Comparative Acute Lethality of 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), 1,2,3,7,8-Pentachlorodibenzo-p-dioxin and 1,2,3,4,7,8-Hexachlorodibenzo-p-dioxin in the Most TCDD-Susceptible and the Most TCDD-Resistant Rat Strain

We have previously demonstrated a more than 300-fold difference in acute LD50 values for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) between male Long-Evans (Turku AB; L-E) and Han/Wistar (Kuopio; H/W) rats after intraperitoneal exposure. In the present study, we compared the acute lethality of TCDD, 1,2,3,7,8-pentachlorodibenzo-p-dioxin (PCDD) and 1,2,3,4,7,8-hexachlorodibenzo-p-dioxin (HCDD) in these strains by intragastric administration. In agreement with previous data, H/W rats proved to be strikingly resistant to TCDD, since even the highest dose tested, 7200 μg/kg, was below the LD50 level for both genders. The corresponding LD50 values for female and male L-E rats were 9.8 and 17.7 μg/kg, respectively. A similar strain difference was discovered for PCDD: the LD50 value was > 1620 μg/kg for female H/W rats and between 20 and 60 μg/kg for female L-E rats. Surprisingly, the acute lethality of HCDD did not follow the same pattern. Female H/W rats turned out to be only about 10 times less susceptible to that congener than female L-E rats (LD50 values 1871 and between 120 and 360 μg/kg, respectively). These findings do not support the widely accepted concept that sufficiently high doses of all dioxin congeners will produce the same effects. Either the higher chlorinated dioxins have toxic effects distinct from those of TCDD or the relative contribution of toxic impacts varies among these compounds.     

Generation of ammodytoxin-anti-cathepsin B immuno-conjugate as a model for delivery of secretory phospholipase A2 into cancer cells.

Ammodytoxin C (AtxC) is a toxic secreted phospholipase A2 (sPLA2) from the venom of Vipera ammodytes snake. To evaluate its potential to kill cancer cells, the toxin was cross-linked to the monoclonal antibody against cathepsin B which endocytoses upon binding to cathepsin B, an antigen overexpressed on the plasma membrane of cancer cells. A photo-reactive derivative of AtxC, possessing the same biological activity as the native toxin, was reacted with antibodies to form a covalent immuno-conjugate. In conditions of the cytosolic redox potential, AtxC was gradually released from the conjugate due to reduction of the disulfide bond in the spacer arm of the cross-linker. The phospholipase activity of the preparation reached maximum in 10min and then decreased gradually. As demonstrated by fluorescence microscopy, the immuno-conjugate targeted Caco-2 colon adenocarcinoma cells but was very slowly internalized, the likely reason of only slight cytotoxicity being observed. Despite the lack of a clear cytotoxic effect of AtxC-antibody conjugate on Caco-2 cells, we demonstrated in this work a new methodology for the targeted delivery of (toxic) sPLA2 into cells, promising in research or therapy.     

Investigations into the toxicology of spirolides, a group of marine phycotoxins

Spirolides are marine phycotoxins produced by the dinoflagellates Alexandrium ostenfeldii and A. peruvianum. Here we report that 13-desmethyl spirolide C shows little cytotoxicity when incubated with various cultured mammalian cell lines. When administered to mice by intraperitoneal (ip) injection, however, this substance was highly toxic, with an LD(50) value of 6.9 μg/kg body weight (BW), showing that such in vitro cytotoxicity tests are not appropriate for predicting the in vivo toxicity of this toxin. Four other spirolides, A, B, C, and 20-methyl spirolide G, were also toxic to mice by ip injection, with LD(50) values of 37, 99, 8.0 and 8.0 μg/kg BW respectively. However, the acute toxicities of these compounds were lower by at least an order of magnitude when administration by gavage and their toxic effects were further diminished when administered with food. These results have implications for future studies of the toxicology of these marine toxins and the risk assessment of human exposure.     

mRNA secondary structure can greatly affect production of recombinant phospholipase A(2) toxins in bacteria.

The neurotoxic activity of ammodytoxin A (AtxA), a phospholipase A(2) from Vipera ammodytes ammodytes venom, has been investigated by protein engineering. With the aim of obtaining AtxA as a non-fused protein in the bacterial cytoplasm and avoiding problems with incomplete cleavage in vivo of the initial Met preceding the first residue (Ser1), a double mutant (S1A/E4Q) was prepared and expressed in Escherichia coli. Immunoblotting of the bacterial lysate showed that the mutant was synthesized at a low level not exceeding 0.5% of total cell protein. Analysis of the potential secondary structure of the mutant mRNA in the translation initiation region suggested that the Ala1 (GCC) and Leu2 (CUG) codons used are likely to be involved in a hairpin structure with the Thr13 (ACG) and Gly14 (GGG) codons, hindering effective translation at the ribosome. To weaken this structure (by DeltaG of about 20 kJ/mol) the same double mutant was prepared using another mutagenic oligonucleotide with silent mutations in the Ala1 (GCU) and Leu2 (UUG) codons. The mutant was successfully produced at a level of approximately 15% of total protein, with the initial Met completely removed in the bacterial cell. Such an approach could be important in solving similar problems in bacterial production of other toxic proteins.     

Interaction of d-pseudoephedrine with water soluble extracts of Platycodi Radix on acute toxicity (author's transl)

Effect of various combinations of Platycodi Radix water soluble extracts (Pla), 1-ephedrine (1-eph), d-pseudoephedrine (d-pseudo) and Ipecacuanhae Radix water soluble extracts (Ipe) on acute toxicity were examined in mice. Oral LD50 of Ipe, d-pseudo and 1-eph was 490 (415--578) mg/kg, 1550 (1360--1767) mg/kg and 1400 (1102--1778) mg/kg, respectively, while that of Pla was over 10 g. LD50 of Pla Ipe, d-pseudo and 1-eph given intraperitoneally was 1400 (1228--1596) mg/kg 235 (210--263) mg/kg, 245 (229--262) mg/kg and 300 (259--348) mg/kg, respectively. The ratio of the predicted LD50 value, which was calculated on the assumption that each component drug would be additively toxic when combined, to the observed LD50 value was used for comparison. The combination of d-pseudo with Pla gave a significantly greater LD50 value than the predicted LD50 value, while the combination of 1-eph with Pla showed a LD50 value which was not significantly different from Finney's additive model. A combination of d-pseudo with 1-eph and Ipe, and of 1-eph with Ipe showed a LD50 value which was not significantly different from that of the additive model.     

Preclinical toxicologic evaluation of chromomycin A3 (NSC-58 514) in mice, dogs and monkeys

The toxicity of chromomycin A3, a potent antitumor antibiotic derived from Streptomyces griseus, was assessed in mice, dogs and monkeys. In mice, the LD50 after 5 daily iv doses was 603 μg/kg. In dogs and monkeys, the single-dose iv minimal lethal dose was 200 and 330 μg/kg, respectively, while this value after 5 daily iv doses was 100 μg/kg for both species. The principal clinicopathologic findings in dogs and monkeys that became moribund or died included emesis, hemorrhagic diarrhea, fever, dehydration, and necrosis of intestinal and lymphoid tissues with occasional necrosis in other organs. Laboratory findings were consistent with clinical signs. Dogs and monkeys that survived treatment exhibited mild, reversible toxic alterations.     

Effects of the organophosphorus compound,O-ethyl-N-dimethyl-phosphoramidocyanidate (tabun), on flavor aversions,locomotor activity,and rotarod performance in rats

An assessment of the behavioral effects of acutely administered anti-cholinesterase compound O-ethyl-N-dimethyl phosphoramidocyanidate (tabun), to male rats was performed in three studies across five dose levels (100–198 μg/kg, sc). Doses of 122 μg/kg or greater produced conditioned flavor aversions. Spontaneous locomotor activity was significantly decreased at doses of 122 μg/kg or greater when compared to vehicle control levels. When administered doses of 168 μg/kg or greater, rats exhibited significant decrements in rotarod performance. In addition, the LD50 of tabun was determined to be 240 μg/kg. Signs of cholinergic intoxication were not apparent until 144 μg/kg tabun or more were given. Behavioral effects were thus obtained at doses that were between 54 and 71% of the LD50.     

Synthesis and antihypertensive activity of novel 3-hydrazino-5-phenyl-1,2,4-triazines.

In an effort to develop antihypertensive agents with peripheral vasodilator activity, a series of 40 novel 3-hydrazino-5-phenyl-1,2,4-triazines (II) were synthesized and evaluated in the spontaneously hypertensive rat assay (SHR assay). Based on the performance of the structurally related standard, hydralazine (I), 15 triazines were active. Thirteen of these hypotensive triazines possessed LD50 values in the mouse greater than I (LD50 = 100 mg/kg); only one active triazine had an LD50 value greater than 300 mg/kg (11d). Four asymmetric triazines had moderate antihypertensive activity and LD50 values greater than 300 mg/kg (6b, 7c, 8f, and 9g). Based on the relationship between toxicity and antihypertensive activity, three triazines (8f, 9g, and 11d) were chosen for dose-responses studies in the SHR assay. None were as efficacious as I, but all three were less toxic, resulting in similar therapeutic indices relative to I.     

Induced toxicity in early-life stage zebrafish (Danio rerio) and its behavioral analysis after exposure to non-doped,nitrogen-doped and nitrogen,sulfur-co doped carbon quantum dots

In this study, the effects of doping of CQDs with alternative functional groups (dopants) were evaluated through embryonic development of zebrafish (Danio rerio). The CQDs were synthesized using simple and low-cost sources: Non-doped (citric acid was used as the carbon source), nitrogen-doped (N-doped) and nitrogen, sulfur-co-doped (N,S-doped). The CQDs induced significant toxicity to zebrafish (>150 μg/mL) and the toxic effects were dose-dependent. The N,S-doped CQDs were the most toxic (LD50 = 149.92 μg/mL), followed by the N-doped CQDs (LD50 = 399.95 μg/mL) while the non-doped CQDs were the least toxic (LD50 = 548.48 μg/mL) of the three. The growth rate (GR) was affected following the toxicity pattern (GR_NS-doped<GR_N-doped<GR_non-doped <GR_blanc), which, in turn, greatly depends on the type of dopant. Morphological malformations, such as pericardial edema, yolk sac edema, tail and spinal curvature were observed to zebrafish embryos as the toxicity, concentration and exposure time to the nanomaterial increased. Behavioral analysis showed that locomotor activity increases as the toxicity of the nanomaterial rises. The differences in toxicity, growth rate and malfunctions of CQDs were attributed to their doping with different heteroatoms. The N,S-doped CQDs, unequivocally, exhibited the most pronounced effects.     

The Cardiovascular Effects of Ricin in Rabbits

Ricin is a toxic lectin from the castor bean. The time course of its toxic effects on the cardiovascular system of rabbits was investigated after determining its LD₅₀ and minimum lethal dose in rabbits by the Up and Down method, as a basis for dosing. Systolic and diastolic arterial pressures, electrocardiogram and heart rate were recorded for 48 hr following administration of either a toxic sublethal (0.22 μg/kg) or minimum lethal dose (0.44 μg/kg) of ricin. After a delayed onset of about 20 hr, the minimum lethal dose (0.44 μg/kg) of ricin caused a significant decrease in both systolic and diastolic pressures (P<0.05). The systolic and diastolic pressures decreased 0.47 and 0.39 mmHg/hr more so than control groups, respectively. A toxic sublethal dose (0.22 μg/kg) of ricin did not significantly alter either systolic or diastolic pressure. Neither dose of ricin caused cardiac arrhythmias or significantly increased heart rate. We conclude that the lethal hypotensive sequelae of ricin toxicity in the rabbit were peripheral in origin and not cardiogenic at these two doses.     

Tissue Distribution,Metabolism, and Excretion of 14C-TCDD in a TCDD-Susceptible and a TCDD-Resistant Rat Straina

A comparative study was carried out in the most TCDD-resistant [Han/Wistar (H/W), LD50 > 3000 μg/kg] and the most TCDD-susceptible [Long-Evans (L-E), LD50 about 10 μg/kg] rat strain to assess the significance of kinetic factors in TCDD toxicity. Young adult males of both strains were administered 5 μg/kg (1.9 μCi/kg) ¹⁴C-TCDD intraperitoneally. Four rats per strain were killed at 4 hr, 1, 4, 8, 16, and 32 days after exposure. A total of 22 tissues along with blood and serum were sampled for liquid scintillation counting. From half of the animals, daily urine and faeces were also analyzed. In addition, 3 rats per strain were given 50 μg/kg (19 μCi/kg) ¹⁴C-TCDD and prepared for whole-body autoradiography after 1, 4 or 8 days. The livers of two rats per strain killed at 4 hr, 4 or 16 days, and the excreta from two rats of both strains collected on days 1-4, 5-8, 13-16, and 29-32 after exposure were analyzed for metabolites of TCDD by high pressure liquid chromatography. The label was mainly excreted in faeces as metabolites of TCDD, and the half-life of elimination was 20.8 (L-E) or 21.9 (H/W) days. A very similar overall distribution pattern was observed in both strains irrespective of dose, and the liver was the major site of accumulation. Practically all liver ¹⁴C-activity was found as the parent compound. Moderate strain-related differences were observed in the thyroid, thymus, prostate, adrenals, and brown and white fat, where lower values were recorded in H/W rats. Site-dependent variation was detected in the brain. The results do not support the view that TCDD metabolism or disposition would have a major impact on the strain difference in TCDD lethality.     

Biochemical and biological activities of the venom of a new species of pitviper from Vietnam, Triceratolepidophis sieversorum.

Biochemical and biological activities of a venom sample from a recently discovered new genus and species of pitviper from Vietnam, Triceratolepidophis sieversorum, were assayed and compared with those of five other viperid snakes (Bothrops asper, Crotalus atrox, Protobothrops flavoviridis, Trimeresurus insularis, and Vipera ammodytes). The venom had high casein hydrolysis, arginine ester hydrolysis and haemorrhagic activities, lacked measurable phosphodiesterase and L-amino acid oxidase activities, and had no procoagulant activity on either bovine fibrinogen or human plasma. Other enzymatic activities (phospholipase A(2), kallikrein) were moderate. The approximate i.p. LD(50) (mice) of the venom is about 5-6 mg/kg.     

Reproductive, acute and subacute toxicity studies with nefopam in laboratory animals.

Nefopam hydrochloride, a compound with non-narcotic analgesic activity, was evaluated for its acute and subacute toxicity in mice, rats, and dogs. Potential teratogenic effects in mice and rabbits and its effect on general reproduction and postnatal development in rats were also studied. The LD50 by various routes of administration showed that in all three species oral LD50 values (80–178 mg/kg) were greater than im LD50 values (30–57 mg/kg) which were higher than iv LD50 values (20–45 mg/kg). A comparative oral acute toxicity study did indicate a strain difference in rats. Repeated daily administration of nefopam hydrochloride to rats and dogs at doses up to 10 mg/kg/day parenterally and 80 mg/kg/day orally revealed no toxic effects except for mild tissue irritation at injection sites and slight weight loss in some dogs receiving the high dose. Reproduction studies did not reveal any effects on fertility, lactation, or pup development and viability.     

Antagonism of cyanide poisoning by dihydroxyacetone

Dihydroxyacetone (DHA) effectively antagonized the lethal effect of cyanide in mice and rabbits, particularly if administered in combination with thiosulfate. Oral DHA (2 and 4 g/kg) given to mice 10 min before injection (i.p.) of cyanide increased the LD50 values of cyanide from 5.7 mg/kg to 12 and 17.6 mg/kg, respectively. DHA prevented cyanide-induced lethality most effectively, if given orally 10-15 min before injection of cyanide. A combination of pretreatment with oral DHA (4 g/kg) and post-treatment with sodium thiosulfate (1 g/kg) increased the LD50 of cyanide by a factor of 9.9. Furthermore, DHA given intravenously to rabbits 5 min after subcutaneous injection of cyanide increased the LD50 of cyanide from 6 mg/kg to more than 11 mg/kg, while thiosulfate (1 g/kg) given intravenously 5 min after cyanide injection increased the LD50 of cyanide only to 8.5 mg/kg. DHA also prevented the convulsions that occurred after cyanide intoxication.     

Evidence for Adrenergic Mediation of Ouabain induced Arrhythmias in the Guinea Pig

Oubain in a dose of 7.5 μg/min. was infused intravenously into urethane anaesthetized guinea pigs weighing from 250–500 g. The incidence of cardiac fibrillation and the cardiotoxic dose (fibrillation, cardiac arrest) were determined in groups of (1) untreated controls. (2) adrenalectomized, (3) reserpinized, 5 mg/kg subcutaneously, (4) reserpinized + adrenalectomized, (5) reserpinized + noradrenaline pretreated animals (100 μg/kg intravenously 15 min. before ouabain). All animals were kept at a constant rectal temperature of 34^°. The pressor response to tyramine (0.1 and 1 mg/kg intravenously) was tested during the same experimental conditions. All animals died in fibrillation except those in (4) in which only 50% showed fibrillation. The sensitivity to ouabain (minimal fibrillatory dose, LD100) was only slightly decreased in (2) while the LD100 was increased from 239 ± 10 μg/kg to 375 ± 15 μg/kg in (3) and 466 ± 17 μg/kg in (4). Infusion of noradrenaline into reserpinized animals caused a partial recovery of the normal ouabain sensitivity as the LD100 decreased from 375 ± 15 μg/kg to 302 ± 11 μg/kg (P < 0.001). The pressor response to tyramine showed features almost similar to the ouabain sensitivity except that the sensitivity to tyramine could not be regained by a noradrenaline infusion in reserpinized guinea pigs. The results indicate that the ouabain cardiotoxicity in the guinea pig is at least to some extent mediated through a release of catecholamines and may explain the species dependent difference in potency of some β-adrenergic blocking agents on ouabain arrhythmias.     

Comparative characterization of two toxic phospholipases A2 from Indian cobra (Naja naja naja) venom.

Indian cobra venom contains many phospholipase A2 (PLA2) toxins. In the present study two toxic PLA2s have been purified from the Indian cobra (Naja naja naja) venom by column chromatography. The NN-XIa-and NN-XIb-PLA2s have mol. wts between 10,700 and 15,000. The NN-XIa-PLA2 induces myotoxic effects, oedema and neurotoxicity in mice and has an i.p. LD50 of 8.5 mg/kg body weight. The NN-XIa-PLA2 is also cytotoxic to Ehrlich ascites tumour cells. The other PLA2, NN-XIb, in contrast has an i.p. LD50 of 0.22 mg/kg body weight, and it induces acute neurotoxicity. The NN-XIb-PLA2 is devoid of the other biological activities which are exhibited by NN-XIa-PLA2.     

Acute toxicity in guinea pigs and rabbits of soot from a polychlorinated biphenyl-containing transformer fire

A fire involving a polychlorinated biphenyl (PCB)-containing transformer extensively contaminated the State Office Building in Binghamton, New York, with a sootlike material containing 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD), 2,3,7,8-tetrachlorodibenzofuran, and high concentrations of numerous other polychlorinated dibenzodioxins, dibenzofurans, and PCBs. The oral LD50s of the soot and of its benzene extract, each administered to female guinea pigs in 0.75% aqueous methyl cellulose, were 410 mg of soot/kg and 327 mg of soot equivalent/kg, respectively. Serum triglycerides were elevated in males at 100 and 500 mg/kg and in females at 500 mg/kg. Alkaline phosphatase was lowered in females at 500 mg/kg. Histopathology revealed dose-related pancreatic duct hyperplasia and salivary gland duct metaplasia in males. Body weight loss was observed in both sexes at 500 mg/kg. Thymus weight decreased in both sexes at 100 and 500 mg/kg, and kidney weights decreased in males at these doses. Dermal application of soot to rabbits for 24 hr caused no overt toxicity, although hepatic centrilobular hypertrophy was observed in both sexes. Similar application of soot extract caused a local serous inflammation in addition to the hepatic centrilobular hypertrophy. The oral LD50 for 2,3,7,8-TCDD in female guinea pigs was 19 μg/kg in aqueous methyl cellulose and 2.5 μg/kg in corn oil. We concluded that the soot matrix alters the dermal but not the oral toxicity of its components, that the toxic effects were consistent with those reported after exposure to dibenzodioxins and dibenzofurans, and the aqueous vehicle markedly diminished the acute toxicity of 2,3,7,8-TCDD relative to that in corn oil vehicle.     

Acute toxicity of nimbolide and nimbic acid in mice, rats and hamsters

Nimbolide and nimbic acid are toxic to mice only when given i.p. and i.v. but they are less toxic to rats and hamsters. The LD50 values of a single i.p. administration of nimbolide to adult male, female and weanling mice were 225, 280 and 240 mg/kg body wt, respectively, and its i.v. LD50 value was decreased to 24 mg/kg body wt in adult male mice. No fatality was observed when nimbolide was given i.g., i.m. and s.c. to adult male mice. Estimated LD50 values of nimbolide in rats and hamsters were somewhat higher than 600 and 500 mg/kg body wt. After 12-23 h i.p. administration of a lethal dose, most animals died of possible dysfunctions in kidney (tubular necrosis), small intestine (hemorrhagic necrosis), pancreas (acinar cell necrosis) and liver (mild fatty infiltration and focal necrosis). In contrast, mice and rats given a lethal dose of nimbolide (i.v.) died of a marked and sudden drop in arterial blood pressure and respiratory paralysis within about 1-18 min. Nimbic acid was less toxic to mice with an i.v. LD50 value of 265 mg/kg body wt and i.p. and i.g. LD50 values of higher than 600 mg/kg body wt. The possible cause of death induced by nimbic acid may be similar to that of nimbolide given i.v. and this is a sudden hypotensive shock.     

Nitroprusside intoxication: protection of alpha-ketoglutarate and thiosulphate.

Protection against the lethal effects of sodium nitroprusside (SNP) was observed in mice after treatment with alpha-ketoglutarate (AKG), either alone or in combination with sodium thiosulphate (STS). The LD50 of SNP was 12.0 (11.0-13.0) mg/kg in mice. Ip injection of AFG (500 mg/kg twice in 20 min) increased the LD50 1.7-fold in mice. STS (1 g/kg, ip) alone increased the LD50 5.5-fold. Furthermore, combined administration of AKG and STS increased the LD50 6.9-fold. SNP elicited increased cyanide levels in blood of mice in a dose-dependent manner. SNP (10 mg/kg, sc) administration gave rise to blood cyanide levels of 73.2 +/- 3.0 microM, 30 min after treatment. Ip injection of AKG significantly decreased blood cyanide levels by 30% in mice 30 min after treatment with 10 mg SNP/kg. A single injection of STS (1 g/kg) or a combination of AKG and STS reduced in blood cyanide levels by 88 or 98%, respectively, in mice after treatment with 10 mg SNP/kg. In addition, the increase in blood cyanide levels induced by injection of 50 mg SNP/kg was markedly inhibited by a combination of AKG and STS or (to a lesser extent) by STS alone. These results suggest that the combined administration of AKG and STS, by preventing the increase in blood cyanide levels induced by SNP, may afford protection against the toxic effects of SNP.