Acute toxicity and cholinesterase inhibition of the nematicide ethoprophos in larvae of gar Atractosteus tropicus (Semionotiformes: Lepisosteidae)

Biomarkers are a widely applied approach in environmental studies. Analyses of cholinesterase (ChE), glutathione S-transferase (GST) and lipid peroxidation (LPO) are biomarkers that can provide information regarding early effects of pollutants at different biochemical levels on an organism. The aim of this study was to evaluate the biomarker approach on a Costa Rican native and relevant species. For this, larvae of gar (Atractosteus tropicus) were exposed to the organophosphorus nematicide, ethoprophos. Acute (96hr) exposure was conducted with pesticide concentrations ranging from 0.1 microg/L to 1 500 microg/L. The 96hr LC50 calculated was 859.7 microg/L. After exposure, three biomarkers (ChE, GST and LPO) were analyzed in fish that survived the acute test. The lowest observed effect concentration (LOEC) regarding ChE activity inhibition was 50 microg/L. This concentration produced a significant inhibition (p<0.05) of the enzyme by 20%. The highest concentration tested without showing any effect on ChE activity and therefore considered as no observed effect concentration (NOEC) was 10 microg/L. Ethoprophos concentration of 400 microg/L caused a ChE inhibition by 79%. In this study, no significant variations (p>0.05) in GST activity and LPO were observed in A. tropicus larvae after exposure to ethoprophos.     

Applying weight gain in Pomacea lineata (SPIX 1824) (Mollusca: Prosobranchia) as a measure of herbicide toxicity.

Pomacea lineata, an extremely ubiquitous snail and pest to rice farmers throughout Asia, holds promise as a valuable resource for monitoring water quality in northeast Brazil. In this paper, we present data demonstrating the rate of weight gain in P. lineata neonates as a consistent measure of the stress imposed by sublethal concentrations of the herbicides Paraquat and Round-up. Our secondary agenda is to demonstrate the feasibility of incorporating bioassay into the standard municipal and state procedure of monitoring water quality. Growth data to assess chronic toxicity were generated in experiments of four and four, eight, twelve and sixteen days for Paraquat and Round-up, respectively. We estimated a 96 h no observed effect concentration (NOEC) and lowest observed effect concentration (LOEC) for Paraquat of 0.12 and 0.25 mg/L. The 96 h Round-up data yielded NOEC and LOEC values, respectively, of 0.25 and 0.5 mg/L. All concentrations of Round-up tested for the 192 h exposure yielded significantly lower growth than the control. Consequently, no NOEC could be derived. The LOEC was < 0.12 mg/L. Furthermore, there was no mortality during the test. At the lowest concentrations of Paraquat tested (0.005 mg/L) there was a significant increase in growth compared with the controls, suggesting a hormetic effect.     

钩虾胆碱酯酶（ChE）和谷胱甘肽转硫酶（GST）的敏感性和特异性比较研究

研究了有机磷农药甲地基嘧啶流磷,有机氯农药林丹,菊酯类农药氯菊酯,表面活性剂直链苯磺酸钠和重金属Zn对钩虾（Gammarus pulex L.)胆碱酯酶（ChE）和谷胱甘肽转硫酶（GST）活性变化以及毒性影响,结果表明,在暴露24h和48h后,仅有机磷农药甲基嘧硫磷显著抑制胆碱酯的酶的活性,在暴露48h后,有机氯农药林丹和菊酯类农药氯菊酯能显著提高谷胱甘肽转硫酶活性,在暴露24h后,仅梵在导致谷胱甘肽转硫酶明显升高,作为生物标志物,胆碱酯酶比谷胱甘肽转硫酶具有更高的特异性,这两种生物标志物较毒性试验方法具有更高的敏感性。     

Responses of Glutathione and Glutathione S-Transferase to Cadmium and Mercury Exposure in Pedunculate Oak (Quercus robur) Leaf Discs

Changes in chlorophyll, non-protein thiol and glutathione (GSH) levels, and the activity of glutathione S-transferase (GST) were investigated in cadmium(ll) and mercury(ll) cchloride treated leaf discs of mature pedunculate oak trees (Quercus robur). Both heavy metals caused decreases in chlorophyll content, but mercury was more toxic than cadmium. Cadmium treatments (30–250μiM) resulted in increasing non-protein thiol levels after 3d, but GSH contents decreased. Mercury (1–20μM) led to a concentration-dependent decline in both non-protein thiol and GSH levels. GST activities were not modified significantly by cadmium, but mercury treatments caused a dose- and time-dependent enzyme induction. Both the phytotoxic- and GST-inducing effect of mercury could be prevented by the cysteine precursor L-2-oxo-4-thiazolidinecarboxylic acid.     

Characterization of cholinesterases in plasma of three Portuguese native bird species: application to biomonitoring

Over the last decades the inhibition of plasma cholinesterase (ChE) activity has been widely used as a biomarker to diagnose organophosphate and carbamate exposure. Plasma ChE activity is a useful and non-invasive method to monitor bird exposure to anticholinesterase compounds; nonetheless several studies had shown that the ChE form(s) present in avian plasma may vary greatly among species. In order to support further biomonitoring studies and provide reference data for wildlife risk-assessment, plasma cholinesterase of the northern gannet (Morus bassanus), the white stork (Ciconia ciconia) and the grey heron (Ardea cinerea) were characterized using three substrates (acetylthiocholine iodide, propionylthiocholine iodide, and S-butyrylthiocholine iodide) and three ChE inhibitors (eserine sulphate, BW284C51, and iso-OMPA). Additionally, the range of ChE activity that may be considered as basal levels for non-exposed individuals was determined. The results suggest that in the plasma of the three species studied the main cholinesterase form present is butyrylcholinesterase (BChE). Plasma BChE activity in non-exposed individuals was 0.48±0.11 SD U/ml, 0.39±0.12 SD U/ml, 0.15±0.04 SD U/ml in the northern gannet, white stork and grey heron, respectively. These results are crucial for the further use of plasma BChE activity in these bird species as a contamination bioindicator of anti-cholinesterase agents in both wetland and marine environments. Our findings also underscore the importance of plasma ChE characterization before its use as a biomarker in biomonitoring studies with birds.     

In vitro exposure of heavy metals on nucleotidase and cholinesterase activities from the digestive gland of Helix aspersa

Zinc, copper and cadmium are important environmental contaminants and differences in purinergic and cholinergic systems of invertebrates have been described when compared to characteristics of these signaling systems in vertebrates. Here we evaluate the effect in vitro of these metals on the ATPase, 5'-nucleotidase and cholinesterase (ChE) activities in the digestive gland of Helix aspersa. Zinc (500 and 1000 microM) promoted a significant decrease in 5'-nucleotidase activity. However, it did not induce changes in ATP hydrolysis. Copper (25 and 50 microM), inhibited significantly ATPase activity, but did not alter 5'-nucleotidase when compared to control (no metal added). In relation to effects of cadmium, an inhibitory effect on ATP hydrolysis has been observed at concentrations of 100, 500 and 1000 microM and a similar decrease of AMP hydrolysis was observed at 500 and 1000 microM. However, there were no significant changes in ChE activity from homogenates of the digestive gland of H. aspersa for all metals tested. This study demonstrated that zinc, cadmium and copper affect ATPase and 5'-nucleotidase in digestive gland, but not ChE, suggesting that the purinergic system may be a target related to toxicity induced by these metals and a possible indicator of biological impact of exposure to these contaminants.     

Effect of cadmium and copper on growth of Bacopa monniera regenerants

The effects of cadmium and copper on in vitro growth of Bacopa monniera (L.) Wettst. was monitored. Cadmium (25 and 50 μM CdCl₂) inhibited plantlet growth and addition of 50 or 100 μm CuSO₄ partially alleviated this negative effect. Cadmium increased both protein and proline contents, but to a lesser extent with the additional supply of CuSO₄. This revised version was published online in July 2006 with corrections to the Cover Date.     

Expression of cholinesterase gene(s) in human brain tissues: translational evidence for multiple mRNA species

To resolve the origin(s) of the molecular heterogeneity of human nervous system cholinesterases (ChEs), we used Xenopus oocytes, which produce biologically active ChE when microinjected with unfractionated brain mRNA. The RNA was prepared from primary gliomas, meningiomas and embryonic brain, each of which expresses ChE activity with distinct substrate specificities and molecular forms. Sucrose gradient fractionation of DMSO-denatured mRNA from these sources revealed three size classes of ChE-inducing mRNAs, sedimenting at approximately 32S, 20S and 9S. The amounts of these different classes of ChE-inducing mRNAs varied between the three tissue sources examined. To distinguish between ChEs produced in oocytes and having different substrate specificities, their activity was determined in the presence of selective inhibitors. Both 'true' (acetylcholine hydrolase, EC 3.1.1.7) and 'pseudo' (acylcholine acylhydrolase, EC 3.1.1.8) multimeric cholinesterase activities were found in the mRNA-injected oocytes. Moreover, human brain mRNAs inducing 'true' and 'pseudo' ChE activities had different size distribution, indicating that different mRNAs might be translated into various types of ChEs. These findings imply that the heterogeneity of ChEs in the human nervous system is not limited to the post-translational level, but extends to the level of mRNA.     

Cholinesterases in development and disease

Cholinesterases (ChEs) including acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) are abundant in the nervous system and other tissues. Here we describe two different aspects of ChEs and the cholinergic system. The first aspect concerns the role of cholinergic transmission in central pattern generation in the neonatal rat spinal cord and the second one describes the involvement of ChEs in the pathologies of dystrophin-deficient mutant (mdx) mice, the animal model of Duchenne muscular dystrophy. Thus, this study is divided into two distinct parts. In the first part we show that AChE is abundant in ventral horn neurons, central canal-adjacent and partition neurons in all the observed segments (L2, L5, S1, and S2). AChE was also found in the intermediolateral and sacral parasympathetic nuclei of L2 and S1, respectively. Blocking the AChE by edrophonium produced non-stationary bursting in spinal cord preparations of developing rats. Cross-wavelet/coherence analyses of the data revealed epochs of locomotor-like activity (left-right and flexor-extensor alternation) followed by other rhythmic or non-rhythmic bursting patterns. Addition of exogenous ACh stabilized the rhythm and increased the incidence of locomotor-like pattern in the preparations. Thus, the cholinergic system in the spinal cord is capable of producing and modulating functional rhythmic bursts. Moreover, bath-applied edrophonium and exogenous ACh were found as potent means of modulation of the locomotor rhythm produced by stimulation of sacrocaudal afferents (SCAs). We show that a subclass of sacral neurons with contralateral funicular projections to the thoracolumbar cord is associated with the cholinergic system. This group of neurons may play a major role in the observed enhancement of the SCA-induced motor rhythm. In the second part we show that adult mdx-muscles are malformed with distorted neuromuscular junctions (nmjs) and impaired regulation of acetylcholine receptors. Examination of circulating ChE levels revealed that in mdx-sera, while AChE activity was elevated, BuChE activity was markedly lower than in wild-type (wt) sera. Thus, BuChE to AChE ratio in mouse sera decreased from 6:1 in wt control to 3:1 in mdx. Because serum ChE levels may be modulated by gonadal steroids, it is possible that lack of dystrophin in mdx-mice may affect this regulation. Further studies are in progress to determine the potential endocrine regulation of ChEs in circulation and at the nmjs of mdx- and wt-mice. These studies will help clarify whether the hormonal regulation is impaired in the mdx mutant, and whether changes in circulating ChE reflect or influence the functional deficits observed in excitable tissues of diseased states.     

Inhibitory effects of cadmium on carbonic anhydrase activity and ionic regulation of the estuarine crab Chasmagnathus granulata (Decapoda,Grapsidae)

This work was aimed at evaluating the gill carbonic anhydrase (CA) activity of the estuarine crab Chasmagnathus granulata exposed in vivo to cadmium, at different salinities. The in vivo effect of the specific inhibitor acetazolamide (AZ) was also assayed. Besides, the inhibition of CA activity by different heavy metals (cadmium, copper, zinc) and AZ were evaluated under in vitro conditions. For the in vivo assays, adult males were acclimated to salinities of 2.5 or 30‰. The corresponding 96-h LC₅₀ of cadmium was 2.69 mg l⁻¹ at 2.5‰, and >50 mg l⁻¹ at 30‰. Cadmium only caused a significant lower CA activity than control at 2.5‰. EC₅₀ for CA inhibition was estimated to be 1.59 mg l⁻¹ at 2.5‰. Statistical differences in Na⁺ hemolymphatic levels (P<0.05) were only detected at 2.5‰, between 0 and 1.25 mg l⁻¹ of cadmium, but no statistical differences were observed for Cl⁻ levels at any assayed salinity. As CA inhibition registered at 2.5‰ was followed by only changes in Na⁺ concentration, it is likely that cadmium exposure could differentially affect ions permeability, among others factors. The concentrations that inhibited in vitro 50% of enzymatic activity (IC₅₀) were 2.15×10⁻⁵, 1.62×10⁻⁵, 3.75×10⁻⁶ and 4.4×10⁻¹⁰ M for cadmium, copper, zinc and AZ, respectively. The comparison with IC₅₀ values of other aquatic species, indicates a higher CA sensitivity for C. granulata to pollutants.     

镉急性染毒对中华稻蝗羧酸酯酶和谷胱甘肽S-转移酶活性的影响

为深入探讨重金属镉(cadmium,Cd)的毒性效应,采用不同浓度氯化镉溶液对中华稻蝗Oxya chinensis Thunberg 4龄若虫进行急性染毒,对处理后24、48、72和96 h虫体内羧酸酯酶(CarE)和谷胱甘肽S-转移酶(GST)活性进行测定。结果表明,随着处理时间的延长,对照组和处理组CarE和GST活性均表现为先升后降的趋势。与对照组相比,以α-NA为底物时高浓度处理组(80 mg.L-1)CarE活性在处理后48 h被激活;以β-NA为底物时中浓度处理组(40 mg.L-1)CarE活性在72 h达到最高;以CDNB为底物时,低浓度处理组(20 mg.L-1)GST活性在24 h被激活达到最大值,之后降低。结果显示,中华稻蝗4龄若虫在Cd胁迫下,体内CarE和GST活性发生了变化,以此来抵御机体所受到的重金属毒害。     

Comparison of methods used for the determination of cholinesterase activity in whole blood

Cholinesterases (ChEs) are classified as either acetylcholinesterase (AChE) or butyrylcholinesterase (BChE) based on their substrate and inhibitor specificity. Organophosphate and carbamate compounds commonly represented by herbicides, pesticides, and nerve gases irreversibly inhibit ChEs. Therefore, exposure to organophosphates and carbamates is normally assessed by measuring ChE activity in blood. There are two approaches for measuring AChE and BChE activity present in whole blood: (1) separating blood into erythrocytes, which contain only AChE, and plasma which contains only BChE, to measure their activity individually, or (2) use a BChE-specific inhibitor to measure the activity of AChE in whole blood. A number of studies have reported the use of different inhibitors for the simultaneous measurement of AChE and BChE activities. However, the inhibitors used for completely inhibiting BChE activity also inhibited AChE activity leading to errors in reported values. The goal of this study was to find the most accurate and simple method for the simultaneous determination of AChE and BChE activity in animal whole blood. Solutions containing human AChE and BChE in various proportions were prepared and AChE and BChE activities were measured using three reported methods. Results demonstrate that ethopropazine and (-) huperzine A appear to be the most specific ChE inhibitors. Preliminary results with human and animal whole blood suggest that 20muM ethopropazine and 500nM (-) huperzine A can be used for measuring AChE and BChE activities across species.     

Effects of ascorbic acid on cadmium-induced oxidative stress and performance of broilers

The effects of cadmium on performance, antioxidant defense system, liver and kidney functions, and cadmium accumulation in selected tissues of broiler chickens were studied. Whether the possible adverse effects of cadmium would reverse with the antioxidant ascorbic acid was also investigated. Hence, 4 treatment groups (3 replicates of 10 chicks each) were designed in the study: control, ascorbic acid, cadmium, and cadmium plus ascorbic acid. Cadmium was given via the drinking water at a concentration of 25 mg/L for 6 wk. Ascorbic acid was added to the basal diet at 200 mg/kg either alone or with cadmium. Cadmium decreased the body weight (BW), body weight gain (BWG), and feed efficiency (FE) significantly at the end of the experiment, wheras its effect on feed consumption (FC) was not significant. Cadmium increased the plasma malondialdehyde (MDA) level as an indicator of lipid peroxidation and lowered the activity of blood superoxide dismutase (SOD). Liver function enzymes, aspartate amino transferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), and gamma glutamyl transferase (GGT) activities were not changed by cadmium. Cadmium ingestion did not alter serum creatinine levels. Although the serum cadmium level was not elevated, cadmium mainly accumulated in the kidneys, liver, pancreas, and muscle. Ascorbic acid supplementation resulted in a reduction of MDA level previously increased by cadmium and a restoration in SOD activity. However, ascorbic acid did not ameliorate the growth inhibitory effect of cadmium nor did it prevent accumulation of cadmium in analyzed tissues. These data indicate that oxidative stress, induced by cadmium, plays a role in decreasing the performance of broilers and that dietary supplementation by ascorbic acid might be useful in reversing the lipid peroxidation induced by cadmium and partly alleviating the adverse effect of cadmium on performance of broilers.     

Cadmium Removal by Lemna minor and Spirodela polyrhiza

The present study investigates the ability of two genus of duckweed (Lemna minor and Spirodela polyrhiza) to phytoremediate cadmium from aqueous solution. Duckweed was exposed to six different cadmium concentrations, such as, 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 mg/L and the experiment was continued for 22 days. Water samples were collected periodically for estimation of residual cadmium content in aqueous solution. At the end of treatment period plant samples were collected and accumulated cadmium content was measured. Cadmium toxicity was observed through relative growth factor and changes in chlorophyll content. Experimental results showed that Lemna minor and Spirodela polyrhiza were capable of removing 42–78% and 52–75% cadmium from media depending upon initial cadmium concentrations. Cadmium was removed following pseudo second order kinetic model. Maximum cadmium accumulation in Lemna minor was 4734.56 mg/kg at 2 mg/L initial cadmium concentration and 7711.00 mg/kg in Spirodela polyrhiza at 3 mg/L initial cadmium concentration at the end of treatment period. Conversely in both cases maximum bio-concentration factor obtained at lowest initial cadmium concentrations, i.e., 0.5 mg/L, were 3295.61 and 4752.00 for Lemna minor and Spirodela polyrhiza respectively. The present study revealed that both Lemna minor and Spirodela polyrhiza was potential cadmium accumulator.     

The tissue disposition and urinary excretion of cadmium, zinc, copper and iron, following repeated parenteral administration of cadmium to rats.

The effect of repeated parenteral administration of cadmium (0.75, 1.5 and 3.0 mg/kg) on tissue disposition and urinary excretion of cadmium, zinc, copper and iron has been studied in the male rat. Cadmium, zinc and copper accumulated in liver and kidney, but the concentration of iron did not alter significantly. The kidney weight relative to body weight showed a dose-related increase in weight of 25--65%. Excretion of cadmium in the urine increased directly with dosage and the increase was most significant when kidney damage had probably occurred. Administration of cadmium also resulted in dose-related increases in the urinary excretion of zinc, copper and iron. The cadmium concentration of blood increased with dosage of cadmium, and the plasma concentrations of zinc and copper were also raised but plasma iron concentration was diminished.     

Ecological and developmental dynamics of a host-parasite system involving a sea anemone and two ctenophores

The lined sea anemone Edwardsiella lineata has evolved a derived parasitic life history that includes a novel body plan adapted for life inside its ctenophore hosts. Reputedly its sole host is the sea walnut, Mnemiopsis leidyi, a voracious planktivore and a seasonally abundant member of many pelagic ecosystems. However, we have observed substantially higher E. lineata prevalence in a second ctenophore species, the ctenophore predator Bero? ovata. The interplay among these 3 species has important conservation consequences as M. leidyi introductions are thought to be responsible for the severe depletion of numerous commercial fisheries in the Mediterranean basin, and both E. lineata and B. ovata have been proposed as biological controls for invasive M. leidyi. Over a 3-yr period (2004-2006), we collected 8,253 ctenophores from Woods Hole, Massachusetts, including M. leidyi, B. ovata, and a third ctenophore, Pleurobrachia pileus, and we recorded E. lineata infection frequencies, parasite load, and parasite location. We also conducted laboratory experiments to determine the likely mechanisms for parasite introduction and the effect of each host on parasite development. We observed peak E. lineata infection frequencies of 0% in P. pileus, 59% in M. leidyi, and 100% in B. ovata, suggesting that B. ovata could be an important natural host for E. lineata. However, in laboratory experiments, E. lineata larvae proved far more successful at infecting M. leidyi than B. ovata, and E. lineata parasites excised from M. leidyi exhibited greater developmental competence than parasites excised from B. ovata. Although we show that E. lineata is efficiently transferred from M. leidyi to B. ovata when the latter preys upon the former, we conclude that E. lineata larvae are not well adapted for parasitizing the latter species and that the E. lineata parasite is not well adapted for feeding in B. ovata; these developmental and ecological factors underlie the host specificity of this recently evolved parasite.     

The effect of environmental parameters on growth,cholinesterase activity and glutathione S-transferase activity in the earthworm (Apporectodea caliginosa)

The effect of age and environmental parameters on growth, cholinesterase (ChE), and glutathione S-transferase (GST) activities were assessed in juvenile Apporectodea caliginosa earthworms. Earthworms were maintained in three types of soil- loam, sandy, and clay- at a range of moisture contents (15-30%), and temperatures (5-20 C). Earthworm age (1-3 months) had no significant affect on ChE activity. Growth rates were influenced by all environmental parameters tested and these effects were interrelated. Optimum conditions for growth appeared to be in loam or sandy soil with 25-30% moisture at 10-15 C. The GST activity was influenced by soil temperature and activity was significantly higher at 15 C than at 5 C and 10 C. Soil type also influenced GST activity and this influence was dependent on moisture content. In sandy soil GST activity was significantly lower at 30% moisture than at lower moisture contents, in loam soil GST activity was significantly higher at 15% moisture than at higher moisture contents, while in clay soil GST activity was not affected by moisture content. These results indicate that in field experiments when evaluating GST activity soil temperature and soil type need to be consistent between control and 'contaminated sites'. ChE activity was only affected by temperature, so this should be considered when comparing control and treatedareas.     

The effect of environmental parameters on growth, cholinesterase activity and glutathione S-transferase activity in the earthworm (Apporectodea caliginosa)

The effect of age and environmental parameters on growth, cholinesterase (ChE), and glutathione S-transferase (GST) activities were assessed in juvenile Apporectodea caliginosa earthworms. Earthworms were maintained in three types of soil- loam, sandy, and clay- at a range of moisture contents (15-30%), and temperatures (5-20 C). Earthworm age (1-3 months) had no significant affect on ChE activity. Growth rates were influenced by all environmental parameters tested and these effects were interrelated. Optimum conditions for growth appeared to be in loam or sandy soil with 25-30% moisture at 10-15 C. The GST activity was influenced by soil temperature and activity was significantly higher at 15 C than at 5 C and 10 C. Soil type also influenced GST activity and this influence was dependent on moisture content. In sandy soil GST activity was significantly lower at 30% moisture than at lower moisture contents, in loam soil GST activity was significantly higher at 15% moisture than at higher moisture contents, while in clay soil GST activity was not affected by moisture content. These results indicate that in field experiments when evaluating GST activity soil temperature and soil type need to be consistent between control and 'contaminated sites'. ChE activity was only affected by temperature, so this should be considered when comparing control and treatedareas.     

鳡鱼仔稚鱼耳石的标记和其日轮的确证

用茜素络合物对鳡鱼仔鱼进行了浸泡标记,耳石上能检测到橘红色荧光标记环。100mg/L茜素络合物溶液的标记效果较差;120mg/L和150mg/L溶液浸泡后微耳石的标记率很高,矢耳石和星耳石的标记率低。标记后耳石上的生长轮数与饲养天数间呈一一对应关系,相关方程为:N=0.2663 0.9276D(n=68,r~2=0.9664)。方程的斜率0.9276与1无显著差异,证明生长轮确系日轮。鱼体长与微耳石、矢耳石及星耳石的直径间呈显著的线性关系,相关方程为:BL=66.8723LD 2.7064(n=73,r~2=0.8867),BL=22.7839SD 6.6066(n=49,r~2=0.8525),BL=47.6079AD 3.5660(n=71,r~2=0.9012)。