Direct Cytotoxicity of Ethylcholine Mustard Aziridinium in Cerebral Microvascular Endothelial Cells

Abstract: The choline analogue ethylcholine mustard aziridinium (AF64A) is a potent and irreversible inhibitor of choline uptake in brain synaptosomes and is used as a neurotoxin to produce animal models of cholinergic hypofunction. However, previous studies have shown that intraocular administration of AF64A in rats not only reduced the number of cholinergic neurons in the retina, but also induced ultrastructural alterations in the microvasculature. The purpose of this study was to investigate whether AF64A has a direct cytotoxic effect on endothelial cells. As revealed by the measurement of lactate dehydrogenase activity in the culture medium, AF64A produced similar concentration-dependent cellular damage in cultures of bovine cerebral endothelial cells and in the human cholinergic neuroblastoma cell line SK-N-MC, but not in bovine cerebral smooth muscle cells. The toxic effect of AF64A correlated well with the affinity of the choline transport system detected in each cell type. The effect of the toxin on endothelial cells was mediated by its interaction with the endothelial cell choline carrier, as demonstrated by the following observations: (a) AF64A inhibited [³H]choline uptake in a concentration-dependent manner in both cultured and freshly isolated cerebral endothelial cells, and (b) the addition of choline or hemicholinium-3 to the culture medium prevented the AF64A-induced toxicity in endothelial cell cultures.     

Ethylcholine mustard aziridinium blocks the axoplasmic transport of acetylcholinesterase in cholinergic nerve fibres of the rat

A cholinotoxin, ethylcholine mustard aziridinium ion, (AF64A) specifically and irreversibly blocks the intraaxonal transport of acetylcholinesterase in the rat. Impairment of the transport of this enzyme in the septo-hippocampal cholinergic fibres and in the sciatic nerve has been studied, using different doses of AF64A. It is demonstrated that the effect on the axonal transport is dose-dependent, but is not related to the mode of drug application. AF64A thus may exert its neurotoxic effects on cholinergic neurons at several target sites of action. In addition to the localized presynaptic mechanisms, it may also be compromising cholinergic function by inhibiting axonal transport in vivo.     

Reversible and Irreversible Inhibition of High-Affinity Choline Transport Caused by Ethylcholine Aziridinium Ion

The effect of ethylcholine aziridinium ion (AF64A) on choline transport in hippocampal, striatal, and cerebrocortical synaptosomes was studied. Synaptosomes prepared from these three brain regions were equally sensitive to AF64A. Low concentrations of AF64A produced a reversible inhibition (IC50 values = 1.35-2.25 microM), whereas higher concentrations produced an irreversible inhibition (IC50 values = 25-30 microM), which started as competitive. The irreversible component of the inhibition was independent of extracellular Na+ concentration, a finding suggesting that the choline transporter is alkylated at its outward position. The kinetics of the inhibition were rapid and similar in the three brain regions examined. The high-affinity choline transport was more sensitive to the toxin than the low-affinity choline transport. Based on these results, we propose a kinetic model that explains the reversible and the irreversible inhibitions induced by AF64A. The possible relationships between the concentrations that in vitro produce reversible and irreversible inhibition and those that in vivo produce selective and nonselective cholinergic hypofunction are discussed.     

Effect of Nerve Growth Factor in Ethylcholine Mustard Aziridinium (AF64A)-Treated Rats: Sensitization of Cholinergic Enzyme Activity in the Septohippocampal Pathway

Abstract: In this study, we examined the effects of nerve growth factor (NGF) administration on cholinergic enzyme activity in both normal and ethylcholine mustard aziridinium (AF64A)-treated rats. Choline acetyltransferase (ChAT) and acetylcholinesterase activity were measured in the hippocampus and septum of rats chronically administered NGF (0.36–2.85 µg/day) into the lateral ventricle for 14 days. In both normal and AF64A-treated rats, NGF increased cholinergic enzyme activity in a dose-dependent manner. Furthermore, although NGF increased ChAT activity in normal rats by 147%, it had a greater effect in AF64A-treated rats, increasing ChAT activity as much as 273%. NGF increased acetylcholinesterase activity in normal rats by only 125% but produced a 221% increase in this activity in AF64A-treated rats. These data indicate that AF64A produces an increased sensitivity to NGF in cholinergic neurons.     

Ethylcholine mustard aziridinium blocks the axoplasmic transport of acetylcholinesterase in cholinergic nerve fibres of the rat

Summary A cholinotoxin, ethylcholine mustard aziridinium ion, (AF64A) specifically and ireversibly blocks the intraaxonal transport of acetylcholinesterase in the rat. Impairment of the transport of this enzyme in the septo-hippocampal cholinergic fibres and in the sciatic nerve has been studied, using different doses of AF64A. It is demonstrated that the effect on the axonal transport is dose-dependent, but is not related to the mode of drug application. AF64A thus may exert its neurotoxic effects on cholinergic neurons at several target sites of action. In addition to the localized presynaptic mechanisms, it may also be compromising cholinergic function by inhibiting axonal transport in vivo.     

DNA sequence analysis of mutations induced by N-2-acetylamino-7-iodofluorene in plasmid pBR322 in Escherichia coli

The spectrum of mutations induced by N-2-acetylamino-7-iodofluorene (AAIF) was analyzed in a forward mutation system based on mutagenesis directed to a small restriction fragment in the tetracycline resistance gene of plasmid pBR322. AAIF was found to induce frameshift mutations and base-pair substitutions at approximately equal frequencies. The frameshift mutations were mostly deletions of single base-pairs, but -2 frameshifts and +1 frameshifts were also detected. With one exception, the substitutions were transversions initiated at a G.C base-pair. Both frameshift mutations and transversions occurred preferentially at sites of repetitive guanine residues. Although AAIF and the related aromatic amines N-2-acetylaminofluorene (AAF) and N-2-aminofluorene (AF) all bind to the C-8 position of guanine, they have different effects on DNA conformation, and these differences are reflected in their mutation spectra. Previous studies have provided evidence that AAF adducts can trigger a B to Z conformational change in alternating GC sequences or displacement of the guanine by the fluorene ring in other sequences; the principal result is two classes of frameshift mutations. AF, whose DNA interaction involves outside binding rather than insertion and denaturation, primarily induces base-pair substitutions. AAIF adducts are chemically similar to AAF adducts, but the iodo group apparently hinders insertion of the fluorene ring into DNA. Consistent with this model, the mutation spectrum of AAIF combines properties of the mutation spectra of both AAF and AF.     

Selective Presynaptic Cholinergic Neurotoxicity Following Intrahippocampal AF64A Injection in Rats

Compound AF64A, ethylcholine mustard aziridinium ion (0.4-8 nmol) was stereotaxically administered into rat dorsal hippocampus, and neurochemical changes were determined 5 days later. AF64A treatment, over an almost 10-fold dose range, resulted in a significant (up to 70%) decline in choline acetyltransferase activity. In the same tissue samples, Na+-dependent choline transport activity was also lowered, with most decreases ranging between 10 and 50% of controls; however, there was no significant correlation (r = 0.39) between these two parameters. Acetylcholinesterase activity was not affected by AF64A treatment when assayed by either histochemical or enzymatic methods. AF64A reduced acetylcholine levels by 43%, but did not alter norepinephrine content or serotonin uptake. These results demonstrate that AF64A can induce a specific, long-term reduction of cholinergic presynaptic biochemical markers in rat hippocampus. Thus, AF64A can serve as a useful new tool to study the cholinergic system and as an important agent to help develop animal models representing disorders of central cholinergic hypofunction.     

Clonidine Prevents Transient Loss of Noradrenaline in Response to Cholinergic Hypofunction Induced by Ethylcholine Aziridinium (AF64A)

Intracerebroventricular injection of ethylcholine aziridinium (AF64A) (2 nmol/ventricle) induced a considerable decrease in the level of acetylcholine (ACh) in hippocampus (from 21.14 +/- 0.84 to 10.04 +/- 0.59 pmol/mg of tissue; p less than 0.001) 4 days after application. The reduction of cholinergic function was accompanied by a decrease in the level of noradrenaline (NA) (from 1.96 +/- 0.08 to 1.41 +/- 0.06 pmol/mg of tissue; p less than 0.001). Two days after administration of AF64A (1 or 2 nmol/ventricle), the dose-dependent decrease in NA level was associated with an increase in the level of its major metabolite, 3-methoxy-4-hydroxyphenylglycol (MHPG), resulting in a considerable increase in the MHPG/NA molar ratio (from 0.84 +/- 0.06 to 1.62 +/- 0.17; p less than 0.002). Chronic treatment of AF64A-injected rats with clonidine (0.02-0.2 mg/kg, i.p., every 8-12 h) had no significant effect on the loss of ACh content, whereas the decrease in NA content in hippocampus was completely prevented. Clonidine induced aggressive behavior in the AF64A-treated rats, in contrast to sedation in vehicle-injected rats. The response to clonidine under these experimental conditions and the increased MHPG/NA molar ratio in response to AF64A suggest that the transient loss of NA content following AF64A administration results from increased NA release. The increased noradrenergic activity in hippocampus may be linked to the reduction of tonic inhibitory cholinergic input. These results are discussed in relation to possible implications for senile dementia of the Alzheimer type.     

通过断裂内含肽介导的反式剪接合成大的蛋白

大肠杆菌难以表达大的蛋白,毒性蛋白以及膜蛋白,“Npu DnaE内含肽表达系统“使这些蛋白的表达成为可能。该系统的基本原理是：在特定位点处将目标基因（编码T7 RNA聚合酶的基因）断裂成两部分,然后分别与Npu DnaE内含肽的N端,C端片段融合,两种融合基因分别表达纯化,在体外将两种融合蛋白等摩尔比混合即可产生有功能的T7 RNA聚合酶。理论上,该体系也可用于合成其他大的蛋白,毒性蛋白或膜蛋白。     

AF64A: An Active Site Directed Irreversible Inhibitor of Choline Acetyltransferase

Ethylcholine mustard aziridinium ion (AF64A, MEChMAz) has been proposed as a cholinergic neuron-specific neurotoxin. We report that in further studies on its mechanism of action incubation of the cholinergic neuroblastoma X glioma cell line, NG-108-15, with 100 microM AF64A resulted in a rapid decrease in cellular choline acetyltransferase (ChAT) activity which preceded cytotoxicity. Thus, a 60-85% decrease in ChAT activity was measured within 5 h of AF64A exposure, whereas cell lysis (measured as the release of the cytosolic enzyme lactate dehydrogenase into the medium) did not become apparent until 18 h of AF64A exposure. This led us to examine the effects of AF64A on partially purified ChAT. We report a concentration- and time-dependent inhibition of partially purified ChAT by AF64A that could not be reversed by dialysis but could be prevented by coincubation of the enzyme and AF64A with choline but not with acetyl-coenzyme A. We present kinetic evidence that choline and AF64A compete for the same site on the enzyme. In addition, thiosulfate, which inactivates the aziridinium ion, eliminated AF64A's capacity to inhibit the enzyme. AF64A also irreversibly inhibited partially purified choline kinase and acetylcholinesterase but not lactate dehydrogenase, alcohol dehydrogenase, carboxypeptidase A, or chymotrypsinogen, enzymes that do not use choline as a substrate or product. Thus, the data suggest that AF64A acts as an irreversible active site directed inhibitor of ChAT and possibly other enzymes recognizing choline.     

Dosage-response relationships for methyl methanesulfonate in Drosophila melanogaster spermatozoa: DNA methylation per nucleotide vs. sex-linked recessive lethal frequency

Two different mechanisms for mutagenesis following treatment with methyl methanesulfonate (MMS) are suggested from the dose-response curve that is best fit by the linear quadratic model where m = 0.130D + 0.038D2 (D = dose measured as alkylations per nucleotide X 10(3), APdN; m = percent sex-linked recessive lethals, SLRL). A predominant component of the dose-response curve at moderate to high dose is the quadratic component which is interpreted as the result of two single-strand breaks. The distribution of methyl adducts in vivo is consistent with the previously determined in vitro distribution of methyl adducts on DNA following treatment with MMS. With the use of HPLC, 82% of the 3H-labeled adducts are found on the N-7 of guanine. It has previously been shown by both in vitro studies and in vivo correlation with mutagenesis that the N-7 alkyl guanine is not itself a predominately genotoxic lesion; however, N-7 alkyl guanine destabilizes guanine resulting in an increased rate of hydrolysis producing apurinic sites. In data presented in this paper, the loss of labeled adducts is shown to be at a rate consistent with hydrolysis of the destabilized alkyl guanine. The apurinic site thus produced should be converted to single-strand breaks by AP endonucleases once sperm has fertilized the egg. Single-strand breaks are repaired by excision repair which is not error-prone; however, multiple breaks producing a proximity effect should lead to double-strand breaks that are repaired by an error-prone process. Mutations that are induced by a proximity effect would account for the quadratic term. It is hypothesized that a proximity effect is produced when two breaks are sufficiently close together to prevent using the complementary strand as a template. The linear component of the dose-response curve is probably due to alkylation of oxygens in the purine or pyrimidine ring leading to mispairing. However, due to the low frequency of ring-oxygen alkylation following treatment with MMS, this important genotoxic site is not the predominant one observed at experimental levels normally used in the laboratory. From the dose-response curve, it is calculated that at mutation frequencies of 10 times the spontaneous frequency or higher, the predominant mechanism is the multi-hit component; however, at mutation induced frequencies of 0.1 of the spontaneous frequency, which are levels more likely to be encountered in man's exposure to environmental mutagens, the dominant mechanism is the linear component.(ABSTRACT TRUNCATED AT 400 WORDS)     

Alkylation of DNA by melphalan in relation to immunoassay of melphalan-DNA adducts: characterization of mono-alkylated and cross-linked products from reaction of melphalan with dGMP and GMP

A product expected to result from cross-linking of guanine bases in DNA by melphalan (4-(2-(di-guanin-7-yl))ethylamino-L-phenylalanine) was obtained from hydrolysis of melphalan-treated sodium deoxyguanylate at pH7 and characterized by U.V. and mass spectra. When tested in a competitive immunoassay using an antibody specific for melphalan-alkylated DNA it showed an affinity intermediate between that of melphalan-alkylated DNA and melphalan. From this and other assays it seemed possible that the cross-linked moiety in DNA was recognised by the antibody, but that its conformation differed from that of the free base tested, sufficiently to account for the discrepancy. It seemed possible that cross-linked guanine nucleotides would provide a better model, and these were therefore isolated, characterised and tested. Products derived from cross-linking of guanylic acid moieties through N-7 and N-7, and through N-7 and phosphate, had higher affinity than the cross-linked base, approximately the same as for alkylated native DNA, but less than for alkylated denatured DNA or RNA.