HI-6, an oxime which is an effective antidote of soman poisoning: A structure-activity study

In contrast to other organophosphates, soman poisoning is resistant to atropine (AT) + oxime therapy. However, new bispyridinium-type oximes + AT are effective antidotes of soman poisoning. In structure-activity studies, T 4925 (1,1′-[oxybis(methylene)]bispyridinium dichloride) had an LD₅₀ of 178 mg/kg and HS-14 (1-[[[pyridinio]methoxy]methyl]-2[(hydroxyiminio)methyl]pyridinium dichloride) had an LD₅₀ of 130 mg/kg. DL-10 (1-[[[3-(aminocarbonyl)pyridinio]methoxy]methyl]pyridinium dichloride) and DL-11 (1-[[[4-(aminocarbonyl)pyridinio]methoxy]methyl]pyridinium dichloride) had LD₅₀'s of 326 and 715 mg/kg, respectively, whereas HS-6 (Reg. No. 22625-23-6) and HI-6 (Reg. No. 34433-31-3) had LD₅₀s of 316 and 514 mg/kg (ip), respectively. T 4925, HS-14, DL-10, and DL-11 at an LD₁ dose + AT (17.4 mg/kg) were relatively ineffective against soman (540 μg/kg; sc) compared to HI-6 and HS-6. Prophylactic ED₅₀s for HI-6 and HS-6 vs soman were 17 and 110 mg/kg, producing safety ratios of 30 and 2.9, respectively. The therapeutic ED₅₀ for HI-6 was 20 mg/kg. Brain cholinesterase (ChE) in mice surviving soman (540 μg/kg; sc) + HI-6 (94 mg/kg) + AT was 0% of control activity at 1 and 2 hr, 5% at 24 hr, and 15% 48 hr later. Mice which died postsoman (540 μg/kg; sc) had 20% brain ChE activity. These results show that HI-6 was one of the least toxic and most efficacious compounds studied and suggest brain ChE inhibition was not the primary lesion in soman poisoning.     

Acute toxicity of berberine and its correlation with the blood concentration in mice

The aim of this study was to investigate the LD₅₀ (median lethal dosage) of berberine (BBR) through three different routes of injection in mice: intravenous (IV) injection, intraperitoneal (IP) injection, and intragastric (IG) oral administration. The concentration of BBR in blood from their IG doses (10.4, 20.8, 41.6, and 83.2 g/kg) and the content relationship of BBR among different injections were analyzed by high-performance liquid chromatography (HPLC). The LD₅₀ of BBR from IV and IP injections is 9.0386 and 57.6103 mg/kg, respectively; but no LD₅₀ was found in the IG group. A significant difference in bioavailability was observed between the different routes. Furthermore, the concentration of BBR in the blood from different IG doses was also significantly different. However, we discovered an interesting phenomenon indicating that the absorption of BBR by oral administration has a limit, therefore, explaining the difficulty in obtaining an LD₅₀ of BBR for IG injection. From the analysis of BBR content in blood after various administrations, we hypothesized that not only does the concentration of BBR in blood contribute to its acute toxicity, but also the routes of administration may be an important facet that affects this toxicity evaluation.     

Rubratoxin B mycotoxicosis in the Syrian hamster

The LD50 for rubratoxin B dissolved in dimethylsulphoxide and administered to Syrian golden hamsters by ip injection was 0.4 (0.2-0.8) mg/kg body weight. The greatest number of deaths occurred 6-24 hr after administration. Gross alterations consisted of congestion of the liver, spleen and kidneys and histopathological alterations involved congestion of the spleen and congestion and mild degenerative changes in hepatocytes. In a second study, rubratoxin B was administered ip daily for 1 wk at doses of 25, 50 and 75% of the ip LD50. Mortality was greatest in the 50 and 75% dose groups. Toxicity was cumulative with multiple doses. Gross alterations were similar to those found in the LD50 study. Histopathological alterations included renal tubular degeneration and necrosis and focal necrosis of hepatocytes. The morphopathogenesis of lesions following a single ip LD50 dose was evaluated in a third study. Histopathological alterations were limited to the kidney and were characterized by renal tubular degeneration and necrosis. Renal lesions were first seen at 2 hr after administration and increased in severity to a maximum at 20 hr. Tubular regeneration was first seen at 24 hr and was found to the end of the test period (72 hr). Serum activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) and serum concentrations of total and indirect bilirubin were increased by 8 hr after dosing and returned to control values by the end of the test period. In a fourth study, rubratoxin B was administered ip daily for 1 wk at a dose of 25% of the ip LD50. Gross alterations were similar to those in the other studies. Histopathological alterations included progressive renal tubular degeneration and necrosis. Serum activities of AST and ALT and concentration of blood urea nitrogen (BUN) were progressively increased with increasing numbers of doses. Urinalysis indicated progressive renal tubular damage.     

Natural Thiols,but Not Thioethers,Attenuate Patulin-Induced Endoplasmic Reticulum Stress in HepG2 Cells

Patulin, a mycotoxin, is known to have cytotoxic effects, but few studies have focused on the involvement of the endoplasmic reticulum (ER) stress response in patulin toxicity and the natural compounds that attenuate it in HepG2 cells. This study tested the ability of patulin to induce ER stress, and that of four thiols and three thioethers to attenuate patulin-induced ER stress in HepG2 cells. Patulin dose-dependently inhibited cell proliferation (IC₅₀, 8.43 μM). Additionally, patulin was found to increase the expression levels of ER stress-related genes and/or protein markers, including BiP, CHOP, and spliced XBP1, in HepG2 cells compared to the vehicle control, indicating its potential in ER stress induction. Patulin-induced cytotoxicity in HepG2 cells was reduced by naturally occurring thiol compounds (glutathione, L-acetyl-L-cysteine, cysteine, and captopril), but not by thioether compounds (sulforaphane, sulforaphene, and S-allyl-L-cysteine). Patulin-thiol co-treatment decreased CHOP expression and BiP and CHOP levels in HepG2 cells but did not alter BiP expression. Spliced XBP1 expression was decreased by patulin-thiol co-treatment. Thus, patulin induced ER stress in HepG2 cells and thiols, but not in thioethers, attenuated patulin-induced ER stress.     

抗癌新药-乙双吗啉(AT-1727)的药理研究

乙双吗啉(AT-1727)是我国合成的一种抗癌新药。它是一种双内酰亚胺化合物,实验证明,乙双吗啉对小鼠肉瘤S₃₇、S₁₈₀有显著抗肿瘤作用,对ECS,HCS,脑瘤B₂₂及L₆₁₅等移植性肿瘤亦有明显抗肿瘤作用。它对S₃₇的50%抑制剂量(ID₅₀)为1.88 mg/kg(ip)及6.61 mg/kg(po)。其抗肿瘤作用与给药方案有一定关系。乙双吗啉对小白鼠毒性LD₅₀为372.8±27.mg/kg(ip)及243.8±26.1 mg/kg(po)。因此,乙双吗啉腹腔注射及口服时,对S₃₇的化疗指数分别为47.5及36.9。给健康犬肌肉注射乙双吗啉25及50 mg/kg/天,连用10天,除出现食量减少,白细胞轻度下降外,对红细胞,血小板、肝、肾功能均无明显影响。乙双吗啉对以溶血素反应为指标的体液免疫有抑制作用;对以移植物抗宿主反应为指标的细胞免疫则无抑制作用。     

云谷霉素的抗肿瘤作用

来自云南省关坪自然保护区土壤的一株链霉菌产生的抗生素云谷霉素经鉴别确定为谷氏菌素。本研究通过口服给药,证明云谷霉素对小鼠结肠癌26和肉瘤180(实体型)有显著疗效;用小鼠可耐受剂量,对小鼠结肠癌26和肉瘤180的抑瘤率分别达85%和83%。提示云谷霉素用于治疗肿瘤的可能性。     

The Relative Bioavailability and Metabolism of Bisphenol A in Rats Is Dependent upon the Route of Administration

Bisphenol A (BPA) is used to produce polymers for food contactapplications, thus there is potential for oral exposure of humansto trace amounts via the diet. BPA was weakly estrogenic inscreening assays measuring uterine weight/response, althoughmuch higher oral doses of BPA were required to elicit a uterotropicresponse as compared to other routes of administration. Theobjective of this study was to determine if a route dependencyexists in the pharmacokinetics and metabolism of ¹⁴C-labeledBPA following single oral (po), intraperitoneal (ip), or subcutaneous(sc) doses of either 10 or 100 mg/kg to Fischer 344 rats. Resultsindicated a marked route dependency in the pharmacokineticsof BPA. The relative bioavailability of BPA and plasma radioactivitywas markedly lower following oral administration as comparedto sc or ip administration. The major fraction of plasma radioactivityfollowing oral dosing was the monoglucuronide conjugate of BPA(68–100% of plasma radioactivity). BPA was the major componentin plasma at Cmax following sc or ip administration exceededonly by BPA-monoglucuronide in females dosed ip. Up to fouradditional unidentified metabolites were present only in theplasma of animals dosed ip or sc. One of these, found only followingip administration, was tentatively identified as the monosulfateconjugate of BPA. The monoglucuronide conjugate was the majorurinary metabolite; unchanged BPA was the principal componentexcreted in feces. These results demonstrated a route dependencyof BPA bioavailability in rats, with oral administration resultingin the lowest bioavailability, and offer an explanation forthe apparent route differences in estrogenic potency observedfor BPA.     

Long-term carcinogenicity and toxicity studies of patulin in the rat

Patulin is a mycotoxin produced by a variety of Penicillium and Aspergillus species which are likely natural contaminants of various foods. The present study was conducted to determine the effects of lifetime administration of patulin in FDRL Wistar rats. Animals received patulin by gastric intubation three times per week at the level of 0.0, 0.1, 0.5 and 1.5 mg per kg body weight. The animals used in this lifetime study were derived from F0 parents exposed to equivalent levels of patulin for 4 weeks before mating, and throughout mating, gestation and lactation. Patulin treatment at 0.5 and 1.5 mg kg-1 to male rats caused a significant decrease in body weight gain in comparison to controls. Body weights of treated female rats were similar to that of control rats. No consistent significant differences among groups were noted in the hematology, clinical chemistry or urine analysis parameters measured during or at the termination of the study. Patulin administered to male and female rats at 1.5 mg kg-1 caused a significantly increased mortality rate as compared to respective control animals. The cause of death appeared to be increased pulmonary and laryngotracheal inflammation. No tumorigenic effect of patulin was observed.     

去甲肾上腺素能系统对埃必定镇痛作用的影响

在大鼠电刺激甩尾测痛模型上,sc或icv埃必定(ipalbidine,Ipa)均具有剂量依赖性的镇痛作用,而脊髓蛛网膜下腔注射Ipa人产生镇痛作用;预先给予利血平可以取消scIpa的镇痛作用,这一作用可被icv补充NE所翻转;电解损毁大鼠叹侧蓝斑,ip二乙基二硫代氨基甲酸钠200mg·kg^-1,酚妥拉明ip10mg·kg^-1或icv150μg和sc哌唑嗪5mg·kg^-1均能使Ipa的镇痛作用明显减弱或消失,而sc育亨宾5mg·kg^-1和ip普萘洛尔10mg·kg^-1对Ipa的镇痛作用无明显影响。上述结果提示Ipa在中枢有镇痛作用,其部位主要在脊髓以上的神经结构,Ipa的镇痛作用可能与去甲肾上腺素能系统的a₁受体有关,而与a₂和β受体无明显关系。     

The mycotoxin patulin increases colonic epithelial permeability in vitro

The gastrointestinal lumen is directly exposed to dietary contaminants, including patulin, a mycotoxin produced by moulds. Patulin is known to increase permeability across intestinal Caco-2 monolayers. This study aimed to determine the effect of patulin on permeability, ion transport and morphology in isolated rat colonic mucosae. Mucosal sheets were mounted in Ussing chambers and voltage clamped. Apical addition of patulin (100–500 μM) rapidly reduced transepithelial electrical resistance (TEER) and increased permeability to [¹⁴C] mannitol (2.9-fold). Patulin also inhibited carbachol-induced electrogenic chloride secretion and histological evidence of mucosal damage was observed. To examine potential mechanisms of action of patulin on colonic epithelial cells, high-content analysis of Caco-2 cells was performed and this novel, quantitative fluorescence-based approach confirmed its cytotoxic effects. With regard to time course, the cytotoxicity determined by high content analysis took longer than the almost immediate reduction of electrical resistance in isolated mucosal sheets. These data indicate patulin is not only cytotoxic to enterocytes but also has the capacity to directly alter permeability and ion transport in intact intestinal mucosae. These data corroborate and extend findings in intestinal cell culture monolayers, and further suggest that safety limits on consumption of patulin may be warranted.     

Correlation between the destruction of tight junction by patulin treatment and increase of phosphorylation of ZO-1 in Caco-2 human colon cancer cells

Patulin is a mycotoxin and its contamination of food has been reported to cause gastrointestinal inflammation, ulcers, and bleeding. The toxicity of patulin is thought to be due to the destruction of tight junctions (TJs) in gastrointestinal tissues. However, the precise mechanism has not been clarified. Here, we investigated the phosphorylation of TJ components. The transepithelial electrical resistance (TER) of Caco-2 human colon cancer cells decreased gradually during the first 24 h of treatment with 50 μM patulin. Immunofluorescence microscopy showed that the TJ proteins ZO-1 and claudin-4, but not occludin, had decreased after 24 h and decreased from the cell-cell contact regions of TJs after 48 h of patulin treatment. Western blotting showed that the level of ZO-1 decreased after 48 h of patulin treatment, but the levels of claudin-4 and occludin remained at the initial level until 72 h. Phosphorylation of ZO-1 was detected by 24 h and increased markedly after 72 h of patulin treatment. However, phosphorylation of claudin-4 and occludin was not detected by probing with anti-phosphotyrosine antibody. Immunoprecipitation showed that interaction of ZO-1 with claudin-4 had decreased after 48 h and was completely absent after 72 h. These results suggest that phosphorylation caused the degradation of ZO-1 protein and the decrease in TER induced by patulin treatment of Caco-2 cells.     

Evaluation of the reproductive toxicity of patulin in growing male rats

Patulin is a mycotoxin produced by several Penicillium, Aspergillus and Byssachlamys species. Patulin can be produced on different food products including fruits, grains, cheese, cured meats, but in natural situations patulin is exclusively found in apple and apple products. Patulin, at dose of 0.1 mg/kg bw/day, was administered by gavage to the growing male rats aged 5–6 week for 60 or 90 days. At the end of the experiment, sperm counts and morphology were investigated. Also, effects of patulin on the epididymis, seminal vesicle and prostate tissues were examined histopathologically and morphologically.

While sperm counts increased in patulin-treated rats for 60 days, sperm counts in patulin-treated rats for 90 days decreased compared to the corresponding control group. Patulin affected sperm morphology of growing male rats. Tail abnormalities like bent and/or coiled tails, and sticking of sperm tails were observed. A significant change was not determined in absolute and relative weights of the seminal vesicle and prostate of patulin-treated rats. While absolute cauda epididymal weights increased in rats treated with patulin for 60 days, absolute and relative cauda epididymal weights reduced in rats treated with patulin for 90 days. In histologic examination, some histopathological changes were observed in the epididymis and prostate tissues of rats in patulin treatment groups.     

Toxicological evaluation of palytoxin in several animal species

Playtoxin, the toxin extracted from the soft coral Palythoa vestitus, Verril 1928, found in the Hawaiian and other Pacific Islands, is a highly lethal substance in several animal species, and is effective by various routes of administration. Its intravenous LD₅₀ in the dog, rabbit, monkey, guinea pig, rat, and mouse range between 0·033 and 0·45 μg/kg. However, when palytoxin is given by the intragastric or intrarectal route, it is relatively non-toxic. Additionally, playtoxin produces marked irritant and tissue damage when topically applied to skin or eyes, as well as having a general necrotizing action on cells when injected.     

In vivo direct patulin-induced fluidization of the plasma membrane of fission yeast Schizosaccharomyces pombe

Patulin is a toxic metabolite produced by various species of Penicillium, Aspergillus and Byssochlamys. In the present study, its effects on the plasma membrane of fission yeast Schizosaccharomyces pombe were investigated. The phase-transition temperature (G) of untreated cells, measured by electron paramagnetic resonance spectrometry proved to be 14.1 °C. Treatment of cells for 20 min with 50, 500, or 1000 μM patulin resulted in a decrease of the G value of the plasma membrane to 13.9, 10.1 or 8.7 °C, respectively. This change in the transition temperature was accompanied by the loss of compounds absorbing light at 260 nm. Treatment of cells with 50, 500 or 1000 μM patulin for 20 min induced the efflux of 25%, 30.5% or 34%, respectively, of these compounds. Besides its cytotoxic effects an adaptation process was observed. This is the first study to describe the direct interaction of patulin with the plasma membrane, a process which could definitely contribute to the adverse toxic effects induced by patulin.     

Toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin in the Golden Syrian hamster

Lethality, pathology, and various clinical chemical parameters were assessed in the hamster following a single ip or po treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). A single dose, 50-day LD₅₀ of greater than 3000 μg TCDD/kg, ip, was obtained for male and female hamsters while the LD₅₀ of orally administered TCDD was found to be 1157 μg/kg. Thus, the hamster appears to be the least sensitive mammalian species to the lethal effect of TCDD that has yet been investigated. TCDD treatment generally reduced the rate of body weight gain, with orally treated hamsters exhibiting the greatest reduction in rate. Thymic atrophy was the most consistent pathologic finding in TCDD treated hamsters. No histopathological changes were seen in the liver, spleen, kidneys, adrenals, or heart. Moderate to severe ileitis and peritonitis were found in many of the hamsters which died following oral treatment with TCDD. This lesion usually affects the distal ileum and consists of a marked hyperplasia of the mucosal epithelium with mild to severe hemorrhaging and necrosis. The intestinal lesion probably contributed in part to the greater lethality of TCDD in orally treated hamsters. A significant increase in serum alkaline phosphatase, bilirubin, protein, iron, cholesterol, and a decrease in serum albumin, chloride, urea nitrogen, and triglycerides were found in hamsters following both ip and po treatment with TCDD.     

Pharmacokinetics of recombinant human granulocyte colony-stimulating factor in the rat. Single and multiple dosing studies.

The pharmacokinetics of recombinant human granulocyte colony-stimulating factor (rhG-CSF) were investigated in male Sprague-Dawley rats at a dose of 5 micrograms/kg. The serum concentrations of rhG-CSF were monitored using a specific sandwich enzyme immunoassay. In single-dose studies, the influence of routes of administration were evaluated. For iv administration, the serum concentration-time data showed the rapid disappearance of rhG-CSF from the systemic blood and a mean residence time (MRT) of 1.341 hr. For sc, im, and ip administration, lower peak serum levels were observed, but after 2 to 3 hr, rhG-CSF levels were higher than those for iv administration. The MRTs after sc, im, and ip injections were 3.918, 2.894, and 3.538 hr, respectively. The serum concentration profiles after extravascular injections showed that an im injection gave slightly faster absorption kinetics of rhG-CSF from the injection site into systemic blood than did sc and ip injections. In multiple-dose studies, rhG-CSF was injected into animals iv and sc at 5 micrograms/kg/day for 7 days. On the day 7 the serum concentration-time profiles after rhG-CSF administration were compared between single and multiple dosing. The AUC after iv multiple dosing decreased by 17.4%, although half-lives and the volume of distribution were not significantly different between single and multiple dosing groups. The AUC after sc multiple dosing decreased by 25.6%; however, the bioavailability and observed maximum serum concentration of rhG-CSF were not significantly different. These results showed that the clearance of rhG-CSF increased after multiple dosing, although the mechanism of increased clearance was not apparent.     

The experimental toxicology of tramadol: an overview

The experimental toxicological findings of tramadol are reviewed and discussed. Tramadol is a centrally acting analgesic. In acute toxicity studies, LD₅₀ values are estimated to be around 300–350 mg/kg body weight (rat, mouse, oral administration). After intravenous administration the LD₅₀ values ranged from 50 to 100 mg/kg body weight. In subacute and chronic toxicity studies, clinical signs of intoxication are mainly behavioural disorders and convulsions, beginning at dose levels of 25 mg/kg. Clinical–pathological alterations or morphological lesions, in particular neuropathological findings were not detected. Overall, the battery of mutagenicity studies shows no evidence of a genotoxic risk to man. Reproductive and developmental toxicity investigations and carcinogenicity studies were without substance-dependent findings. Toxicological and toxicokinetical data of both enantiomers did not show biologically relevant deviations in comparison to the data on tramadol. The toxicological characteristic of this compound is demonstrated.     

Quantitative determination of biological activity of botulinum toxins utilizing compound muscle action potentials (CMAP), and comparison of neuromuscular transmission blockage and muscle flaccidity among toxins

The biological activity of various types of botulinum toxin has been evaluated using the mouse intraperitoneal LD₅₀ test (ip LD₅₀). This method requires a large number of mice to precisely determine toxin activity, and so has posed a problem with regard to animal welfare. We have used a direct measure of neuromuscular transmission, the compound muscle action potential (CMAP), to evaluate the effect of different types of botulinum neurotoxin (NTX), and we compared the effects of these toxins to evaluate muscle relaxation by employing the digit abduction scoring (DAS) assay.This method can be used to measure a broad range of toxin activities the day after administration. Types A, C, C/D, and E NTX reduced the CMAP amplitude one day after administration at below 1 ip LD₅₀, an effect that cannot be detected using the mouse ip LD₅₀ assay. The method is useful not only for measuring toxin activity, but also for evaluating the characteristics of different types of NTX. The rat CMAP test is straightforward, highly reproducible, and can directly determine the efficacy of toxin preparations through their inhibition of neuromuscular transmission. Thus, this method may be suitable for pharmacology studies and the quality control of toxin preparations.     

Immunological evaluation of the mycotoxin patulin in female b6C3F1 mice

Patulin is a mycotoxin produced by many fungal species of the genera Penicillium, Aspergillus and Bryssochamys. Previous literature reports have suggested that patulin is toxic to the immune system. The studies presented were conducted to provide a comprehensive assessment of the effects of patulin on the immune system. Unlike previous reports, the doses of patulin used (0.08, 0.16, 0.32, 0.64, 1.28 and 2.56 mg/kg) were based on predicted human exposure levels. Female B6C3F₁ mice were exposed orally to patulin for 28 days. Effects were not observed on final body weight or body weight gain. Relative weight of the liver, spleen, thymus, kidneys with adrenals, and lungs was not affected. Peripheral blood leucocyte and lymphocyte counts were decreased by approximately 30% in the two highest dose groups. The leucocyte differential was not altered. Total spleen cell, total T-cell (CD3⁺), helper T-cell (CD4⁺CD8⁻), B-cell (surface immunoglobulin⁺) and monocyte (MAC-3⁺) counts were not changed. Cytotoxic T-cell (CD8⁺CD4⁻) counts were increased 50% only by the highest dose. Natural killer cell (NK1.1⁺CD3⁻) and monocyte (MAC-1⁺) counts were increased 30% and 24%, respectively, only in the 0.08 mg/kg group. Humoral immune function as assessed by antibody-forming cell response and serum IgM titre to sheep erythrocytes, and cell-mediated immune function evaluated utilizing natural killer cell activity and the mixed lymphocyte reaction were not altered. Oral exposure to patulin for 28 days did not alter the ability of female B6C3F₁ mice to mount either a cell-mediated or humoral immune response.     

The value of acute toxicity testing of pharmaceuticals for estimation of human response

The determination of single high doses of active pharmaceutical ingredients (API) is used mostly to fulfill regulatory demands. Oral LD₅₀ values in animals for over 300 API were compared to the minimal effective therapeutic doses (METD) in humans in order to find a correlation between animal and human data. The highest correlation between human METD and animal LD₅₀ was found for the dog (R = 0.323), the lowest for the rat (0.287). It was determined that acute oral LD₅₀ of rats have poor correlation with the METD, and cannot be used as a classification criteria into official acute toxic categories. Only 13% of API has been classified as fatal if swallowed according to the EU CLP regulation, none of the substances with very low therapeutic dose have been identified as EU CLP acute toxicity category 1. Substances with very low therapeutic doses, which could potentially have toxic effects in humans, are not identified with the use of oral LD₅₀ and current classification system. We propose that the acute toxicity based on rat LD₅₀ dose is not used as a basis for classification of pharmaceuticals, and that the METD is applied as basis for classification.