In vitro brain acetylcholinesterase response among three inbred strains of mice to monocrotophos

Abstract

The strain differences in the neurotoxic potential of monocrotophos (HCP) were assessed by determining the inhibition of brain acetylcholinesterase (AChE) in BALB/cAnN, DBA/2J and C57BL/6J in vitro. MCP being a competitive inhibitor for AChE, alters the Km values widely among these inbred strains. Comparatively least alterations in Km were found in BALB/cAnN and maximum in DBA/2J. Based on the Ki values DBA/2J was found to be the most sensitive strain to HCP inhibition followed by C57BL/6J and BALB/cAnN.     

Species differences in brain acetylcholinesterase and neuropathic target esterase response to monocrotophos

The species differences in the neurotoxic and delayed neurotoxic potential of monocrotophos (MCP) were assessed by determining the in vitro inhibition of brain Acetylcholinesterase (AChE) and Neuropathic target esterase (NTE) in rat, mice, chicken and pigeon. Based on I50 values, chicken brain AChE was found to be most sensitive to inhibition by MCP followed by rat whereas mice and pigeon were almost equally sensitive to AChE inhibition by MCP. The data on NTE inhibition by MCP in all the four species indicate the non-delayed neurotoxic nature of MCP. The results show that although there are many common features of the brain AChE and NTE of the four non-target organisms studied, certain species characteristics exist in their inhibition responses to MCP.     

Inhibitory effects on esterase enzymes buche and ache in rainbow trout (Oncorhyncus mykiss) produced by the slow release insecticide chitosan diethyl phosphate

The study on the toxicity of chitosan diethyl phosphate (ChDP), a controlled release insecticide, on the activities of butyrylcholinesterase (BuChE) and acetylcholinesterase (AChE) in rainbow trout exposed to this pesticide was carried out. It was found that ChDP reduced BuChE activity in O. mykiss by a factor of eight at 6 days, with high fluctuation to the end of the exposition time at 12 days. The in vitro analysis of brain AChE treated with ChDP and Phenamiphos showed that it was competitively inhibited by both organophosphates. The values obtained for Km and Vmax for the AChE-ChDP (Km: 21.23 microM; Vmax: 43.10 micromol/min/g) and AChE-Phenamiphos (Km: 38.62 microM; Vmax: 38.91 micromol/min/g) systems were relatively low compared to values of the AChE (control) system (Km: 62.99 microM; Vmax: 63.29 micromol/min/g). Results reported in this study confirmed that chitosan diethyl phosphate performs similarly to organophosphate pesticides, producing inhibition in cholinesterases in rainbow trout.     

The effect of pesticides and metals on acetylcholinesterase (AChE) in various tissues of blue mussel (Mytilus trossulus L.) in short-term in vivo exposures at different temperatures

The purpose of the study was to assess the impact of short-term exposure to selected toxicants as well as metal accumulation upon acetylcholinesterase (AChE) in the blue mussel, Mytilus trossulus L., in laboratory in vivo experiments. Mussels were exposed for up to 48 hours to a mixture of copper (Cu^{2 +}, 400 μ g L^?1) and cadmium (Cd^{2 +}, 200 μ g L^?1), to dichlorvos (DDVP, 100 μ g L^?1), and to carbaryl (100 μ g L^?1) at two temperatures: 5°C and 20°C. Samples were collected after 0, 6, 12, 24, and 48 hours of exposure, and AChE activity and metal concentration (where applicable) were analysed in gills, digestive gland, mantle+muscles, and the whole soft tissue. Very strong AChE inhibition was observed in response to the dichlorvos treatment, mainly in gills. Carbaryl and the metals caused a short-term inhibition effect. Considerable differences in AChE activity between the two temperatures were noticed. In particular, the metals were accumulated much faster at 20°C than at 5°C, especially in gills. No correlation between AChE activity and metal concentration was found. Gills turned out to be the optimal tissue for AChE activity analysis in short-term studies.     

Sublethal effects of monocrotophos on locomotor behavior and gill architecture of the mosquito fish, Gambusia affinis

Subacute studies of monocrotophos [Dimethyl (E)-1-methyl-2-(methyl-carbamoyl) vinyl phosphate] on mosquito fish, Gambusia affinis, were carried out in vivo for 24 days to assess the locomotor behavior, structural integrity of gill, and targeted enzyme acetylcholinesterase (AChE, EC: 3.1.1.7) interactions. Monocrotophos (MCP) can be rated as moderately toxic to G. affinis, with a median lethal concentration (LC(50)) of 20.49 +/- 2.45 mgL(-1). The fish exposed to sublethal concentration of LC(10) (7.74 mgL(-1)) were under stress and altered their locomotor behavior, such as distance traveled per unit time (m min(-1)) and swimming speed (cm sec(-1)) with respect to the length of exposure. Inhibition in the activity of brain AChE and deformities in the primary and secondary lamellae of gill may have resulted in failure of exchange of gases. The maximum inhibition of 95% of AChE activity was observed on days 20 and 24.Morphological aberrations in the gills were also studied during exposure to the sublethal concentration of monocrotophos for a period ranging from 8 to 24 days. The extent of damage in gill was dependent on the duration of exposure. The findings revealed that inhibition in brain AChE activity and structural alteration in gill were responsible for altering the locomotor behavior of exposed fish.     

Inhibitory Effects on Esterase Enzymes Buche and Ache in Rainbow Trout (Oncorhyncus mykiss) Produced by the Slow Release Insecticide Chitosan Diethyl Phosphate

The study on the toxicity of chitosan diethyl phosphate (ChDP), a controlled release insecticide, on the activities of butyrylcholinesterase (BuChE) and acetylcholinesterase (AChE) in rainbow trout exposed to this pesticide was carried out. It was found that ChDP reduced BuChE activity in O. mykiss by a factor of eight at 6 days, with high fluctuation to the end of the exposition time at 12 days. The in vitro analysis of brain AChE treated with ChDP and Phenamiphos showed that it was competitively inhibited by both organophosphates. The values obtained for K_m and V_max for the AChE-ChDP (K_m: 21.23 μ M; V_max: 43.10 μ mol/min/g) and AChE-Phenamiphos (K_m: 38.62 μ M; V_max: 38.91 μ mol/min/g) systems were relatively low compared to values of the AChE (control) system (K_m: 62.99 μ M; V_max: 63.29 μ mol/min/g). Results reported in this study confirmed that chitosan diethyl phosphate performs similarly to organophosphate pesticides, producing inhibition in cholinesterases in rainbow trout.     

Effects of repeated fumonisin B1 exposure on markers of oxidative stress in liver,kidneys, and lungs of C57BL/6 mice

Abstract

Fu monisin B₁ (FB₁) is a mycotoxin commonly found in maize and maize-based products. Ingestion of FB₁-contaminated causes a myriad of dose- and species-dependent toxic effects to human and animal health. In the present study we evaluated the effects of FB₁ (8?mg/kg, i.p. for 4?days) on body weight and oxidative stress parameters in the liver, kidney and lung of C57BL/6 male mice. No changes in the organ-to-body weight ratio, organ-to-adrenal gland weight ratio or organ-to-brain weight ratio were found. On the other hand, FB₁ exposure increased NPSH levels in liver and lungs whereas decreased FRAP content in liver and kidneys. Levels of TBARS, ascorbic acid and NOx content were not altered by FB₁. In summary, four days of FB₁ exposure are sufficient to disrupt antioxidant defenses in liver, kidneys and lungs of C57BL/6 male mice without concomitant changes in organs weight.     

Sublethal Effects of Monocrotophos on Locomotor Behavior and Gill Architecture of the Mosquito Fish,Gambusia affinis

Subacute studies of monocrotophos [Dimethyl (E)-1-methyl-2-(methyl-carbamoyl) vinyl phosphate] on mosquito fish, Gambusia affinis, were carried out in vivo for 24 days to assess the locomotor behavior, structural integrity of gill, and targeted enzyme acetylcholinesterase (AChE, EC: 3.1.1.7) interactions. Monocrotophos (MCP) can be rated as moderately toxic to G. affinis, with a median lethal concentration (LC₅₀) of 20.49 ± 2.45 mgL^?1. The fish exposed to sublethal concentration of LC₁₀ (7.74 mgL^?1) were under stress and altered their locomotor behavior, such as distance traveled per unit time (m min^?1) and swimming speed (cm sec^?1) with respect to the length of exposure. Inhibition in the activity of brain AChE and deformities in the primary and secondary lamellae of gill may have resulted in failure of exchange of gases. The maximum inhibition of 95% of AChE activity was observed on days 20 and 24.

Morphological aberrations in the gills were also studied during exposure to the sublethal concentration of monocrotophos for a period ranging from 8 to 24 days. The extent of damage in gill was dependent on the duration of exposure. The findings revealed that inhibition in brain AChE activity and structural alteration in gill were responsible for altering the locomotor behavior of exposed fish.     

Delayed neuropathy in sheep by the phosphonothioate insecticide cyanofenphos.

Cyanofenphos (surecide)(R), 25% E.C., O-ethyl O-(4-cyanophenyl) phenylphosphonothioate, was orally administered to one year old lambs at sublethal doses of 1 mg, 2 mg and 4 mg active ingredient kg-1 day-1 for time intervals 60, 45 and 30 days respectively. Irreversible paralytic ataxia symptoms of delayed neuropathy appeared at about 80, 50 and 30 days respectively. In weekly blood samples, AChE (acetylcholine-sterase) and MAO (monoamine oxidase) activities were inhibited depending upon level of dosing and time interval. However no significant correlation was found between the extent of plasma AChE and MAO inhibition and the onset of ataxia symptoms. In brain samples from ataxiated animals, AChE, MAO and NTE (neurotoxic esterase) activities were assayed simultaneously with untreated animal. Direct correlation was shown between in vivo NTE inhibition and the occurrence of delayed neuropathy. Cyanofenphos is the third compound of the phenyl phosphonothioate type on the market showing delayed neuropathy together with Leptophos and EPN.     

Effect of carbaryl (carbamate insecticide) on acetylcholinesterase activity of two strains of Daphnia magna (Crustacea,Cladocera)

The present study was designed to investigate the effect of carbaryl (carbamate insecticide) on the acetylcholinesterase activity in two strains (same clone A) of the crustacean cladoceran Daphnia magna. Four carbaryl concentrations (0.4, 0.9, 1.8 and 3.7 µg L^?1) were compared against control AChE activity. Our results showed that after 48 h of carbaryl exposure, all treatments induced a significant decrease of AChE activities whatever the two considered strains. However, different responses were registered in terms of lowest observed effect concentrations (LOEC: 0.4 µg L^?1 for strain 1 and 0.9 µg L^?1 for strains 2) revealing differences in sensitivity among the two tested strains of D. magna. These results suggest that after carbaryl exposure, the AChE activity responses can be also used as a biomarker of susceptibility. Moreover, our results show that strain1 is less sensitive than strain 2 in terms of IC50-48 h of AChE activity. Comparing the EC50-48 h of standard ecotoxicity test and IC50-48 h of AChE inhibition, there is the same order of sensitivity with both strains.     

Influence of paraquat, glyphosate, and cadmium on the activity of some serum enzymes and protein electrophoretic behavior (in vitro)

In vitro study for the determination of the toxicity of some pesticides (glyphospate and paraquat) and cadmium chloride (CdCl2) on the activities of serum acetylcholinesterase (AChE), lactate dehydrogenase (LDH), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (AlP), and acid phosphatase (AcP) is described. Changes in electrophoretic patterns of serum proteins were also tested. Results revealed that glyphosate was effective on all enzymes except AcP. Its IC50 values (the concentration of compound that inhibits 50% of the enzyme activity in 1 h at 37 degrees C) were 714.3, 750, 54.2, 270.8, and 71.4 mM for AChE, LDH, AST, ALT, and AlP, respectively. The inhibitory effect of paraquat varied markedly among all enzymes. The IC50 values of paraquat were 321.4 and 750 mM for AST and ALT, respectively. It had mild effect on AChE and LDH; and no effect on the activities of AlP and AcP. The effect of CdCl2 was pronounced with AChE, ALT, AlP, and AcP, and no effect on LDH and AST was found. The corresponding IC50 values were 77.7, 22.2, 33.3, and 83.3 mM for AChE, ALT, AlP, and AcP, respectively. Polyacrylamide gel electrophoretic patterns of serum proteins showed marked differences with glyphosate and CdCl2 but not with paraquat. The results suggest that the in vitro enzyme-activity test seems to have a potential for the assessment of pesticide and heavy metal toxicity.     

Genetic susceptibility to nickel-induced acute lung injury

Human exposure to insoluble and soluble nickel compounds is extensive. Besides wide usage in many industries, nickel compounds are contained in cigarette smoke and, in low levels, in ambient particulate matter. Soluble nickel particulate, especially nickel sulfate (NiSO₄), has been associated with acute lung injury. To begin identifying genes controlling susceptibility to NiSO₄, mean survival times (MSTs) of eight inbred mouse strains were determined after aerosol exposure. Whereas A/J (A) mice were sensitive, C57BL/6J (B6) mice survived nearly twice as long (resistant). Their offspring were similarly resistant, demonstrating heritability as a dominant trait. Quantitative trait locus (QTL) analysis of backcross mice generated from these strains identified a region on chromosome 6 significantly linked to survival time. Regions on chromosomes 1 and 12 were suggestive of linkage and regions on chromosomes 8, 9, and 16 contributed to the response. Haplotype analysis demonstrated that QTLs on chromosomes 6, 9, 12, and 16 could explain the MST difference between the parental strains. To complement QTL analysis results, cDNA microarray analysis was assessed following NiSO₄ exposure of A and B6 mice. Significant expression changes were identified in one or both strains for >100 known genes. Closer evaluation of these changes revealed a temporal pattern of increased cell proliferation, extracellular matrix repair, hypoxia, and oxidative stress, followed by diminished surfactant proteins. Certain expressed sequence tags clustered with known genes, suggesting possible co-regulation and novel roles in pulmonary injury. Together, results from QTL and microarray analyses of nickel-induced acute lung injury survival allowed us to generate a short list of candidate genes.     

Screening of POP pollution by AChE and EROD activities in Zebra mussels from the Italian Great Lakes

The increase of ethoxyresorufin dealkylation (EROD) and the inhibition of acetylcholinesterase (AChE) as biomarkers have been commonly used in vertebrates for the persistent organic pollutants (POPs) biomonitoring of aquatic environments, but very few studies have been performed for invertebrates. Previous researches demonstrated the interference due to some chemicals on EROD and AChE activities of the freshwater bivalve Zebra mussel (Dreissena polymorpha) in laboratory and field studies, showing its possible use for the screening of POP effects. We investigated the contamination of the Italian sub-alpine great lakes (Maggiore, Lugano, Como, Iseo, Garda) by the biomarker approach on Zebra mussel specimens collected at 17 sampling sites with different morphometric characteristics and anthropization levels. Results showed a homogeneous contamination of AChE inhibitors in Lake Garda, Maggiore, Como and Iseo with values ranging from 0.5 to 3 nmol/min/mg proteins and with an average inhibition of about 66% to controls. The planar compounds pollution, able to activate the EROD activity, seems higher in some sampling stations of Lake Garda, Como and Iseo (2-4 pmol/min/mg proteins) than that measured in Lake Lugano (1.5-3 pmol/min/mg proteins). On the contrary, the enzyme activity in Lake Maggiore showed an interesting opposite effect of AhR-binding compounds and trace metals. Finally, the possible use of Zebra mussel specimens maintained at laboratory conditions as controls against the selection of the less polluted sampling site is discussed.     

Inhibition of brain AChE in brook trout by aminocarb and its toxic metabolites

In-vitro inhibitions of brain AChE in brook trout, Salvelinus fontinalis (Mitchill), by aminocarb (4-dimethylamino-m-tolyl N-methylcarbamate) and its toxic metabolites, MAA (4-methylamino-m-tolyl N-methylcarbamate), AA(4-amino-m-tolyl N-methylcarbamate), MFA (4-methylformamido-m-tolyl N-methylcarbamate) and FA (4-formamido-m-tolyl N-methylcarbamate) were investigated. The molar concentrations of inhibitors causing 50% inhibition (I50s) were AA (3.62 X 10(-6] less than MAA (7.92 X 10(-6] less than aminocarb (1.01 X 10(-5] less than MFA (4.29 X 10(-5] less than FA (7.11 X 10(-5]. After exposure of fish to various concentrations of aminocarb (25, 250 and 2500 ppb) and MAA (25, 250, 500 and 2500 ppb) at 9 degrees C in dechlorinated tap water for 96 h, inhibitions of brain AChE ranged from 13 to 77%. Mortality occurred only in fish exposed to 500 ppb (22%) and 2500 ppb (100%) of MAA. Enzyme activities recovered to the control levels 12 to 96 h after the fish had been transferred to clean water for clearing; in survivors of the 500-ppb MAA exposure, however, AChE activities decreased again thereafter.     

Impact of chemical exposure on the fish Pomatoschistus microps Krøyer (1838) in estuaries of the Portuguese Northwest coast

Juveniles of the estuarine fish Pomatoschistus microps were collected from autumn 2001 to summer 2002 in five stations along the Portuguese Northwest coast with different types and/or levels of environmental contamination: two reference sites with low levels of contamination (R1 and R2) and three differently impacted areas with higher levels of contamination. UI is located in an estuary under the influence of urban and industrial effluents, AA in a channel that receives intensive agriculture run-off and IE in a highly impacted industrial area. The activity of the enzymes acetylcholinesterase (AChE), lactate dehydrogenase (LDH), 7-ethoxyresorufin O-deethylase (EROD) and glutathione S-transferases (GST) were used as environmental biomarkers on P. microps. A significant seasonality effect on all the enzymatic activities was found, lower levels being registered in winter and spring on AChE, in autumn on LDH, and in winter on GST and EROD. The battery of biomarkers used was capable of discriminating sites with different types and/or levels of contamination, R1 and UI being the highest discriminated (91.7% and 66.7%, respectively). At R1 significantly lower levels of AChE and LDH were found, and EROD was significantly inducted at UI. Furthermore, IE presented higher levels of GST, and R2 and AA an inhibition of AChE in winter and spring. The results indicated that the battery of biomarkers used in this study seems to be a useful tool to distinguish between different types of environmental contamination in estuarine systems, and that P. microps is a suitable species to be used as bioindicator.     

Disposition, elimination and enzyme induction of 1,2,3,7,8-PnCDD, 1,2,3,6,7,8-HxCDD and 2,3,4,7,8-PnCDF in the liver of the mouse after a single oral dose

The disposition, elimination and cytochrome P450 related enzyme induction of 1,2,3,7,8-pentachlorodibenzo-p-dioxin (PnCDD), 1,2,3,6,7,8-hexachlorodibenzo-p-dioxin (HxCDD) and 2,3,4,7,8-pentachlorodibenzofuran (PnCDF) were studied in the liver of the male C57BL/6 mouse. Elimination of all compounds from the liver followed first-order kinetics. Half-lives for PnCDD, HxCDD and PnCDF were 15, 52 and 65 days respectively. 7-Ethoxyresorufin-O-deethylation (EROD) activity in the livers of the treated animals showed a good correlation with the liver tissue concentrations but the slopes of these liver concentration-response curves were not equal for all three compounds.     

Polycyclic aromatic hydrocarbons air levels in Florence,Italy, and their correlation with other air pollutants

Benzo(a)pyrene [B(a)P] air levels were measured in Florence (Italy) in the period 1992-2001. For the period 1999-2000 seven polycyclic aromatic hydrocarbons (PAH) (benzo(a)anthracene, crysene, benzo(a)pyrene (B(a)P), benzo(b)fluoranthene (B(b)F), benzo(k)fluoranthene, dibenzo(a,h)anthracene (DBA) and benzo(g,h,i)perylene (BGP)), were measured in the air in four different sites (one with heavy traffic (A), one in a park (B), one in a residential area (C) and one in a hill area (D)). B(a)P levels were elevated in 1992-1998 (maximum average value of winter months: 5.8 ng/ m3) but a decreasing trend was observed in the following years, probably due to improvement in vehicle emissions. The sum of PAH in the air in the period 1999-2000 was about one order of magnitude lower in the hill site (D) relative to the urban sites, and residential areas (B and C) had values 2.5-3 times lower compared to site A with a heavy traffic. PAH concentrations decreased in the warmer seasons of 2000 in all sites. A negative correlation was found between PAH levels and ozone. A positive correlation with carbon monoxide (CO) (r = 0.862, P < 0.001) and low B(a)P/BGP ratios, ranging from 0.44 to 0.51, indicated that vehicular traffic was the major PAH source in all monitored sites. Using B(a)P(TEF) values (toxic equivalency factors) for evaluating the biological activity of PAH, we found that the highest PAH contributors in terms of potential air carcinogenic activity were B(a)P and DBA. Therefore, in addition to B(a)P, DBA concentration should be considered in the evaluation of air quality in terms of PAH contamination.     

Photodegradation of organophosphorus insecticides - investigations of products and their toxicity using gas chromatography-mass spectrometry and AChE-thermal lens spectrometric bioassay

Four organophosphorus compounds: azinphos-methyl, chlorpyrifos, malathion and malaoxon in aqueous solution were degraded by using a 125 W xenon parabolic lamp. Gas chromatography-mass spectrometry (GC-MS) was used to monitor the disappearance of starting compounds and formation of degradation products as a function of time. AChE-thermal lens spectrometric bioassay was employed to assess the toxicity of photoproducts. The photodegradation kinetics can be described by a first-order degradation curve C=C0e(-kt), resulting in the following half lives: 2.5min for azinphos-methyl, 11.6 min for malathion, 13.3 min for chlorpyrifos and 45.5 min for malaoxon, under given experimental conditions. During the photoprocess several intermediates were identified by GC-MS suggesting the pathway of OP degradation. The oxidation of chlorpyrifos results in the formation of chlorpyrifos-oxon as the main identified photoproduct. In case of malathion and azinphos-methyl the corresponding oxon analogues were not detected. The formation of diethyl (dimethoxy-phosphoryl) succinate in traces was observed during photodegradation of malaoxon and malathion. Several other photoproducts including trimethyl phosphate esters, which are known to be AChE inhibitors and 1,2,3-benzotriazin-4(3H)-one as a member of triazine compounds were identified in photodegraded samples of malathion, malaoxon, and azinphos-methyl. Based on this, two main degradation pathways can be proposed, both result of the (P-S-C) bond cleavage taking place at the side of leaving group. The enhanced inhibition of AChE observed with the TLS bioassay during the initial 30 min of photodegradation in case of all four OPs, confirmed the formation of toxic intermediates. With the continuation of irradiation, the AChE inhibition decreased, indicating that the formed toxic compounds were further degraded to AChE non-inhibiting products. The presented results demonstrate the importance of toxicity monitoring during the degradation of OPs in processes of waste water remediation, before releasing it into the environment.     

Mice exposure to tannery effluents changes their olfactory capacity,and their response to predators and to the inhibitory avoidance test

Environmental Science and Pollution Research - The current study has assessed whether the oral and/or dermal exposure of C57Bl/6 J mice to tannery effluent (a complex pollutant consisting of...