Muscle Acetylcholinesterase Research Articles

Reactivation by novel pyridinium oximes of rat serum and skeletal muscle acetylcholinesterase inhibited by organophosphates.

The treatment of organophosphate (OP) anticholinesterases currently lacks an effective oxime reactivator of OP-inhibited acetylcholinesterase (AChE) which can penetrate the blood-brain barrier (BBB). Our laboratories have synthesized novel substituted phenoxyalkyl pyridinium oximes and tested them for their ability to promote survival of rats challenged with lethal doses of nerve agent surrogates. These previous studies demonstrated the ability of some of these oximes to promote 24-h survival to rats challenged with a lethal level of highly relevant surrogates for sarin and VX. The reactivation of OP-inhibited AChE in peripheral tissues was likely to be a major contributor to their efficacy in survival of lethal OP challenges. In the present study, twenty of these novel oximes were screened in vitro for reactivation ability for AChE in rat skeletal muscle and serum using two nerve agent surrogates: phthalimidyl isopropyl methylphosphonate (PIMP, a sarin surrogate) and 4-nitrophenyl ethyl methylphosphonate (NEMP, a VX surrogate). The oximes demonstrated a range of 23%-102% reactivation of AChE in vitro across both tissue types. Some of the novel oximes tested in the present study demonstrated the ability to more effectively reactivate AChE in serum than the currently approved oxime, 2-PAM. Therefore, some of these novel oximes have the potential to reverse AChE inhibition in peripheral target tissues and contribute to survival efficacy.

B-esterases activities in several tissues of the marine fish: sea bass and hake, as potential biomarkers of bisphenol A derivatives

In the marine environment, a new threat linked to plastic pollution relates to plastic additives. This threat encompasses multiple chemical compound groups with a high bioaccumulation potential for these chemical mixtures. Hence, informative biomarkers are needed to indicate the effects of environmentally realistic mixtures of these additives. This study proposes an in vitro approach using tissue homogenates of two marine fish, the European sea bass and hake, which are both of interest in aquaculture and fisheries. The selected biomarkers are B-esterase activities comprising acetylcholinesterase (AChE) and carboxylesterases (CEs). The physiological role of AChE in brain and muscle is mainly neural transmission, while CEs participate in liver detoxification processes. However, B-esterases are also widely distributed in other tissues/organs, where their role is yet to be determined, but their inhibition may have undesired biological consequences. Here, we compared the interaction of the plastic additives, bisphenol A and some of its derivatives, like tetrabromobisphenol A (TBBPA), with B-esterase activities. We particularly focused not only on the robust and broadly distributed CE enzymes in brain, gonad, liver, kidney, and plasma tissues of two marine fish, but also on the use of two commercial substrates, p-nitrophenyl butyrate and α-naphthyl butyrate, tentatively representing two CE isoforms. The results evidenced specific species and tissue responses that could be due to a diverse isoform composition. They identified sea bass as better protected against neurotoxic exposures, at least in terms of B-esterase composition. The flame retardant TBBPA was the most reactive to B-esterases inhibition, although Bisphenol A bis (2,3-dihydroxy propyl) ether and Bisphenol F bis (3-chloro-2-hydroxypropyl) ether warrant further toxicology assessments.

Fungicides from rice cultivation (tebuconazole and azoxystrobin) alters biochemical and histological markers of hammertoad tadpoles (Boanafaber).

Tebuconazole (TBZ) and azoxystrobin (AZX) are fungicides frequently used in rice cultivation. Despite protecting crops against fungal diseases, these compounds can contaminate the natural environments close to the crops, exerting negative effects on non-target organisms, the present study aimed to characterize the contamination by fungicides of a river that flows in an area dominated by rice cultivation in the north of the state of Santa Catarina, SC, Brazil. Concentrations of TBZ and AZX found in the field were used to evaluate their negative effects on development, biochemical biomarkers and histopatology of the liver of a native tadpole species, the hammerfrog (Boana faber). Tadpoles were exposed for 16 days to the lowest (1.20 μg/L) and highest (2.60 μg/L) concentration of TBZ, lowest (0.70 μg/L) and highest (1.60 μg/L) concentration of AZX, and the mix of both fungicides at lowest and highest concentration of each found in field analyses. Exposure to the lower TBZ concentration and both concentrations of the Mix accelerated the development of tadpoles. AZX caused an increase in the activities of glutathione S-transferase (GST), carboxylesterase (CbE) and glucose-6-phosphate dehydrogenase (G6PDH) in the liver, an increase in the levels of protein carbonyls (PC) in the liver and an increase in the activity of acetylcholinesterase (AChE) in muscle of tadpoles. TBZ, on the other hand, generated an increase in GST, G6PDH, PC and histopathological severity scores in liver and in muscle AChE activity. The effects were more intense in the groups exposed to the Mix of contaminants. No treatment altered brain AChE. The data showed that the fungicides from in rice cultivation found in natural aquatic environments around the crops pose risks to the health of the animals, compromising their metabolism and development.

UK long-term monitoring dataset: assessing factors affecting biomarkers commonly used in environmental programmes

Long-term programmes like the UK Clean Seas Environmental Monitoring Programme (CSEMP) rely on biological effects techniques, including biomarkers, to assess if chemical pollutants are affecting sentinel species in the aquatic environment. In this study, we have applied mixed and fixed effect linear models to the long-term CSEMP dataset (2005-2018) to evaluate if factors such as region (location), sex, age, gonadosomatic index (GSI) and condition factor (CF), contribute to the variability observed in the levels of 3 well established biomarkers (inhibition of acetylcholinesterase in muscle-AChE, induction of hepatic 7-ethoxyresorufin-O-deethylase-EROD and presence of biliary 1-hydroxypyrene-1-OH pyrene) used to monitor biological effects of contaminants in dab (Limanda limanda) around UK waters. Regional differences (location) were a significant explanatory variable for the 3 biomarkers. Substantial differences were found in regional average muscle AChE activity, overall indicating the East Coast regions as likely more impacted by acetylcholinesterase inhibitors (e.g. organophosphates and carbamates). We report for the first time that, while accounting for region, sex and gonadosomatic index are significant predictors of muscle acetylcholinesterase isoform in dab (R2 = 0.13-0.15), and therefore advise the future analysis of this marker should be done by sex when reporting for marine environmental purposes. Dab condition factor is also a significant predictor for both enzymatic markers (AChE and EROD) but not for 1-OH pyrene. Sex and age, and their interaction, were the strongest variables (R2 = 0.46) influencing hepatic EROD, showing that although overall females had higher hepatic EROD, male dab hepatic EROD increased with age. Correlations between the three biomarkers might assist in discerning types of prevalent regional contamination over the studied period. Our results highlight the factors to account for when assessing if environmental contamination is contributing to the responses of these 3 biomarkers in dab, and other flatfish species, used in marine environmental monitoring programmes.

Donepezil inhibits neuromuscular junctional acetylcholinesterase and enhances synaptic transmission and function in isolated skeletal muscle.

Donepezil, a piperidine inhibitor of acetylcholinesterase (AChE) prescribed for treatment of Alzheimer's disease, has adverse neuromuscular effects in humans, including requirement for higher concentrations of non-depolarising neuromuscular blockers during surgery. Here, we examined the effects of donepezil on synaptic transmission at neuromuscular junctions (NMJs) in isolated nerve-muscle preparations from mice. We measured effects of therapeutic concentrations of donepezil (10 nM to 1 μM) on AChE enzymic activity, muscle force responses to repetitive stimulation, and spontaneous and evoked endplate potentials (EPPs) recorded intracellularly from flexor digitorum brevis muscles from CD01 or C57BlWldS mice. Donepezil inhibited muscle AChE with an approximate IC50 of 30 nM. Tetanic stimulation in sub-micromolar concentrations of donepezil prolonged post-tetanic muscle contractions. Preliminary Fluo4-imaging indicated an association of these contractions with an increase and slow decay of intracellular Ca2+ transients at motor endplates. Donepezil prolonged spontaneous miniature EPP (MEPP) decay time constants by about 65% and extended evoked EPP duration almost threefold. The mean frequency of spontaneous MEPPs was unaffected but the incidence of 'giant' MEPPs (gMEPPs), some exceeding 10 mV in amplitude, was increased. Neither mean MEPP amplitude (excluding gMEPPs), mean EPP amplitude, quantal content or synaptic depression during repetitive stimulation were significantly altered by concentrations of donepezil up to 1 μM. Adverse neuromuscular signs associated with donepezil therapy, including relative insensitivity to neuromuscular blockers, are probably due to inhibition of AChE at NMJs, prolonging the action of ACh on postsynaptic nicotinic acetylcholine receptors but without substantively impairing evoked ACh release.

Acetylcholinesterase Inhibition of Marine Fish (Rastrelliger kanagurta) by Organophosphorus Pesticides as Biomarker of Neurotoxicants

This paper evaluated the inhibitory effects of various phosphorothioates (Ethyl parathion and Chlorpyriphos) and phosphates (DDVP, Monocrotophos and Phosphamidon) on acetylcholinesterase (AChE) of marine Mackerel fish (Rastrelliger kanagurta) from the Goa coast. The characteristics of the AChE enzymes isolated from different tissues of the fishes for their affinity towards the substrate were determined by the Michaelis Menten constants. The pseudocholinesterase activity was also measured in the muscle tissues in terms of Butyryl-cholinesterase (BChE) activity of the fish. The AChE activity was found to be predominant in the Brain, gills, liver and muscle tissues. However, no significant BChE activity was observed in the muscle tissue. Among the five organophosphorus pesticides, DDVP was found to be most toxic with least I50 values (0.99±0.04nM) followed by Chlorpyriphos (I50, 24.59±2.86 nM), Ethyl Parathion (I50, 183.21±11.68 nM), Monocrotophos (I50, 13536.93±1292.94 nM) and Phosphamidon (I50, 21433.1±2651.96 nM) in the decreasing order. The I50 values of all other pesticides decreased considerably with respect to DDVP. The relative neurotoxicity of the pesticides decreased by an order of magnitude in the case of Chlorpyriphos followed by Ethyl Parathion by an order of magnitude whereas in the case of Monocrotophos and Phosphamidon the toxicity decreased significantly by an order of magnitude. The variation in the muscle AChE inhibition by the organophosphorus pesticides can be attributed to the sensitivity of the enzyme for each of the pesticides as observed by the bimolecular inhibition constants (KII). The muscle AChE inhibition of Rastrelliger kanagurta can be used as biomarker for bio- monitoring of neurotoxic contaminants in the marine environment.

Response of biomarkers to metals, hydrocarbons and organochlorine pesticides contamination in crabs (Callinectes ornatus and C. bocourti) from two tropical estuaries (São José and São Marcos bays) of the Maranhão State (northeastern Brazil)

Response of biomarkers to chemical contamination was evaluated in crabs of the Callinectes genus (Callinectes ornatus and C. bocourti) from two tropical estuaries (São José and São Marcos bays) of the Maranhão State (northeastern Brazil). Biomarkers evaluated included hepatopancreatic metallothionein-like proteins (MTLP) and lipid peroxidation (LPO), as well as muscle acetylcholinesterase (AChE). Tissue concentrations of metals (pereiopod muscle and hepatopancreas), hydrocarbons (hepatopancreas) and organochlorine pesticides (hepatopancreas) were also evaluated. Crab samples were collected in three sites of each estuary (São Marcos Bay and São José Bay). Sampling was performed in August/2012 (dry season), January/2013 (rainy season), August/2013 (dry season), and January/2014 (rainy season). Concentrations of chemical contaminants and responses of biomarkers showed significant spatial (São Marcos Bay and São José Bay) and/or seasonal (dry and rainy seasons) and annual (2012–2014) variability. However, a general higher Zn concentration was observed in hepatopancreas of crabs from São José Bay. In turn, a general higher Cd concentration paralleled by oxidative damage (LPO) was observed in hepatopancreas of crabs from São Marcos Bay. As expected, these findings support the idea that this bay is more intensively or chronically impacted by industrial activities while the São José Bay is likely more affected by domestic activities. Interestingly, LPO level in crab hepatopancreas showed to be the most reliable and adequate biomarker to distinguish the two bays.

Bioconcentration and toxicological impacts of fipronil and 2,4-D commercial formulations (single and in mixture) in the tropical fish, Danio rerio.

The insecticide fipronil and the herbicide 2,4-D are the most applied pesticides in sugarcane crops leading to aquatic contamination. The whole-body bioconcentration of fipronil and 2,4-D, single and in mixture, was evaluated in Danio rerio after 96-h exposure. The activities of catalase (CAT) and glutathione S-transferase(GST) in whole body and in the gills and the acetylcholinesterase (AChE) in muscle were determined. The gill histopathology and the morphology of the pavement (PVC) and the mitochondria-rich(MRC) cells at gill surface were analyzed. Bioconcentration occurred after exposure to fipronil (2.69 L kg-1) and 2,4-D (1.73 L kg-1) single and in mixture of fipronil (3.10 L kg-1) and 2,4-D (1.27 L kg-1). Whole-body CAT activity was unchanged, and its activity decreased in the gills after exposure to fipronil and increased after exposure to 2,4-D and mixture. GST and AChE increased after single exposure to each pesticide and mixture of both. Fish exposed to mixture increased the MRC fractional area (MRCFA) which suggested possible ionic regulation disturbance and reduced the microridge of the PVC surface. Synergistic interactions occurred in the CAT activity and MRCFA after exposure to mixture of pesticides. The results indicate that the recommended application dose of fipronil and 2,4-D, single or in mixture, for sugarcane crops affects this fish species altering its homeostasis.

"In-vitro Effects of Chlorpyrifos and Monocrotophos on the Activity of Acetylcholinesterase (AChE) in Different Tissues of Apple Snail Pila globosa (Swainson, 1822)"

The impact of two organophosphorus insecticides [Chlorpyrifos (CPF) and Monocrotophos (MCP)] on non-target wild natural gastropod, Pila globosa (apple snail) from the paddy fields was studied. The activity of acetylcholinesterase (AChE) was monitored on foot-muscle and hepatopancreas tissues of control and exposed snails. In the foot- muscle AChE inhibition progressed and reached 54.19% and 63.13% of the control, whereas, the AChE inhibition in the hepatopancreas reached 46.96% and 53.67% over control after 48 hours of exposure to 1.5 mL.L-1 and 2.5 mL.L-1 CPF respectively. After 48 hours of MCP exposure at 1.5 mL.L-1 and 2.5 mL.L-1 separately, the AChE inhibition of foot muscle was 49.07% and 57.59% respectively while in hepatopancreas it was 44.65% and 48.84% respectively. Our results show more inhibition of AChE activities on the foot-muscle than hepatopancreas in a concentration and time-dependent manner with greater severity by CPF in comparison to MCP. AChE inhibition increased with the increasing exposure time.

Toxicity of silver nanoparticles on different tissues in adult Danio rerio.

Although silver nanoparticles (AgNP) are among the most studied nanomaterials by virtue of their broad application in many areas, little is known about their overall toxicity to aquatic organisms after their contamination of the water environment. This study aimed to analyze the effect of the exposure (96 h) to different AgNP concentrations on Danio rerio (zebrafish) tissues. AgNP were synthesized and characterized by transmission electron microscopy (TEM), showing spherical AgNP of 30.00 ± 16.80 nm size. The effects of different AgNP concentrations (1, 3, and 5 μg L-1) on brain, muscle, gill, and liver tissues of zebrafish were investigated. The results show a significant decrease in brain and muscle acetylcholinesterase (AChE) activity. Liver and gill catalase (CAT) activity also decreased significantly. At the highest exposure concentration, muscle AChE was more inhibited (37.3%) than brain AChE (26.4%) and gill CAT was more inhibited (67.4%) than liver CAT (51.2%). D. rerio also showed gill morphological changes such as fusion of secondary lamellae, curvature, dilated marginal channel, and epithelial lifting. This study indicates that gill CAT together with morphological studies are potential biomarkers for AgNP.

In vivo cholinesterase sensitivity of gilthead seabream (Sparus aurata) exposed to organophosphate compounds: Influence of biological factors

Two cholinesterases have been found in vertebrates, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). These enzymes are present in the gilthead seabream, AChE in the brain and muscle and BChE in the muscle. Cholinesterases have been used as biomarker of effect in environmental monitoring studies. However, there are few studies about the influence of biometric parameters on ChE. This paper studies the possible influence of biological factors on brain and muscle cholinesterase (ChE) in Sparus aurata. Our results show that ChE activity in brain and muscle tissues changes depending on several biological variables. ChE activity in these tissues decreased when the age (48–152 week), body length (14.15–28.95 cm) and body weight (42.73–380.74 g) of the fishes studied increased. The relationships between brain and muscle ChE activity and several biometric factors were curvilinear.On the other hand, in vivo sensitivity of cholinesterase in the gilthead seabream exposed to organophosphorus pesticides (azinphosmethyl, dimethoate and dichlorvos) was studied in order to learn about recovery from cholinesterase present (brain and muscle) after exposure to a sublethal dose. The recovery of muscle AChE was similar to cerebral AChE, while muscular BChE showed a slower recovery.

Mugil liza (Valenciennes, 1836) biomarker responses in a hypersaline lagoon in southeastern Brazil before and after a microalgal bloom

Araruama lagoon is one of the largest permanent hypersaline lagoons in the world and has been impacted for twenty years by sewage dumping. In early 2005, a Prasinophyceae (Pseudoscourfieldia sp.) phytoplankton bloom occurred, leading to fish mortality during subsequent summers (2006 and 2007). In this context, the aim of the present study was to accompany this historical period through the use of biochemical biomarkers in mullet (Mugil liza) during and after the algal bloom. Mullet were collected from Araruama Lagoon in June and July 2005 and August 2007, as well as from a reference lagoon with no algal blooms, Saquarema Lagoon. Several enzymatic activities in liver were analyzed, namely glutathione peroxidase (GPX), catalase (CAT), total glutathione S-transferase (GSTt), specific GSTπ (pi) and GSTμ (mi) classes and ethoxyresorufin-O-deethylase (EROD), as well as muscle acetylcholinesterase activity (AChE). In 2005, only EROD activity was significantly increased at Araruama Lagoon, indicating induction of the biotransformation enzyme system (CYP 1A1). GSTπ, on the other hand, was lower in the Araruama Lagoon, especially in 2005, when compared to mullet from Saquarema lagoon (reference area). These results highlight the importance of developing actions that go beyond the physical-chemical monitoring of these environments, since the study of ecological relationships and the physiology of organisms affected by algal blooms also include biochemical parameters to evaluate new approaches to icthyotoxicity.

Diclofenac and caffeine inhibit hepatic antioxidant enzymes in the freshwater fish Astyanax altiparanae (Teleostei: Characiformes)

Although concentrations of pharmaceutical compounds in aquatic ecosystems are low, they can cause toxic effects on organisms. The aim of this study was to evaluate the effects of diclofenac (DCF), a non-steroidal anti-inflammatory drug, and caffeine (CAF), a central nervous system stimulant, both alone or combined, in Astyanax altiparanae males under acute exposure (96 h), measuring neurotoxicity biomarkers, antioxidant response and damage at biochemical and cellular levels. DCF concentration in water, separated and combined, was 3.08 mg L−1 and that of CAF was 9.59 mg L−1. To assess neurotoxicity, brain and muscle acetylcholinesterase (AChE) activities were measured. To evaluate oxidative stress, the enzymatic activities of superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT) and glutathione S-transferase (GST), as well as lipoperoxidation (LPO), were analyzed in liver and gills. Activity of hepatic cyclooxygenase (COX) was also evaluated. Genotoxicity was assessed in blood using comet assay and micronucleus test, as well as nuclear abnormalities. DCF and CAF, alone or combined, had neither effect on AChE activity, nor in the activity of SOD, CAT, GPx and GST in gills. In liver, DCF inhibited SOD and GPx activity, CAF inhibited CAT activity, the mixture inhibited SOD and GST activity; although only fish exposed to CAF showed increased hepatic LPO. Under these experimental conditions, no effect on COX activity was observed, nor cytotoxic and genotoxic damage. The most pronounced effects were caused by the drugs separately, since both compounds altered the enzymes, but only CAF triggered LPO, showing more harmful effects.

Integrated ecotoxicological assessment of the complex interactions between chlorpyrifos and glyphosate on a non-target species Cnesterodon decemmaculatus (Jenyns, 1842)

Pesticide mixtures are frequent in freshwaters systems around the world, threatening the biota exposed to these conditions. The aim of this study was to determine the single and joint effect of two widely used pesticides in southern South America on a widely distributed fish species. In a 96-h assay, individuals of Cnesterodon decemmaculatus were exposed to 0.84 nL/L and 8.4 nL/L of Clorfox and 0.2 mg/L and 2 mg/L of Roundup Max, commercial formulations of chlorpyrifos and glyphosate, respectively. Also, there were four mixture treatments with all the possible combinations of both pesticides. A multi-level approach was carried out to assess their effects covering the following relevant biomarkers: behavior (immobile time, line crossings and average speed), somatic conditions (Fulton condition factor and hepatosomatic index), serum parameters (cortisol levels, lactate dehydrogenase (LDH), and creatine phosphokinase activity (CPK)), brain and muscle acetylcholinesterase and cytological characteristics (micronuclei frequency and nuclear abnormalities in erythrocytes). Our results showed that Clorfox exposures affect behavioral parameters, serum cortisol, and nuclear characteristics of erythrocytes. Roundup Max affects only the cortisol levels whereas mixture treatments have an effect on behavioral parameters, cortisol levels, LDH and CPK activities, and nuclear characteristics of erythrocytes. Potentiation was the main interaction at the lowest concentrations of both pesticides whereas antagonism occurred at the highest concentrations of both pesticides. These results are highly significant since they arise from an integrated ecotoxicological assessment at several levels of biological organization but even more important is that the potentiated effects of the mixtures we registered are environmentally relevant concentrations.

Acute toxicity of the pesticide trichlorfon and inhibition of acetylcholinesterase in Colossoma macropomum (Characiformes: Serrasalmidae)

In Brazilian fish farms, trichlorfon has been widely used to control acanthocephalan infections in Colossoma macropomum. Toxicity tests were conducted to estimate the median lethal concentration (LC50–96 h) and evaluate the effects of trichlorfon on acetylcholinesterase (AChE) and glutathione S-transferase (GST) from different tissues of C. macropomum. The LC50–96 h of trichlorfon was estimated to be 0.87 mg L−1. In the sublethal toxicity tests, concentrations of 0.26 mg L−1 (30% of LC50–96 h) and 0.43 mg L−1 (50% of LC50–96 h) were used. AChE and GST activities were measured in the brain, muscle, intestine, and liver. In vitro studies were conducted to estimate the kinetic properties and half-maximal inhibitory concentration (IC50) values of AChE in the brain and muscle for trichlorfon. In the sublethal toxicity experiments, inhibition of more than 90% of AChE in the brain, muscle, and intestine was observed. However, the activity of GST did not vary in any of the tissues studied. This finding suggests that trichlorfon is not metabolised by this enzyme. The in vitro assay results suggest that trichlorfon tends to be a classic uncompetitive inhibitor of AChE in both the brain and muscle, since Km and Vmax values decrease, while the slope remains unchanged. The IC50 values ​of muscle AChE are lower than those of the brain. All these results show that C. macropomum has low tolerance to this pesticide and suggest that brain AChE can be used as a biochemical biomarker, while muscle AChE may be used as an indicator of mortality in toxicological studies.

Characterization and kinetic study of the brain and muscle acetylcholinesterase from Danio rerio

Acetylcholinesterase (AChE) plays an important role in the therapy of Alzheimer's disease and in the detection of pesticides such as organophosphates which are also widely used in chemical warfare. The aim of this study is the physicochemical and kinetic characterization of brain and muscle ChE from Danio rerio (Zebrafish). Optimal activity was found for brain ChE at alkaline pH 9.0 at 30 °C, and for muscle ChE at alkaline pH 8.5 at temperatures between 20 °C and 35 °C. The apparent kinetic constants, Kmapp and Vmaxapp, for brain ChE were determined as 0.191 ± 0.024 mM and 0.566 ± 0.028 U/mg protein, and for muscle ChE as 0.230 ± 0.030 mM and 0.677 ± 0.039 U/mg protein. Both brain and muscle ChE showed inhibition at high substrate concentrations. Brain and muscle ChE showed IC50 values for physostigmine of 0.61 μM and 0.37 μM, respectively. The ChE activity in brain was significantly inhibited by BW254c51 in all concentrations tested, but not by Iso-OMPA, while muscle ChE presented a moderate decrease (13 to 29%) in the activity values, indicating that BuChE is present.

Prophylactic potential of memantine against soman poisoning in rats

BackgroundCarbamates physostigmine and pyridostigmine have been used as a pretreatment against poisoning with nerve agents in order to reversibly inhibit and thus protect from irreversible inhibition a portion of acetylcholinesterase (AChE) in brain and respiratory muscles that is crucial for survival. Memantine, an adamantine derivative, has emerged as a promising alternative to carbamates, since it prevented the fasciculations and skeletal muscle necrosis induced by carbamates and organophosphates, including nerve agents. AimThis experimental study was undertaken in order to investigate and compare the protective and behavioural effects of memantine and standard carbamates physostigmine and pyridostigmine in rats poisoned with soman and treated with atropine, oxime HI-6 and diazepam. Another goal was to elucidate the mechanisms of the antidotal effect of memantine and its potential synergism with standard antidotes against nerve agents. Materials and methodsMale Wistar rats were used throughout the experiments. In dose-finding experiments memantine was administered at dose interval 0–72 mg/kg sc 60 min before sc injection of soman. In time-finding experiments memantine was injected 18 mg/kg sc 0–1440 min before soman. Standard treatment antidotes - atropine 10 mg/kg, HI-6 50 mg/kg and diazepam 2.5 mg/kg – were administered im within 15 s post-exposure. Soman 0.75 LD50 was used to study its inhibitions of neuromuscular transmission on the phrenic nerve-diaphragm preparation in situ and of tissue AChE activity. Behavioural effects of the prophylactic antidotes were investigated by means of the rotarod test. Based on these data therapeutic index and therapeutic width was calculated for all three prophylactic agents. ResultsMemantine pretreatment (18 mg/kg sc) produced in rats poisoned with soman significantly better protective ratios (PRs) than the two carbamates – 1.25 when administered alone and 2.3 when combined with atropine pretreatment and 6.33 and 7.23 with atropine/HI-6 and atropine/HI-6/diazepam post-exposure therapy, respectively. The highest PR of 10.11 obtained in Atr/HI-6-treated rats was achieved after pretreatment with memantine 36 mg/kg. This additional protection lasted for 8 h. All three prophylactic regimens antagonised the soman-induced neuromuscular blockade, but the effect of memantine was fastest. Pretreatment with memantine assured higher AChE activity in brain and diaphragm than in unpretreated rats (46% vs 28% and 68% vs. 38%, respectively). All three prophylactic regimens affected the rotarod performance in rats, but the effect of memantine was relatively strongest. Memantine and pyridostigmine had lowest and highest therapeutic index and therapeutic width, respectively. ConclusionsAlthough memantine assures better and longer-lasting protection against soman poisoning in rats than the two carbamates, its small therapeutic index and narrow therapeutic width seriously limit its potential as a pretreatment agent. Despite its behavioural effects, memantine seems to be beneficial antidote when administered after soman, along with atropine/HI-6/diazepam therapy. Mechanism of the antidotal effect of memantine against soman poisoning appears to be a combination of AChE-protecting and NMDA receptor-blocking action.

Efficacy of antidotes and their combinations in the treatment of acute carbamate poisoning in rats

BackgroundPhysostigmine and its analogues neostigmine, pyridostigmine and rivastigmine are carbamates nowadays used in many indications, including antidotal effects against antimuscarinic poisonings, reversal of competitive neuromuscular block, myasthenia gravis, Alzheimer’s disease and prophylaxis against nerve agent intoxications. Use of these medicinal carbamates, but also of carbamate insecticides, created need for research into the potential and mechanisms of action of several antidotes against carbamate poisonings, including anticholinergics and oximes. AimThe goal of this experimental study was to ascertain the life-preserving potential of anticholinergics atropine, hexamethonium and d-tubocurarine, oxime HI-6 and their combinations in rats poisoned with physostigmine or pyridostigmine. Materials and methodsExperiments were performed in Wistar rats. Carbamates were injected subcutaneously (sc) and antidotes intramuscularly (im). Median lethal dose (LD50) in animals treated with antidotes were compared to the ones in saline-treated rats and protective ratios (PRs) were calculated. Atropine (5, 10 and 20 mg/kg), hexamethonium (5, 10 and 20 mg/kg), d-tubocurarine (0.005, 0.010 and 0.020 mg/kg) and oxime HI-6 (25, 50 and 100 mg/kg) were used as monotherapies and in dual combinations, where atropine was the obligatory antidote. Biochemical experiments consisted in measuring of the cholinesterase activities in brain, whole blood and diaphragm in rats 5, 15, 30, 60, 120 and 240 min after poisoning with 0.8 LD50 of physostigmine or pyridostigmine. ResultsAll the tested antidotes assured some degree of protection against the two carbamates. Atropine and hexamethonium produced better protection in physostigmine-poisoned rats, while d-tubocurarine and HI-6 were more efficacious in pyridostigmine-intoxicated animals. Oxime HI-6 50 mg/kg reactivated acetylcholinesterase (AChE) in brain inhibited by physostigmine and in diaphragm inhibited by pyridostigmine. ConclusionsMechanism of physostigmine-induced lethal effect is predominantly central and it involves inhibition of brain AChE, while pyridostigmine produces the same effect exclusively outside the central nervous system, by inhibiting AChE in the respiratory muscles. As a consequence, increasing doses of atropine and their combination with hexamethonium assure excellent protection against physostigmine toxicity, while the best protection against pyridostigmine is provided by a strictly peripherally acting antinicotinic d-tubocurarine and bispyridinium oxime HI-6. The oxime acts as antidote against physostigmine and pyridostigmine poisoning by reactivating AChE in the brain and diaphragm, respectively.

Selection of biochemical and physiological parameters in the croaker Micropogonias furnieri as biomarkers of chemical contamination in estuaries using a generalized additive model (GAM)

Biochemical and physiological parameters in the croaker Micropogonias furnieri were evaluated as biomarkers of chemical contaminants in estuaries. Juvenile croakers (10–20 cm total body length) were collected in summer and winter (2011 and 2012), in two sites at the Lagoa dos Patos estuary (southern Brazil). Fish were also collected in summer (2011 and 2012) in one site at the Barra do Chui estuary (southern Brazil). Tissue (gills, muscle or liver) samples were dissected and analyzed for contaminants [metals, polycyclic aromatic hydrocarbons (PAHs) and organochlorine pesticides (OCPs)] and selected biochemical and physiological parameters [metallothionein-like proteins (MTLP), acetylcholinesterase (AChE) activity, ethoxyresorufin-O-deethylase (EROD) activity, and lipid peroxidation (LPO)]. Additionally, water samples were collected for water chemical analyses (salinity, pH, temperature, dissolved oxygen, total alkalinity, and sulfate concentration). Data obtained were integrated and analyzed, using Principal Component Analysis (PCA) and Generalized Additive Model (GAM) approaches. Results showed that changes in concentrations of chemical contaminants and responses of biochemical and physiological parameters did not show any pattern according to the site, season and year of fish collection. However, they were influenced by fish body length and water temperature and salinity. Liver LPO and EROD activity were not responsive to PAHs. However, liver LPO was responsive to HCB, p,p-DDD, p,p-DDT and endosulfan sulfate. In turn, gill MTLP concentration, muscle AChE activity and liver EROD activity were responsive to non-essential metals (Pb, Cd and Ag). Considering that the ecotoxicological modeling approach adopted (GAM) accounted for biological, spatial and temporal variability of data associated with fish body size and site, season and year of fish collection, gill MTLP concentration, muscle AChE activity, as well as liver LPO and EROD activity can be considered as reliable biomarkers of fish exposure to chemical contaminants in estuaries.

Spatial and temporal analysis of biliary 1-hydroxypyrene, hepatic ethoxyresorufin-O-deethylase and muscle acetylcholinesterase activity in UK flatfish

Chemical pollution of the marine environment from anthropogenic sources is a global concern due to the potential for long term effects on the ecosystem. Chemical monitoring schemes can detect harmful substances in water, sediment or biota. However, the presence of specific determinants gives no information on how individual species or the ecosystem are impacted. Biological effects techniques are therefore used in environmental monitoring schemes to provide a clearer picture of whether the chemicals present are causing deleterious effects on the species sampled, or whether organisms have been exposed to certain classes of organic contaminants that they may be able to metabolise. Using these techniques, we can provide an assessment of the health of our marine environment (Thain et al., 2008).