inhibition and spontaneous reactivation of esterases by organophosphorus compounds in presence of...

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S320 Abstracts / Toxicology Letters 196S (2010) S37–S351 Therefore, combination ratios should be predicated very carefully and further risk assessment studies should be performed in other pyrethroids with different doses. doi:10.1016/j.toxlet.2010.03.1009 P308-025 Evaluation of liquid–liquid extraction and different solid phase extraction cartridges for determination of selected synthetic pyrethroid insecticides in whole blood O. Yavuz, A. Aksoy, Y.K. Das, E. Atmaca Ondokuz Mayis University, Turkey Synthetic pyrethroid insecticides are widely used for control of many insects in agriculture, forestry, veterinary medicine, public health and in similar contexts. The presence of residual concentra- tions of the pyrethroids in environment due to the use of different formulations may possibly contribute to humans and domestic animals exposure either by inhalation or skin resorption. The evaluation of residues in blood and body fluids gives an indica- tion about the extent of exposure. Therefore, typical human and animal exposure assessment approach is to measure metabolic biomarkers in blood, urine or other biological fluids. In this study, comparison of classical LLE and different SPE cartridges for deter- mination of some synthetic pyrethroid insecticides in whole blood was aimed. Six synthetic pyrethroids (alphacypermethrin, perme- thrin, S-bioallethrin, cyphenothrin, cyfluthrin and tetramethrin) were spiked at three different concentration levels (250, 500 and 1000 ng/ml) in 1 ml rabbit blood and each dilution of the insecti- cides was applied classical LLE and three different SPE procedures (florisil, C18 and silica). Final solutions were analysed with gas chro- matography equipped with electron capture detector. Detection limits of the insecticides were found between 3.55 and 16.55 ng/ml. Recovery percentages were determined generally higher than 100% and matrix effect was observed in LLE procedures of all pyrethroids. SPE cartridges decreased this failure and SPE applications may eval- uate beneficial for synthetic pyrethroid analysis in whole blood. However, the reduction of the matrix effect was occurred different grades for used SPE cartridges and it was insufficient in some pro- cedures. Florisil cartridges were determined better than the other sorbents for providing good recoveries. This study may be analytical data for pyrethroid analysis using combinations of classical LLE and different SPE sorbents. Also, it was concluded that further studies should be performed with different LLE procedures and solutions of elution and extraction for determination of the optimal technique. doi:10.1016/j.toxlet.2010.03.1010 P308-026 Dual effects of lindane (gamma-hexachlorocyclohexane) on calcium homeostasis and exocytosis in rat PC12 cells H.J. Heusinkveld 1 , G.O. Thomas 2 , I. Lamot 1 , M. Van Den Berg 1 , A.B. Kroese 1 , R.H. Westerink 1 1 Institute for Risk Assessment Sciences, Neurotoxicology Research Group, UK, 2 Department of Environmental Sciences, Lancaster University, UK Persistent organochlorine pesticides, including lindane, are still abundant in the environment and often found in human and ani- mal tissues. Lindane has been shown to induce a wide range of adverse health effects, including alterations in neurobehavior. These adverse effects are mediated, at least partly, via inhibition of postsynaptic GABAA and glycine receptors. However, lindane- induced increases in the basal intracellular calcium concentration ([Ca 2+ ] i ) have also been reported. Calcium is the trigger for many cellular processes, including apoptosis, gene expression and exo- cytosis. We therefore investigated effects of lindane on dopamine exocytosis and calcium homeostasis in neuroendocrine PC12 cells using respectively amperometry and the calcium-sensitive fluores- cent ratio dye Fura-2. Mechanisms underlying changes in [Ca 2+ ] i were investigated using specific voltage-gated calcium channel (VGCC) blockers. Amperometric recordings demonstrated that lindane (>10 M) rapidly induced exocytosis in PC12 cells. Lindane-induced exocy- tosis was paralleled by a dose-dependent increase in [Ca 2+ ] i , which depended largely on influx of extracellular calcium. It was further shown that blocking L-type VGCCs did not prevent the increase in basal [Ca 2+ ] i , which appeared mainly mediated by N-, and P/Q-type VGCCs. In addition to increasing basal exocytosis and [Ca 2+ ] i , lin- dane (>10 M) dose-dependently inhibited depolarization-evoked exocytosis. As expected, this coincided with lindane-induced inhibition of the depolarization-evoked increase in [Ca 2+ ] i . However, in contrast to the increase in basal [Ca 2+ ] i , lindane- induced effects on depolarization-evoked [Ca 2+ ] i appeared VGCC a-specific. Lindane induces differential effects on basal and depolarization- evoked [Ca 2+ ] i , resulting in parallel effects on exocytosis. Considering the nature of these effects and the threshold concen- tration (>10 M), it can be concluded that the adverse health effects of lindane exposure are not uniquely due to postsynaptic effects but that presynaptic effects play a role as well. doi:10.1016/j.toxlet.2010.03.1011 P308-027 Inhibition and spontaneous reactivation of esterases by organophosphorus compounds in presence of the substrate: Paraoxon as a model J. Estévez, I. Mangas, E. Vilanova Universidad Miguel Hernández, Spain The kinetic analysis of the inhibition of esterases by organophos- phorus compounds is sometimes unable to yield consistent results by fitting simple inhibition kinetic models to experimental data of complex systems. In vitro experiments involve pre-incubating the enzyme preparation with an inhibitor concentration (I) dur- ing the inhibition times (t), and then incubating with a substrate during the enzyme–substrate reaction time (ts) to measure the residual enzyme activity (E). During the substrate reaction, inhi- bition is not usually significant due to the dilution of the inhibitor and the protective effect of the substrate. However some inhibi- tion may occur for the high potent inhibitors. In this work, this is known as “ongoing inhibition”. The kinetic data were obtained for 0.026 nM concentration of paraoxon incubated for up to two hours forty-three minutes with soluble fraction of chicken periph- eral nerve in presence of phenylvalerate as substrate. At 18 min of inhibition at 37 C the percentage of activity was around 61% and the percentage of residual activity at long time was around 35%. This inhibition is clearly time-progressive. The spontaneous reac- tivation of paraoxon-inhibited phenylvalerate esterases has also been observed in the chicken peripheral nerve soluble fraction in the presence of phenylvalerate. The kinetic data were obtained for preincubated samples with 10 nM of paraoxon and then they were diluted with buffer and phenylvalerate up to the residual concen-

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Page 1: Inhibition and spontaneous reactivation of esterases by organophosphorus compounds in presence of the substrate: Paraoxon as a model

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320 Abstracts / Toxicology L

herefore, combination ratios should be predicated very carefullynd further risk assessment studies should be performed in otheryrethroids with different doses.

oi:10.1016/j.toxlet.2010.03.1009

308-025valuation of liquid–liquid extraction and different solid phasextraction cartridges for determination of selected syntheticyrethroid insecticides in whole blood

. Yavuz, A. Aksoy, Y.K. Das, E. Atmaca

Ondokuz Mayis University, Turkey

ynthetic pyrethroid insecticides are widely used for control ofany insects in agriculture, forestry, veterinary medicine, public

ealth and in similar contexts. The presence of residual concentra-ions of the pyrethroids in environment due to the use of differentormulations may possibly contribute to humans and domesticnimals exposure either by inhalation or skin resorption. Thevaluation of residues in blood and body fluids gives an indica-ion about the extent of exposure. Therefore, typical human andnimal exposure assessment approach is to measure metaboliciomarkers in blood, urine or other biological fluids. In this study,omparison of classical LLE and different SPE cartridges for deter-ination of some synthetic pyrethroid insecticides in whole bloodas aimed. Six synthetic pyrethroids (alphacypermethrin, perme-

hrin, S-bioallethrin, cyphenothrin, cyfluthrin and tetramethrin)ere spiked at three different concentration levels (250, 500 and

000 ng/ml) in 1 ml rabbit blood and each dilution of the insecti-ides was applied classical LLE and three different SPE proceduresflorisil, C18 and silica). Final solutions were analysed with gas chro-

atography equipped with electron capture detector. Detectionimits of the insecticides were found between 3.55 and 16.55 ng/ml.ecovery percentages were determined generally higher than 100%nd matrix effect was observed in LLE procedures of all pyrethroids.PE cartridges decreased this failure and SPE applications may eval-ate beneficial for synthetic pyrethroid analysis in whole blood.owever, the reduction of the matrix effect was occurred differentrades for used SPE cartridges and it was insufficient in some pro-edures. Florisil cartridges were determined better than the otherorbents for providing good recoveries. This study may be analyticalata for pyrethroid analysis using combinations of classical LLE andifferent SPE sorbents. Also, it was concluded that further studieshould be performed with different LLE procedures and solutions oflution and extraction for determination of the optimal technique.

oi:10.1016/j.toxlet.2010.03.1010

308-026ual effects of lindane (gamma-hexachlorocyclohexane) onalcium homeostasis and exocytosis in rat PC12 cells

.J. Heusinkveld 1, G.O. Thomas 2, I. Lamot 1, M. Van Den Berg 1,.B. Kroese 1, R.H. Westerink 1

Institute for Risk Assessment Sciences, Neurotoxicology Researchroup, UK, 2 Department of Environmental Sciences, Lancasterniversity, UK

ersistent organochlorine pesticides, including lindane, are stillbundant in the environment and often found in human and ani-al tissues. Lindane has been shown to induce a wide range

f adverse health effects, including alterations in neurobehavior.

196S (2010) S37–S351

These adverse effects are mediated, at least partly, via inhibitionof postsynaptic GABAA and glycine receptors. However, lindane-induced increases in the basal intracellular calcium concentration([Ca2+]i) have also been reported. Calcium is the trigger for manycellular processes, including apoptosis, gene expression and exo-cytosis. We therefore investigated effects of lindane on dopamineexocytosis and calcium homeostasis in neuroendocrine PC12 cellsusing respectively amperometry and the calcium-sensitive fluores-cent ratio dye Fura-2. Mechanisms underlying changes in [Ca2+]iwere investigated using specific voltage-gated calcium channel(VGCC) blockers.

Amperometric recordings demonstrated that lindane (>10 �M)rapidly induced exocytosis in PC12 cells. Lindane-induced exocy-tosis was paralleled by a dose-dependent increase in [Ca2+]i, whichdepended largely on influx of extracellular calcium. It was furthershown that blocking L-type VGCCs did not prevent the increase inbasal [Ca2+]i, which appeared mainly mediated by N-, and P/Q-typeVGCCs. In addition to increasing basal exocytosis and [Ca2+]i, lin-dane (>10 �M) dose-dependently inhibited depolarization-evokedexocytosis. As expected, this coincided with lindane-inducedinhibition of the depolarization-evoked increase in [Ca2+]i.However, in contrast to the increase in basal [Ca2+]i, lindane-induced effects on depolarization-evoked [Ca2+]i appeared VGCCa-specific.

Lindane induces differential effects on basal and depolarization-evoked [Ca2+]i, resulting in parallel effects on exocytosis.Considering the nature of these effects and the threshold concen-tration (>10 �M), it can be concluded that the adverse health effectsof lindane exposure are not uniquely due to postsynaptic effects butthat presynaptic effects play a role as well.

doi:10.1016/j.toxlet.2010.03.1011

P308-027Inhibition and spontaneous reactivation of esterases byorganophosphorus compounds in presence of the substrate:Paraoxon as a model

J. Estévez, I. Mangas, E. Vilanova

Universidad Miguel Hernández, Spain

The kinetic analysis of the inhibition of esterases by organophos-phorus compounds is sometimes unable to yield consistent resultsby fitting simple inhibition kinetic models to experimental dataof complex systems. In vitro experiments involve pre-incubatingthe enzyme preparation with an inhibitor concentration (I) dur-ing the inhibition times (t), and then incubating with a substrateduring the enzyme–substrate reaction time (ts) to measure theresidual enzyme activity (E). During the substrate reaction, inhi-bition is not usually significant due to the dilution of the inhibitorand the protective effect of the substrate. However some inhibi-tion may occur for the high potent inhibitors. In this work, thisis known as “ongoing inhibition”. The kinetic data were obtainedfor 0.026 nM concentration of paraoxon incubated for up to twohours forty-three minutes with soluble fraction of chicken periph-eral nerve in presence of phenylvalerate as substrate. At 18 min ofinhibition at 37 ◦C the percentage of activity was around 61% andthe percentage of residual activity at long time was around 35%.This inhibition is clearly time-progressive. The spontaneous reac-tivation of paraoxon-inhibited phenylvalerate esterases has also

been observed in the chicken peripheral nerve soluble fraction inthe presence of phenylvalerate. The kinetic data were obtained forpreincubated samples with 10 nM of paraoxon and then they werediluted with buffer and phenylvalerate up to the residual concen-
Page 2: Inhibition and spontaneous reactivation of esterases by organophosphorus compounds in presence of the substrate: Paraoxon as a model

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ration of paraoxon was 0.026 nM. After that the samples werencubated for up to two hours forty-three minutes. At 43 min ofeactivation at 37 ◦C the percentage of activity was around 9% and atong time was around 32%. This spontaneous reactivation is clearlyime-progressive. The results show the “ongoing inhibition” andongoing reactivation” may be taken into account in the kineticodels to analyze the interaction by paraoxon of soluble chicken

eripheral nerve phenylvalerate esterases.

oi:10.1016/j.toxlet.2010.03.1012

308-028econdary (nosocomial) poisoning from organophosphates inealth workers, fact or fiction?

. Anderson 1, B. Rajapakse 2, N. Buckley 1

Prince of Wales Clinical School, University of New South Wales, UK,South Asian Clinical Toxicology Research Collaboration, UK

here is international debate regarding the risk to health work-rs treating pesticide poisoning. Multiple papers have suggestedisks from organophosphates (OP) in health workers but provideittle evidence in support. In contrast, Sri Lankan health workersreat thousands of OP poisonings each year with limited protec-ion and no reports of nosocomial poisoning. This ongoing studys prospectively assessing poisoning from organophosphates inealth workers in Sri Lanka. So far 11 subjects (clinical assistants,esearchers and doctors) employed from 6 to 18 months have beenecruited from 2 sites. Baseline data includes previous history ofxposure, pulse, BP, oximetry, spirometry, urine and blood sam-les (n = 11). Acetylcholinesterase (AChE), plasma cholinesteraseBChE) and dialkylphosphate urinary metabolites are measured asiomarkers of OP exposure. The same measurements as for baselinere collected for post-exposure/treatment data at 1, 4, and 24 h.

Current data indicate all health workers had frequently treatedP poisonings and wore gloves but no protective gown or mask./11 health workers reported occasions of mild symptoms afterrevious exposures including vertigo, tightness of chest and res-iratory tract irritation, but none sought medical attention or tookedication. Prospective findings to date include post exposure data

rom just one health worker who treated a deliberate chlorpyri-os ingestion. There was no significant change to either AChE orChE, both sensitive biomarkers of exposure. [AChE;BChE % change

rom baseline at 1 h + 5.56; −11.7%; 4 h −3.7; −4.79%; 24 hrs −6.8;4.79%]. There were no reported symptoms or change in observa-

ions.Health workers in Sri Lanka are at high risk of exposure to

atients with severe organophosphate poisoning. To date thisngoing study has found no evidence to suggest this risk translatesnto any measurable adverse health effects that warrant greaterrecautions.

oi:10.1016/j.toxlet.2010.03.1013

308-029rotective effects of saffron and crocin against biochemicalarkers of diazinon subacute toxicity

.A. Moallem, H. Hosseinzadeh, A. Timcheh-Hariri

Mashhad University of Medical Sciences, Iran

iazinon is one of the most common organophosphate insecticidesn the world. In addition to its inhibitory effect on choline esterase

196S (2010) S37–S351 S321

(ChE), it produces reactive oxygen species. Saffron, a commonspice with many health benefits along with its main ingredient,crocin have been shown to possess potent antioxidant activity.The purpose of this study was to evaluate the protective effectsof saffron and crocin against biochemical markers of diazinonsubacute toxicity. Oral administration of diazinon (20 mg/kg/day)and intraperitonneal injection of saffron aqueous extract or crocin(50, 100 and 200 mg/kg/day) were used for four weeks. Diazi-non resulted in reductions of serum total protein and albumin.In contrast to saffron, the high dose of crocin was able to pre-vent these reductions. Enzymatic studies showed that diazinontreatment caused increases in aspartate aminotransferase (AST),lactate dehydrogenase (LDH), alkaline phosphatase (ALP), creatinephosphokinase (CPK), creatine phosphokinase MB (CPK-MB) andgamma glutamyl transferase (GGT). Crocin at doses of 100 and200 mg/kg/day reduced AST and at all doses reduced LDH, ALP,CPK and CPK-MB. Crocin at 50 and 100 mg/kg/day reduced GGTlevels. Saffron did not affect AST, ALT and ALP but at 100 and200 mg/kg/day reduced LDH, CPK and CPK-MB. Diazinon increasedserum S100�, TNF-� and F2�-Isoprostan. Crocin at 100 mg/kg/dayand saffron at 200 mg/kg/day were able to significantly reduce thelevels of these biomarkers. As expected, diazinon caused a signif-icant reduction in the activity of ChE. Crocin and saffron were noteffective against this effect. Therefore, it is evident crocin and tosome extent saffron were able to normalize changes induced insome biochemical markers as result of subacute toxicity of diazi-non. As ChE was not affected in these studies, it is suggested thatthe antioxidant activities of crocin and saffron mediate their effectsin protecting against diazinon toxicity.

doi:10.1016/j.toxlet.2010.03.1014

P308-030Determination of tissue blood partition coefficients fornon-volatile herbicides and fungicides using negligibledepletion solid phase microextraction (nd-SPME)

R. Tremblay 1, D. Kim 2, J. Fisher 1

1 Environmental Health Science Department, University of Georgia,Athens, GA, USA, 2 Syngenta Crop Protection Inc., Greensboro, NC, USA

Partition coefficients (PC) are parameters used in physiologicallybased pharmacokinetic models to estimate the free concentra-tion of chemicals in specific tissues and organs. PC values havebeen quantified for volatile compounds using air to blood or tissueratios; however, the same method cannot be applied for non-volatile compounds. The objective of this research project wasto develop and apply an analytical method for measuring PCsof non-volatile compounds. Buffered water was used instead ofair. Ultra-filtration filters used to separate the blood and tissuesfrom the liquid were found to significantly retain compounds withKow > 1. Instead, a negligible depletion solid phase microextrac-tion (nd-SPME) method, not requiring filtration, was implemented.Only the free-phase compounds and not tissue-bound can partitionto the SPME fiber coating. PC values were measured for a series oflow volatility pesticides with water solubility ranging from 0.07to 990 mg/L and Kow between 1.9 and 5.5. Tissue:blood PC val-ues were quantified for liver, brain, skin, fat, kidneys and muscleof male Sprague–Dawley rats. Tissue and blood (0.01–0.5 mL) wereadded to 8.5 mL solutions spiked with the chemicals (0.07–20 ppm

in phosphate buffered saline) and allowed to equilibrate for 4+h at 37 ◦C. Free concentrations were measured (3–8 replicates)by inserting a polydimethylsiloxane fiber (PDMS, 100 �m thick,1–4 mm length) directly in the solutions for 1 min. The extracted