Research ArticleBioconcentration and Acute Intoxication ofBrazilian Freshwater Fishes by the Methyl ParathionOrganophosphate Pesticide
Joatildeo Bosco de Salles1 Renato Matos Lopes2
Cristiane M C de Salles3 Vicente P F Cassano4 Manildo Marciatildeo de Oliveira5
Vera L F Cunha Bastos6 and Jayme Cunha Bastos6
1Universidade Estadual da Zona Oeste Rio de Janeiro RJ Brazil2Laboratorio de Comunicacao Celular Fundacao Oswaldo Cruz Instituto Oswaldo Cruz Avenue Brasil 4365 Manguinhos21045-900 Rio de Janeiro RJ Brazil3Setor de Bioquımica Departamento de Quımica Universidade Federal Rural do Rio de Janeiro Seropedica RJ Brazil4Department of Biology Loyola Marymount University Los Angeles CA USA5Laboratorio de Ecotoxicologia eMicrobiologiaAmbiental (LEMAM) Instituto Federal de Educacao Ciencia e Tecnologia FluminenseCabo Frio RJ Brazil6Departamento de Bioquımica Instituto de Biologia Roberto Alcantara Gomes Universidade do Estado do Rio de JaneiroRio de Janeiro RJ Brazil
Correspondence should be addressed to Renato Matos Lopes rmatoslopesgmailcom
Received 14 November 2014 Accepted 26 February 2015
Academic Editor Sunil Kumar
Copyright copy 2015 Joao Bosco de Salles et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited
Three species of freshwater Brazilian fishes (pacu Piaractus mesopotamicus piavussu Leporinus macrocephalus and curimbataProchilodus lineatus) were exposed to an acute dose of 5 ppmmethyl parathion organophosphate pesticideThree to five individualsper species were exposed one at a time to 40 liters tap water spiked with Folidol 600 Pesticide concentrations and cholinesterase(ChE) activities were evaluated in serum liver brain heart and muscle The bioconcentration of methyl parathion was similar forall studied fishes Brain tissue showed the highest pesticide concentration reaching 80 ppm after exposure for 30min to methylparathionThree to 5 hours of 5 ppmmethyl parathion exposure provoked the death of all P lineatus at 92 brain AChE inhibitionwhereas fish from the other two species survived for up to 78 hours with less than 80 brain AChE inhibition Our results indicatethat acute toxic effects of methyl parathion to fish are correlated with brain AChE sensitivity to methyl paraoxon
1 Introduction
Pesticides are a group of toxic compounds with a deep effecton aquatic life and water quality Organophosphates (OP)are a group of pesticides widely used in Latin America Forinstance in Brazil methyl parathion (OO-dimethyl O-p-nitrophenylphosphorothioate) has extensively been appliedin agriculture food storage shelters pest control programsand aquaculture ponds to control aquatic insect larvae whichare predators of fingerling [1 2] The development of the
Brazilian freshwater fish industry was accompanied by theextensive use of methyl parathion to control ectoparasiteinfestations in fish This practice resulted in the discharge oflarge amounts of methyl parathion-treated waters into thenearby area Since OP has no selectivity for any specific targetorganism discharged waters might cause the intoxication ofnatural populations of aquatic organisms [3 4]
An important mechanism of acute toxicity by methylparathion is the inhibition of acetylcholinesterase (AChE)activity Methyl parathion is a weak acetylcholinesterase
Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 197196 9 pageshttpdxdoiorg1011552015197196
2 BioMed Research International
inhibitor but it can be activated into a more potent metabo-lite its oxon derivative methyl paraoxon (OO-dimethylO-p-nitrophenyl phosphate) by a desulfuration reactioncatalyzed by cytochrome P-450 [5] Methyl parathion ismetabolized by both plants and animals and it is not expectedto persist or bioconcentrate However studies have reportedthat Girardinichthys multiradiatus captured in the IgnacioRamirez Dam (Mexico) accumulated methyl parathion morethan 13000 times in relation to water levels of this compound[6 7] Methyl parathion can be detoxified by dealkylationby glutathione S-transferases (GST) [8] whereas methylparaoxon can be removed from blood by scavenger enzymessuch as carboxylesterase and cholinesterases or degraded byparaoxonase yielding 4-nitrophenol and dimethylphospho-ric acid [9]
Intoxication mechanisms of aquatic organisms by OP arenot completely understood Dembele and coworkers haveattributed fish death to asphyxia resulting from gill irritationnot dependent on brainAChE inhibition [10] Patil andDavid[11] reported that sublethal OP levels could induce oxidativestress and inferred that oxidative damages might be relatedto the death of exposed animals Nevertheless OP toxicitycan be attributed to AChE inhibition In neuromuscularjunctions and the central nervous system AChE inhibitioninduces excessive cholinergic stimulation producing in coor-dination fatigue involuntary muscle contractions and even-tually paralysis of the body extremities and the respiratorymuscles [12] Accordingly kinetic characterization of AChEand butyrylcholinesterase (BChE) (another cholinesteraseform) allows the determination of sensitivity differencesbetween both enzymes to organophosphate pesticides whichis essential for environmental monitoring programs [4 13]Therefore the aim of this work was to study the effects oforganophosphate methyl parathion on ChE activities fromdifferent tissues and organs from three freshwater fish speciesthat dwell in Brazilian waters
2 Material and Methods
21 Chemicals Acetylthiocholine iodide (ASCho) buty-rylthiocholine iodide (BSCho) propionylthiocholine iodide(PrSCho) p-nitrophenol (PNP) 551015840-dithiobis (2-nitrob-enzoic acid) (DTNB) and methyl paraoxon (OO-dimethylO-p-nitrophenyl phosphate) were obtained from Sigma (StLouis MO USA) Triton X-100 was acquired from Riedel ofHaen AG (Hannover) Isooctane HPLC grade was acquiredfromMerck Folidol 600 (methyl parathion 600 g Lminus1 Bayer-Brazil) was purchased at the local market All other reagentswere of analytical grade
22 Animals and Experimental Procedures Common andscientific names geographical origin and size of fishesexamined in this work are shown in Table 1 Specimensof Piaractus mesopotamicus (Holmberg 1887) commonlynamed pacu and Leporinus macrocephalus Garavello ampBritski 1988 (piavussu) were supplied by the Morro GrandeFish Farm RJ Brazil Specimens of curimbata Prochilo-dus lineatus (Valenciennes 1836) were supplied by the Sol
Nascente Fish Farm RJ Brazil Experimental procedureswere carried out according to the ethical principles of animalexperimentation elaborated by the Brazilian College forAnimal Experimentation (COBEA) which is in agreementwith the uniform requirements for manuscript submissionsto biomedical journals
Prior to the assays the fish were separated by species andkept into 500 L tanks with dechlorinated water at 25 plusmn 2∘Cfor 15 daysWater was constantly aerated through a biologicalfiltering system pushed by common pumps to produce5mgO
2Lminus1 (measured with an oximeter) Fish were always
on a 14 10-h lightdark cycle and fedwith a commercial pelletonce a day at 9 orsquoclock AM Water pH was 64 plusmn 02
In order to be exposed fish were removed from the largertanks and individually kept in 40 L aquaria filled with waterobtained from the 500 L tanks Methyl parathion (as Folidol)was added once to a final 5 ppm concentration All fish werenot fed 24 h prior to and during the experiments in orderto limit organic matter in the water Moreover the waterduring fish exposure was not filtered to avoid removal of thepesticide Control fishwere submitted to the same conditionswithout Folidol
The concentration of 5 ppmmethyl parathion was chosento elicit a quick response and to correlate the inhibitionof cholinesterase with the bioconcentration of pesticide inseveral tissues and organs of the fish
The intervals of 30min 24 hours and 78 hours werechosen to carry out laboratory analyses Curimbatas showedmore sensitivity to methyl parathion This was noticed whenthey stopped moving their opercula Once this happenedtheywere immediately removed and laboratory analyses wereimmediately carried out
23 Tissue Samples Blood was collected by puncture ofthe dorsal aorta Then the fish were euthanized by quicklysectioning their spinal cord Liver brain and heart wereremoved using scissors and tongs Also a portion of epaxialmuscle (1 g wet tissue) was excised All tissues were separatelywashed quickly with 50mL of ice-cold saline (09 NaCl)and placed into cryogenic vials that were dropped intoliquid nitrogen for storage They were thawed separately bysuspension in four volumes of ice-cold 01mol Lminus1 potassiumphosphate buffer pH 70 They were minced with scissorsand homogenized with 20 strokes in a Potter-Elvehjemapparatus while being maintained in an ice bath at 5-6∘CChE assay in brain and liver tissue were carried out usingthis crude homogenate as sample Samples for assaying heartandmuscle ChEwere produced bymixing their homogenateswith three volumes of 10mmol Lminus1 Tris-HCl buffer pH 70containing 3 Triton X-100 and 1mol Lminus1 sodium chlorideAfter centrifuging these mixtures at 3000timesg for 10min at5∘C enzyme assays were carried out in the resulting heart andmuscle Triton X-100 soluble supernatant fractions
24 Methyl Parathion Determinations Serum and homoge-nate samples (200 120583L) were extracted by using a mixture of6mL of isooctane 200 120583L methanol and 200120583L saturatedsodium chloride solution The extracts were centrifuged
BioMed Research International 3
Table 1 Common and scientific name suppliers and average size of fishes examined
Common name Scientific name Suppliera Size and SDb (cm)
Curimbata Prochilodus lineatus(Valenciennes 1836) Sol Nascente Fish Farm 18 plusmn 5
Pacu Piaractus mesopotamicus(Holmberg 1887) Morro Grande Fish Farm 18 plusmn 4
Piavussu Leporinus macrocephalusGaravello and Britski 1988 Morro Grande Fish Farm 20 plusmn 5
aAll suppliers are located in the state of Rio de Janeiro BrazilbSD standard deviation
at 3000timesg for 10min at 5∘C Three milliliters from eachsupernatant fraction was collected and evaporated undera gentle nitrogen stream The residue was reconstitutedin 200120583L of acetonitrile From this reconstituted sample50120583L were injected onto a 200mm times 46mm ODS HypersilRP-18 5 120583m particle size HPLC column using (50 50 vv)acetonitrileultrapure water as mobile phase with a flow-rate of 1mLminminus1 and examined under UV light withthe detector set at 270 nm Under these conditions methylparathion recovery was estimated at approximately 92following quantification based on a standard prepared witha 98 methyl parathion pure sample previously obtained bythin-layer chromatography
25 Cholinesterase Assays Serum and tissue homogenatesamples were placed in a medium containing 18mmol Lminus1from one of the three substrates (acetylthiocholine propi-onylthiocholine or butyrylthiocholine) with 032mmol Lminus1DTNB Enzyme activitywas continuously recordedup to 90 sat 412 nm using a Shimadzu spectrophotometer model UV-160A according to the Ellman method [14] The reactionwas carried out in microcuvettes All reagents were dissolvedto 200120583L final volume with a 01mmol Lminus1 sodium phos-phate buffer pH 75 at 25∘C and the thionitrobenzoate ionconcentration was estimated using an extinction coefficientof 14150Mminus1 cmminus1 One unit (1 U) of enzyme activity wasdefined as the amount that hydrolyzes 10 120583mol of substrateper minute
3 Results
31 Tissue Distribution of ChE Activity The studied fishspecies (control groups) exhibited remarkable differencesin tissue-specific cholinesterase activity (Figure 1) The ChEactivity measured in the serum of curimbata and pacuspecimens was lower than the activity measured in brainliver heart and muscle By contrast in serum of piavussuthe ChE activity was similar to that measured in liver brainheart and muscle
32 Methyl Parathion Bioconcentration Muscle heart brainliver and serum from pacu piavussu and curimbata exhib-ited similar methyl parathion bioconcentration patterns(Figure 2) Brain tissue showed the highest capacity formethyl parathion bioconcentration reaching 80 ppm (16-fold
increase) after 30 minutes exposure to methyl parathion inwater (Figure 2) The maximum methyl parathion concen-tration in muscle heart brain and serum was achieved after30minutes with one exception methyl parathion concentra-tions in piavussu showed an increase after 24 hours exposurereaching a maximum of 120 ppm in liver corresponding to a24-fold increase (Figure 3) Methyl parathion was not foundin samples from control animals
33 Cholinesterase Inhibition Heart liver and serum cho-linesterase activities for pacu piavussu and curimbata weregreatly inhibited after 30 minutes exposure to Folidol Bycontrast skeletal muscle ChE activity for the three speciesand brain AChE activity for pacu showed no inhibitionwithin this time period (Figure 4) Exposure of fishes to waterwith Folidol for 78 h resulted in 70 brain AChE activityinhibition in pacu and 60 brain AChE activity inhibitionin piavussu
All curimbatas stopped moving their opercula between 3and 5 hours of exposure to 5 ppm Folidol showingmore than90 inhibition of brain AChE activity and no inhibition ofskeletal muscle ChE activity (Figure 4) Increasing the timeof exposure from 30min to 78 hours did not considerablymodify muscle heart brain liver or serum ChE activities inpiavussu (Figure 4)
4 Discussion
Brain tissue showed the highest pesticide bioconcentrationreaching 80 ppm after 30 minutes of exposure to methylparathion (Figure 2) The three fish species selected for thisstudy present different brain cholinesterase sensitivities tointoxication by methyl paraoxon [15] In these fishes thesensitivity of brain ChE to intoxication bymethyl paraoxon isinversely related to the activity of the brain enzyme forASCho[15] in contrast to what was described for trout and rat byKemp and Wallace [16]
The results demonstrate that the fishes exposed to Folidolrapidly absorbed methyl parathion since concentrations ofmethyl parathion in the tissues and serum peaked within30min of exposure to Folidol This concentration contin-ued to increase only in liver in which the amount ofmethyl parathion doubled after 24 hours (Figure 3) Suchfindings can be ascribed to the promptness with whichmethyl parathion undergoes biotransformation into methyl
Figure 1 Tissue distribution of cholinesterase activity in pacu piavussu and curimbata (control fish) The box shows the substrates used inenzyme assays Results are expressed as 120583mol of products formed per minute per gram of wet tissue (U gminus1) or per mL of serum (UmLminus1)Liver and brain ChE activities were assayed in homogenates while heart and muscle ChE activities were assayed in the enzymatic fractionsolubilized with Triton and NaCl Results are average values with the corresponding SEM of assays carried out in five individuals of eachspecies
BioMed Research International 5
0 50 100
Serum
Liver
Brain
Heart
Muscle
Pacu
0 50 100Methyl parathion (ppm)
Methyl parathion (ppm)
Methyl parathion (ppm)
Piavussu
0 50 100
Curimbataacute
Serum
Liver
Brain
Heart
Muscle
Serum
Liver
Brain
Heart
Muscle
Figure 2 Concentration of methyl parathion in muscle heart brain and liver and serum from pacu piavussu and curimbata Each one offour animals per species was placed in separated tanks containing 40 L of water with Folidol to produce 5 ppm methyl parathion After 30minutes exposure blood samples were collected and the fishes were euthanized Tissues were dissected and homogenized Methyl parathionwas extracted from serum and tissue homogenates and quantified by HPLC
paraoxon inside liver cells as compared to other organsSince P-450 can be inhibited by the sulphur removed fromparathion [17 18] it is plausible that metabolism of methylparathion molecules occurred in liver up to 30min and thena consequent inhibition of liver P-450 allowed that moremethyl parathion could accumulate and be extracted Whenrainbow trout was exposed to the 75 ngmLminus1 ethyl parathionAbbas and coworkers [19] observed a peak concentration ofthis pesticide in plasma up to 45 hours In the experiments
conducted in the present study pesticide bioconcentrationoccurred over a shorter period probably due to the sig-nificantly higher pesticide concentration used Our resultsindicate that methyl parathion can bioconcentrate in brainof the tested fish up to approximately 80 ppm (4 times itssolubility in water) (Figure 2)This bioconcentration capacityis probably due to high lipid solubility of this OP compoundThe half-life of parathion in fish has been reported as 5 timeslonger than in rats [19]This suggests that this pesticide could
6 BioMed Research International
0 70 140
Serum
Liver
Brain
Heart
Muscle
Methyl parathion (ppm)
Pesticide bioconcentration
0 50 100ChE activity ()
Enzyme inhibition
Serum
Liver
Brain
Heart
Muscle
05h24h
05h24h
Figure 3 Piavussumethyl parathion concentrations and cholinesterase inhibition after exposure Each one of eight piavussuswas individuallyplaced in separated tanks containing 40 L of water with Folidol to produce 5 ppm methyl parathion Incubation happened for the showedtimes After exposure blood was collected fishes were submitted to euthanasia and tissues were dissected and homogenized Cholinesteraseactivity was assayed in serum and in homogenates tissues with acetylthiocholine and expressed as percentage of control activity Methylparathion was extracted from serum and tissue homogenates and quantified by HPLC
persist for a relatively long time in fish raising concernsregarding human consumption of fish previously exposed toOP
The results reported herein demonstrated that ChEinhibition in fish exposed to Folidol does not seem todepend on the methyl parathion bioconcentration in theirtissues No ChE inhibition in pacu brain was observed up to30min of exposure despite the fact that this organ presented80 ppm of methyl parathion at that time Therefore thisdata is important for further investigations Tissues in whichChE inhibition occurred more quickly such as pacu liver(Figure 4) showing higher BChE activity in comparison toAChE activity (Figure 1) BChE has been shown to be moresensitive to methyl paraoxon and is probably more quicklyinhibited [20] In addition activation of OP compoundsoccurs mainly in the liver because of the high concentrationsof P-450 in that tissue Thus liver ChE undergoes inhibitionby methyl paraoxon locally generated in tissue before thismetabolite leaks out to general blood circulation and reachesother target tissues
In their work de Aguiar and coworkers [2] described an87 brain AChE inhibition in matrinxa (Brycon cephalus)exposed to water with 2 ppm Folidol 600 for 96 hours Thepresent study indicates that piavussu and pacumight bemoretolerant to Folidol than matrinxa since piavussu presented66 and pacu 74 brain AChE inhibition when exposed to
water with 5 ppmmethyl parathion for 78 hours On the otherhand curimbata showed a 92 brain AChE inhibition withonly 5 hours of exposure to 5 ppmmethyl parathion probablydue to the higher sensitivity of this species AChE to inhibitionby methyl paraoxon [15]
Mammals poisoned by OP usually die by asphyxiaHowever fishes have been reported to be more resistant topoisoning by high levels of organophosphates compoundsthan rats [21] The actual causes and mechanisms of fishpoisoning by OP are not fully understood Our findingsreinforce that interspecific differences in AChE inhibition bytheir oxon derivatives should be considered Physiologicaland behavioral disturbances start at 50 AChE inhibitionand death usually follows when inhibition exceeds 80 inmammals and birds [22] We found here that curimbatas alsoperished when brain AChE inhibition reached above 80
This study suggests that a major contributing factor toacute fish toxicity by Folidol is brain AChE sensitivity tomethyl paraoxon the oxon derivative of methyl parathionThe same explanation that is the sensitivity of brain AChEto oxon derivatives has been suggested for chlorpyrifosparathion and methyl parathion toxicity in mosquito fish(Gambusia affinis) [23] Although it was clearly establishedthat curimbatas possess the most sensitive brain AChEamong the studied species the death of curimbatas exposedto 5 ppm methyl parathion cannot be solely credited to brain
BioMed Research International 7
0 50 100
Serum
Liver
Brain
Heart
Muscle
Pacu
0 50 100
Piavussu
0 50 100
Curimbataacute
05h78h
05h78h
05h5h
ChE activity( relative to controls)
ChE activity( relative to controls)
Serum
Liver
Brain
Heart
Muscle
Serum
Liver
Brain
Heart
Muscle
ChE activity( relative to controls)
Figure 4 Cholinesterase activity in tissues of pacu piavussu and curimbata exposed to 5 ppm methyl parathion Each one of eight animalsby species was individually treated with Folidol 600 for the indicated time in a 40 L aquarium After 30min of exposure blood was collectedfrom four fishes by species and these fishes were submitted to euthanasia Then of the remaining fish four pacus and four piavussus wereeuthanized after 78 h exposure while four curimbatas were collected immediately after they showed no movement of opercula (from 3 to 5 hof exposure) Tissues were dissected and homogenized Cholinesterase was assayed in serum and homogenates with acetylthiocholine andexpressed as percentage of the activity found in controls
8 BioMed Research International
AChE inhibition Other possible causes of this fish deathsuch as Na+ K+ ATPase inhibition [24] and tissue hypoxiawhich compromises heart function need to be examined
5 Conclusion
Prochilodus lineatus (curimbata) Piaractus mesopotamicus(pacu) and Leporinus macrocephalus (piavussu) studied hereshowed similar capacities to bioconcentratemethyl parathionin their tissues after exposure to 5 ppm in water Howeveronly curimbata with the highest brain AChE sensitivity tomethyl paraoxon died after 5 hours of exposure to FolidolPacu and piavussu are more resistant to methyl paraoxonand were alive up to 78 hours of exposure to 5 ppm ofmethyl parathion BrainAChE sensitivity tomethyl paraoxonmight be a decisive factor for determining the sensitivityof these species to poisoning by high concentrations oforganophosphate compounds The present study indicatesthat fishes whose brain acetylcholinesterase activity is moresensitive to oxon derivatives will suffer more severe impactsfrom environmental contamination by organophosphate pes-ticides
Measures reducing the use of organophosphate pesticidesin fish culture should be adopted in order to minimizethe discharge and consequent impact of these chemicals tonatural communities inhabiting rivers and lakes of which thesensitivity to intoxication by pesticides is largely unknown
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors are grateful to Morro Grande Fish Farm andSol Nascente Fish Farm for supplying the fishes This paperis dedicated to Dr Moacelio Veranio Silva Filho (in memo-riam)
References
[1] E Barbieri and L A A Ferreira ldquoEffects of the organophos-phate pesticide Folidol 600 on the freshwater fish Nile Tilapia(Oreochromis niloticus)rdquo Pesticide Biochemistry and Physiologyvol 99 no 3 pp 209ndash214 2011
[2] L H de Aguiar G Moraes I M Avilez A E Altran and CF Correa ldquoMetabolical effects of Folidol 600 on the neotrop-ical freshwater fish matrinxa Brycon cephalusrdquo EnvironmentalResearch vol 95 no 2 pp 224ndash230 2004
[3] D Hernandez-Moreno M Perez-Lopez F Soler C Gravatoand L Guilhermino ldquoEffects of carbofuran on the sea bass(Dicentrarchus labrax L) study of biomarkers and behaviouralterationsrdquo Ecotoxicology and Environmental Safety vol 74 no7 pp 1905ndash1912 2011
[4] C RD Assis A G Linhares VMOliveira et al ldquoComparativeeffect of pesticides on brain acetylcholinesterase in tropical fishrdquoScience of the Total Environment vol 441 pp 141ndash150 2012
[5] L G Sultatos ldquoMammalian toxicology of organophosphoruspesticidesrdquo Journal of Toxicology and Environmental Health vol43 no 3 pp 271ndash289 1994
[6] O Arellano-Aguilar and C M Garcia ldquoEffects of methylparathion exposure on development and reproduction in theviviparous fish girardinichthys multiradiatusrdquo EnvironmentalToxicology vol 24 no 2 pp 178ndash186 2009
[7] M Y de la Vega Salazar L Martınez Tabche and C MacIasGarcıa ldquoBioaccumulation of methyl parathion and its toxicol-ogy in several species of the freshwater community in IgnacioRyamirez dam in Mexicordquo Ecotoxicology and EnvironmentalSafety vol 38 no 1 pp 53ndash62 1997
[8] J E Chambers and H W Chambers ldquoBiotransformationof organophosphorous insecticides in mammalsrdquo in PesticideTransformation Products Fate and Significance in the Envi-ronment L Somasundaram and J R Coats Eds pp 32ndash42American Chemistry Society Washington DC USA 1991
[9] B N LaDu ldquoHuman serum paraoxonasearylesteraserdquo in Phar-macogenetics of DrugMetabolism Pergamon W Kalow Ed pp51ndash91 Pergamon New York NY USA 1992
[10] K Dembele E Haubruge and C Gaspar ldquoConcentrationeffects of selected insecticides on brain acetylcholinesterasein the common carp (Cyprinus carpio L)rdquo Ecotoxicology andEnvironmental Safety vol 45 no 1 pp 49ndash54 2000
[11] V K Patil and M David ldquoOxidative stress in freshwaterfish Labeo rohita as a biomarker of malathion exposurerdquoEnvironmental Monitoring and Assessment vol 185 no 12 pp10191ndash10199 2013
[12] M Jokanovic ldquoCurrent understanding of the mechanismsinvolved in metabolic detoxification of warfare nerve agentsrdquoToxicology Letters vol 188 no 1 pp 1ndash10 2009
[13] R M Lopes M V S Filho J B de Salles V L F C Bastosand J C Bastos ldquoCholinesterase activity of muscle tissue fromfreshwater fishes characterization and sensitivity analysis to theorganophosphate methyl-paraoxonrdquo Environmental Toxicologyand Chemistry vol 33 no 6 pp 1331ndash1336 2014
[14] G L Ellman K D Courtney V Andres Jr and R MFeatherstone ldquoA new and rapid colorimetric determination ofacetylcholinesterase activityrdquo Biochemical Pharmacology vol 7no 2 pp 88ndash95 1961
[15] M V S Filho M M Oliveira J B Salles V L F C BastosV P F Cassano and J C Bastos ldquoMethyl-paraoxon compara-tive inhibition kinetics for acetylcholinesterases from brain ofneotropical fishesrdquo Toxicology Letters vol 153 no 2 pp 247ndash254 2004
[16] J R Kemp and K B Wallace ldquoMolecular determinants ofthe species-selective inhibition of brain acetylcholinesteraserdquoToxicology and Applied Pharmacology vol 104 no 2 pp 246ndash258 1990
[17] J Halpert D Hammond and R A Neal ldquoInactivation ofpurified rat liver cytochrome P-450 during the metabolismof parathion (diethyl p-nitrophenyl phosphorothionate)rdquo TheJournal of Biological Chemistry vol 255 no 3 pp 1080ndash10891980
[18] B J Norman R E Poore and R A Neal ldquoStudies ofthe binding of sulfur released in the mixed-function oxi-dase-catalyzed metabolism of diethyl p-nitrophenyl phos-phorothionate(parathion) to diethyl p-nitrophenyl phosphate(paraoxon)rdquo Biochemical Pharmacology vol 23 no 12 pp1733ndash1744 1974
[19] R Abbas I R Schultz S Doddapaneni and W L HaytonldquoToxicokinetics of parathion and paraoxon in rainbow trout
BioMed Research International 9
after intravascular administration and water exposurerdquo Toxicol-ogy and Applied Pharmacology vol 136 no 1 pp 194ndash199 1996
[20] V L F Cunha Bastos A Rossini L F Ribeiro Pinto etal ldquoDifferent sensitivities to paraoxon of brain and serumcholinesterases from pacu an indigenous Brazilian fishrdquo Bul-letin of Environmental Contamination and Toxicology vol 60no 1 pp 1ndash8 1998
[21] J S Boone and U E Chambers ldquoTime course of inhibitionof cholinesterase and aliesterase activities and nonproteinsulfhydryl levels following exposure to organophosphorusinsecticides in mosquitofish (Gambusia affinis)rdquo Fundamentaland Applied Toxicology vol 29 no 2 pp 202ndash207 1996
[22] C HWalker ldquoThe use of biomarkers to measure the interactiveeffects of chemicalsrdquo Ecotoxicology and Environmental Safetyvol 40 no 1-2 pp 65ndash70 1998
[23] J S Boone and J E Chambers ldquoBiochemical factors contribut-ing to toxicity differences among chlorpyrifos parthion andmethyl parathion in mosquitofish (Gambusia affinis)rdquo AquaticToxicology vol 39 no 3-4 pp 333ndash343 1997
[24] J Blasiak ldquoAllosteric inhibition of the (Na+ + K+)-ATPaseby parathion and methylparathionrdquo Pesticide Biochemistry andPhysiology vol 54 no 1 pp 40ndash47 1996
inhibitor but it can be activated into a more potent metabo-lite its oxon derivative methyl paraoxon (OO-dimethylO-p-nitrophenyl phosphate) by a desulfuration reactioncatalyzed by cytochrome P-450 [5] Methyl parathion ismetabolized by both plants and animals and it is not expectedto persist or bioconcentrate However studies have reportedthat Girardinichthys multiradiatus captured in the IgnacioRamirez Dam (Mexico) accumulated methyl parathion morethan 13000 times in relation to water levels of this compound[6 7] Methyl parathion can be detoxified by dealkylationby glutathione S-transferases (GST) [8] whereas methylparaoxon can be removed from blood by scavenger enzymessuch as carboxylesterase and cholinesterases or degraded byparaoxonase yielding 4-nitrophenol and dimethylphospho-ric acid [9]
Intoxication mechanisms of aquatic organisms by OP arenot completely understood Dembele and coworkers haveattributed fish death to asphyxia resulting from gill irritationnot dependent on brainAChE inhibition [10] Patil andDavid[11] reported that sublethal OP levels could induce oxidativestress and inferred that oxidative damages might be relatedto the death of exposed animals Nevertheless OP toxicitycan be attributed to AChE inhibition In neuromuscularjunctions and the central nervous system AChE inhibitioninduces excessive cholinergic stimulation producing in coor-dination fatigue involuntary muscle contractions and even-tually paralysis of the body extremities and the respiratorymuscles [12] Accordingly kinetic characterization of AChEand butyrylcholinesterase (BChE) (another cholinesteraseform) allows the determination of sensitivity differencesbetween both enzymes to organophosphate pesticides whichis essential for environmental monitoring programs [4 13]Therefore the aim of this work was to study the effects oforganophosphate methyl parathion on ChE activities fromdifferent tissues and organs from three freshwater fish speciesthat dwell in Brazilian waters
2 Material and Methods
21 Chemicals Acetylthiocholine iodide (ASCho) buty-rylthiocholine iodide (BSCho) propionylthiocholine iodide(PrSCho) p-nitrophenol (PNP) 551015840-dithiobis (2-nitrob-enzoic acid) (DTNB) and methyl paraoxon (OO-dimethylO-p-nitrophenyl phosphate) were obtained from Sigma (StLouis MO USA) Triton X-100 was acquired from Riedel ofHaen AG (Hannover) Isooctane HPLC grade was acquiredfromMerck Folidol 600 (methyl parathion 600 g Lminus1 Bayer-Brazil) was purchased at the local market All other reagentswere of analytical grade
22 Animals and Experimental Procedures Common andscientific names geographical origin and size of fishesexamined in this work are shown in Table 1 Specimensof Piaractus mesopotamicus (Holmberg 1887) commonlynamed pacu and Leporinus macrocephalus Garavello ampBritski 1988 (piavussu) were supplied by the Morro GrandeFish Farm RJ Brazil Specimens of curimbata Prochilo-dus lineatus (Valenciennes 1836) were supplied by the Sol
Nascente Fish Farm RJ Brazil Experimental procedureswere carried out according to the ethical principles of animalexperimentation elaborated by the Brazilian College forAnimal Experimentation (COBEA) which is in agreementwith the uniform requirements for manuscript submissionsto biomedical journals
Prior to the assays the fish were separated by species andkept into 500 L tanks with dechlorinated water at 25 plusmn 2∘Cfor 15 daysWater was constantly aerated through a biologicalfiltering system pushed by common pumps to produce5mgO
2Lminus1 (measured with an oximeter) Fish were always
on a 14 10-h lightdark cycle and fedwith a commercial pelletonce a day at 9 orsquoclock AM Water pH was 64 plusmn 02
In order to be exposed fish were removed from the largertanks and individually kept in 40 L aquaria filled with waterobtained from the 500 L tanks Methyl parathion (as Folidol)was added once to a final 5 ppm concentration All fish werenot fed 24 h prior to and during the experiments in orderto limit organic matter in the water Moreover the waterduring fish exposure was not filtered to avoid removal of thepesticide Control fishwere submitted to the same conditionswithout Folidol
The concentration of 5 ppmmethyl parathion was chosento elicit a quick response and to correlate the inhibitionof cholinesterase with the bioconcentration of pesticide inseveral tissues and organs of the fish
The intervals of 30min 24 hours and 78 hours werechosen to carry out laboratory analyses Curimbatas showedmore sensitivity to methyl parathion This was noticed whenthey stopped moving their opercula Once this happenedtheywere immediately removed and laboratory analyses wereimmediately carried out
23 Tissue Samples Blood was collected by puncture ofthe dorsal aorta Then the fish were euthanized by quicklysectioning their spinal cord Liver brain and heart wereremoved using scissors and tongs Also a portion of epaxialmuscle (1 g wet tissue) was excised All tissues were separatelywashed quickly with 50mL of ice-cold saline (09 NaCl)and placed into cryogenic vials that were dropped intoliquid nitrogen for storage They were thawed separately bysuspension in four volumes of ice-cold 01mol Lminus1 potassiumphosphate buffer pH 70 They were minced with scissorsand homogenized with 20 strokes in a Potter-Elvehjemapparatus while being maintained in an ice bath at 5-6∘CChE assay in brain and liver tissue were carried out usingthis crude homogenate as sample Samples for assaying heartandmuscle ChEwere produced bymixing their homogenateswith three volumes of 10mmol Lminus1 Tris-HCl buffer pH 70containing 3 Triton X-100 and 1mol Lminus1 sodium chlorideAfter centrifuging these mixtures at 3000timesg for 10min at5∘C enzyme assays were carried out in the resulting heart andmuscle Triton X-100 soluble supernatant fractions
24 Methyl Parathion Determinations Serum and homoge-nate samples (200 120583L) were extracted by using a mixture of6mL of isooctane 200 120583L methanol and 200120583L saturatedsodium chloride solution The extracts were centrifuged
BioMed Research International 3
Table 1 Common and scientific name suppliers and average size of fishes examined
Common name Scientific name Suppliera Size and SDb (cm)
Curimbata Prochilodus lineatus(Valenciennes 1836) Sol Nascente Fish Farm 18 plusmn 5
Pacu Piaractus mesopotamicus(Holmberg 1887) Morro Grande Fish Farm 18 plusmn 4
Piavussu Leporinus macrocephalusGaravello and Britski 1988 Morro Grande Fish Farm 20 plusmn 5
aAll suppliers are located in the state of Rio de Janeiro BrazilbSD standard deviation
at 3000timesg for 10min at 5∘C Three milliliters from eachsupernatant fraction was collected and evaporated undera gentle nitrogen stream The residue was reconstitutedin 200120583L of acetonitrile From this reconstituted sample50120583L were injected onto a 200mm times 46mm ODS HypersilRP-18 5 120583m particle size HPLC column using (50 50 vv)acetonitrileultrapure water as mobile phase with a flow-rate of 1mLminminus1 and examined under UV light withthe detector set at 270 nm Under these conditions methylparathion recovery was estimated at approximately 92following quantification based on a standard prepared witha 98 methyl parathion pure sample previously obtained bythin-layer chromatography
25 Cholinesterase Assays Serum and tissue homogenatesamples were placed in a medium containing 18mmol Lminus1from one of the three substrates (acetylthiocholine propi-onylthiocholine or butyrylthiocholine) with 032mmol Lminus1DTNB Enzyme activitywas continuously recordedup to 90 sat 412 nm using a Shimadzu spectrophotometer model UV-160A according to the Ellman method [14] The reactionwas carried out in microcuvettes All reagents were dissolvedto 200120583L final volume with a 01mmol Lminus1 sodium phos-phate buffer pH 75 at 25∘C and the thionitrobenzoate ionconcentration was estimated using an extinction coefficientof 14150Mminus1 cmminus1 One unit (1 U) of enzyme activity wasdefined as the amount that hydrolyzes 10 120583mol of substrateper minute
3 Results
31 Tissue Distribution of ChE Activity The studied fishspecies (control groups) exhibited remarkable differencesin tissue-specific cholinesterase activity (Figure 1) The ChEactivity measured in the serum of curimbata and pacuspecimens was lower than the activity measured in brainliver heart and muscle By contrast in serum of piavussuthe ChE activity was similar to that measured in liver brainheart and muscle
32 Methyl Parathion Bioconcentration Muscle heart brainliver and serum from pacu piavussu and curimbata exhib-ited similar methyl parathion bioconcentration patterns(Figure 2) Brain tissue showed the highest capacity formethyl parathion bioconcentration reaching 80 ppm (16-fold
increase) after 30 minutes exposure to methyl parathion inwater (Figure 2) The maximum methyl parathion concen-tration in muscle heart brain and serum was achieved after30minutes with one exception methyl parathion concentra-tions in piavussu showed an increase after 24 hours exposurereaching a maximum of 120 ppm in liver corresponding to a24-fold increase (Figure 3) Methyl parathion was not foundin samples from control animals
33 Cholinesterase Inhibition Heart liver and serum cho-linesterase activities for pacu piavussu and curimbata weregreatly inhibited after 30 minutes exposure to Folidol Bycontrast skeletal muscle ChE activity for the three speciesand brain AChE activity for pacu showed no inhibitionwithin this time period (Figure 4) Exposure of fishes to waterwith Folidol for 78 h resulted in 70 brain AChE activityinhibition in pacu and 60 brain AChE activity inhibitionin piavussu
All curimbatas stopped moving their opercula between 3and 5 hours of exposure to 5 ppm Folidol showingmore than90 inhibition of brain AChE activity and no inhibition ofskeletal muscle ChE activity (Figure 4) Increasing the timeof exposure from 30min to 78 hours did not considerablymodify muscle heart brain liver or serum ChE activities inpiavussu (Figure 4)
4 Discussion
Brain tissue showed the highest pesticide bioconcentrationreaching 80 ppm after 30 minutes of exposure to methylparathion (Figure 2) The three fish species selected for thisstudy present different brain cholinesterase sensitivities tointoxication by methyl paraoxon [15] In these fishes thesensitivity of brain ChE to intoxication bymethyl paraoxon isinversely related to the activity of the brain enzyme forASCho[15] in contrast to what was described for trout and rat byKemp and Wallace [16]
The results demonstrate that the fishes exposed to Folidolrapidly absorbed methyl parathion since concentrations ofmethyl parathion in the tissues and serum peaked within30min of exposure to Folidol This concentration contin-ued to increase only in liver in which the amount ofmethyl parathion doubled after 24 hours (Figure 3) Suchfindings can be ascribed to the promptness with whichmethyl parathion undergoes biotransformation into methyl
Figure 1 Tissue distribution of cholinesterase activity in pacu piavussu and curimbata (control fish) The box shows the substrates used inenzyme assays Results are expressed as 120583mol of products formed per minute per gram of wet tissue (U gminus1) or per mL of serum (UmLminus1)Liver and brain ChE activities were assayed in homogenates while heart and muscle ChE activities were assayed in the enzymatic fractionsolubilized with Triton and NaCl Results are average values with the corresponding SEM of assays carried out in five individuals of eachspecies
BioMed Research International 5
0 50 100
Serum
Liver
Brain
Heart
Muscle
Pacu
0 50 100Methyl parathion (ppm)
Methyl parathion (ppm)
Methyl parathion (ppm)
Piavussu
0 50 100
Curimbataacute
Serum
Liver
Brain
Heart
Muscle
Serum
Liver
Brain
Heart
Muscle
Figure 2 Concentration of methyl parathion in muscle heart brain and liver and serum from pacu piavussu and curimbata Each one offour animals per species was placed in separated tanks containing 40 L of water with Folidol to produce 5 ppm methyl parathion After 30minutes exposure blood samples were collected and the fishes were euthanized Tissues were dissected and homogenized Methyl parathionwas extracted from serum and tissue homogenates and quantified by HPLC
paraoxon inside liver cells as compared to other organsSince P-450 can be inhibited by the sulphur removed fromparathion [17 18] it is plausible that metabolism of methylparathion molecules occurred in liver up to 30min and thena consequent inhibition of liver P-450 allowed that moremethyl parathion could accumulate and be extracted Whenrainbow trout was exposed to the 75 ngmLminus1 ethyl parathionAbbas and coworkers [19] observed a peak concentration ofthis pesticide in plasma up to 45 hours In the experiments
conducted in the present study pesticide bioconcentrationoccurred over a shorter period probably due to the sig-nificantly higher pesticide concentration used Our resultsindicate that methyl parathion can bioconcentrate in brainof the tested fish up to approximately 80 ppm (4 times itssolubility in water) (Figure 2)This bioconcentration capacityis probably due to high lipid solubility of this OP compoundThe half-life of parathion in fish has been reported as 5 timeslonger than in rats [19]This suggests that this pesticide could
6 BioMed Research International
0 70 140
Serum
Liver
Brain
Heart
Muscle
Methyl parathion (ppm)
Pesticide bioconcentration
0 50 100ChE activity ()
Enzyme inhibition
Serum
Liver
Brain
Heart
Muscle
05h24h
05h24h
Figure 3 Piavussumethyl parathion concentrations and cholinesterase inhibition after exposure Each one of eight piavussuswas individuallyplaced in separated tanks containing 40 L of water with Folidol to produce 5 ppm methyl parathion Incubation happened for the showedtimes After exposure blood was collected fishes were submitted to euthanasia and tissues were dissected and homogenized Cholinesteraseactivity was assayed in serum and in homogenates tissues with acetylthiocholine and expressed as percentage of control activity Methylparathion was extracted from serum and tissue homogenates and quantified by HPLC
persist for a relatively long time in fish raising concernsregarding human consumption of fish previously exposed toOP
The results reported herein demonstrated that ChEinhibition in fish exposed to Folidol does not seem todepend on the methyl parathion bioconcentration in theirtissues No ChE inhibition in pacu brain was observed up to30min of exposure despite the fact that this organ presented80 ppm of methyl parathion at that time Therefore thisdata is important for further investigations Tissues in whichChE inhibition occurred more quickly such as pacu liver(Figure 4) showing higher BChE activity in comparison toAChE activity (Figure 1) BChE has been shown to be moresensitive to methyl paraoxon and is probably more quicklyinhibited [20] In addition activation of OP compoundsoccurs mainly in the liver because of the high concentrationsof P-450 in that tissue Thus liver ChE undergoes inhibitionby methyl paraoxon locally generated in tissue before thismetabolite leaks out to general blood circulation and reachesother target tissues
In their work de Aguiar and coworkers [2] described an87 brain AChE inhibition in matrinxa (Brycon cephalus)exposed to water with 2 ppm Folidol 600 for 96 hours Thepresent study indicates that piavussu and pacumight bemoretolerant to Folidol than matrinxa since piavussu presented66 and pacu 74 brain AChE inhibition when exposed to
water with 5 ppmmethyl parathion for 78 hours On the otherhand curimbata showed a 92 brain AChE inhibition withonly 5 hours of exposure to 5 ppmmethyl parathion probablydue to the higher sensitivity of this species AChE to inhibitionby methyl paraoxon [15]
Mammals poisoned by OP usually die by asphyxiaHowever fishes have been reported to be more resistant topoisoning by high levels of organophosphates compoundsthan rats [21] The actual causes and mechanisms of fishpoisoning by OP are not fully understood Our findingsreinforce that interspecific differences in AChE inhibition bytheir oxon derivatives should be considered Physiologicaland behavioral disturbances start at 50 AChE inhibitionand death usually follows when inhibition exceeds 80 inmammals and birds [22] We found here that curimbatas alsoperished when brain AChE inhibition reached above 80
This study suggests that a major contributing factor toacute fish toxicity by Folidol is brain AChE sensitivity tomethyl paraoxon the oxon derivative of methyl parathionThe same explanation that is the sensitivity of brain AChEto oxon derivatives has been suggested for chlorpyrifosparathion and methyl parathion toxicity in mosquito fish(Gambusia affinis) [23] Although it was clearly establishedthat curimbatas possess the most sensitive brain AChEamong the studied species the death of curimbatas exposedto 5 ppm methyl parathion cannot be solely credited to brain
BioMed Research International 7
0 50 100
Serum
Liver
Brain
Heart
Muscle
Pacu
0 50 100
Piavussu
0 50 100
Curimbataacute
05h78h
05h78h
05h5h
ChE activity( relative to controls)
ChE activity( relative to controls)
Serum
Liver
Brain
Heart
Muscle
Serum
Liver
Brain
Heart
Muscle
ChE activity( relative to controls)
Figure 4 Cholinesterase activity in tissues of pacu piavussu and curimbata exposed to 5 ppm methyl parathion Each one of eight animalsby species was individually treated with Folidol 600 for the indicated time in a 40 L aquarium After 30min of exposure blood was collectedfrom four fishes by species and these fishes were submitted to euthanasia Then of the remaining fish four pacus and four piavussus wereeuthanized after 78 h exposure while four curimbatas were collected immediately after they showed no movement of opercula (from 3 to 5 hof exposure) Tissues were dissected and homogenized Cholinesterase was assayed in serum and homogenates with acetylthiocholine andexpressed as percentage of the activity found in controls
8 BioMed Research International
AChE inhibition Other possible causes of this fish deathsuch as Na+ K+ ATPase inhibition [24] and tissue hypoxiawhich compromises heart function need to be examined
5 Conclusion
Prochilodus lineatus (curimbata) Piaractus mesopotamicus(pacu) and Leporinus macrocephalus (piavussu) studied hereshowed similar capacities to bioconcentratemethyl parathionin their tissues after exposure to 5 ppm in water Howeveronly curimbata with the highest brain AChE sensitivity tomethyl paraoxon died after 5 hours of exposure to FolidolPacu and piavussu are more resistant to methyl paraoxonand were alive up to 78 hours of exposure to 5 ppm ofmethyl parathion BrainAChE sensitivity tomethyl paraoxonmight be a decisive factor for determining the sensitivityof these species to poisoning by high concentrations oforganophosphate compounds The present study indicatesthat fishes whose brain acetylcholinesterase activity is moresensitive to oxon derivatives will suffer more severe impactsfrom environmental contamination by organophosphate pes-ticides
Measures reducing the use of organophosphate pesticidesin fish culture should be adopted in order to minimizethe discharge and consequent impact of these chemicals tonatural communities inhabiting rivers and lakes of which thesensitivity to intoxication by pesticides is largely unknown
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors are grateful to Morro Grande Fish Farm andSol Nascente Fish Farm for supplying the fishes This paperis dedicated to Dr Moacelio Veranio Silva Filho (in memo-riam)
References
[1] E Barbieri and L A A Ferreira ldquoEffects of the organophos-phate pesticide Folidol 600 on the freshwater fish Nile Tilapia(Oreochromis niloticus)rdquo Pesticide Biochemistry and Physiologyvol 99 no 3 pp 209ndash214 2011
[2] L H de Aguiar G Moraes I M Avilez A E Altran and CF Correa ldquoMetabolical effects of Folidol 600 on the neotrop-ical freshwater fish matrinxa Brycon cephalusrdquo EnvironmentalResearch vol 95 no 2 pp 224ndash230 2004
[3] D Hernandez-Moreno M Perez-Lopez F Soler C Gravatoand L Guilhermino ldquoEffects of carbofuran on the sea bass(Dicentrarchus labrax L) study of biomarkers and behaviouralterationsrdquo Ecotoxicology and Environmental Safety vol 74 no7 pp 1905ndash1912 2011
[4] C RD Assis A G Linhares VMOliveira et al ldquoComparativeeffect of pesticides on brain acetylcholinesterase in tropical fishrdquoScience of the Total Environment vol 441 pp 141ndash150 2012
[5] L G Sultatos ldquoMammalian toxicology of organophosphoruspesticidesrdquo Journal of Toxicology and Environmental Health vol43 no 3 pp 271ndash289 1994
[6] O Arellano-Aguilar and C M Garcia ldquoEffects of methylparathion exposure on development and reproduction in theviviparous fish girardinichthys multiradiatusrdquo EnvironmentalToxicology vol 24 no 2 pp 178ndash186 2009
[7] M Y de la Vega Salazar L Martınez Tabche and C MacIasGarcıa ldquoBioaccumulation of methyl parathion and its toxicol-ogy in several species of the freshwater community in IgnacioRyamirez dam in Mexicordquo Ecotoxicology and EnvironmentalSafety vol 38 no 1 pp 53ndash62 1997
[8] J E Chambers and H W Chambers ldquoBiotransformationof organophosphorous insecticides in mammalsrdquo in PesticideTransformation Products Fate and Significance in the Envi-ronment L Somasundaram and J R Coats Eds pp 32ndash42American Chemistry Society Washington DC USA 1991
[9] B N LaDu ldquoHuman serum paraoxonasearylesteraserdquo in Phar-macogenetics of DrugMetabolism Pergamon W Kalow Ed pp51ndash91 Pergamon New York NY USA 1992
[10] K Dembele E Haubruge and C Gaspar ldquoConcentrationeffects of selected insecticides on brain acetylcholinesterasein the common carp (Cyprinus carpio L)rdquo Ecotoxicology andEnvironmental Safety vol 45 no 1 pp 49ndash54 2000
[11] V K Patil and M David ldquoOxidative stress in freshwaterfish Labeo rohita as a biomarker of malathion exposurerdquoEnvironmental Monitoring and Assessment vol 185 no 12 pp10191ndash10199 2013
[12] M Jokanovic ldquoCurrent understanding of the mechanismsinvolved in metabolic detoxification of warfare nerve agentsrdquoToxicology Letters vol 188 no 1 pp 1ndash10 2009
[13] R M Lopes M V S Filho J B de Salles V L F C Bastosand J C Bastos ldquoCholinesterase activity of muscle tissue fromfreshwater fishes characterization and sensitivity analysis to theorganophosphate methyl-paraoxonrdquo Environmental Toxicologyand Chemistry vol 33 no 6 pp 1331ndash1336 2014
[14] G L Ellman K D Courtney V Andres Jr and R MFeatherstone ldquoA new and rapid colorimetric determination ofacetylcholinesterase activityrdquo Biochemical Pharmacology vol 7no 2 pp 88ndash95 1961
[15] M V S Filho M M Oliveira J B Salles V L F C BastosV P F Cassano and J C Bastos ldquoMethyl-paraoxon compara-tive inhibition kinetics for acetylcholinesterases from brain ofneotropical fishesrdquo Toxicology Letters vol 153 no 2 pp 247ndash254 2004
[16] J R Kemp and K B Wallace ldquoMolecular determinants ofthe species-selective inhibition of brain acetylcholinesteraserdquoToxicology and Applied Pharmacology vol 104 no 2 pp 246ndash258 1990
[17] J Halpert D Hammond and R A Neal ldquoInactivation ofpurified rat liver cytochrome P-450 during the metabolismof parathion (diethyl p-nitrophenyl phosphorothionate)rdquo TheJournal of Biological Chemistry vol 255 no 3 pp 1080ndash10891980
[18] B J Norman R E Poore and R A Neal ldquoStudies ofthe binding of sulfur released in the mixed-function oxi-dase-catalyzed metabolism of diethyl p-nitrophenyl phos-phorothionate(parathion) to diethyl p-nitrophenyl phosphate(paraoxon)rdquo Biochemical Pharmacology vol 23 no 12 pp1733ndash1744 1974
[19] R Abbas I R Schultz S Doddapaneni and W L HaytonldquoToxicokinetics of parathion and paraoxon in rainbow trout
BioMed Research International 9
after intravascular administration and water exposurerdquo Toxicol-ogy and Applied Pharmacology vol 136 no 1 pp 194ndash199 1996
[20] V L F Cunha Bastos A Rossini L F Ribeiro Pinto etal ldquoDifferent sensitivities to paraoxon of brain and serumcholinesterases from pacu an indigenous Brazilian fishrdquo Bul-letin of Environmental Contamination and Toxicology vol 60no 1 pp 1ndash8 1998
[21] J S Boone and U E Chambers ldquoTime course of inhibitionof cholinesterase and aliesterase activities and nonproteinsulfhydryl levels following exposure to organophosphorusinsecticides in mosquitofish (Gambusia affinis)rdquo Fundamentaland Applied Toxicology vol 29 no 2 pp 202ndash207 1996
[22] C HWalker ldquoThe use of biomarkers to measure the interactiveeffects of chemicalsrdquo Ecotoxicology and Environmental Safetyvol 40 no 1-2 pp 65ndash70 1998
[23] J S Boone and J E Chambers ldquoBiochemical factors contribut-ing to toxicity differences among chlorpyrifos parthion andmethyl parathion in mosquitofish (Gambusia affinis)rdquo AquaticToxicology vol 39 no 3-4 pp 333ndash343 1997
[24] J Blasiak ldquoAllosteric inhibition of the (Na+ + K+)-ATPaseby parathion and methylparathionrdquo Pesticide Biochemistry andPhysiology vol 54 no 1 pp 40ndash47 1996
Table 1 Common and scientific name suppliers and average size of fishes examined
Common name Scientific name Suppliera Size and SDb (cm)
Curimbata Prochilodus lineatus(Valenciennes 1836) Sol Nascente Fish Farm 18 plusmn 5
Pacu Piaractus mesopotamicus(Holmberg 1887) Morro Grande Fish Farm 18 plusmn 4
Piavussu Leporinus macrocephalusGaravello and Britski 1988 Morro Grande Fish Farm 20 plusmn 5
aAll suppliers are located in the state of Rio de Janeiro BrazilbSD standard deviation
at 3000timesg for 10min at 5∘C Three milliliters from eachsupernatant fraction was collected and evaporated undera gentle nitrogen stream The residue was reconstitutedin 200120583L of acetonitrile From this reconstituted sample50120583L were injected onto a 200mm times 46mm ODS HypersilRP-18 5 120583m particle size HPLC column using (50 50 vv)acetonitrileultrapure water as mobile phase with a flow-rate of 1mLminminus1 and examined under UV light withthe detector set at 270 nm Under these conditions methylparathion recovery was estimated at approximately 92following quantification based on a standard prepared witha 98 methyl parathion pure sample previously obtained bythin-layer chromatography
25 Cholinesterase Assays Serum and tissue homogenatesamples were placed in a medium containing 18mmol Lminus1from one of the three substrates (acetylthiocholine propi-onylthiocholine or butyrylthiocholine) with 032mmol Lminus1DTNB Enzyme activitywas continuously recordedup to 90 sat 412 nm using a Shimadzu spectrophotometer model UV-160A according to the Ellman method [14] The reactionwas carried out in microcuvettes All reagents were dissolvedto 200120583L final volume with a 01mmol Lminus1 sodium phos-phate buffer pH 75 at 25∘C and the thionitrobenzoate ionconcentration was estimated using an extinction coefficientof 14150Mminus1 cmminus1 One unit (1 U) of enzyme activity wasdefined as the amount that hydrolyzes 10 120583mol of substrateper minute
3 Results
31 Tissue Distribution of ChE Activity The studied fishspecies (control groups) exhibited remarkable differencesin tissue-specific cholinesterase activity (Figure 1) The ChEactivity measured in the serum of curimbata and pacuspecimens was lower than the activity measured in brainliver heart and muscle By contrast in serum of piavussuthe ChE activity was similar to that measured in liver brainheart and muscle
32 Methyl Parathion Bioconcentration Muscle heart brainliver and serum from pacu piavussu and curimbata exhib-ited similar methyl parathion bioconcentration patterns(Figure 2) Brain tissue showed the highest capacity formethyl parathion bioconcentration reaching 80 ppm (16-fold
increase) after 30 minutes exposure to methyl parathion inwater (Figure 2) The maximum methyl parathion concen-tration in muscle heart brain and serum was achieved after30minutes with one exception methyl parathion concentra-tions in piavussu showed an increase after 24 hours exposurereaching a maximum of 120 ppm in liver corresponding to a24-fold increase (Figure 3) Methyl parathion was not foundin samples from control animals
33 Cholinesterase Inhibition Heart liver and serum cho-linesterase activities for pacu piavussu and curimbata weregreatly inhibited after 30 minutes exposure to Folidol Bycontrast skeletal muscle ChE activity for the three speciesand brain AChE activity for pacu showed no inhibitionwithin this time period (Figure 4) Exposure of fishes to waterwith Folidol for 78 h resulted in 70 brain AChE activityinhibition in pacu and 60 brain AChE activity inhibitionin piavussu
All curimbatas stopped moving their opercula between 3and 5 hours of exposure to 5 ppm Folidol showingmore than90 inhibition of brain AChE activity and no inhibition ofskeletal muscle ChE activity (Figure 4) Increasing the timeof exposure from 30min to 78 hours did not considerablymodify muscle heart brain liver or serum ChE activities inpiavussu (Figure 4)
4 Discussion
Brain tissue showed the highest pesticide bioconcentrationreaching 80 ppm after 30 minutes of exposure to methylparathion (Figure 2) The three fish species selected for thisstudy present different brain cholinesterase sensitivities tointoxication by methyl paraoxon [15] In these fishes thesensitivity of brain ChE to intoxication bymethyl paraoxon isinversely related to the activity of the brain enzyme forASCho[15] in contrast to what was described for trout and rat byKemp and Wallace [16]
The results demonstrate that the fishes exposed to Folidolrapidly absorbed methyl parathion since concentrations ofmethyl parathion in the tissues and serum peaked within30min of exposure to Folidol This concentration contin-ued to increase only in liver in which the amount ofmethyl parathion doubled after 24 hours (Figure 3) Suchfindings can be ascribed to the promptness with whichmethyl parathion undergoes biotransformation into methyl
Figure 1 Tissue distribution of cholinesterase activity in pacu piavussu and curimbata (control fish) The box shows the substrates used inenzyme assays Results are expressed as 120583mol of products formed per minute per gram of wet tissue (U gminus1) or per mL of serum (UmLminus1)Liver and brain ChE activities were assayed in homogenates while heart and muscle ChE activities were assayed in the enzymatic fractionsolubilized with Triton and NaCl Results are average values with the corresponding SEM of assays carried out in five individuals of eachspecies
BioMed Research International 5
0 50 100
Serum
Liver
Brain
Heart
Muscle
Pacu
0 50 100Methyl parathion (ppm)
Methyl parathion (ppm)
Methyl parathion (ppm)
Piavussu
0 50 100
Curimbataacute
Serum
Liver
Brain
Heart
Muscle
Serum
Liver
Brain
Heart
Muscle
Figure 2 Concentration of methyl parathion in muscle heart brain and liver and serum from pacu piavussu and curimbata Each one offour animals per species was placed in separated tanks containing 40 L of water with Folidol to produce 5 ppm methyl parathion After 30minutes exposure blood samples were collected and the fishes were euthanized Tissues were dissected and homogenized Methyl parathionwas extracted from serum and tissue homogenates and quantified by HPLC
paraoxon inside liver cells as compared to other organsSince P-450 can be inhibited by the sulphur removed fromparathion [17 18] it is plausible that metabolism of methylparathion molecules occurred in liver up to 30min and thena consequent inhibition of liver P-450 allowed that moremethyl parathion could accumulate and be extracted Whenrainbow trout was exposed to the 75 ngmLminus1 ethyl parathionAbbas and coworkers [19] observed a peak concentration ofthis pesticide in plasma up to 45 hours In the experiments
conducted in the present study pesticide bioconcentrationoccurred over a shorter period probably due to the sig-nificantly higher pesticide concentration used Our resultsindicate that methyl parathion can bioconcentrate in brainof the tested fish up to approximately 80 ppm (4 times itssolubility in water) (Figure 2)This bioconcentration capacityis probably due to high lipid solubility of this OP compoundThe half-life of parathion in fish has been reported as 5 timeslonger than in rats [19]This suggests that this pesticide could
6 BioMed Research International
0 70 140
Serum
Liver
Brain
Heart
Muscle
Methyl parathion (ppm)
Pesticide bioconcentration
0 50 100ChE activity ()
Enzyme inhibition
Serum
Liver
Brain
Heart
Muscle
05h24h
05h24h
Figure 3 Piavussumethyl parathion concentrations and cholinesterase inhibition after exposure Each one of eight piavussuswas individuallyplaced in separated tanks containing 40 L of water with Folidol to produce 5 ppm methyl parathion Incubation happened for the showedtimes After exposure blood was collected fishes were submitted to euthanasia and tissues were dissected and homogenized Cholinesteraseactivity was assayed in serum and in homogenates tissues with acetylthiocholine and expressed as percentage of control activity Methylparathion was extracted from serum and tissue homogenates and quantified by HPLC
persist for a relatively long time in fish raising concernsregarding human consumption of fish previously exposed toOP
The results reported herein demonstrated that ChEinhibition in fish exposed to Folidol does not seem todepend on the methyl parathion bioconcentration in theirtissues No ChE inhibition in pacu brain was observed up to30min of exposure despite the fact that this organ presented80 ppm of methyl parathion at that time Therefore thisdata is important for further investigations Tissues in whichChE inhibition occurred more quickly such as pacu liver(Figure 4) showing higher BChE activity in comparison toAChE activity (Figure 1) BChE has been shown to be moresensitive to methyl paraoxon and is probably more quicklyinhibited [20] In addition activation of OP compoundsoccurs mainly in the liver because of the high concentrationsof P-450 in that tissue Thus liver ChE undergoes inhibitionby methyl paraoxon locally generated in tissue before thismetabolite leaks out to general blood circulation and reachesother target tissues
In their work de Aguiar and coworkers [2] described an87 brain AChE inhibition in matrinxa (Brycon cephalus)exposed to water with 2 ppm Folidol 600 for 96 hours Thepresent study indicates that piavussu and pacumight bemoretolerant to Folidol than matrinxa since piavussu presented66 and pacu 74 brain AChE inhibition when exposed to
water with 5 ppmmethyl parathion for 78 hours On the otherhand curimbata showed a 92 brain AChE inhibition withonly 5 hours of exposure to 5 ppmmethyl parathion probablydue to the higher sensitivity of this species AChE to inhibitionby methyl paraoxon [15]
Mammals poisoned by OP usually die by asphyxiaHowever fishes have been reported to be more resistant topoisoning by high levels of organophosphates compoundsthan rats [21] The actual causes and mechanisms of fishpoisoning by OP are not fully understood Our findingsreinforce that interspecific differences in AChE inhibition bytheir oxon derivatives should be considered Physiologicaland behavioral disturbances start at 50 AChE inhibitionand death usually follows when inhibition exceeds 80 inmammals and birds [22] We found here that curimbatas alsoperished when brain AChE inhibition reached above 80
This study suggests that a major contributing factor toacute fish toxicity by Folidol is brain AChE sensitivity tomethyl paraoxon the oxon derivative of methyl parathionThe same explanation that is the sensitivity of brain AChEto oxon derivatives has been suggested for chlorpyrifosparathion and methyl parathion toxicity in mosquito fish(Gambusia affinis) [23] Although it was clearly establishedthat curimbatas possess the most sensitive brain AChEamong the studied species the death of curimbatas exposedto 5 ppm methyl parathion cannot be solely credited to brain
BioMed Research International 7
0 50 100
Serum
Liver
Brain
Heart
Muscle
Pacu
0 50 100
Piavussu
0 50 100
Curimbataacute
05h78h
05h78h
05h5h
ChE activity( relative to controls)
ChE activity( relative to controls)
Serum
Liver
Brain
Heart
Muscle
Serum
Liver
Brain
Heart
Muscle
ChE activity( relative to controls)
Figure 4 Cholinesterase activity in tissues of pacu piavussu and curimbata exposed to 5 ppm methyl parathion Each one of eight animalsby species was individually treated with Folidol 600 for the indicated time in a 40 L aquarium After 30min of exposure blood was collectedfrom four fishes by species and these fishes were submitted to euthanasia Then of the remaining fish four pacus and four piavussus wereeuthanized after 78 h exposure while four curimbatas were collected immediately after they showed no movement of opercula (from 3 to 5 hof exposure) Tissues were dissected and homogenized Cholinesterase was assayed in serum and homogenates with acetylthiocholine andexpressed as percentage of the activity found in controls
8 BioMed Research International
AChE inhibition Other possible causes of this fish deathsuch as Na+ K+ ATPase inhibition [24] and tissue hypoxiawhich compromises heart function need to be examined
5 Conclusion
Prochilodus lineatus (curimbata) Piaractus mesopotamicus(pacu) and Leporinus macrocephalus (piavussu) studied hereshowed similar capacities to bioconcentratemethyl parathionin their tissues after exposure to 5 ppm in water Howeveronly curimbata with the highest brain AChE sensitivity tomethyl paraoxon died after 5 hours of exposure to FolidolPacu and piavussu are more resistant to methyl paraoxonand were alive up to 78 hours of exposure to 5 ppm ofmethyl parathion BrainAChE sensitivity tomethyl paraoxonmight be a decisive factor for determining the sensitivityof these species to poisoning by high concentrations oforganophosphate compounds The present study indicatesthat fishes whose brain acetylcholinesterase activity is moresensitive to oxon derivatives will suffer more severe impactsfrom environmental contamination by organophosphate pes-ticides
Measures reducing the use of organophosphate pesticidesin fish culture should be adopted in order to minimizethe discharge and consequent impact of these chemicals tonatural communities inhabiting rivers and lakes of which thesensitivity to intoxication by pesticides is largely unknown
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors are grateful to Morro Grande Fish Farm andSol Nascente Fish Farm for supplying the fishes This paperis dedicated to Dr Moacelio Veranio Silva Filho (in memo-riam)
References
[1] E Barbieri and L A A Ferreira ldquoEffects of the organophos-phate pesticide Folidol 600 on the freshwater fish Nile Tilapia(Oreochromis niloticus)rdquo Pesticide Biochemistry and Physiologyvol 99 no 3 pp 209ndash214 2011
[2] L H de Aguiar G Moraes I M Avilez A E Altran and CF Correa ldquoMetabolical effects of Folidol 600 on the neotrop-ical freshwater fish matrinxa Brycon cephalusrdquo EnvironmentalResearch vol 95 no 2 pp 224ndash230 2004
[3] D Hernandez-Moreno M Perez-Lopez F Soler C Gravatoand L Guilhermino ldquoEffects of carbofuran on the sea bass(Dicentrarchus labrax L) study of biomarkers and behaviouralterationsrdquo Ecotoxicology and Environmental Safety vol 74 no7 pp 1905ndash1912 2011
[4] C RD Assis A G Linhares VMOliveira et al ldquoComparativeeffect of pesticides on brain acetylcholinesterase in tropical fishrdquoScience of the Total Environment vol 441 pp 141ndash150 2012
[5] L G Sultatos ldquoMammalian toxicology of organophosphoruspesticidesrdquo Journal of Toxicology and Environmental Health vol43 no 3 pp 271ndash289 1994
[6] O Arellano-Aguilar and C M Garcia ldquoEffects of methylparathion exposure on development and reproduction in theviviparous fish girardinichthys multiradiatusrdquo EnvironmentalToxicology vol 24 no 2 pp 178ndash186 2009
[7] M Y de la Vega Salazar L Martınez Tabche and C MacIasGarcıa ldquoBioaccumulation of methyl parathion and its toxicol-ogy in several species of the freshwater community in IgnacioRyamirez dam in Mexicordquo Ecotoxicology and EnvironmentalSafety vol 38 no 1 pp 53ndash62 1997
[8] J E Chambers and H W Chambers ldquoBiotransformationof organophosphorous insecticides in mammalsrdquo in PesticideTransformation Products Fate and Significance in the Envi-ronment L Somasundaram and J R Coats Eds pp 32ndash42American Chemistry Society Washington DC USA 1991
[9] B N LaDu ldquoHuman serum paraoxonasearylesteraserdquo in Phar-macogenetics of DrugMetabolism Pergamon W Kalow Ed pp51ndash91 Pergamon New York NY USA 1992
[10] K Dembele E Haubruge and C Gaspar ldquoConcentrationeffects of selected insecticides on brain acetylcholinesterasein the common carp (Cyprinus carpio L)rdquo Ecotoxicology andEnvironmental Safety vol 45 no 1 pp 49ndash54 2000
[11] V K Patil and M David ldquoOxidative stress in freshwaterfish Labeo rohita as a biomarker of malathion exposurerdquoEnvironmental Monitoring and Assessment vol 185 no 12 pp10191ndash10199 2013
[12] M Jokanovic ldquoCurrent understanding of the mechanismsinvolved in metabolic detoxification of warfare nerve agentsrdquoToxicology Letters vol 188 no 1 pp 1ndash10 2009
[13] R M Lopes M V S Filho J B de Salles V L F C Bastosand J C Bastos ldquoCholinesterase activity of muscle tissue fromfreshwater fishes characterization and sensitivity analysis to theorganophosphate methyl-paraoxonrdquo Environmental Toxicologyand Chemistry vol 33 no 6 pp 1331ndash1336 2014
[14] G L Ellman K D Courtney V Andres Jr and R MFeatherstone ldquoA new and rapid colorimetric determination ofacetylcholinesterase activityrdquo Biochemical Pharmacology vol 7no 2 pp 88ndash95 1961
[15] M V S Filho M M Oliveira J B Salles V L F C BastosV P F Cassano and J C Bastos ldquoMethyl-paraoxon compara-tive inhibition kinetics for acetylcholinesterases from brain ofneotropical fishesrdquo Toxicology Letters vol 153 no 2 pp 247ndash254 2004
[16] J R Kemp and K B Wallace ldquoMolecular determinants ofthe species-selective inhibition of brain acetylcholinesteraserdquoToxicology and Applied Pharmacology vol 104 no 2 pp 246ndash258 1990
[17] J Halpert D Hammond and R A Neal ldquoInactivation ofpurified rat liver cytochrome P-450 during the metabolismof parathion (diethyl p-nitrophenyl phosphorothionate)rdquo TheJournal of Biological Chemistry vol 255 no 3 pp 1080ndash10891980
[18] B J Norman R E Poore and R A Neal ldquoStudies ofthe binding of sulfur released in the mixed-function oxi-dase-catalyzed metabolism of diethyl p-nitrophenyl phos-phorothionate(parathion) to diethyl p-nitrophenyl phosphate(paraoxon)rdquo Biochemical Pharmacology vol 23 no 12 pp1733ndash1744 1974
[19] R Abbas I R Schultz S Doddapaneni and W L HaytonldquoToxicokinetics of parathion and paraoxon in rainbow trout
BioMed Research International 9
after intravascular administration and water exposurerdquo Toxicol-ogy and Applied Pharmacology vol 136 no 1 pp 194ndash199 1996
[20] V L F Cunha Bastos A Rossini L F Ribeiro Pinto etal ldquoDifferent sensitivities to paraoxon of brain and serumcholinesterases from pacu an indigenous Brazilian fishrdquo Bul-letin of Environmental Contamination and Toxicology vol 60no 1 pp 1ndash8 1998
[21] J S Boone and U E Chambers ldquoTime course of inhibitionof cholinesterase and aliesterase activities and nonproteinsulfhydryl levels following exposure to organophosphorusinsecticides in mosquitofish (Gambusia affinis)rdquo Fundamentaland Applied Toxicology vol 29 no 2 pp 202ndash207 1996
[22] C HWalker ldquoThe use of biomarkers to measure the interactiveeffects of chemicalsrdquo Ecotoxicology and Environmental Safetyvol 40 no 1-2 pp 65ndash70 1998
[23] J S Boone and J E Chambers ldquoBiochemical factors contribut-ing to toxicity differences among chlorpyrifos parthion andmethyl parathion in mosquitofish (Gambusia affinis)rdquo AquaticToxicology vol 39 no 3-4 pp 333ndash343 1997
[24] J Blasiak ldquoAllosteric inhibition of the (Na+ + K+)-ATPaseby parathion and methylparathionrdquo Pesticide Biochemistry andPhysiology vol 54 no 1 pp 40ndash47 1996
Figure 1 Tissue distribution of cholinesterase activity in pacu piavussu and curimbata (control fish) The box shows the substrates used inenzyme assays Results are expressed as 120583mol of products formed per minute per gram of wet tissue (U gminus1) or per mL of serum (UmLminus1)Liver and brain ChE activities were assayed in homogenates while heart and muscle ChE activities were assayed in the enzymatic fractionsolubilized with Triton and NaCl Results are average values with the corresponding SEM of assays carried out in five individuals of eachspecies
BioMed Research International 5
0 50 100
Serum
Liver
Brain
Heart
Muscle
Pacu
0 50 100Methyl parathion (ppm)
Methyl parathion (ppm)
Methyl parathion (ppm)
Piavussu
0 50 100
Curimbataacute
Serum
Liver
Brain
Heart
Muscle
Serum
Liver
Brain
Heart
Muscle
Figure 2 Concentration of methyl parathion in muscle heart brain and liver and serum from pacu piavussu and curimbata Each one offour animals per species was placed in separated tanks containing 40 L of water with Folidol to produce 5 ppm methyl parathion After 30minutes exposure blood samples were collected and the fishes were euthanized Tissues were dissected and homogenized Methyl parathionwas extracted from serum and tissue homogenates and quantified by HPLC
paraoxon inside liver cells as compared to other organsSince P-450 can be inhibited by the sulphur removed fromparathion [17 18] it is plausible that metabolism of methylparathion molecules occurred in liver up to 30min and thena consequent inhibition of liver P-450 allowed that moremethyl parathion could accumulate and be extracted Whenrainbow trout was exposed to the 75 ngmLminus1 ethyl parathionAbbas and coworkers [19] observed a peak concentration ofthis pesticide in plasma up to 45 hours In the experiments
conducted in the present study pesticide bioconcentrationoccurred over a shorter period probably due to the sig-nificantly higher pesticide concentration used Our resultsindicate that methyl parathion can bioconcentrate in brainof the tested fish up to approximately 80 ppm (4 times itssolubility in water) (Figure 2)This bioconcentration capacityis probably due to high lipid solubility of this OP compoundThe half-life of parathion in fish has been reported as 5 timeslonger than in rats [19]This suggests that this pesticide could
6 BioMed Research International
0 70 140
Serum
Liver
Brain
Heart
Muscle
Methyl parathion (ppm)
Pesticide bioconcentration
0 50 100ChE activity ()
Enzyme inhibition
Serum
Liver
Brain
Heart
Muscle
05h24h
05h24h
Figure 3 Piavussumethyl parathion concentrations and cholinesterase inhibition after exposure Each one of eight piavussuswas individuallyplaced in separated tanks containing 40 L of water with Folidol to produce 5 ppm methyl parathion Incubation happened for the showedtimes After exposure blood was collected fishes were submitted to euthanasia and tissues were dissected and homogenized Cholinesteraseactivity was assayed in serum and in homogenates tissues with acetylthiocholine and expressed as percentage of control activity Methylparathion was extracted from serum and tissue homogenates and quantified by HPLC
persist for a relatively long time in fish raising concernsregarding human consumption of fish previously exposed toOP
The results reported herein demonstrated that ChEinhibition in fish exposed to Folidol does not seem todepend on the methyl parathion bioconcentration in theirtissues No ChE inhibition in pacu brain was observed up to30min of exposure despite the fact that this organ presented80 ppm of methyl parathion at that time Therefore thisdata is important for further investigations Tissues in whichChE inhibition occurred more quickly such as pacu liver(Figure 4) showing higher BChE activity in comparison toAChE activity (Figure 1) BChE has been shown to be moresensitive to methyl paraoxon and is probably more quicklyinhibited [20] In addition activation of OP compoundsoccurs mainly in the liver because of the high concentrationsof P-450 in that tissue Thus liver ChE undergoes inhibitionby methyl paraoxon locally generated in tissue before thismetabolite leaks out to general blood circulation and reachesother target tissues
In their work de Aguiar and coworkers [2] described an87 brain AChE inhibition in matrinxa (Brycon cephalus)exposed to water with 2 ppm Folidol 600 for 96 hours Thepresent study indicates that piavussu and pacumight bemoretolerant to Folidol than matrinxa since piavussu presented66 and pacu 74 brain AChE inhibition when exposed to
water with 5 ppmmethyl parathion for 78 hours On the otherhand curimbata showed a 92 brain AChE inhibition withonly 5 hours of exposure to 5 ppmmethyl parathion probablydue to the higher sensitivity of this species AChE to inhibitionby methyl paraoxon [15]
Mammals poisoned by OP usually die by asphyxiaHowever fishes have been reported to be more resistant topoisoning by high levels of organophosphates compoundsthan rats [21] The actual causes and mechanisms of fishpoisoning by OP are not fully understood Our findingsreinforce that interspecific differences in AChE inhibition bytheir oxon derivatives should be considered Physiologicaland behavioral disturbances start at 50 AChE inhibitionand death usually follows when inhibition exceeds 80 inmammals and birds [22] We found here that curimbatas alsoperished when brain AChE inhibition reached above 80
This study suggests that a major contributing factor toacute fish toxicity by Folidol is brain AChE sensitivity tomethyl paraoxon the oxon derivative of methyl parathionThe same explanation that is the sensitivity of brain AChEto oxon derivatives has been suggested for chlorpyrifosparathion and methyl parathion toxicity in mosquito fish(Gambusia affinis) [23] Although it was clearly establishedthat curimbatas possess the most sensitive brain AChEamong the studied species the death of curimbatas exposedto 5 ppm methyl parathion cannot be solely credited to brain
BioMed Research International 7
0 50 100
Serum
Liver
Brain
Heart
Muscle
Pacu
0 50 100
Piavussu
0 50 100
Curimbataacute
05h78h
05h78h
05h5h
ChE activity( relative to controls)
ChE activity( relative to controls)
Serum
Liver
Brain
Heart
Muscle
Serum
Liver
Brain
Heart
Muscle
ChE activity( relative to controls)
Figure 4 Cholinesterase activity in tissues of pacu piavussu and curimbata exposed to 5 ppm methyl parathion Each one of eight animalsby species was individually treated with Folidol 600 for the indicated time in a 40 L aquarium After 30min of exposure blood was collectedfrom four fishes by species and these fishes were submitted to euthanasia Then of the remaining fish four pacus and four piavussus wereeuthanized after 78 h exposure while four curimbatas were collected immediately after they showed no movement of opercula (from 3 to 5 hof exposure) Tissues were dissected and homogenized Cholinesterase was assayed in serum and homogenates with acetylthiocholine andexpressed as percentage of the activity found in controls
8 BioMed Research International
AChE inhibition Other possible causes of this fish deathsuch as Na+ K+ ATPase inhibition [24] and tissue hypoxiawhich compromises heart function need to be examined
5 Conclusion
Prochilodus lineatus (curimbata) Piaractus mesopotamicus(pacu) and Leporinus macrocephalus (piavussu) studied hereshowed similar capacities to bioconcentratemethyl parathionin their tissues after exposure to 5 ppm in water Howeveronly curimbata with the highest brain AChE sensitivity tomethyl paraoxon died after 5 hours of exposure to FolidolPacu and piavussu are more resistant to methyl paraoxonand were alive up to 78 hours of exposure to 5 ppm ofmethyl parathion BrainAChE sensitivity tomethyl paraoxonmight be a decisive factor for determining the sensitivityof these species to poisoning by high concentrations oforganophosphate compounds The present study indicatesthat fishes whose brain acetylcholinesterase activity is moresensitive to oxon derivatives will suffer more severe impactsfrom environmental contamination by organophosphate pes-ticides
Measures reducing the use of organophosphate pesticidesin fish culture should be adopted in order to minimizethe discharge and consequent impact of these chemicals tonatural communities inhabiting rivers and lakes of which thesensitivity to intoxication by pesticides is largely unknown
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors are grateful to Morro Grande Fish Farm andSol Nascente Fish Farm for supplying the fishes This paperis dedicated to Dr Moacelio Veranio Silva Filho (in memo-riam)
References
[1] E Barbieri and L A A Ferreira ldquoEffects of the organophos-phate pesticide Folidol 600 on the freshwater fish Nile Tilapia(Oreochromis niloticus)rdquo Pesticide Biochemistry and Physiologyvol 99 no 3 pp 209ndash214 2011
[2] L H de Aguiar G Moraes I M Avilez A E Altran and CF Correa ldquoMetabolical effects of Folidol 600 on the neotrop-ical freshwater fish matrinxa Brycon cephalusrdquo EnvironmentalResearch vol 95 no 2 pp 224ndash230 2004
[3] D Hernandez-Moreno M Perez-Lopez F Soler C Gravatoand L Guilhermino ldquoEffects of carbofuran on the sea bass(Dicentrarchus labrax L) study of biomarkers and behaviouralterationsrdquo Ecotoxicology and Environmental Safety vol 74 no7 pp 1905ndash1912 2011
[4] C RD Assis A G Linhares VMOliveira et al ldquoComparativeeffect of pesticides on brain acetylcholinesterase in tropical fishrdquoScience of the Total Environment vol 441 pp 141ndash150 2012
[5] L G Sultatos ldquoMammalian toxicology of organophosphoruspesticidesrdquo Journal of Toxicology and Environmental Health vol43 no 3 pp 271ndash289 1994
[6] O Arellano-Aguilar and C M Garcia ldquoEffects of methylparathion exposure on development and reproduction in theviviparous fish girardinichthys multiradiatusrdquo EnvironmentalToxicology vol 24 no 2 pp 178ndash186 2009
[7] M Y de la Vega Salazar L Martınez Tabche and C MacIasGarcıa ldquoBioaccumulation of methyl parathion and its toxicol-ogy in several species of the freshwater community in IgnacioRyamirez dam in Mexicordquo Ecotoxicology and EnvironmentalSafety vol 38 no 1 pp 53ndash62 1997
[8] J E Chambers and H W Chambers ldquoBiotransformationof organophosphorous insecticides in mammalsrdquo in PesticideTransformation Products Fate and Significance in the Envi-ronment L Somasundaram and J R Coats Eds pp 32ndash42American Chemistry Society Washington DC USA 1991
[9] B N LaDu ldquoHuman serum paraoxonasearylesteraserdquo in Phar-macogenetics of DrugMetabolism Pergamon W Kalow Ed pp51ndash91 Pergamon New York NY USA 1992
[10] K Dembele E Haubruge and C Gaspar ldquoConcentrationeffects of selected insecticides on brain acetylcholinesterasein the common carp (Cyprinus carpio L)rdquo Ecotoxicology andEnvironmental Safety vol 45 no 1 pp 49ndash54 2000
[11] V K Patil and M David ldquoOxidative stress in freshwaterfish Labeo rohita as a biomarker of malathion exposurerdquoEnvironmental Monitoring and Assessment vol 185 no 12 pp10191ndash10199 2013
[12] M Jokanovic ldquoCurrent understanding of the mechanismsinvolved in metabolic detoxification of warfare nerve agentsrdquoToxicology Letters vol 188 no 1 pp 1ndash10 2009
[13] R M Lopes M V S Filho J B de Salles V L F C Bastosand J C Bastos ldquoCholinesterase activity of muscle tissue fromfreshwater fishes characterization and sensitivity analysis to theorganophosphate methyl-paraoxonrdquo Environmental Toxicologyand Chemistry vol 33 no 6 pp 1331ndash1336 2014
[14] G L Ellman K D Courtney V Andres Jr and R MFeatherstone ldquoA new and rapid colorimetric determination ofacetylcholinesterase activityrdquo Biochemical Pharmacology vol 7no 2 pp 88ndash95 1961
[15] M V S Filho M M Oliveira J B Salles V L F C BastosV P F Cassano and J C Bastos ldquoMethyl-paraoxon compara-tive inhibition kinetics for acetylcholinesterases from brain ofneotropical fishesrdquo Toxicology Letters vol 153 no 2 pp 247ndash254 2004
[16] J R Kemp and K B Wallace ldquoMolecular determinants ofthe species-selective inhibition of brain acetylcholinesteraserdquoToxicology and Applied Pharmacology vol 104 no 2 pp 246ndash258 1990
[17] J Halpert D Hammond and R A Neal ldquoInactivation ofpurified rat liver cytochrome P-450 during the metabolismof parathion (diethyl p-nitrophenyl phosphorothionate)rdquo TheJournal of Biological Chemistry vol 255 no 3 pp 1080ndash10891980
[18] B J Norman R E Poore and R A Neal ldquoStudies ofthe binding of sulfur released in the mixed-function oxi-dase-catalyzed metabolism of diethyl p-nitrophenyl phos-phorothionate(parathion) to diethyl p-nitrophenyl phosphate(paraoxon)rdquo Biochemical Pharmacology vol 23 no 12 pp1733ndash1744 1974
[19] R Abbas I R Schultz S Doddapaneni and W L HaytonldquoToxicokinetics of parathion and paraoxon in rainbow trout
BioMed Research International 9
after intravascular administration and water exposurerdquo Toxicol-ogy and Applied Pharmacology vol 136 no 1 pp 194ndash199 1996
[20] V L F Cunha Bastos A Rossini L F Ribeiro Pinto etal ldquoDifferent sensitivities to paraoxon of brain and serumcholinesterases from pacu an indigenous Brazilian fishrdquo Bul-letin of Environmental Contamination and Toxicology vol 60no 1 pp 1ndash8 1998
[21] J S Boone and U E Chambers ldquoTime course of inhibitionof cholinesterase and aliesterase activities and nonproteinsulfhydryl levels following exposure to organophosphorusinsecticides in mosquitofish (Gambusia affinis)rdquo Fundamentaland Applied Toxicology vol 29 no 2 pp 202ndash207 1996
[22] C HWalker ldquoThe use of biomarkers to measure the interactiveeffects of chemicalsrdquo Ecotoxicology and Environmental Safetyvol 40 no 1-2 pp 65ndash70 1998
[23] J S Boone and J E Chambers ldquoBiochemical factors contribut-ing to toxicity differences among chlorpyrifos parthion andmethyl parathion in mosquitofish (Gambusia affinis)rdquo AquaticToxicology vol 39 no 3-4 pp 333ndash343 1997
[24] J Blasiak ldquoAllosteric inhibition of the (Na+ + K+)-ATPaseby parathion and methylparathionrdquo Pesticide Biochemistry andPhysiology vol 54 no 1 pp 40ndash47 1996
Figure 2 Concentration of methyl parathion in muscle heart brain and liver and serum from pacu piavussu and curimbata Each one offour animals per species was placed in separated tanks containing 40 L of water with Folidol to produce 5 ppm methyl parathion After 30minutes exposure blood samples were collected and the fishes were euthanized Tissues were dissected and homogenized Methyl parathionwas extracted from serum and tissue homogenates and quantified by HPLC
paraoxon inside liver cells as compared to other organsSince P-450 can be inhibited by the sulphur removed fromparathion [17 18] it is plausible that metabolism of methylparathion molecules occurred in liver up to 30min and thena consequent inhibition of liver P-450 allowed that moremethyl parathion could accumulate and be extracted Whenrainbow trout was exposed to the 75 ngmLminus1 ethyl parathionAbbas and coworkers [19] observed a peak concentration ofthis pesticide in plasma up to 45 hours In the experiments
conducted in the present study pesticide bioconcentrationoccurred over a shorter period probably due to the sig-nificantly higher pesticide concentration used Our resultsindicate that methyl parathion can bioconcentrate in brainof the tested fish up to approximately 80 ppm (4 times itssolubility in water) (Figure 2)This bioconcentration capacityis probably due to high lipid solubility of this OP compoundThe half-life of parathion in fish has been reported as 5 timeslonger than in rats [19]This suggests that this pesticide could
6 BioMed Research International
0 70 140
Serum
Liver
Brain
Heart
Muscle
Methyl parathion (ppm)
Pesticide bioconcentration
0 50 100ChE activity ()
Enzyme inhibition
Serum
Liver
Brain
Heart
Muscle
05h24h
05h24h
Figure 3 Piavussumethyl parathion concentrations and cholinesterase inhibition after exposure Each one of eight piavussuswas individuallyplaced in separated tanks containing 40 L of water with Folidol to produce 5 ppm methyl parathion Incubation happened for the showedtimes After exposure blood was collected fishes were submitted to euthanasia and tissues were dissected and homogenized Cholinesteraseactivity was assayed in serum and in homogenates tissues with acetylthiocholine and expressed as percentage of control activity Methylparathion was extracted from serum and tissue homogenates and quantified by HPLC
persist for a relatively long time in fish raising concernsregarding human consumption of fish previously exposed toOP
The results reported herein demonstrated that ChEinhibition in fish exposed to Folidol does not seem todepend on the methyl parathion bioconcentration in theirtissues No ChE inhibition in pacu brain was observed up to30min of exposure despite the fact that this organ presented80 ppm of methyl parathion at that time Therefore thisdata is important for further investigations Tissues in whichChE inhibition occurred more quickly such as pacu liver(Figure 4) showing higher BChE activity in comparison toAChE activity (Figure 1) BChE has been shown to be moresensitive to methyl paraoxon and is probably more quicklyinhibited [20] In addition activation of OP compoundsoccurs mainly in the liver because of the high concentrationsof P-450 in that tissue Thus liver ChE undergoes inhibitionby methyl paraoxon locally generated in tissue before thismetabolite leaks out to general blood circulation and reachesother target tissues
In their work de Aguiar and coworkers [2] described an87 brain AChE inhibition in matrinxa (Brycon cephalus)exposed to water with 2 ppm Folidol 600 for 96 hours Thepresent study indicates that piavussu and pacumight bemoretolerant to Folidol than matrinxa since piavussu presented66 and pacu 74 brain AChE inhibition when exposed to
water with 5 ppmmethyl parathion for 78 hours On the otherhand curimbata showed a 92 brain AChE inhibition withonly 5 hours of exposure to 5 ppmmethyl parathion probablydue to the higher sensitivity of this species AChE to inhibitionby methyl paraoxon [15]
Mammals poisoned by OP usually die by asphyxiaHowever fishes have been reported to be more resistant topoisoning by high levels of organophosphates compoundsthan rats [21] The actual causes and mechanisms of fishpoisoning by OP are not fully understood Our findingsreinforce that interspecific differences in AChE inhibition bytheir oxon derivatives should be considered Physiologicaland behavioral disturbances start at 50 AChE inhibitionand death usually follows when inhibition exceeds 80 inmammals and birds [22] We found here that curimbatas alsoperished when brain AChE inhibition reached above 80
This study suggests that a major contributing factor toacute fish toxicity by Folidol is brain AChE sensitivity tomethyl paraoxon the oxon derivative of methyl parathionThe same explanation that is the sensitivity of brain AChEto oxon derivatives has been suggested for chlorpyrifosparathion and methyl parathion toxicity in mosquito fish(Gambusia affinis) [23] Although it was clearly establishedthat curimbatas possess the most sensitive brain AChEamong the studied species the death of curimbatas exposedto 5 ppm methyl parathion cannot be solely credited to brain
BioMed Research International 7
0 50 100
Serum
Liver
Brain
Heart
Muscle
Pacu
0 50 100
Piavussu
0 50 100
Curimbataacute
05h78h
05h78h
05h5h
ChE activity( relative to controls)
ChE activity( relative to controls)
Serum
Liver
Brain
Heart
Muscle
Serum
Liver
Brain
Heart
Muscle
ChE activity( relative to controls)
Figure 4 Cholinesterase activity in tissues of pacu piavussu and curimbata exposed to 5 ppm methyl parathion Each one of eight animalsby species was individually treated with Folidol 600 for the indicated time in a 40 L aquarium After 30min of exposure blood was collectedfrom four fishes by species and these fishes were submitted to euthanasia Then of the remaining fish four pacus and four piavussus wereeuthanized after 78 h exposure while four curimbatas were collected immediately after they showed no movement of opercula (from 3 to 5 hof exposure) Tissues were dissected and homogenized Cholinesterase was assayed in serum and homogenates with acetylthiocholine andexpressed as percentage of the activity found in controls
8 BioMed Research International
AChE inhibition Other possible causes of this fish deathsuch as Na+ K+ ATPase inhibition [24] and tissue hypoxiawhich compromises heart function need to be examined
5 Conclusion
Prochilodus lineatus (curimbata) Piaractus mesopotamicus(pacu) and Leporinus macrocephalus (piavussu) studied hereshowed similar capacities to bioconcentratemethyl parathionin their tissues after exposure to 5 ppm in water Howeveronly curimbata with the highest brain AChE sensitivity tomethyl paraoxon died after 5 hours of exposure to FolidolPacu and piavussu are more resistant to methyl paraoxonand were alive up to 78 hours of exposure to 5 ppm ofmethyl parathion BrainAChE sensitivity tomethyl paraoxonmight be a decisive factor for determining the sensitivityof these species to poisoning by high concentrations oforganophosphate compounds The present study indicatesthat fishes whose brain acetylcholinesterase activity is moresensitive to oxon derivatives will suffer more severe impactsfrom environmental contamination by organophosphate pes-ticides
Measures reducing the use of organophosphate pesticidesin fish culture should be adopted in order to minimizethe discharge and consequent impact of these chemicals tonatural communities inhabiting rivers and lakes of which thesensitivity to intoxication by pesticides is largely unknown
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors are grateful to Morro Grande Fish Farm andSol Nascente Fish Farm for supplying the fishes This paperis dedicated to Dr Moacelio Veranio Silva Filho (in memo-riam)
References
[1] E Barbieri and L A A Ferreira ldquoEffects of the organophos-phate pesticide Folidol 600 on the freshwater fish Nile Tilapia(Oreochromis niloticus)rdquo Pesticide Biochemistry and Physiologyvol 99 no 3 pp 209ndash214 2011
[2] L H de Aguiar G Moraes I M Avilez A E Altran and CF Correa ldquoMetabolical effects of Folidol 600 on the neotrop-ical freshwater fish matrinxa Brycon cephalusrdquo EnvironmentalResearch vol 95 no 2 pp 224ndash230 2004
[3] D Hernandez-Moreno M Perez-Lopez F Soler C Gravatoand L Guilhermino ldquoEffects of carbofuran on the sea bass(Dicentrarchus labrax L) study of biomarkers and behaviouralterationsrdquo Ecotoxicology and Environmental Safety vol 74 no7 pp 1905ndash1912 2011
[4] C RD Assis A G Linhares VMOliveira et al ldquoComparativeeffect of pesticides on brain acetylcholinesterase in tropical fishrdquoScience of the Total Environment vol 441 pp 141ndash150 2012
[5] L G Sultatos ldquoMammalian toxicology of organophosphoruspesticidesrdquo Journal of Toxicology and Environmental Health vol43 no 3 pp 271ndash289 1994
[6] O Arellano-Aguilar and C M Garcia ldquoEffects of methylparathion exposure on development and reproduction in theviviparous fish girardinichthys multiradiatusrdquo EnvironmentalToxicology vol 24 no 2 pp 178ndash186 2009
[7] M Y de la Vega Salazar L Martınez Tabche and C MacIasGarcıa ldquoBioaccumulation of methyl parathion and its toxicol-ogy in several species of the freshwater community in IgnacioRyamirez dam in Mexicordquo Ecotoxicology and EnvironmentalSafety vol 38 no 1 pp 53ndash62 1997
[8] J E Chambers and H W Chambers ldquoBiotransformationof organophosphorous insecticides in mammalsrdquo in PesticideTransformation Products Fate and Significance in the Envi-ronment L Somasundaram and J R Coats Eds pp 32ndash42American Chemistry Society Washington DC USA 1991
[9] B N LaDu ldquoHuman serum paraoxonasearylesteraserdquo in Phar-macogenetics of DrugMetabolism Pergamon W Kalow Ed pp51ndash91 Pergamon New York NY USA 1992
[10] K Dembele E Haubruge and C Gaspar ldquoConcentrationeffects of selected insecticides on brain acetylcholinesterasein the common carp (Cyprinus carpio L)rdquo Ecotoxicology andEnvironmental Safety vol 45 no 1 pp 49ndash54 2000
[11] V K Patil and M David ldquoOxidative stress in freshwaterfish Labeo rohita as a biomarker of malathion exposurerdquoEnvironmental Monitoring and Assessment vol 185 no 12 pp10191ndash10199 2013
[12] M Jokanovic ldquoCurrent understanding of the mechanismsinvolved in metabolic detoxification of warfare nerve agentsrdquoToxicology Letters vol 188 no 1 pp 1ndash10 2009
[13] R M Lopes M V S Filho J B de Salles V L F C Bastosand J C Bastos ldquoCholinesterase activity of muscle tissue fromfreshwater fishes characterization and sensitivity analysis to theorganophosphate methyl-paraoxonrdquo Environmental Toxicologyand Chemistry vol 33 no 6 pp 1331ndash1336 2014
[14] G L Ellman K D Courtney V Andres Jr and R MFeatherstone ldquoA new and rapid colorimetric determination ofacetylcholinesterase activityrdquo Biochemical Pharmacology vol 7no 2 pp 88ndash95 1961
[15] M V S Filho M M Oliveira J B Salles V L F C BastosV P F Cassano and J C Bastos ldquoMethyl-paraoxon compara-tive inhibition kinetics for acetylcholinesterases from brain ofneotropical fishesrdquo Toxicology Letters vol 153 no 2 pp 247ndash254 2004
[16] J R Kemp and K B Wallace ldquoMolecular determinants ofthe species-selective inhibition of brain acetylcholinesteraserdquoToxicology and Applied Pharmacology vol 104 no 2 pp 246ndash258 1990
[17] J Halpert D Hammond and R A Neal ldquoInactivation ofpurified rat liver cytochrome P-450 during the metabolismof parathion (diethyl p-nitrophenyl phosphorothionate)rdquo TheJournal of Biological Chemistry vol 255 no 3 pp 1080ndash10891980
[18] B J Norman R E Poore and R A Neal ldquoStudies ofthe binding of sulfur released in the mixed-function oxi-dase-catalyzed metabolism of diethyl p-nitrophenyl phos-phorothionate(parathion) to diethyl p-nitrophenyl phosphate(paraoxon)rdquo Biochemical Pharmacology vol 23 no 12 pp1733ndash1744 1974
[19] R Abbas I R Schultz S Doddapaneni and W L HaytonldquoToxicokinetics of parathion and paraoxon in rainbow trout
BioMed Research International 9
after intravascular administration and water exposurerdquo Toxicol-ogy and Applied Pharmacology vol 136 no 1 pp 194ndash199 1996
[20] V L F Cunha Bastos A Rossini L F Ribeiro Pinto etal ldquoDifferent sensitivities to paraoxon of brain and serumcholinesterases from pacu an indigenous Brazilian fishrdquo Bul-letin of Environmental Contamination and Toxicology vol 60no 1 pp 1ndash8 1998
[21] J S Boone and U E Chambers ldquoTime course of inhibitionof cholinesterase and aliesterase activities and nonproteinsulfhydryl levels following exposure to organophosphorusinsecticides in mosquitofish (Gambusia affinis)rdquo Fundamentaland Applied Toxicology vol 29 no 2 pp 202ndash207 1996
[22] C HWalker ldquoThe use of biomarkers to measure the interactiveeffects of chemicalsrdquo Ecotoxicology and Environmental Safetyvol 40 no 1-2 pp 65ndash70 1998
[23] J S Boone and J E Chambers ldquoBiochemical factors contribut-ing to toxicity differences among chlorpyrifos parthion andmethyl parathion in mosquitofish (Gambusia affinis)rdquo AquaticToxicology vol 39 no 3-4 pp 333ndash343 1997
[24] J Blasiak ldquoAllosteric inhibition of the (Na+ + K+)-ATPaseby parathion and methylparathionrdquo Pesticide Biochemistry andPhysiology vol 54 no 1 pp 40ndash47 1996
Figure 3 Piavussumethyl parathion concentrations and cholinesterase inhibition after exposure Each one of eight piavussuswas individuallyplaced in separated tanks containing 40 L of water with Folidol to produce 5 ppm methyl parathion Incubation happened for the showedtimes After exposure blood was collected fishes were submitted to euthanasia and tissues were dissected and homogenized Cholinesteraseactivity was assayed in serum and in homogenates tissues with acetylthiocholine and expressed as percentage of control activity Methylparathion was extracted from serum and tissue homogenates and quantified by HPLC
persist for a relatively long time in fish raising concernsregarding human consumption of fish previously exposed toOP
The results reported herein demonstrated that ChEinhibition in fish exposed to Folidol does not seem todepend on the methyl parathion bioconcentration in theirtissues No ChE inhibition in pacu brain was observed up to30min of exposure despite the fact that this organ presented80 ppm of methyl parathion at that time Therefore thisdata is important for further investigations Tissues in whichChE inhibition occurred more quickly such as pacu liver(Figure 4) showing higher BChE activity in comparison toAChE activity (Figure 1) BChE has been shown to be moresensitive to methyl paraoxon and is probably more quicklyinhibited [20] In addition activation of OP compoundsoccurs mainly in the liver because of the high concentrationsof P-450 in that tissue Thus liver ChE undergoes inhibitionby methyl paraoxon locally generated in tissue before thismetabolite leaks out to general blood circulation and reachesother target tissues
In their work de Aguiar and coworkers [2] described an87 brain AChE inhibition in matrinxa (Brycon cephalus)exposed to water with 2 ppm Folidol 600 for 96 hours Thepresent study indicates that piavussu and pacumight bemoretolerant to Folidol than matrinxa since piavussu presented66 and pacu 74 brain AChE inhibition when exposed to
water with 5 ppmmethyl parathion for 78 hours On the otherhand curimbata showed a 92 brain AChE inhibition withonly 5 hours of exposure to 5 ppmmethyl parathion probablydue to the higher sensitivity of this species AChE to inhibitionby methyl paraoxon [15]
Mammals poisoned by OP usually die by asphyxiaHowever fishes have been reported to be more resistant topoisoning by high levels of organophosphates compoundsthan rats [21] The actual causes and mechanisms of fishpoisoning by OP are not fully understood Our findingsreinforce that interspecific differences in AChE inhibition bytheir oxon derivatives should be considered Physiologicaland behavioral disturbances start at 50 AChE inhibitionand death usually follows when inhibition exceeds 80 inmammals and birds [22] We found here that curimbatas alsoperished when brain AChE inhibition reached above 80
This study suggests that a major contributing factor toacute fish toxicity by Folidol is brain AChE sensitivity tomethyl paraoxon the oxon derivative of methyl parathionThe same explanation that is the sensitivity of brain AChEto oxon derivatives has been suggested for chlorpyrifosparathion and methyl parathion toxicity in mosquito fish(Gambusia affinis) [23] Although it was clearly establishedthat curimbatas possess the most sensitive brain AChEamong the studied species the death of curimbatas exposedto 5 ppm methyl parathion cannot be solely credited to brain
BioMed Research International 7
0 50 100
Serum
Liver
Brain
Heart
Muscle
Pacu
0 50 100
Piavussu
0 50 100
Curimbataacute
05h78h
05h78h
05h5h
ChE activity( relative to controls)
ChE activity( relative to controls)
Serum
Liver
Brain
Heart
Muscle
Serum
Liver
Brain
Heart
Muscle
ChE activity( relative to controls)
Figure 4 Cholinesterase activity in tissues of pacu piavussu and curimbata exposed to 5 ppm methyl parathion Each one of eight animalsby species was individually treated with Folidol 600 for the indicated time in a 40 L aquarium After 30min of exposure blood was collectedfrom four fishes by species and these fishes were submitted to euthanasia Then of the remaining fish four pacus and four piavussus wereeuthanized after 78 h exposure while four curimbatas were collected immediately after they showed no movement of opercula (from 3 to 5 hof exposure) Tissues were dissected and homogenized Cholinesterase was assayed in serum and homogenates with acetylthiocholine andexpressed as percentage of the activity found in controls
8 BioMed Research International
AChE inhibition Other possible causes of this fish deathsuch as Na+ K+ ATPase inhibition [24] and tissue hypoxiawhich compromises heart function need to be examined
5 Conclusion
Prochilodus lineatus (curimbata) Piaractus mesopotamicus(pacu) and Leporinus macrocephalus (piavussu) studied hereshowed similar capacities to bioconcentratemethyl parathionin their tissues after exposure to 5 ppm in water Howeveronly curimbata with the highest brain AChE sensitivity tomethyl paraoxon died after 5 hours of exposure to FolidolPacu and piavussu are more resistant to methyl paraoxonand were alive up to 78 hours of exposure to 5 ppm ofmethyl parathion BrainAChE sensitivity tomethyl paraoxonmight be a decisive factor for determining the sensitivityof these species to poisoning by high concentrations oforganophosphate compounds The present study indicatesthat fishes whose brain acetylcholinesterase activity is moresensitive to oxon derivatives will suffer more severe impactsfrom environmental contamination by organophosphate pes-ticides
Measures reducing the use of organophosphate pesticidesin fish culture should be adopted in order to minimizethe discharge and consequent impact of these chemicals tonatural communities inhabiting rivers and lakes of which thesensitivity to intoxication by pesticides is largely unknown
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors are grateful to Morro Grande Fish Farm andSol Nascente Fish Farm for supplying the fishes This paperis dedicated to Dr Moacelio Veranio Silva Filho (in memo-riam)
References
[1] E Barbieri and L A A Ferreira ldquoEffects of the organophos-phate pesticide Folidol 600 on the freshwater fish Nile Tilapia(Oreochromis niloticus)rdquo Pesticide Biochemistry and Physiologyvol 99 no 3 pp 209ndash214 2011
[2] L H de Aguiar G Moraes I M Avilez A E Altran and CF Correa ldquoMetabolical effects of Folidol 600 on the neotrop-ical freshwater fish matrinxa Brycon cephalusrdquo EnvironmentalResearch vol 95 no 2 pp 224ndash230 2004
[3] D Hernandez-Moreno M Perez-Lopez F Soler C Gravatoand L Guilhermino ldquoEffects of carbofuran on the sea bass(Dicentrarchus labrax L) study of biomarkers and behaviouralterationsrdquo Ecotoxicology and Environmental Safety vol 74 no7 pp 1905ndash1912 2011
[4] C RD Assis A G Linhares VMOliveira et al ldquoComparativeeffect of pesticides on brain acetylcholinesterase in tropical fishrdquoScience of the Total Environment vol 441 pp 141ndash150 2012
[5] L G Sultatos ldquoMammalian toxicology of organophosphoruspesticidesrdquo Journal of Toxicology and Environmental Health vol43 no 3 pp 271ndash289 1994
[6] O Arellano-Aguilar and C M Garcia ldquoEffects of methylparathion exposure on development and reproduction in theviviparous fish girardinichthys multiradiatusrdquo EnvironmentalToxicology vol 24 no 2 pp 178ndash186 2009
[7] M Y de la Vega Salazar L Martınez Tabche and C MacIasGarcıa ldquoBioaccumulation of methyl parathion and its toxicol-ogy in several species of the freshwater community in IgnacioRyamirez dam in Mexicordquo Ecotoxicology and EnvironmentalSafety vol 38 no 1 pp 53ndash62 1997
[8] J E Chambers and H W Chambers ldquoBiotransformationof organophosphorous insecticides in mammalsrdquo in PesticideTransformation Products Fate and Significance in the Envi-ronment L Somasundaram and J R Coats Eds pp 32ndash42American Chemistry Society Washington DC USA 1991
[9] B N LaDu ldquoHuman serum paraoxonasearylesteraserdquo in Phar-macogenetics of DrugMetabolism Pergamon W Kalow Ed pp51ndash91 Pergamon New York NY USA 1992
[10] K Dembele E Haubruge and C Gaspar ldquoConcentrationeffects of selected insecticides on brain acetylcholinesterasein the common carp (Cyprinus carpio L)rdquo Ecotoxicology andEnvironmental Safety vol 45 no 1 pp 49ndash54 2000
[11] V K Patil and M David ldquoOxidative stress in freshwaterfish Labeo rohita as a biomarker of malathion exposurerdquoEnvironmental Monitoring and Assessment vol 185 no 12 pp10191ndash10199 2013
[12] M Jokanovic ldquoCurrent understanding of the mechanismsinvolved in metabolic detoxification of warfare nerve agentsrdquoToxicology Letters vol 188 no 1 pp 1ndash10 2009
[13] R M Lopes M V S Filho J B de Salles V L F C Bastosand J C Bastos ldquoCholinesterase activity of muscle tissue fromfreshwater fishes characterization and sensitivity analysis to theorganophosphate methyl-paraoxonrdquo Environmental Toxicologyand Chemistry vol 33 no 6 pp 1331ndash1336 2014
[14] G L Ellman K D Courtney V Andres Jr and R MFeatherstone ldquoA new and rapid colorimetric determination ofacetylcholinesterase activityrdquo Biochemical Pharmacology vol 7no 2 pp 88ndash95 1961
[15] M V S Filho M M Oliveira J B Salles V L F C BastosV P F Cassano and J C Bastos ldquoMethyl-paraoxon compara-tive inhibition kinetics for acetylcholinesterases from brain ofneotropical fishesrdquo Toxicology Letters vol 153 no 2 pp 247ndash254 2004
[16] J R Kemp and K B Wallace ldquoMolecular determinants ofthe species-selective inhibition of brain acetylcholinesteraserdquoToxicology and Applied Pharmacology vol 104 no 2 pp 246ndash258 1990
[17] J Halpert D Hammond and R A Neal ldquoInactivation ofpurified rat liver cytochrome P-450 during the metabolismof parathion (diethyl p-nitrophenyl phosphorothionate)rdquo TheJournal of Biological Chemistry vol 255 no 3 pp 1080ndash10891980
[18] B J Norman R E Poore and R A Neal ldquoStudies ofthe binding of sulfur released in the mixed-function oxi-dase-catalyzed metabolism of diethyl p-nitrophenyl phos-phorothionate(parathion) to diethyl p-nitrophenyl phosphate(paraoxon)rdquo Biochemical Pharmacology vol 23 no 12 pp1733ndash1744 1974
[19] R Abbas I R Schultz S Doddapaneni and W L HaytonldquoToxicokinetics of parathion and paraoxon in rainbow trout
BioMed Research International 9
after intravascular administration and water exposurerdquo Toxicol-ogy and Applied Pharmacology vol 136 no 1 pp 194ndash199 1996
[20] V L F Cunha Bastos A Rossini L F Ribeiro Pinto etal ldquoDifferent sensitivities to paraoxon of brain and serumcholinesterases from pacu an indigenous Brazilian fishrdquo Bul-letin of Environmental Contamination and Toxicology vol 60no 1 pp 1ndash8 1998
[21] J S Boone and U E Chambers ldquoTime course of inhibitionof cholinesterase and aliesterase activities and nonproteinsulfhydryl levels following exposure to organophosphorusinsecticides in mosquitofish (Gambusia affinis)rdquo Fundamentaland Applied Toxicology vol 29 no 2 pp 202ndash207 1996
[22] C HWalker ldquoThe use of biomarkers to measure the interactiveeffects of chemicalsrdquo Ecotoxicology and Environmental Safetyvol 40 no 1-2 pp 65ndash70 1998
[23] J S Boone and J E Chambers ldquoBiochemical factors contribut-ing to toxicity differences among chlorpyrifos parthion andmethyl parathion in mosquitofish (Gambusia affinis)rdquo AquaticToxicology vol 39 no 3-4 pp 333ndash343 1997
[24] J Blasiak ldquoAllosteric inhibition of the (Na+ + K+)-ATPaseby parathion and methylparathionrdquo Pesticide Biochemistry andPhysiology vol 54 no 1 pp 40ndash47 1996
Figure 4 Cholinesterase activity in tissues of pacu piavussu and curimbata exposed to 5 ppm methyl parathion Each one of eight animalsby species was individually treated with Folidol 600 for the indicated time in a 40 L aquarium After 30min of exposure blood was collectedfrom four fishes by species and these fishes were submitted to euthanasia Then of the remaining fish four pacus and four piavussus wereeuthanized after 78 h exposure while four curimbatas were collected immediately after they showed no movement of opercula (from 3 to 5 hof exposure) Tissues were dissected and homogenized Cholinesterase was assayed in serum and homogenates with acetylthiocholine andexpressed as percentage of the activity found in controls
8 BioMed Research International
AChE inhibition Other possible causes of this fish deathsuch as Na+ K+ ATPase inhibition [24] and tissue hypoxiawhich compromises heart function need to be examined
5 Conclusion
Prochilodus lineatus (curimbata) Piaractus mesopotamicus(pacu) and Leporinus macrocephalus (piavussu) studied hereshowed similar capacities to bioconcentratemethyl parathionin their tissues after exposure to 5 ppm in water Howeveronly curimbata with the highest brain AChE sensitivity tomethyl paraoxon died after 5 hours of exposure to FolidolPacu and piavussu are more resistant to methyl paraoxonand were alive up to 78 hours of exposure to 5 ppm ofmethyl parathion BrainAChE sensitivity tomethyl paraoxonmight be a decisive factor for determining the sensitivityof these species to poisoning by high concentrations oforganophosphate compounds The present study indicatesthat fishes whose brain acetylcholinesterase activity is moresensitive to oxon derivatives will suffer more severe impactsfrom environmental contamination by organophosphate pes-ticides
Measures reducing the use of organophosphate pesticidesin fish culture should be adopted in order to minimizethe discharge and consequent impact of these chemicals tonatural communities inhabiting rivers and lakes of which thesensitivity to intoxication by pesticides is largely unknown
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors are grateful to Morro Grande Fish Farm andSol Nascente Fish Farm for supplying the fishes This paperis dedicated to Dr Moacelio Veranio Silva Filho (in memo-riam)
References
[1] E Barbieri and L A A Ferreira ldquoEffects of the organophos-phate pesticide Folidol 600 on the freshwater fish Nile Tilapia(Oreochromis niloticus)rdquo Pesticide Biochemistry and Physiologyvol 99 no 3 pp 209ndash214 2011
[2] L H de Aguiar G Moraes I M Avilez A E Altran and CF Correa ldquoMetabolical effects of Folidol 600 on the neotrop-ical freshwater fish matrinxa Brycon cephalusrdquo EnvironmentalResearch vol 95 no 2 pp 224ndash230 2004
[3] D Hernandez-Moreno M Perez-Lopez F Soler C Gravatoand L Guilhermino ldquoEffects of carbofuran on the sea bass(Dicentrarchus labrax L) study of biomarkers and behaviouralterationsrdquo Ecotoxicology and Environmental Safety vol 74 no7 pp 1905ndash1912 2011
[4] C RD Assis A G Linhares VMOliveira et al ldquoComparativeeffect of pesticides on brain acetylcholinesterase in tropical fishrdquoScience of the Total Environment vol 441 pp 141ndash150 2012
[5] L G Sultatos ldquoMammalian toxicology of organophosphoruspesticidesrdquo Journal of Toxicology and Environmental Health vol43 no 3 pp 271ndash289 1994
[6] O Arellano-Aguilar and C M Garcia ldquoEffects of methylparathion exposure on development and reproduction in theviviparous fish girardinichthys multiradiatusrdquo EnvironmentalToxicology vol 24 no 2 pp 178ndash186 2009
[7] M Y de la Vega Salazar L Martınez Tabche and C MacIasGarcıa ldquoBioaccumulation of methyl parathion and its toxicol-ogy in several species of the freshwater community in IgnacioRyamirez dam in Mexicordquo Ecotoxicology and EnvironmentalSafety vol 38 no 1 pp 53ndash62 1997
[8] J E Chambers and H W Chambers ldquoBiotransformationof organophosphorous insecticides in mammalsrdquo in PesticideTransformation Products Fate and Significance in the Envi-ronment L Somasundaram and J R Coats Eds pp 32ndash42American Chemistry Society Washington DC USA 1991
[9] B N LaDu ldquoHuman serum paraoxonasearylesteraserdquo in Phar-macogenetics of DrugMetabolism Pergamon W Kalow Ed pp51ndash91 Pergamon New York NY USA 1992
[10] K Dembele E Haubruge and C Gaspar ldquoConcentrationeffects of selected insecticides on brain acetylcholinesterasein the common carp (Cyprinus carpio L)rdquo Ecotoxicology andEnvironmental Safety vol 45 no 1 pp 49ndash54 2000
[11] V K Patil and M David ldquoOxidative stress in freshwaterfish Labeo rohita as a biomarker of malathion exposurerdquoEnvironmental Monitoring and Assessment vol 185 no 12 pp10191ndash10199 2013
[12] M Jokanovic ldquoCurrent understanding of the mechanismsinvolved in metabolic detoxification of warfare nerve agentsrdquoToxicology Letters vol 188 no 1 pp 1ndash10 2009
[13] R M Lopes M V S Filho J B de Salles V L F C Bastosand J C Bastos ldquoCholinesterase activity of muscle tissue fromfreshwater fishes characterization and sensitivity analysis to theorganophosphate methyl-paraoxonrdquo Environmental Toxicologyand Chemistry vol 33 no 6 pp 1331ndash1336 2014
[14] G L Ellman K D Courtney V Andres Jr and R MFeatherstone ldquoA new and rapid colorimetric determination ofacetylcholinesterase activityrdquo Biochemical Pharmacology vol 7no 2 pp 88ndash95 1961
[15] M V S Filho M M Oliveira J B Salles V L F C BastosV P F Cassano and J C Bastos ldquoMethyl-paraoxon compara-tive inhibition kinetics for acetylcholinesterases from brain ofneotropical fishesrdquo Toxicology Letters vol 153 no 2 pp 247ndash254 2004
[16] J R Kemp and K B Wallace ldquoMolecular determinants ofthe species-selective inhibition of brain acetylcholinesteraserdquoToxicology and Applied Pharmacology vol 104 no 2 pp 246ndash258 1990
[17] J Halpert D Hammond and R A Neal ldquoInactivation ofpurified rat liver cytochrome P-450 during the metabolismof parathion (diethyl p-nitrophenyl phosphorothionate)rdquo TheJournal of Biological Chemistry vol 255 no 3 pp 1080ndash10891980
[18] B J Norman R E Poore and R A Neal ldquoStudies ofthe binding of sulfur released in the mixed-function oxi-dase-catalyzed metabolism of diethyl p-nitrophenyl phos-phorothionate(parathion) to diethyl p-nitrophenyl phosphate(paraoxon)rdquo Biochemical Pharmacology vol 23 no 12 pp1733ndash1744 1974
[19] R Abbas I R Schultz S Doddapaneni and W L HaytonldquoToxicokinetics of parathion and paraoxon in rainbow trout
BioMed Research International 9
after intravascular administration and water exposurerdquo Toxicol-ogy and Applied Pharmacology vol 136 no 1 pp 194ndash199 1996
[20] V L F Cunha Bastos A Rossini L F Ribeiro Pinto etal ldquoDifferent sensitivities to paraoxon of brain and serumcholinesterases from pacu an indigenous Brazilian fishrdquo Bul-letin of Environmental Contamination and Toxicology vol 60no 1 pp 1ndash8 1998
[21] J S Boone and U E Chambers ldquoTime course of inhibitionof cholinesterase and aliesterase activities and nonproteinsulfhydryl levels following exposure to organophosphorusinsecticides in mosquitofish (Gambusia affinis)rdquo Fundamentaland Applied Toxicology vol 29 no 2 pp 202ndash207 1996
[22] C HWalker ldquoThe use of biomarkers to measure the interactiveeffects of chemicalsrdquo Ecotoxicology and Environmental Safetyvol 40 no 1-2 pp 65ndash70 1998
[23] J S Boone and J E Chambers ldquoBiochemical factors contribut-ing to toxicity differences among chlorpyrifos parthion andmethyl parathion in mosquitofish (Gambusia affinis)rdquo AquaticToxicology vol 39 no 3-4 pp 333ndash343 1997
[24] J Blasiak ldquoAllosteric inhibition of the (Na+ + K+)-ATPaseby parathion and methylparathionrdquo Pesticide Biochemistry andPhysiology vol 54 no 1 pp 40ndash47 1996
AChE inhibition Other possible causes of this fish deathsuch as Na+ K+ ATPase inhibition [24] and tissue hypoxiawhich compromises heart function need to be examined
5 Conclusion
Prochilodus lineatus (curimbata) Piaractus mesopotamicus(pacu) and Leporinus macrocephalus (piavussu) studied hereshowed similar capacities to bioconcentratemethyl parathionin their tissues after exposure to 5 ppm in water Howeveronly curimbata with the highest brain AChE sensitivity tomethyl paraoxon died after 5 hours of exposure to FolidolPacu and piavussu are more resistant to methyl paraoxonand were alive up to 78 hours of exposure to 5 ppm ofmethyl parathion BrainAChE sensitivity tomethyl paraoxonmight be a decisive factor for determining the sensitivityof these species to poisoning by high concentrations oforganophosphate compounds The present study indicatesthat fishes whose brain acetylcholinesterase activity is moresensitive to oxon derivatives will suffer more severe impactsfrom environmental contamination by organophosphate pes-ticides
Measures reducing the use of organophosphate pesticidesin fish culture should be adopted in order to minimizethe discharge and consequent impact of these chemicals tonatural communities inhabiting rivers and lakes of which thesensitivity to intoxication by pesticides is largely unknown
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors are grateful to Morro Grande Fish Farm andSol Nascente Fish Farm for supplying the fishes This paperis dedicated to Dr Moacelio Veranio Silva Filho (in memo-riam)
References
[1] E Barbieri and L A A Ferreira ldquoEffects of the organophos-phate pesticide Folidol 600 on the freshwater fish Nile Tilapia(Oreochromis niloticus)rdquo Pesticide Biochemistry and Physiologyvol 99 no 3 pp 209ndash214 2011
[2] L H de Aguiar G Moraes I M Avilez A E Altran and CF Correa ldquoMetabolical effects of Folidol 600 on the neotrop-ical freshwater fish matrinxa Brycon cephalusrdquo EnvironmentalResearch vol 95 no 2 pp 224ndash230 2004
[3] D Hernandez-Moreno M Perez-Lopez F Soler C Gravatoand L Guilhermino ldquoEffects of carbofuran on the sea bass(Dicentrarchus labrax L) study of biomarkers and behaviouralterationsrdquo Ecotoxicology and Environmental Safety vol 74 no7 pp 1905ndash1912 2011
[4] C RD Assis A G Linhares VMOliveira et al ldquoComparativeeffect of pesticides on brain acetylcholinesterase in tropical fishrdquoScience of the Total Environment vol 441 pp 141ndash150 2012
[5] L G Sultatos ldquoMammalian toxicology of organophosphoruspesticidesrdquo Journal of Toxicology and Environmental Health vol43 no 3 pp 271ndash289 1994
[6] O Arellano-Aguilar and C M Garcia ldquoEffects of methylparathion exposure on development and reproduction in theviviparous fish girardinichthys multiradiatusrdquo EnvironmentalToxicology vol 24 no 2 pp 178ndash186 2009
[7] M Y de la Vega Salazar L Martınez Tabche and C MacIasGarcıa ldquoBioaccumulation of methyl parathion and its toxicol-ogy in several species of the freshwater community in IgnacioRyamirez dam in Mexicordquo Ecotoxicology and EnvironmentalSafety vol 38 no 1 pp 53ndash62 1997
[8] J E Chambers and H W Chambers ldquoBiotransformationof organophosphorous insecticides in mammalsrdquo in PesticideTransformation Products Fate and Significance in the Envi-ronment L Somasundaram and J R Coats Eds pp 32ndash42American Chemistry Society Washington DC USA 1991
[9] B N LaDu ldquoHuman serum paraoxonasearylesteraserdquo in Phar-macogenetics of DrugMetabolism Pergamon W Kalow Ed pp51ndash91 Pergamon New York NY USA 1992
[10] K Dembele E Haubruge and C Gaspar ldquoConcentrationeffects of selected insecticides on brain acetylcholinesterasein the common carp (Cyprinus carpio L)rdquo Ecotoxicology andEnvironmental Safety vol 45 no 1 pp 49ndash54 2000
[11] V K Patil and M David ldquoOxidative stress in freshwaterfish Labeo rohita as a biomarker of malathion exposurerdquoEnvironmental Monitoring and Assessment vol 185 no 12 pp10191ndash10199 2013
[12] M Jokanovic ldquoCurrent understanding of the mechanismsinvolved in metabolic detoxification of warfare nerve agentsrdquoToxicology Letters vol 188 no 1 pp 1ndash10 2009
[13] R M Lopes M V S Filho J B de Salles V L F C Bastosand J C Bastos ldquoCholinesterase activity of muscle tissue fromfreshwater fishes characterization and sensitivity analysis to theorganophosphate methyl-paraoxonrdquo Environmental Toxicologyand Chemistry vol 33 no 6 pp 1331ndash1336 2014
[14] G L Ellman K D Courtney V Andres Jr and R MFeatherstone ldquoA new and rapid colorimetric determination ofacetylcholinesterase activityrdquo Biochemical Pharmacology vol 7no 2 pp 88ndash95 1961
[15] M V S Filho M M Oliveira J B Salles V L F C BastosV P F Cassano and J C Bastos ldquoMethyl-paraoxon compara-tive inhibition kinetics for acetylcholinesterases from brain ofneotropical fishesrdquo Toxicology Letters vol 153 no 2 pp 247ndash254 2004
[16] J R Kemp and K B Wallace ldquoMolecular determinants ofthe species-selective inhibition of brain acetylcholinesteraserdquoToxicology and Applied Pharmacology vol 104 no 2 pp 246ndash258 1990
[17] J Halpert D Hammond and R A Neal ldquoInactivation ofpurified rat liver cytochrome P-450 during the metabolismof parathion (diethyl p-nitrophenyl phosphorothionate)rdquo TheJournal of Biological Chemistry vol 255 no 3 pp 1080ndash10891980
[18] B J Norman R E Poore and R A Neal ldquoStudies ofthe binding of sulfur released in the mixed-function oxi-dase-catalyzed metabolism of diethyl p-nitrophenyl phos-phorothionate(parathion) to diethyl p-nitrophenyl phosphate(paraoxon)rdquo Biochemical Pharmacology vol 23 no 12 pp1733ndash1744 1974
[19] R Abbas I R Schultz S Doddapaneni and W L HaytonldquoToxicokinetics of parathion and paraoxon in rainbow trout
BioMed Research International 9
after intravascular administration and water exposurerdquo Toxicol-ogy and Applied Pharmacology vol 136 no 1 pp 194ndash199 1996
[20] V L F Cunha Bastos A Rossini L F Ribeiro Pinto etal ldquoDifferent sensitivities to paraoxon of brain and serumcholinesterases from pacu an indigenous Brazilian fishrdquo Bul-letin of Environmental Contamination and Toxicology vol 60no 1 pp 1ndash8 1998
[21] J S Boone and U E Chambers ldquoTime course of inhibitionof cholinesterase and aliesterase activities and nonproteinsulfhydryl levels following exposure to organophosphorusinsecticides in mosquitofish (Gambusia affinis)rdquo Fundamentaland Applied Toxicology vol 29 no 2 pp 202ndash207 1996
[22] C HWalker ldquoThe use of biomarkers to measure the interactiveeffects of chemicalsrdquo Ecotoxicology and Environmental Safetyvol 40 no 1-2 pp 65ndash70 1998
[23] J S Boone and J E Chambers ldquoBiochemical factors contribut-ing to toxicity differences among chlorpyrifos parthion andmethyl parathion in mosquitofish (Gambusia affinis)rdquo AquaticToxicology vol 39 no 3-4 pp 333ndash343 1997
[24] J Blasiak ldquoAllosteric inhibition of the (Na+ + K+)-ATPaseby parathion and methylparathionrdquo Pesticide Biochemistry andPhysiology vol 54 no 1 pp 40ndash47 1996
after intravascular administration and water exposurerdquo Toxicol-ogy and Applied Pharmacology vol 136 no 1 pp 194ndash199 1996
[20] V L F Cunha Bastos A Rossini L F Ribeiro Pinto etal ldquoDifferent sensitivities to paraoxon of brain and serumcholinesterases from pacu an indigenous Brazilian fishrdquo Bul-letin of Environmental Contamination and Toxicology vol 60no 1 pp 1ndash8 1998
[21] J S Boone and U E Chambers ldquoTime course of inhibitionof cholinesterase and aliesterase activities and nonproteinsulfhydryl levels following exposure to organophosphorusinsecticides in mosquitofish (Gambusia affinis)rdquo Fundamentaland Applied Toxicology vol 29 no 2 pp 202ndash207 1996
[22] C HWalker ldquoThe use of biomarkers to measure the interactiveeffects of chemicalsrdquo Ecotoxicology and Environmental Safetyvol 40 no 1-2 pp 65ndash70 1998
[23] J S Boone and J E Chambers ldquoBiochemical factors contribut-ing to toxicity differences among chlorpyrifos parthion andmethyl parathion in mosquitofish (Gambusia affinis)rdquo AquaticToxicology vol 39 no 3-4 pp 333ndash343 1997
[24] J Blasiak ldquoAllosteric inhibition of the (Na+ + K+)-ATPaseby parathion and methylparathionrdquo Pesticide Biochemistry andPhysiology vol 54 no 1 pp 40ndash47 1996
![Cyanide Intoxication after Ingestion of Wild Cherry (Prunus Avium) · cause acute cyanide intoxication or some chronic diseases [14]. Several cyanide intoxication cases has been reported](https://static.documents.pub/doc/80x56/613b2037f8f21c0c8268d3d6/cyanide-intoxication-after-ingestion-of-wild-cherry-prunus-avium-cause-acute-cyanide.jpg)
