Cholinesterase activities in the Antarctic scallop Adamussium colbecki: Tissue expression and effect of ZnCl2 exposure

Biochemical characterization of cholinesterase activity (ChE) was carried out on the Antarctic scallop Adamussium colbecki collected in winter 2000 from Campo Icaro (Ross Sea, Antarctica) in order to increase its suitability as a sentinel organism for monitoring the Antarctic environment. The digestive gland, gills and adductor muscle were investigated for substrate specificity and inhibitors sensitivity using acetylthiocholine iodide (ASCh) and butyrylthiocholine iodide (BSCh) as substrates and tetra (monoisopropyl)pyrophosphor-tetramide (Iso-OMPA), 1,5-bis(4-allyldimethylammoniumphenyl)-penthan-3-one dibromide (BW284c51) and the insecticide chlorpyrifos as inhibitors. Effect of in vivo exposure to ZnCl₂ was also investigated. All the tissues expressed ChE activity (gill > adductor muscle > digestive gland) and low substrates specificity throughout the hydrolysis of both ASCh and BSCh substrates. Partial (25–29%) and total inhibition (100%) of ChE activity in gills was demonstrated following in vitro incubation with Iso-OMPA and BW284c51 (3 mM), respectively. Concentration-dependent inhibition was also evident with chlorpyrifos in the range 10⁻⁴–10⁻¹⁰ M (IC₅₀ 10⁻⁶) while in vivo exposure to ZnCl₂ did not seem to affect ChE activity in the scallop. The potential use of ChE in the A. colbecki as biomarker for monitoring water contamination in the marine Antarctic environment is discussed.     

Cholinesterase activities in the Antarctic scallop Adamussium colbecki: tissue expression and effect of ZnCl2 exposure

Biochemical characterization of cholinesterase activity (ChE) was carried out on the Antarctic scallop Adamussium colbecki collected in winter 2000 from Campo Icaro (Ross Sea, Antarctica) in order to increase its suitability as a sentinel organism for monitoring the Antarctic environment. The digestive gland, gills and adductor muscle were investigated for substrate specificity and inhibitors sensitivity using acetylthiocholine iodide (ASCh) and butyrylthiocholine iodide (BSCh) as substrates and tetra (monoisopropyl)pyrophosphor-tetramide (Iso-OMPA), 1,5-bis(4-allyldimethylammoniumphenyl)-penthan-3-one dibromide (BW284c51) and the insecticide chlorpyrifos as inhibitors. Effect of in vivo exposure to ZnCl(2) was also investigated. All the tissues expressed ChE activity (gill > adductor muscle > digestive gland) and low substrates specificity throughout the hydrolysis of both ASCh and BSCh substrates. Partial (25-29%) and total inhibition (100%) of ChE activity in gills was demonstrated following in vitro incubation with Iso-OMPA and BW284c51 (3 mM), respectively. Concentration-dependent inhibition was also evident with chlorpyrifos in the range 10(-4)-10(-10) M (IC(50) 10(-6)) while in vivo exposure to ZnCl(2) did not seem to affect ChE activity in the scallop. The potential use of ChE in the A. colbecki as biomarker for monitoring water contamination in the marine Antarctic environment is discussed.     

毒死蜱和重金属对近海沉积物碱性磷酸酶和过氧化氢酶活性的影响

选择连云港近海临洪河口河心浅滩沉积物,采用不同浓度毒死蜱和重金属于室内培养,研究了毒死蜱和重金属对沉积物碱性磷酸酶和过氧化氢酶酶活性及其动力学特征的影响。研究结果表明:1)碱性磷酸酶活性受毒死蜱影响显著,峰值出现在10-200mg/kg浓度区间,培养第三周时V_max显著下降,酶活性抑制作用最显著。2)毒死蜱对过氧化氢酶激活作用的峰值出现在50mg/kg处。培养三周,过氧化氢酶活性逐渐回归正常值。3)Cd处理对沉积物碱性过氧化氢酶和过氧化氢酶抑制作用显著,明显强于Pb处理以及Cd、Pb复合处理,而Pb在相当程度上缓解了Cd对两种酶的抑制作用。4)酶活性可以用来指示沉积物的早期污染状况。     

In vitro and in vivo inhibition of acetylcholinesterase and carboxylesterase by metals in zebrafish (Danio rerio)

Metals are natural components in ecosystems; however, if these elements are in excess, they can have adverse effects on living organisms. This study analyzes the interference of copper, lead, iron and cadmium in acetylcholinesterase (AChE) and carboxylesterase (CbE) activities in zebrafish. AChE was significantly inhibited in vitro by copper, iron, lead and cadmium at higher concentrations (10 and 20 mmol/L), whereas CbE was inhibited only at a concentration of 20 mmol/L. In vivo, only lead and cadmium were able to cause AChE inhibition at higher concentrations, while iron didn't cause any changes, and copper promoted an increase in AChE activity at a concentration of 0.06 mg/L. CbE activity did not change at any of the times (two and seven days) and concentrations tested, except in the case of copper exposure, which resulted in a decrease in CbE activity. Indeed, iodoacetamide treatment didn't changed AChE neither CbE activities, results which indicate that the metal inhibiting effect is probably not due to its biding to thiol groups close the active site of the enzyme. This outcome reveals that metals are important esterase inhibitors in zebrafish, and should be considered in environmental monitoring studies that use esterase inhibition as exposure biomarkers of organophosphate and carbamate pesticides.     

海鱼AChE监测海水有机磷农药的可行性研究

有机磷农药(OPs)对乙酰胆碱酯酶(AChE EC 3.1.1.7)具有专一性抑制作用,因此AChE常作为淡水和土壤OPs污染的特异性标识物(biomarker).本文在海水背景下,通过测定两种有机磷农药(马拉硫磷和甲基对硫磷)对海洋鱼类-鲈鱼[Lateolabrax japonicus(cuvier)]脑组织AChE的抑制效应,分析了海鱼AChE监测海水OPs的可行性.结果显示,抑制时间为20min时,抑制程度达到相对稳定;在低浓度OPs抑制下,纯酶液比粗酶液表现出更好的敏感性,对马拉硫磷和甲基对硫磷的灵敏度分别达到0.28μg*L-1和0.2μg*L-1;在一定浓度范围内,AChE活力对OPs具有显著的线性响应.研究表明,利用鲈鱼AChE指示和监测海水OPs的微污染是完全可行的.     

Role of biotransformation in determining aldicarb toxicity in fish

The carbamate pesticide, aldicarb, demonstrates significant acute toxicity in fish, and is readily biotransformed by most organisms studied. In fish, both the cytochrome P450 (CYP) and the flavin monooxygenase systems (FMO) are involved in bioactivating aldicarb to aldicarb sulfoxide, which is a more potent inhibitor of acetylcholinesterase (AChE). Channel catfish (Ictalurus punctatus), along with many other fresh water species, do not express FMO and are relatively resistant to the effects of aldicarb. This project examined the toxicity, AChE inhibition, metabolism, and toxicokinetic of aldicarb in channel catfish, and compared these values with an aldicarb-sensitive species, rainbow trout, which expresses FMO. Studies of in vitro and in vivo aldicarb biotransformation in catfish suggest that a low rate of bioactivation (10 times slower V_max), resulting in less initial conversion to the activated metabolite, aldicarb sulfoxide, may be a contributing factor to resistance of channel catfish to aldicarb toxicity. These data are supported by toxicokinetic and enzyme inhibition studies. This work demonstrates that differences in FMO expression among fish species may have significant influence on toxicity resulting from exposure to some xenobiotics.     

Characterisation of choline esterases and their tissue and subcellular distribution in mussel (Mytilus edulis)

Acetylcholinesterase in mussel is potentially a useful biomarker of exposure to organophosphates (OP) in the marine environment. This study looked at cholinesterase activity in subcellular fractions of various tissues from the common mussel, Mytilus edulis. Measurement of enzyme rates demonstrated that although highest specific activity was found in foot 'mitochondrial' fraction, recovery of activity was very low. Gill 'microsomal' fraction had the second highest specific activity with a useful level of recovery and therefore was the most suitable tissue fraction for biomarker applications. Comparative studies of alternative alkylthiocholine substrates and competitive inhibitors suggest there is a single cholinesterase enzyme type present in this fraction. Inhibition of alkylcholine hydrolysis by BW284C51, specific to acetylcholinesterase in vertebrates, showed that cholinesterase activity in gill 'microsomal' fraction is inhibited by this compound but to a lesser extent than in vertebrate AChE. Inhibition of cholinesterase activity by azamethiphos in gill 'microsomal' fraction gave an IC50 of approximately 100 microM and showed both time and concentration dependence. However this indicates a lower potency compared to other animals and it is debatable whether mussel cholinesterase activity is useful as a biomarker of exposure in the field.     

Modulation of trout 7-ethoxyresorufin-O-deethylase (EROD) activity by estradiol and octylphenol.

Estrogens appear to have a modulating effect on the expression of cytochrome P4501A (CYP1A) in fish. A number of in vivo studies have demonstrated that hepatic CYP1A expression in females decrease during sexual maturation when plasma levels of 17 beta-estradiol (E2) increase, or in cases when the fish in injected with E2. Since a number of environmental contaminants have weak estrogen-like activities, the question arises if these compounds are able to modulate CYP1A expression as well. In the present study, we used in vitro monolayer cultures of rainbow trout, Oncorhynchus mykiss, liver cells to compare concentration-dependent (10(-9) to 10(-5) M) effects of the natural steroid E2 and the non-steroidal xenoestrogen 4-tert-octylphenol (OP) on CYP1A-catalyzed 7-ethoxyresorufin-O-deethylase (EROD) activity. The concentration dependency of the estrogenic activity of the two test compounds was assessed by determination of hepatocellular vitellogenin (Vg) release into the culture medium. Exposure of hepatocytes to E2 concentrations of 10(-8) M and higher led to a significant inhibition of basal cellular EROD activity. On the contrary, exposure to OP did not result in an inhibition of EROD activity, even at OP concentrations (10(-6) M, 10(-5) M) which were associated with a significant induction of Vg synthesis.     

Brain acetylcholinesterase as a marine pesticide biomarker using Brazilian fishes

Brain acetylcholinesterase (AChE) of some fishes from the coast of Rio de Janeiro State was studied as a possible pesticide biomarker in marine environmental monitoring. AChE specific activity in brain varied from 145 to 530 U/g of proteins and the Michaelis-Menten constant (K(M)) for acetylthiocholine varied from 104 to 291 microM among the 20 species studied. The enzyme sensitivity to methyl paraoxon, evaluated by the inhibition kinetic constants, shows that some species (Paralonchurus brasiliensis and Genidens genidens) are more sensitive (IC50-30 min=455 and 468 nM, respectively). The less sensitive Merluccius hubbsi and Percophis brasiliensis (IC50-30 min=3339 and 3259 nM, respectively) belong to the super-order Paracanthopterygii, which includes the more ancient species. On the other hand, more susceptible species belong to the super-order Acanthopterygii, which includes more recent species. These results suggest a possible evolutionary linkage for AChE sensitivity to methyl paraoxon. The application of inhibition kinetic constants for fish brain AChE in phylogenetic studies is still being investigated. The results have shown that a fish sentinel species should have the highest brain AChE level among the more sensitive ones.     

海黍子和脆江蓠对重金属锌、镉富集的比较研究

采用室内培养实验方法,研究了褐藻海黍子(Sargassum muticum)和红藻脆江蓠(Gracilaria chouae)在不同质量浓度的锌和镉溶液里15 d,藻体的生长和体内金属离子含量的变化。结果表明:两种藻的生长速率与暴露溶液金属质量浓度呈负相关,藻体内积累金属离子的量和溶液的金属质量浓度呈正相关,藻体内金属离子的含量均在培养3 d后显著增加,且随着培养时间的延长而持续增加。海黍子和脆江蓠对重金属Zn~(2+)的富集能力明显高于对Cd~(2+)的富集能力,在相同条件下,海黍子对重金属Zn~(2+),Cd~(2+)的积累量明显高于脆江蓠。以上研究结果对利用藻类修复重金属污染的海洋水体能起到一定的指导和参考作用。     

Characterization of cholinesterases in marbled sole, Limanda yokohamae, and their inhibition in vitro by the fungicide iprobenfos

Cholinesterases (ChEs) have been characterized in marbled sole (Limanda yokohamae) for use as a possible biomarker of pollution exposure. In brain, ChEs existed almost exclusively (>95%) as acetylcholinesterase (AChE) whereas in muscle, about 20-30% of ChE activity was in the form of butyrylcholinesterase (BChE; pseudocholinesterase). Acetylthiocholine and butyrylthiocholine (identified in mammalian studies as diagnostic substrates for AChE and BChE respectively) were hydrolyzed mainly, but not exclusively, by these enzymes. The inhibitors BW284C51 and iso-OMPA (identified in mammalian studies as diagnostic inhibitors of AChE and BChE respectively) were not specific for these enzymes in marbled sole. Brain AChE and muscle AChE and BChE were characterized in terms of their kinetic properties (KM etc.) and optimal conditions (substrate concentration, protein concentration, pH etc.) were established to allow routine assays of ChE activity to proceed under pseudo-first order conditions. The sensitivity of ChEs to a locally significant pesticide, iprobenfos (IBP; kitazin) was established in terms of IC50 concentrations. Brain AChE was relatively insensitive to IBP, but muscle AChE and BChE were sensitive to IBP concentrations in the high nM range. However, ambient IBP concentrations in Korean coastal waters are usually not high enough to cause detectable ChE inhibition in this species.     

联苯菊酯暴露对雄性褐菖鲉精巢发育的干扰

Bifenthrin(BF) is a pyrethroid insecticide that is widely used in agriculture, horticulture, and for residential purposes. However, few studies addressing the reproductive toxicity of BF on fishes are available. The present study was conducted to investigate the effects of BF on testicular development in Sebastiscus marmoratus and to gain insight into its mechanism. After exposure to 1, 10 and 100 ng/L BF for 50 days, there was a reduced number of mature sperm and an abundance of the late stages of spermatocysts in the testes. The levels of 17β-estradiol and testosterone were decreased significantly after BF exposure. The activity of caspase-3 was increased in a dosedependent manner after BF exposure, TUNEL assay indicated that BF exposure resulted in the occurrence of apoptosis in the testes, which might be main reason for the inhibition of spermatogenesis.     

Environmental monitoring using acetylcholinesterase inhibition in vitro. A case study in two Mexican lagoons.

Cholinesterase inhibition is considered a specific biomarker of exposure and effect for organophosphorous pesticides. Its use for monitoring has been hindered, particularly in tropical countries where organophosphates are widely used for malaria and dengue control, because of the frequent lack of suitable controls. An in vitro technique is proposed as a biochemical method for monitoring pollutant mixtures in sediment toxicity tests. Brain homogenate from the fish Oreochromis niloticus is used as the enzyme source. Optimum incubation time, extraction solvent and effect of crude oil on acetylcholinesterase (AChE) are reported. The method described was used in sediments from two Mexican lagoons, located in an oil extraction area where pesticides are used in agriculture and vector control campaigns. AChE inhibitions from 3 to 21% were found in these lagoons, even in the presence of high concentrations of petroleum.     

鼠尾藻对重金属锌、镉富集及排放作用的研究

将鼠尾藻(Sargassum thunbergii)暴露在不同质量浓度锌和镉溶液里40 d,以及暴露20 d后,移入自然海水恢复20 d,分析了藻体内金属离子含量的变化。富集实验结果表明,鼠尾藻积累金属离子的量和暴露溶液金属质量浓度正相关、与暴露时间具有线性关系;暴露3 d,藻体的金属离子含量(除了暴露在Cd2+质量浓度为0.1 mg/L处理组)均显著增加;在Zn2+处理组,溶液里Zn2+质量浓度越大,线性关系越显著,而在Cd2+处理组,Cd2+质量浓度5.0 mg/L处理组藻体内Cd2+含量与暴露时间的线性关系则最低。排放试验结果表明,排放20 d后与排放0 d比较,藻体内的金属离子质量浓度均显著降低,但是均显著高于暴露前水平;在Zn2+溶液里,鼠尾藻暴露Zn2+质量浓度0.1 mg/L和Zn2+质量浓度5.0 mg/L 20 d后移入自然海水5 d后,藻体内的Zn2+含量与排放0 d相比显著降低,而在Cd2+溶液里,藻体排放5 d后,藻体内的Cd2+含量与排放0 d相比均显著降低。最终结果表明,鼠尾藻潜在地可作为一种指示生物,用于监测海洋环境重金属污染状况。同时,鼠尾藻对Zn2+和Cd2+具有较强的富集能力,可以利用鼠尾藻修复受重金属污染的海洋水体,尤其是锌的污染。     

Biomarker responses to pollution in two invertebrate species: Scrobicularia plana and Nereis diversicolor from the Cádiz bay (SW Spain)

The clam Scrobicularia plana and the polychaete worm Nereis diversicolor were collected in several sites from a littoral enclosure in SW Spain. The aim of our study was to relate various biomarker responses in these species to a pollution gradient caused by untreated domestic discharges and to verify the adequacy of the selected species as sentinels in this habitat. The biomarkers selected were the antioxidant enzymes catalase (CAT), glutathione peroxidase (GPX) and DT-diaphorase (DT-D). In addition, the activities of cytochrome P450-dependent ethoxyresorufin O-deethylase (EROD) activity, the phase II detoxifying enzyme glutathione S-transferase (GST) and the neurotoxicity marker acetylcholinesterase (AChE) were measured. Metallothionein levels were selected as biomarkers of heavy metals exposure in both species. The results suggest a different response in the water filtering organism (clam) and the sediment eater (polychaete), probably as a consequent of different pollution exposure and that samples from the “Caño Sancti-Petri” were exposed to biologically active compounds that altered some of their biochemical responses. AChE was the most sensitive biomarker in both species and N. diversicolor proved to be a more robust sentinel in this ecosystem.     

水体Hg^2＋对中华绒螯蟹（Eriocheir sinensis）血淋巴抗氧化酶活力和丙二醛含量的影响

采用单因子实验设计方法,进行了水体汞（Hg^2＋）在不同暴露时间对中华绒螯蟹血淋巴超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GPX）和过氧化氢酶（CAT）活力以及脂质过氧化产物丙二醛（MDA）含量的影响研究。结果表明,较低浓度Hg^2＋（0．01和0．05mg／L）处理组中华绒螯蟹血淋巴SOD、GPX、CAT活力在...     

Pb2+对青蛤的急性毒性及对其血淋巴液中免疫相关酶活性的影响

采用静态急性毒性实验研究了重金属Pb~(2+)对青蛤(Cyclina sinensis)的生物急性毒性效应,测定了在96 h Pb~(2+)半致死浓度的1/10(TC组)和1/100(SC组)两个浓度胁迫下,血淋巴液中酸性磷酸酶(ACP)、碱性磷酸酶(AKP)和溶菌酶(LSZ)活性的变化。结果显示:Pb~(2+)的96 h LC50为7.938 mg/L; SC组ACP活性表现为诱导-抑制趋势,除4 d外均与对照组差异显著(P0.05), TC组为抑制趋势,为3个组中的最低,与对照组差异显著(P0.05);实验组SC组和TC组的AKP、LSZ活性均表现为前期为诱导中后期受抑制的趋势,与对照组差异显著(P0.05),且TC组活性始终低于SC组,表现出Pb~(2+)的胁迫浓度越高酶活性受到的抑制作用越大。以上结果表明,重金属Pb~(2+)对青蛤的毒性级别为高毒级,能造成青蛤免疫相关酶的活性受到抑制,影响青蛤的免疫能力,而且这种抑制作用随着环境中Pb~(2+)浓度增加而增加。     

Lead and Aroclor 1254 disrupt reproductive neuroendocrine function in Atlantic croaker

We have previously shown that lead (lead chloride) and a polychlorinated biphenyl (PCB) mixture (Aroclor 1254) can alter hypothalamic serotonin (5-hydroxytryptamine, 5-HT) content, and pituitary gonadotropin II (GTH II) release in vitro, in the Atlantic croaker (Micropogonias undulatus). In the present study we investigated whether impairment of the hypothalamic 5-HT pathway involves alterations in tryptophan hydroxylase (TPH), the rate-limiting enzyme in 5-HT biosynthesis, or monoamine oxidase (MAO), the catabolic enzyme. Aroclor 1254 (1 mg/kg body wt. for 30 days) significantly inhibited hypothalamic TPH activity without altering MAO activity, and caused a significant decline in 5-HT content. On the other hand, lead exposure (15 mg/kg body wt. for 30 days) only induced a slight decrease in hypothalamic 5-HT content and TPH activity, and a minor increase in MAO activity. However, both Aroclor 1254 and lead significantly inhibited the GTH II response to stimulation by a luteinizing hormone-releasing hormone analog (LHRHa) in vivo and caused reduced gonadal growth. These results demonstrate that impairment of hypothalamic serotonin metabolism by Aroclor 1254 involves inhibition of 5-HT synthesis, whereas lead does not exert a profound influence on 5-HT metabolism. The decline in 5-HT availability due to reduced 5-HT synthesis in the PCB-exposed fish may result in disruption of the stimulatory 5-HT-GnRH pathway controlling GTH II secretion leading to impairment of gonadal growth.