cont. was estimated in mice pretreated with olive oil and LD₅₀-CCl₄ in CCl₄-pretreated mice. Multiple regression analyses revealed a significant correlation between log 1/LD₅₀ and log P as follows: log (1/LD₅₀–cont.) = – 0.120 (log P)²+0.4871og P–1.182, and log (1/LD₅₀-CCl₄) = –0.128 (log P)²+0.5661og P–1.157. There was no significant correlation either between ED₅₀ and LD₅₀ or between ED₅₀ for N18TG-2 and ED₅₀ for C6. The results suggest that metabolic activation might not occur during acute toxicity from the ethers, and that hydrophobicity, expressed as log P, plays an important role in acute toxicity.     

Effect of norathyriol,isolated from Tripterospermum lanceolatum,on A 23 187-induced pleurisy and analgesia in mice

A 23 187-induced pleurisy in the mouse was demonstrated in this study. The protein leakage, leukocyte accumulation, LTB₄ and PGE₂ production in the pleural cavity of mice were increased by A 23 187 in a dose-dependent manner. At 7.5 nmole A 23 187 intrapleural injection, the protein level peaked at 0.5–2 h, PMN leukocytes accumulation peaked at 3–4 h, and LTB₄ and PGE₂ production peaked at 0.5–1 h. In this in vivo model we investigated the anti-inflammatory effect of norathyriol, isolated from Tripterospermum lanceolatum. A 23 187-induced protein leakage was reduced by norathyriol (ID₅₀ was about 30.6 mg/kg i.p.), indomethacin and BW 755 C. A 23 187-induced PMN leukocytes accumulation was suppressed by norathyriol (ID₅₀ was about 16.8 mg/kg, i.p.) and BW 755 C, while enhanced by indomethacin. Like BW 755 C, norathyriol reduced both LTB₄ and PGE₂ production (ID₅₀ was about 18.6and 29.1 mg/kg i.p., respectively), while indomethacin reduced PGE₂ but not LTB₄ generation. We also demonstrated the analgesic effect of norathyriol on the acetic acid-induced writhing response. Acetic acid-induced writhing response was depressed by norathyriol (ID₅₀ was about 27.9 mg/kg i.p.), indomethacin and ibuprofen. These results suggest that norathyriol, like BW 755 C, might be a dual, yet weak, cyclooxygenase and lipoxygenase pathway blocker. The inhibitory effect of norathyriol on the A 23 187-induced pleurisy and acetic acid-induced writhing response in mice is proposed to be dependent on the reduction of eicosanoids mediators formation in the inflammatory site. Correspondence to: J.-P. Wang at the above address