索曼中毒运动终板乙酰胆碱酯酶活性和接头传递功能的关系

用大鼠的在体膈肌局部索曼(Soman)中毒法,观察了乙酰胆碱酯酶(ACHE)在运动终板的再生和肌接头传递功能的恢复过程,以及肟类药物(HI-6)对两过程的促进作用。提出终板 AChE 活性与高频间接刺激(100次/秒)引起膈肌强直收缩幅度有一定的关系。中毒早期(30分钟以内)HI-6对索曼抑制的膈肌终板 AChE 有一定的重活化作用,并能相应地恢复肌接头的传递功能。     

Genetic Factors in Susceptibility: Serum PON1 Variation Between Individuals and Species

In mammals, serum paraoxonase (PON1) is tightly associated with high-density lipoprotein (HDL) particles. In human populations, PON1 exhibits a substrate dependent activity polymorphism determined by an Arg/Gln (R/Q) substitution at amino acid residue 192. The physiological role of this protein appears to be involvement in the metabolism of oxidized lipids. Several studies have suggested that the PON1_R192 allele may be a risk factor in coronary artery disease. PON1 also plays an important role in the metabolism of organophosphates including insecticides and nerve agents. The PON1_R192 isoform hydrolyzes paraoxon rapidly, but diazoxon, soman and sarin slowly compared with the PON1_Q192 isoform. Both PON1 isoforms hydrolyze phenylacetate at approximately the same rate, while PON1_R192 hydrolyzes chlorpyrifos oxon slightly faster than PON_Q192. Animal model studies involving injection of purified rabbit PON1 into mice clearly demonstrated the ability of PON1 to protect cholinesterases from inhibition by OP compounds. The consequence of having low PON1 levels has been addressed with toxicology studies in PON1 knockout mice. These mice showed dramatically increased sensitivity to chlorpyrifos oxon, diazoxon and some increased sensitivity to the respective parent compounds. These observations are consistent with earlier studies that showed a good correlation between high rates of OP hydrolysis by serum PON1 and resistance to specific OP compounds. They are also consistent with the observations that newborns have an increased sensitivity to OP toxicity, due in part to their not expressing adult PON1 levels for weeks to months after birth, depending on the species. Together, these studies point out the importance of considering the genetic variability of PON1₁₉₂ isoforms and levels as well as the developmental time course of PON1 appearance in serum in developing risk assessment models     

梭曼对大鼠应激性体温过高的作用及中枢和外周胆碱能受体阻断剂对其效应的影响

目的：观察梭曼对大鼠应激性体温过高的抑制作用以及中枢和外周胆碱能受体阻断剂对其效应的影响。方法：用无线遥测技术测量大鼠的体温,观察皮下注射梭曼、东莨菪碱、甲基东莨菪碱和吡啶斯的明对开放环境中大鼠应激性体温过高的影响。用分光光度技术测定血浆中胆碱酯酶活性。结果：①对照组大鼠在开放实验箱中体温升高达0.96℃,而注射梭曼动物体温只升高了0.55℃。中枢性胆碱能受体阻断剂东莨菪碱几乎完全阻断梭曼对应激体温过高的抑制作用,而外周胆碱能受体阻断剂甲基东莨菪碱则能明显增强梭曼对应激性体温过高的抑制作用。②外周抗胆碱酯酶剂吡啶斯的明能使血浆胆碱酯酶的活性降低至52%,同时明显提高开放环境中大鼠应激性体温过高的反应。甲基东莨菪碱几乎可以阻断吡啶斯的明对应激体温过高反应的影响。结论：神经毒剂梭曼可改变大鼠在开放环境中应激性体温过高的反应能力,其作用主要是通过中枢毒蕈碱型胆碱能通路所致。此外,外周胆碱能神经参与大鼠开放应激性体温过高的形成过程。     

梭曼对大鼠摄食中枢的抑制作用

采用双侧下丘脑外例区（摄食中枢）局部给药的方法,观察梭曼对大鼠摄食中枢的作用。实验结果表明,双侧下丘脑摄食中枢各注入梭曼３μｇ,给药当日大鼠平均摄食量下降６０．９％,抑制作用持续３ｄ,与给药前比较,差别显著（Ｐ＜０．０１或０．０５）;各注入阿托品０．１ｍｇ,平均摄食量下降４１．０％,第２天后恢复正常。胆碱酪酶重活化剂ＨＩ－６与梭曼同时注入下丘脑摄食中枢,或者中枢注入梭曼后立即肌注阿托品、美加明,均可部分对抗梭曼引起的中枢性摄食抑制。说明梭曼抑制大鼠摄食中枢与乙酰胆碱酯酶及乙酰胆碱受体确实有密切关系。     

DNA aptamers developed against a soman derivative cross‐react with the methylphosphonic acid core but not with flanking hydrophobic groups

Twelve rounds of systematic evolution of ligands by exponential enrichment (SELEX) were conducted against a magnetic bead conjugate of the para‐aminophenylpinacolylmethylphosphonate (PAPMP) derivative of the organophosphorus (OP) nerve agent soman (GD). The goal was to develop DNA aptamers that could scavenge GD in vivo, thereby reducing or eliminating the toxic effects of this dangerous compound. Aptamers were sequenced and screened in peroxidase‐based colorimetric plate assays after rounds 8 and 12 of SELEX. The aptamer candidate sequences exhibiting the highest affinity for the GD derivative from round 8 also reappeared in several clones from round 12. Each of the highest affinity PAPMP‐binding aptamers also bound methylphosphonic acid (MPA). In addition, the aptamer with the highest overall affinity for PAPMP carried a sequence motif (TTTAGT) thought to bind MPA based on previously published data (J. Fluoresc 18: 867–876, 2008). This sequence motif was found in several other relatively high affinity PAPMP aptamer candidates as well. In studies with the nerve agent GD, pre‐incubation of a large molar excess of aptamer candidates failed to protect human butyrylcholinesterase (BuChE) from inhibition. With the aid of three‐dimensional molecular modeling of the GD derivative it appears that a hydrophilic cleft sandwiched between the pinacolyl group and the p‐aminophenyl ring might channel nucleotide interactions to the phosphonate portion of the immobilized GD derivative. However, bona fide GD free in solution may be repulsed by the negative phosphate backbone of aptamers and rotate its phosphonate and fluorine moieties away from the aptamer to avoid being bound. Future attempts to develop aptamers to GD might benefit from immobilizing the pinacolyl group of bona fide GD to enhance exposure of the phosphonate and fluorine to the random DNA library. Copyright © 2008 John Wiley & Sons, Ltd.     

Electrophoretic study of aged butyrylcholinesterase after inhibition by soman

The following states of purified tetrameric form (C₄) of human plasma butyrylcholinesterase were studied by electrophoretic techniques: native, inhibited by soman and by methane sulfonyl fluoride and soman-aged.In order to detect a significant conformational change of the aged cholinesterase as compared to the non-inhibited (native) species, enzymes were treated with a set of bifunctional reagents (diimidates) of different chain lengths. After denaturation, the cross-link products were subjected to sodium dodecyl-sulfate polyacrylamide gel electrophoresis. The peak areas of the cross-linked species and the parameters of cross-linkability were calculated from densitometric data, versus the maximal effective reagent length.The effect of occupancy of the esteratic site by substituted phosphonyl group and by methyl-sulfonyl residue on the binding activity of the anionic site was studied by affinity electrophoresis at varying temperatures with immobilized-procaïnamide as ligand. Apparent dissociation-constants of the enzyme-ligand complexes were estimated from measurement of mobilities versus ligand concentration. Corresponding thermodynamic quantities were calculated from Van't Hoff plots and basic thermodynamic equations.The reactivity of aged-cholinesterase with diimidates was similar to that of the native enzyme. Affinity for immobilized-procaïnamide was slightly lowered in aged and inhibited enzymes as compared to the native and sulfonylated enzymes. As for the ligand-induced isomerization of anionic site (A → B), revealed by affinity electrophoresis, the ligand concentration at the midpoint of transition ($\text{A}\text{= 0.5}$) was slightly greater for the aged enzyme than for the native one.From these results, the following conclusions can be drawn: the dealkylation of somancholinesterase conjugate (aging) does not seem to induce structural changes detectable in the cross-linkability of lysyle residues at the subunit interfaces andthe surface of the tetrameric enzyme. On the other hand, the affinity of the anionic site and ligand-induced isomerization process are altered in soman-inhibited and aged enzymes. These data suggest the occurence of a weak conformational change of the active center and/or the formation of non-covalent bonds between the methylphosphonyl residue and side chain groups as a result of the dealkylation reaction.     

Deriving Toxicity Values for Organophosphate Nerve Agents: A Position Paper in Support of the Procedures and Rationale for Deriving Oral RfDs for Chemical Warfare Nerve Agents

During the process of deriving oral Reference Dose (RfDs) values for chemical warfare agents, several issues arose regarding the identification of adverse effect levels and the application of uncertainty factors. For those agents that function as cholinesterase inhibitors (e.g., agents VX, GA, GB, and GD), these issues included the following: (1) Is the endpoint of blood cholinesterase inhibition an indicator of toxicity or a biomarker of exposure? (2) Can an experimental animal species be more sensitive than humans, thereby eliminating the need for an animal-to-human uncertainty factor? (3) Can the uncertainty factor that is used to extrapolate from a lowest-observed adverse-effect-level (LOAEL) to a no-observed-adverse-effect-level (NOAEL) be less than the default value of 10? (4) Can an oral RfD be derived from non-oral toxicity data? (5) Can an uncertainty factor of less than 10 be used to extrapolate from subchronic to chronic exposure (e.g., is the critical effect adequately described by the subchronic exposure data)? (6) What constitutes an adequate data base for organophosphate cholinesterase inhibitors, and what uncertainty factor should be used for an incomplete data base? Analysis of relevant data resulted in the following selection and justifications of uncertainty factors. For uncertainty associated with intraspecies extrapolation (UF_H), physiologic and pathologic conditions affecting cholinest-erase activity levels justified maintaining a UF_H of 10 for all of the nerve agents. Because available data indicated that humans tended to be more sensitive than rats regarding anticholinesterase effects, an interspecies variability (UF_A) factor of 10 was retained for agents GA, GB, and GD. For agent VX, however, the available data revealed that the domestic sheep test species exhibited sensitivity equivalent to or greater than that of humans thereby justifying a UF_A of 1. For uncertainties regarding extrapolation from subchronic-to- chronic exposure data, consideration of information on the physiology of cholinergic systems and the available toxicity data for the nerve agents and other cholinest-erase inhibitors indicated that a UF_S of 3 was justified for all four of the nerve agents. For uncertainties regarding LOAEL-to- NOAEL extrapolation (UF_L), the selection of agent GB, GD, and VX doses resulting in cholinesterase inhibition in the absence of clinical signs of toxicity (biomarker of exposure) justified this endpoint as a minimal LOAEL and a UF_L of 3. For agent GA, a NOAEL was used, and therefore no UF_L was required. The uncertainty factor for data base completeness (UF_D), was based upon several considerations. Of primary concern was the fact that chronic toxicity studies are not considered an essential component of the data base requirements for cholinesterase inhibitors because of the unlikelihood that the endpoint will change with an increase in exposure time beyond that defined as a subchronic exposure. Additionally, limited data regarding reproductive and developmental toxicity were not considered to represent critical toxicity endpoints for the nerve agents or cholinesterase inhibitors in general. Although the data base for agents GA, GB, and GD were lacking reproductive and developmental toxicity data to some extent, a UF_D of 3 was justified for the aforementioned reasons. The data base for agent VX was considered complete and a UF_D of 1 was selected for development of the RfD for this agent. A modifying factor (MF) to reflect qualitative assessment of additional uncertainties in the critical study or data base that are not addressed by uncertainty factors was limited to agent GA due to the route-to-route (i.e., intraperitoneal to oral) extrapolation and to insure the equivalent oral NOAEL was not overestimated. This article provides a brief overview of the nerve agents, information on cholinergic systems that is pertinent to deriving toxicity values for nerve agents and other organophosphate cholinesterase inhibitors, and a discussion of key issues regarding the use of uncertainty factors in RfD derivations.     

不同性别及性腺功能对梭曼引起大鼠低温的影响

目的：研究不同性别及性腺功能对梭曼引起大鼠低温的影响。方法：用数字体温计测量大鼠的结肠温度,观察梭曼引起正常雄性和雌性大鼠低温反应的性别差异以及切除性腺后对其作用的影响。结果：①雌性大鼠对梭曼引起的低温反应比雄鼠更敏感。②切除雄性大鼠睾丸后能明显提高对梭曼低温反应的敏感性,而切除卵巢的雌性大鼠对梭曼的低温反应与模拟手术组无明显差异。结论：雄性和雌性大鼠对梭曼敏感性的性别差异主要取决于睾丸的功能。     

Competitive Irreversible Inhibition of Enzymes in the Presence of A Substrate: Scope and Limitations

Abstract

Rapid irreversible inhibition of enzymes constitutes a difficult problem and demands sophisticated techniques to meet contemporary expectations of accuracy and precision. Modern computerized, analytical techniques now allow inhibition to be measured in the presence of a chromogenic substrate, the decomposition product of which can be followed by a conventional method and in a continuous mode. This article has been written to fulfill a need for guidelines to aid the designer of experiments for the irreversible inhibition of enzymes. Thus the scope and limitations of the continuous competitive method for the irreversible inhibition of enzymes is examined here. Examples of acetylcholinesterase inhibition by two diagonally different phosphonate inhibitors are used for illustrating accuracy and precision of the competitive irreversible inhibition technique at different levels of enzyme saturation with inhibitor and substrate.     

Inhalation toxicity of soman vapor in non-anesthetized rats: A preliminary assessment of inhaled bronchodilator or steroid therapy

Respiratory toxicity, injury and treatment following vapor inhalational exposure to the chemical warfare nerve agent (CWNA) soman (GD) were examined in non-anesthetized rats. This study exposed male Sprague–Dawley rats (250–300 g) to 520, 560, 600, 825 or 1410 mg × min/m³ of soman in a customized head-out inhalation system. Signs of CWNA-induced cholinergic crises were observed in all soman-exposed animals. The LCt50 of vaporized soman as determined by probit analysis was 593.1 mg × min/m³. All animals exposed to 825 and 1410 mg × min/m³ developed severe convulsions and died within 4–8 min post-exposure. Edema measured by wet/dry weight ratio of the left lung lobe increased in a dose-dependent manner in all soman-exposed animals. Bronchoalveolar lavage (BAL) fluid and blood acetylcholinesterase (AChE) activities were inhibited dose-dependently in soman-exposed groups at 24 h. A significant increase in total BAL protein was observed in soman-exposed animals at all doses. AChE activity was inhibited in lung and whole brain tissues in all soman-exposed animals. Histopathological analysis of the lungs of animals exposed to 600 mg × min/m³ of soman revealed prominent morphological changes including alveolar histiocytosis, hemorrhage and inflammation consisting of neutrophilic exudate. Exposure of animals to 600 mg × min/m³ of soman followed by treatment with two actuations for 10 s of Combivent (21 μg of ipratropium bromide and 120 μg of albuterol sulfate) and Symbicort (80 μg budesonide and 4.5 μg formoterol) by inhalation into a modified metered dose inhaler (MDI) 10 min post-exposure resulted in increased minute volume, but did not decrease mortality. These results indicate that inhalation exposure to soman vapor causes acute respiratory toxicity and injury in untreated, un-anesthetized rats and that inhalation treatment with Combivent or Symbicort did improve the respiratory outcomes, but did not influence lethality.