Oxidative stress on land snail Helix aspersa as a sentinel organism for ecotoxicological effects of urban pollution with heavy metals.

The oxidative stress in the digestive gland of the land snail Helix aspersa was considered as a bioindicator for atmospheric pollution with heavy metals from several industries and vehicular traffic in Kafr El-Zayat city. Regional means of heavy metals concentration of all sites were 0.71, 7.09, 0.71, 2.68, 41.44 and 18.01 mg kg(-1) wet mass for Cd, Mn, Ni, Pb, Zn and Cu, respectively. In addition, the highest values of Cd concentrations were found 1.22 and 1.73 mg kg(-1) wet mass in S1 (Potato International Center) and S4 (The Nile bank), respectively. Lactate dehydrogenase (D-LDH(and recorded lipid peroxidation (LPO) levels were significantly high in S1 and S2 (Traffic station). On the other hand, the highest activity of catalase (CAT) was found in S2 (194.04% of control), while the activity of glutathione peroxidase (GPx) reached the highest significant value in S1. As a matter of fact, glutathione-S-transferase (GST) and glutathione reductase (GR) activities were significantly higher in polluted sites than in reference zone. In contrast, the glutathione (GSH) concentration of exposed animals showed significant decrease in all sites, with the lowest value in S1 (57.61% of control). However, metallothioneins concentration (MT) showed no significant difference in all sites except in S1 which accounted for 127.81% of control. Therefore, the overall results of this study showed the importance of H. aspersa as a sentinel organism for biomonitoring the biologic impact of atmospheric pollution in urban areas.

Organophosphorus pollutants (OPP) in aquatic environment at Damietta Governorate, Egypt: implications for monitoring and biomarker responses.

The study was carried out from spring 1999 to spring 2001 to monitor the residue levels of organophosphorus pollutants (OPP) in aquatic environment of the drainage canal surrounding a pesticide factory at Damietta Governorate. Water, sediment, and fish samples were collected at six different seasonal periods. OPPs were analyzed by GLC and confirmed using GC-MS. Chlorpyrifos, chlorpyrifos-methyl, malathion, diazinon, pirimiphos-methyl and profenofos were detected in most samples. Chlorpyrifos was dominant in all water and sediment samples. It was ranged from 24.5 to 303.8 and 0.9 to 303.8 ppb in water and sediment samples, respectively. Diazinon level was slightly similar to chlorpyrifos in fish samples. Data based on the grand total concentration of OPP showed that the most polluted samples were collected either at spring 1999 or autumn 2000. They were 675.5 and 303.8 ppb in water samples and 43.0 and 52.2 ppb in fish collected at spring 1999 and autumn 2000, respectively. The obtained results are in parallel to that found in case of cholinesterase activity where the activity of both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) was declined at these seasonal period. The activity levels of AChE and BuChE were found to be 77.18% and 59.67% of control at spring 1999 and 78.62% and 85.80% of control, at autumn 2000, respectively. Thus, AChE and BuChE could be used as biomarkers for tracing and biomonitoring OPP pollution.

Characterization of a vertebrate neuromuscular junction that demonstrates selective resistance to botulinum toxin.

Botulinum toxin blocks transmitter release by proceeding through a series of four steps: binding to cell surface receptors, penetration of the cell membrane by receptor-mediated endocytosis, penetration of the endosome membrane by pH-induced translocation, and intracellular proteolysis of substrates that govern exocytosis. Each of these steps is essential for toxin action on intact cells. Therefore, alterations in cell structure or cell function that impede any of these steps should confer resistance to toxin. In the present study, screening for susceptibility to four serotypes of botulinum toxin revealed that the cutaneous-pectoris nerve-muscle preparation of Rana pipiens is resistant to type B botulinum toxin. Resistance was demonstrated both by electrophysiologic techniques and by dye-staining techniques. In addition, resistance to serotype B was demonstrated at toxin concentrations that were 2 orders of magnitude higher than those associated with blockade produced by other serotypes. In experiments on broken cell preparations, type B toxin cleaved synaptobrevin from frog brain synaptosomes. However, the toxin did not bind to frog nerve membranes. These findings suggest that resistance is due to an absence of cell surface receptors for botulinum toxin type B. The fact that cutaneous-pectoris preparations were sensitive to other botulinum toxin serotypes (A, C, and D), as well as other neuromuscular blocking agents (alpha-latrotoxin, beta-bungarotoxin), indicates that botulinum toxin type B receptors are distinct.

Expression of botulinum toxin binding sites in Xenopus oocytes.

The binding of iodinated botulinum toxin type B to nerve membranes was studied by using rat and mouse preparations. The toxin was examined both in the single-chain and in the proteolytically processed dichain form, and binding sites both in the spinal cord and in various brain regions were assayed. Rat and mouse brains possessed specific binding sites for botulinum toxin type B. The average Kd values for the various rat and mouse membrane preparations examined were 4.2 +/- 0.7 nM and 3.7 +/- 0.9 nM, respectively. The average Bmax values for the same tissue preparations were 7.3 +/- 0.7 pmol/mg of protein and 7.5 +/- 1.9 pmol/mg protein, respectively. The binding of botulinum toxin type B to rat brain membranes was not antagonized by a polyclonal antibody against the cytosolic domain of synaptotagmin 1 or by a monoclonal antibody directed against the luminal domain of synaptotagmin 1. In addition, these antibodies did not protect the mouse phrenic nerve-hemidiaphragm from toxin-induced neuromuscular blockade. Extraction of whole-brain mRNA and injection into Xenopus oocytes led to expression of binding sites for botulinum toxin. Extraction and injection of cerebellar mRNA led to expression of a higher density of binding sites. The number of binding sites was not diminished when oocytes were pretreated with antibodies against the cytosolic and luminal domains of synaptotagmin 1. These findings are likely to aid in the isolation, characterization, and reconstitution of toxin binding sites.

Actions of the insecticide 2(nitromethylene)tetrahydro-1,3-thiazine on insect and vertebrate nicotinic acetylcholine receptors.

The nitromethylene heterocyclic compound 2(nitromethylene)tetrahydro)1,3-thiazine (NMTHT) inhibits the binding of [125I]alpha-bungarotoxin to membranes prepared from cockroach (Periplaneta americana) nerve cord and fish (Torpedo californica) electric organ. Electrophysiological studies on the cockroach fast coxal depressor motorneuron (Df) reveal a dose-dependent depolarization in response to bath-applied NMTHT. Responses to ionophoretic application of NMTHT onto the cell-body membrane of motorneuron Df are suppressed by bath-applied mecamylamine (1.0 x 10(-4) M) and alpha-bungarotoxin (1.0 x 10(-7) M). These findings, together with the detection of a reversal potential close to that estimated for acetylcholine, provide evidence for an agonist action of this nitromethylene on an insect neuronal nicotinic acetylcholine receptor. The binding of [3H]H12-histrionicotoxin to Torpedo membranes was enhanced in the presence of NMTHT indicating an agonist action at this vertebrate peripheral nicotinic acetylcholine receptor. NMTHT is ineffective in radioligand binding assays for rat brain GABAA receptors, rat brain L-glutamate receptors and insect (Musca domestica) L-glutamate receptors. Partial block of rat brain muscarinic acetylcholine receptors is detected at millimolar concentrations of NMTHT. Thus nitromethylenes appear to exhibit selectivity for acetylcholine receptors and exhibit an agonist action at nicotinic acetylcholine receptors.

Desensitization of the nicotinic acetylcholine receptor by diisopropylfluorophosphate.

The interaction of diisopropylfluorophosphate (DFP) with the nicotinic acetylcholine (ACh) receptor of Torpedo electric organ was studied, using [3H]-phencyclidine ([3H]-PCP) as a reporter probe. Phencyclidine binds with different kinetics to resting, activated, and desensitized receptor conformations. Although DFP did not inhibit binding of [3H]-ACh or 125I-alpha-bungarotoxin (BGT) to the receptor recognition sites and potentiated in a time-dependent manner [3H]-PCP binding to the receptor's high-affinity allosteric site, it inhibited the ACh- or carbamylcholine-stimulated [3H]-PCP binding. This suggested that DFP bound to a third kind of site on the receptor and affected receptor conformation. Preincubation of the membranes with DFP increased the receptor's affinity for carbamylcholine by eightfold and raised the pseudo-first-order rate of [3H]-PCP binding to that of an agonist-desensitized receptor. Accordingly, it is suggested that DFP induces receptor desensitization by binding to a site that is distinct from the recognition or high-affinity noncompetitive sites.

Direct actions of organophosphate anticholinesterases on nicotinic and muscarinic acetylcholine receptors.

Four nerve agents and one therapeutic organophosphate (OP) anticholinesterase (anti-ChE) bind to acetylcholine (ACh) receptors, inhibit or modulate binding of radioactive ligands to these receptors, and modify events regulated by them. The affinity of nicotinic (n) ACh receptors of Torpedo electric organs and most muscarinic (m) ACh receptors of rat brain and N1E-115 neuroblastoma cultures for the OP compounds was usually two to three orders of magnitude lower than concentrations required to inhibit 50% (IC-50) of ACh-esterase activity. However, a small population of m-ACh receptors had an affinity as high as that of ACh-esterase for the OP compound. This population is identified by its high-affinity [3H]-cis-methyldioxolane ([3H]-CD) binding. Although sarin, soman, and tabun had no effect, (O-ethyl S[2-(diisopropylamino)ethyl)] methyl phosphonothionate (VX) and echothiophate inhibited competitively the binding of [3H]-quinuclidinyl benzilate ([3H]-QNB) and [3H]-pirenzepine ([3H]-PZ) to m-ACh receptors. However, VX was more potent than echothiophate in inhibiting this binding and 50-fold more potent in inhibiting carbamylcholine (carb)-stimulated [3H]-cGMP synthesis in N1E-115 neuroblastoma cells--both acting as m receptor antagonist. All five OPs inhibited [3H]-CD binding, with IC-50s of 3, 10, 40, 100, and 800 nM for VX, soman, sarin, echothiophate, and tabun, respectively. The OP anticholinesterases also bound to allosteric sites on the n-ACh receptor (identified by inhibition of [3H]-phencyclidine binding), but some bound as well to the receptor's recognition site (identified by inhibition of [125I]-alpha-bungarotoxin binding). Soman and echothiophate in micromolar concentrations acted as partial agonists of the n-ACh receptor and induced receptor desensitization. On the other hand, VX acted as an open channel blocker of the activated receptor and also enhanced receptor desensitization. It is suggested that the toxicity of OP anticholinesterases may include their action on n-ACh as well as m-ACh receptors if their concentrations in circulation rise above micromolar levels. At nanomolar concentrations their toxicity is due mainly to their inhibition of ACh-esterase. However, at these low concentrations, many OP anticholinesterases (eg, VX and soman) may affect a small population of m-ACh receptors, which have a high affinity for CD. Such effects on m-ACh receptors may play an important role in the toxicity of certain OP compounds.

Oxadiazolidinones: irreversible inhibition of cholinesterases and effects on acetylcholine receptors.

Inhibition of four acetylcholinesterases (AChE) and a butyrylcholinesterase (BuChE) by 3-(2,3-dihydro-2,2-dimethyl-benzofuran-'7-yl)-5-methoxy-1,3,4-oxadiaz ol-2(3H)-one (DBOX) and 3-(2-methoxyphenyl)-5-methoxy-1,3,4-oxadiazol-2(3H)-one (MPOX) was measured by the Ellman spectrophotometric method. Both oxadiazolidinones inhibited AChE and BuChE irreversibly and with quasi first order kinetics. DBOX was 2-3 orders of magnitude more potent than MPOX. Housefly brain AChE and horse serum BuChE were more sensitive than AChEs of red blood cells or eel and Torpedo electric organs. Aldicarb, a carbamate anticholinesterase, which protected Torpedo AChE against irreversible phosphorylation by DFP, also protected it against irreversible inhibition by DBOX and MPOX. It is suggested that the nonesteratic oxadiazolidinones are converted to carbanillates on the surface of the enzyme, then acylate the active site of ChEs, producing carbanillated enzymes. At higher concentrations, the two oxadiazolidinones also affected the specific binding of (125I) alpha-bungarotoxin (alpha-BGT) and [3H]perhydrohistrionicotoxin (H12-HTX) to Torpedo nicotinic ACh-receptors, but did not affect the specific binding of [3H]quinuclidinyl benzilate (QNB) to rat brain muscarinic ACh-receptors.

Sequential thin-layer chromatography of phosfolan, mephosfolan and related compounds.

A sequential thin-layer chromatographic method (STLC) has been developed to analyze the two organophosphorus insecticides phosfolan (O,O-diethyl 1,3-dithiolan-2-ylidenephosphoramidate) and mephospholan (O,O-diethyl 4-methyl-1,3-dithiolan-2-ylidenephosphoramidate) and some of their possible degradation products. Gelman type SA, Instant Thin Layer Chromatography (ITLC) silicic acid-impregnated glass fiber sheets were first developed up to 6 cm with the primary solvent 2-butanone-1-butanol-water (9:3:1), then after drying, to 16 cm with the secondary solvent acetonitrile-n-hexane-benzene-acetic acid (80:40:40:1). This two-solvent sequential system separated each insecticide from its corresponding metabolites. The R1 values were: phosfolan, 0.69; ethylene dithioimido-carbonate hydrochloride, 0.08; ethylene dithiocarbonate, 0.90; potassium thiocyanate, 0.40; mephosfolan, 0.76; O,O-diethyl 4-hydroxymethyl-1,3-dithiolan-2-amidate, 0.83; propylene dithioimidocarbonate hydrochloride; 0.15 and propylene dithiocarbonate, 0.87.

Activation, inactivation, and desensitization of acetylcholine receptor channel complex detected by binding of perhydrohistrionicotoxin.

The effects of receptor activation were studied on the interaction of perhydrohistrionicotoxin (H(12)-HTX) with the ionic channel of the nicotinic acetylcholine (AcCho) receptor in membranes from the electric organ of Torpedo ocellata and with the endplate region of the soleus muscle of the rat. In Torpedo membranes, the initial rate (i.e., within 30 sec) of [(3)H]H(12)-HTX bindings to the ionic channel of the AcCho receptor was accelerated 10(2)- to 10(3)-fold in the presence of carbamoylcholine (Carb). H(12)-HTX also inhibited Carb-activated (22)Na(+) influx, over 95% inhibition at 10 muM H(12)-HTX. At this concentration H(12)-HTX did not inhibit [(3)H]AcCho binding to the AcCho-receptor sites. There was good correspondence between the degree of acceleration of [(3)H]H(12)-HTX binding and the stimulation of (22)Na(+) influx over a wide range of Carb concentrations (up to 100 muM). Preincubation of Torpedo membranes with Carb decreased the initial rate of [(3)H]H(12)-HTX binding, as well as the rate of (22)Na(+) influx, which may reflect desensitization of the AcCho-receptor. d-Tubocurarine inhibited the agonist-mediated acceleration of [(3)H]H(12)-HTX binding and (22)Na(+) influx. In the soleus muscle endplate, H(12)-HTX inhibited the transient depolarization induced by microiontophoretic application of AcCho; the more receptors activated and channels opened, the stronger was the inhibition by H(12)-HTX. These findings suggest that H(12)-HTX binds to closed and open ionic channels, with a preference for the latter conformation. It is also suggested that the conformational changes associated with activation or desensitization of the receptor can be monitored by studying binding of [(3)H]H(12)-HTX to the ionic channel sites as well as by the AcCho-receptor-regulated (22)Na(+) influx.

Sorption-desorption characteristics of methyl parathion by clays.

Methyl parathion (O,O-dimethyl O-(4-nitrophenyl) phosphorothioate) adsorption was studied on montmorillonite, kaolinite, halloysite, natural zeolite, ion exchange resins and calcium carbonate. Methyl parathion was highly adsorbed by montmorillonite, followed by zeolite, and very little adsorption was obtained on kaolinite and halloysite. Calcium carbonate did not exhibit any adsorption. The values of the partial molar free energy "delta G" were calculated for all systems. For the montmorillonite-methyl parathion system, the calculated partial molar heat of adsorption "delta H", and the conformity of the data to Freundlich equation indicated a possible physical mechanism of adsorption. Increasing acetone concentration decreases methyl parathion adsorption and dehydration increased adsorption in the nonaqueous system. Moreover, the increased adsorption on the swollen clay indicated that methyl parathion was adsorbed on the interlamellar surfaces of the clay. This was also in agreement with the results of the desorption studies, since the insecticide was not desorbed using the same aqueous acetone solution. This indicated that methyl parathion was adsorbed as a water-insoluble organic compound.

Steroidal derivatives. Part 1: some novel steroidal thiosemicarbazones. Their synthesis, anticancer and endocrinological activities.

The synthesis of several novel thiosemicarbazone derivatives of steroids, including estrogens and androgens, is described. Evaluation of the products in P 388 Lymphocytic Leukemia indicated no anticancer activity. The endocrinological screening showed that estrogenicity is slightly reduced but not abolished in the thiosemicarbazones derived from estrone-3-methyl ether (compounds 1, 2 and 4). The androgenic activity of the thiosemicarbazones derived from testosterone (compounds 7--9) was more pronounced than that of testosterone. Among the same thiosemicarbazone derivatives 7--9, only o-tolyl derivative 8 exhibited anabolic activity.