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Date Created: 09/17/15
s lnstructor Allan Felsut Wrglmrgggggw sustained Wu quot Fall mus Represe control Witn rnodulation frorn tne endocrlrle s rn nts integration of nervous systern nat benavior is affected only VWEH anotner fundarnental pnysiological rnecnanisrn is affecte 7 All exogenous stirnuli are perceived tnrougn tne sensory systerntnat cornrnu icatesvvitn tne brain 7 l lovveveri We can study benaviors as DXlEDnglEal endpoints Presumption The myriad Jnctions collectively defning behavior can be isolated for st Selectlve eTnus different benaviors can be observed ly by appropriate investigative nnioues Historically many neurotoxi compounds end c ed up as environmental contaminants e insecticides 7 Heavy rnetals copper lead mercury e n a g e tnat OP insectioid o more tnaniust innibit aoetylonolinesteras 7 Because it can be shown tnat certain doses affect neurobenavioral functioning of neonataliiuvenilei and adult ratsquot 7 Tnen cnildren can be adversely affected in terrns of intellectual develop ent Tne co ce n also extends to PCBS and Hg and Pb e Tne Jacobson studies ept r We now Knutv that a etylcnolinetiteris irnportantin nelp structure brain cnlorpyrifos in particular nas been SHINn to alter neurite uutgmvvtns in cell culture Tne rnecnanisrn is ndttnougritto be directly due to acetylcnolinesterase innibinon e e i cnlorpyrifos as Well as its nydrolytic metabolite are e ecnve and tnus may work at a site distal to tne enzyme pernaps a rec o rfterac c tne patterning oftne nerves intne developing 7 However basc morpnolooical s1udles snuwlnal acetvlcnoline i sterase itselfrnav also be irnn tnat nign s H emit hln htah t t n w w n m as Btgheeet at 1999 t r i ash tsnvou 075099 IHGvIQJ 39 gtV as I rut sear 0 E hummus use 0590M urswv Hwtza anam Elwsma VD seas WW mt r 25 Effects eh thtetteetuat devetoprheht through eany PCB ex 0 ure tfthey are 00 urhhg at aH are t ety 0 have been rhedtated through endocrme System e ects e Orgahtzatmhat vs asttvatmhat Effects A recent hypothests ts that honecoptahar PCBs sttrhutate catcturh th ux at herve terrhthats e oeute cause premature retease nr excesstve retease er neurntransmtner dun g ehtreat d2 etepmehtat permds h the fetus a ct OE tct mphehyt 2 tetmcmamhtphenv C23be 2 edtcmambtphenw surrehmaphenh Neh Enrptanar PCB EnngEnErs Functtona Observatton Battertes F085 7 Destghed tn determthe peteh tat heuretextstty e Caveat umeesteneeteeueuateuheewhetemetuereat meehahsms Mus1 ensuve rehahhtv mmeasuvememsbv havmu smet preteens 1m eehststem behavtuvat ubsewattuns e Examptes nfbehavmr ttmetnftrststep gatt re ex fnrehmb ghp strength Motor Activity 7 Observe activitv ieveis or ammais as thev move m an open space or Within a caged ehvirohmeht mazes rohmhg Wheels unencumbered uperi e Errects could be dueto other systems hot ecessarilv neurotoxicity Naturalistic Behaviors e Agresssiori ma mg behavior sedior assessing enducririe system eiiecls Ethological analysis 7 Observing ahimais in natural or quasirriatural environment Sensory fun Ion r acoustic startie response Cognitive func ion oid e behavior 7 Maze ieamihg ror spatiai ieamihg Operant Behavior 7 Measure of learned or acquired behavior 7 Scheduled learriirig behavior Schooling Feeding including predation Migration Aggression Fear Learning Rheotropism orientation toward the current Attraction or 39 e Breathing frequency may be considered metabolic effect Swimming activity spontaneous locomotory behavior ii lliiii i 92 Fish placed in tanks divided into chambers 7 Record rish eht into each chamber as measure o locomotory activity perhaps ieamihg Out t tanks with electrodes or other types of detectors that can detect change in water ion 0 record swimming activity sual observation 7 Divide chamber in hair separated ov a barrier With holes iri it to aiiovvrish to pass through i e rnuvirig lhruugh the barrier per unit uHirne 7 Use or video cameras ror continuous visual mohitormg Ammonia affects swimming behavior of coho salmon 7 Wicks et ai 2 02 Measured critical swimming velocity of coho salmon alter exposure to four levels of ammonia in water 7 0 0 02 0 04 0 8 rngL Related plasma levels to swimming velocity Observed that behavior affected susceptibility Wicks elai Aquatic rovicui as 55 39 o 08mgL V i i Cohtroi 39i o rngL o 04 rngL Critical Swimming veiocitv Lengthssec Dan on mu m m min mgL Water ammonia w wrenwm Ilil 2 39a 2 g 0 002 mgL 33 I l E M 39 000 mgL m s 5 39 39 V l v we Control Tu 0m IL V 39 g g 004 mgL U 0 0 u 2 a l 5 Plasma ammonia mM Ptal nuatir Tuxlcul 5a 55 A Mortality Res ing 207 mgL Swimming 32 mgL m 400 m min Total Ammonia mgL w Ptal nuatir Tuxlcul 5955 Diminishing Returns Salmon Decline and Pesticides Lethal Lawns Diazinnn Use Threatens Salmon Survival Diazinon disrupts antipredator and homing behaviors in chinook salmonquot Scholz N L et al 2000 Can J Fish Aquatic Sciences 5719111918 Observed alarm pheromone response ofsalmon exposed to low levels ofdiazinon formerly a major urban use insecticide When in the presence of a predator salmon release an alarm pheromone that should decrease their activity level N N 5 AU OM r snimzeiai 2000 39 Salmon to Stimulation wim Alann Pheromone of Time Spent in Motion I Pre Stimulus I Post Stimulus 20 10 0 0 1 1 Diazinon Concentration ppb amnion to Stimulation wim Alann Pheromone Food Strikes Per Minute 14 I Pre Stimulus I Post Stimulus 1 2 10 ones 10 01 1 Smozeia 2000 Diazinon Concentration ppb Effect of Diazinon on Homing Behavior of Chinook Salmon I Returns Relative to I Total Returns Control 0 ppb Relative to Released 100 0 0 1 1 10 Diazinon Concentration ppb Sm oz er a 2000 Food searching capabilities Ability to avoid predators Ability to Withstand extremes in cold Interestingly birds are known to avoid contaminated seedsll instructur Aiian Feisut areisettneity Wsu euu WMHiNc rON ST 433 UN IVERSITY Faii 2nn5 orheaith nas become an issue n nas increased but arabie ii 39i M9 s SOiiS r Funhermure erusiun iackufwater and u a u u running Soii quaiity den 7 Ca I Lecture 17 Microbial and Plant Toxicity er SEIii te rune ien as Vitai iiving system Water Envirunments and suppert human neaitn amp nanitatien Winding etai ZUUE Ecutux Envian sarety 62 23a Micro iai communities and their bioche ecoiogy is or micai uciai to tne optimai ecoiogicai mnctions or Soii Soiidi waten air phases Soiids Ciays2 urn 39 Siits gt2 umr u urn Sands gt5u urn e inurgani ininerais and puiymers iays y a e Determines huw much Waterthe SEIii can huiu Mineiaisuii94 DIVZQM E OYganiEMaiiEiE a 5 m1 LEW mil 3 ii mi Living Rants 8 5 i gtii ii39iiisigir its N t39 tC I39 EdaphunE5 a Hen ycmg e rboni nitrogen phosphorus suifur Contribute to structure of soquot through organic matter decomposition and Bacteria synthesis aninumyeeies 5m Fun i 25 Degrade contaminants Cumpusitiun er a Grassianu XXUW i2 Suii Diyvveignt Basis 3quot 3 quota Dunkeretai iaao WWW 25 i eta 35 ij mm immimviii m Recncigi i995 i 5mm Win mums mom i imamWit StDw nmmiz uniqu Emir Waste situations including spills Routine application of pesticides rFumigant rFui39igicideS Hei bicides pH dependent eqmlibnum i l illrig in Vitro Tests 7 Culture microbes ii i liquid or agar culture Can count colony forming units ems agarpia d togtltii i to culture arid look at effect Ol i turbidity oi CFUS on In Vivo Tests Whoe soils tested for microbial activity and num ers Respiration dehydrogenase enzyme activity indicative of general oxidative metabolic activity Carbon mineralization COZ evolution indicative of biomass Nitrification Specific enzyme tests nitrogenase urease phosphatase Most probable number methods Ecological Tests Decomposition of litter Use of leaf material in buried litter bags Weigh biomass a er period oftime Examine appearance of material Work of CM Tu circa 25 years ago Measured dehydrogenase activity general respiratory enzyme for overall bioactivity Nitrogen fixation nitrogenase Denitrification Carbon Mineralization CO2 evolution Most Probable Number method No effect beyond a few days Plot delineated and treated with single or multiple combinations of herbicides and insecticides Crop cultivated for 20 years After the last application of herbicides monitored microbial activity and function right after application and in the fall Found no differences among plots due to pesticide use One exception microbial biomass was augmented in association with use of he pesticide aldicarb Hart and Brookes 1996 K lg pasxmm I Pashade 56 Days m m Benomyl Triadimefon k Glyphosate Benomyl Aldicarb Triadimefon Glyphosate L Chlorfenvinphos Chlorfenvinphos Hart and Brookes 1996 0Days 077 7 7 sna 057 z E1 mm 8 In 05 3 Equot 56 Days E 03 I 2 D2 Aldicarb Triadimefon Benomyl Aldicarb Triadimefon Glyphosate Chlorfenvinphos m o c o E gt S t 2 c O Hart and Brookes 1996 56 Days i5 0 Days NOSN w yquot sail a 5 o o N o a a quotr ar 4 H Triadimefon Triadimefon Glyphosate Chlorfenvinphos Chlorfenvinphos Chemical spills and waste produce extremely high concentrations that are toxic At lower concentrations the same chemicals are not toxic Petroleum hydrocarbons Pesticides Concerns Agricultural use of pesticides especially herbicides Restoration of waste sites Air pollution acid rain in the US is a more minor concern now than 15 years ago Herbicides not likely to be selective for native species Problems with drift of sprays onto native species natural lands as well as drift into nontarge crops Historical problems of 24D use in cereals and consequent effects on grapes Although herbicides tend to be biodegradable in soil low levels of residues could affect subsequent susceptible crops grown in rotation with tolerant crop carryoverquot Sulfonylurea herbicides amp imidazolinone herbicides Inhibit acetolactate synthase in pathway for branc ed chain amino ac39d synthesis Valine leucine isoleucine Application rates gha ouncesacre F cl 1 a O O N N s LNJLNk H S O O HO 2NAN lhifensulfuvun N d H chluvsulfumn Wheat i 0 o o N N N O O N diNJKNJLNJ O diNlNAN H H N H H F Wheat 39 pvusulfumn nicusulfumn Sulfonylurea Herbicides 0 do NJl g ANJLN 0 0 Oil LN a Wheat Hamp SUllDNE Umquot metsulfuvun math l 0 OH 0 N N H HN N HN imazethapyr imazapyr imazaquiri Use in soybeans Glyphosate Inhibits EPSPS enzyme enolpyruvylshikimic acid phosphate synthase in biosynthetic pathway for aromatic aci s tryptophan phenylalanine tyrosine Use rates kg a I sacre Used in Roundup Ready Crops Soybean cotton canola corn OH HO OH P N c HY glyphosate Benzoic Acid 0 OH Cl 0 Auxm agonists Cl Mimic indole 3 acetic aCId ct Control cell elongation and growth MD mewpmp MCPP Mamba Includes phenoxyacetic acids 24 D and picolinic acids picloram clopyralid 0 OH HO 0 j Application rates kgha O Ho 0 O N C N Ga HZN Cl Cl Cl 3 Cl piclorarn clopyralid WOW Picolinic Acids Photosynthesis inhibitors Atrazine Most frequently detected pesticide residue in water Cl Cl N LN N kN ANJLNANN ANANANA 0 u S H H H H NH2 alrazine simazine melribuzin Symmetrical triazines Asymmetrical triazine Acylanilides chloroacetamides des alachlor and metolachlor 39equently found in a e Acetocnlorwas marketed in the 90 s as a substitute for alachlor that was supposed to be iess likely to be found in Water 7 Affect lipid metabolism 7 Rates kgna Cl Cl Cl on 0 2 on N o N v NVOV 1 0 alacmor acetochlor metolachlor Mt Bipyridiniiiums ationic paraquatamp diquat 7 Generate free radicais oxidizing the ceii 7 Contact toxicant not active from 50H sea as a rnrduwn amp defuiiatur herbicide putatues 7 Rates Kgna mm 1 1 mmdNMa bivviidimum mun Dinitroaniiines tniuraiin oryzaiin pendimetnaiin r inn bit mitotic Spindie formation ceiis don t divide roots do n ate 7 Rates sub kgna 0mg 2 Own H 0M YaNsA EMA me F p r 5 p M b g ivmuvaiin aryzaiin pendwe hahn Germination tests Root elongation test Growth response tests Under consideration reproductive studies mm 1 mm I Explessiun m DoseRespunse Function var Piaanoxmity 7eiling Grawm As Biomass or ngm 50 m m Cnniml gage Eoiuiion Cnncemraltmy instructur Allan Felsut 1mm afelsuttrlllty Wsu edu Fall Zuus WASIHNGTDN STATE a Ule Lecture 2 Biotic Phase Transfers Bioconcentration Bioaccumulation Biomagni cation Bioavailability Originally referred to the amount of chemical residue accumulated by an organism by adsorption le through the integument or other routes of entry including food ingestion esu ts in inc eased concentration of pesticide relative to envir t matrix includes food water amp air Bioconcentration is commonly thought of as being adverse Bear in mind that all nutrients and secondary metabolites are bioconcentrated uatlc eludes terrestrial orgarllsrnsl but also lrl orga lSmS especlallylfsoll dwelllrlg and lants Uptake of contaminants via bioconcentration as well as by food ingestion Most appropriately used if cannot distinguish between the two mechanisms of uptake Ratio of concentration of contaminant in the tissue orwhole bod or biochemical compartment like lipids relative to the concentration of contaminant in environmental phase or matrix BCF Clurganlsm or BAF Clphase BCF amp BAF can argey be exp a ned by nvok ng the same thermodynam c ru esquot used to exp a n phys cochem ca propert es and other phase transfers Thus uptake of contam nants by aquat c organ sms from water and sed ment s often exp a ned by nvok ng the phrase equ br um part t on ng theoryquot Given chemical x amp y x has a lower fugacity than y in water Is the BCF in the common test invertebrate Daphm39a higher for x or lower than fory In fact this question cannot be definitively answered until an important piece of the puzzle is placed on the board BCF or BAF should only be determined when the system is at equilibrium or forfield measurements at least in an empirical steady state BCF measured here tlowng water clean water Pesticide Ppm l l l l l l l 1 concentration in fish water concentration l In other words chemicals are metabolized at different rates which will affect their actual BCF or BAF DDT Log Kow 57 7 Log BCFwater log 5 Permethn39n Log Kow 3 6 Log BCF water log 2 dCA dt CA5 Cw BCF39CW k2 k1 Cw kz39CA Where CA concentration in the animal CA5 concentration in animal at steady state CW concentration in water k1 rate constant for uptake from water day39l k2 rate constant for elimination 39om animal day39l dieldn I lnfluralin 1000 39 lindane BCF 1m 39diazinon Fish 10 carbaryl 1 10 I 0000 I 00000 10 a Water Solubility ugL dieldrln lrlfluralll i 39 39 lindane 39 diazino I carbaryl 1000 10000 100000 KOW Waxy layers on invertebrate cuticle amp plant leaves Mucilaginous layers on plant roots Lipid bilayer of cell membranes Possible movement alon junctions between cells into interstitial spaces Gastrointestinal physiology of animals m mullw umwu mum mmum Inlmumlr m L i w Her n9 gull GuH Eggs 353mm pph mznnn thoughtto be a majormechamsm ha exp ams comparatwe y mgher concen ofcontammants m orgamsms from mg e t trat ons her ems have proven ncreasmg y mgher concentranons m nssues as tropmc eve moreases Emmam ca m quotquot355 Iquot r x e fuud sham magm catmn quot399 Lake quotm39 Ewsynem DDT mgkg WE WE EM DDT and PCBS ef c ent y mgested and I Wha e Eudy R eswdues food absored m assoc at on W th However Contammants depurated at a rate S oWerthan the consumpt on ofbwomass Needed forenergy reqmremen 5 Thus the Contammants are stored m hpophmo twssues at much mgherconcentranons than occurm the food P ankmn w n h y Fxshrea ng was 92mm ubservatmns mum hypn hesws repur ed m N m h P DDT d WuudweHeN 19E7 DDTvesMuesm anEas1 Cuaslesmavy A E 9 WWWquot was 3995 95 WW case uVbm ugma cuncenlvauunnapevswstenhnsemmde Smence Unphm eveHu We next 153 8217824 EMFW nmmagnmcatmn facturs Auuauc F s n We nebva es a I Mamma s a Ke yet a 2mm ETAC 2 2m algae invenehrates sh LEEWanE 1995 ESKT 29154 LEEHam 1995 argues that ab Expenments shuvvfevv substances amuaHy bmmagmiy Dwfferences m hpuphmc nntammant cuncentratmn between truphn Eve s ean be EXp amEd by me nntent ewereneesfmm ene truphu have m the next Larger urgamsms sh have s uvvar depuratmn ratesthan smaHer urgamsms phytup anktun mvenebrates The SCHan fund thaw Effect has 3 DW prubabmty uf eeeurrenee re auve m passwe emsmn mm the Envwrunment e Dwfferences m mm nntent and depuratmn amung urgamsms at uwerenurepme Eve s ean accuuntfur uwerenees m ECF ur EAF x e y Lament 1995 ESKT 29 m Odacmammhenmdmxm Mcmmahenzene m 1 m We the mam umevs emaemmee dmevence m meaccummauen YaE DY EAFECF bE1WEEHYDDd mam Msu the Emma s genevauy lt1 Data based an ab expenmems EAFeWater FemWater 314mm FEW Fund 1D Leaxanc 1995 ESKT 29 m F39CEHngQL hme budy LEE EMUBBE ADDD SDDD ZDDD mun Lemancm e x w o 4 K1depuvahun camera Leuranctw t r 2 EAFrfnnd ta ECF Ratrn Frsntnvertebrates a Leatanc taaa EM Auuatr A tmenemates Fmax btnm agntftcattnn factan rllll Ill g 3 Apptrcabte to mammats brte sattrntceHeS Secreted through gaH btadderassoctate thh hydrophobtc on tnants m gut tumen r ans mecnamsm nnnnaHy rsutates munugtycenues and nee fatty actds frnrn undtgested fat gtubutes Mtcettes move to a water boundary tayer near brusn border membrane r e gut ttssue ceH evagtnattons thh a mgnerprt tnan m the tumen 39 MtceHes break up and contamtnant t5 reteased 0 05 t the ceH WaH Where tt dtfmses atong a concentratton gradtent tnto the ceH and from there tne generat crrcutatron g A smclw r Wrtmrr each was ts a neIWuvk ut anevtutes aptttavtes venutes arm tympn duds entrat tadeat e Nutvtentsabsumed ya 5 cans uNtHus and Uanspuned mt netwuvk utmuuu vessets tlrrt Eutk Pnase Lumen nsttnedWatevLa er EurtnetratCEtt MtceHe Dtssuctattun nytnmtcrnn Brtesattmrcette m 0 Wu 03 3 353437523725 39 a fquot w um ased an Kettvet at 2am Er 2 2m The tract on of the contacted dose that s transferred from the s te of contact or adm n strat on nto the genera c rcu at on or In so andor water because of sorpt on of contam nants to part cu ate matter not a of c e present w be taken up or cross the ntegument of the organ sm Bwatve ctams RadtotabeHed Ch orpynfoswUptake amp Depurattoh Uptake mugs Ehmmattoh Many meta s occur as cat ons n so and water Thus metats cah bmd thtehsety to 50H or sedtmeht parttctes espeoaHy tr they are hch m da s Bmdmg makes them tess btoavattabte thah just ah et ehtat ahatysts or totat oohcehtrattoh woutd uggest Note however that as concentrat on of meta n so water goes up BCF wou d not go up t may decrease Reason metats have Specmc membrane transport systems such as mmer protems WASHI NG I ON STATE instructor Aiian Felsot areisottncity wsu eou 1 UNIVERSIH I Fall 2005 Neurotoxicity Pan The pharmacooynamic action of most insecticides is at the ievei ornerve function te biochemicai receptors are at different sites ofthe nerve ceii Some insecticides are extremeiy persistent in environment and in tissue 7 These are banneorrom commerciai use but resioues stiii bioaccumuiate in the rooo web h insecticides are easiiy biodegradable in the environment and easiiy metaboiizeo umber or commoniy useo insecticioes have very high acute toxicities against nontarget invertebrates aquatic and terrestrial vertebrates in addition to the historical issues of i ieui otoxic ll l ecti ides a d acute toxicityto birds arid fishy concerns about i ieui od eveiopmentai toxicity have grown e inoeeoi these have been addressed ror humans in the Food auaiity Protection Act or i996 a maior amendment to the Federal insecticioe Fungicide Rndenticide Act FlFRA Historicai concerns overneurotoxicoiogicai ff r ead and mercury New concerns overpotentiai neurooeveiopmentai effects of PCBs and PBDEs Nerve function is remarkably conserved r a oss both invertebrates and vertebrates e Eariiest knowledge ofhovv nerv nrnents usingthe squid giant axons which was severai mm in Thus toxicants that act at the level 0 he n t erve can potentially have adverse effects across a wide spectrum of organisms Rapid coordination in the be y Electrically excitable cells neurons Generate electrical signals long distances without degrading strength of signal Function similarly among all animals Basic structure a cell body maintenance of c e Dend sorna contains nucleus metaboiic eii rites receive amp carry signai toward rAxoris carry signai away from sorna 7 Supporting ceiis for ex myeiin sheath Sensory neurons 7 Transmit inrormation coiiecteo rrom externai stimuii e esbonoto stimuii insioe b o For ex blood oxygen ie orientation or the head lnterneurnns e Linx otherneurons N n v vel position or a inirit within he centrai nervous system etworxeo to exchange inrormation ano berrorm com biex computations ieaoing to behavior orthought Rahhn Retina lmerneumns Neuruns gruuped mm smsters m annust aH phy a TypmaHy the 2 bumes ur same are untamed wnnrn the entrax nannus system CNS wmch unsrsts ur a7 Muturneuruns mutuneumns 7 Carry mstrunmna srgnaxs tn erresmr urgans An enesmns a new nssuer ur urgan that acts m nange the unmtmn at an urgamsm w W respunse m neurana ur hurmuna srgnaxs 7 smnacmm on W Fur Ex r untractmn ur musmes ur secretmns by S A V g ands m 2 saw m we enunv amnth budv mmhnE dwansavneumna samata caHed gangha s u y sensurv Wuvmatmn 7 Tu dehvevmmuv S Ena smat cuntquhe acmw unnusues m menus Suppumve EHs structurak rnsmauve and metabuhc 7 Gha ceHs A39ferem neuron carries sensory Informatlon to Imerneurons In the cus man nrmmnw 7 Arman nanspumngm cunuucungm a cemva regmn L v 9 rThuvacm a gangha more 7 menu nanspumng m r cunductmmume 4 m rmrpnmrmmm t penpherv umacemva commew7nnunmna 39 vegmn quotW quot m lvfu39 In H quot3 NE 0T Mamedwam RandaHeta zuuz Ewen sAmma thsmagv aniw Fmg W Neuron single cell 7 Ce body contams nuc eus 7Brancnrng fbers Adapkdham anda eu 2m2 V 765p amenetermma scaHedthesynapse J Axum WW DE dHtES 39 7 nsu atmg membrane orsheath caHed the myehn r murmrmnmmv WWW LMivmmns 51 Ed Synapse Picture from Scienti c American the axon hillock a region signals produce a graded response Nerve terminals from other neurons Dendritesd Integration Impulse Conduction AP T r S ike Initiation Direction of signal conductance Pvesynapllc terminals Glial Cells lt Forming Insulating Soma Myelin Sheath Plasma membrane of soma and its dendrites receive signals from the terminals of other neurons The spikeinitiating zone usually located atjunction of the soma and axon a ka axon hillock integrates the input signals from many neurons Spikeinitiating zone with sufficient input will initiate a change in plasma membrane voltage potential and generate an action potential AP Action potentials are what are commonly thought of as the nerve impulse orsidna Picture modi ed from Randall et al 2002 Eckert s Animal Physiology Axon terminals Nucleus Because of the synapse nerve signals only travel one direction down the axon Nerve signal is actually an electric current that moves along the axon until it reaches the synapse which separates the axon and the dendrite Ability to conduct electrical current is due to two factors Membrane electrical potential Permeability ofthe membrane to ions A measurable electrical potential difference exists across cell membrane ie potential difference on outside vs inside of cell membrane Inside of cell is negative with respect to outside of cell Poten iaI difference results from distribution of ions on each side of a selectively permeable membrane When nerve cell not stimulated potential difference is called the resting potentialquot Charge separation or potential difference across the membrane inside surface the cytoplasm or axoplasm side and the outside surface actually the space in between the cell membrane and the myelin or the cell membrane and the blood Inside is negative with respect to the outside The electrical potential priorto nerve signal conduction is called he membrane resting potential Resting potential value 70 mv The nerve cell membrane is semipermeable Allows potassium ions to leak slowly across the membrane but sodium and chloride cannot cross the membrane The cell membrane has protein embedded along its surface Proteins act like channels or gates allowing ions to pass freely Protein gates are closed until the nerve is stimulated The difference in distr bution of K and Na on either side of the membrane at equil brium creates the resting potential Unequal distribution of K and Na on each side ofthe membrane Axoplasm Blood Sea Water Inside Cell Outside Cell Potassium K 400 20 10 Sodium Na 50 450 470 Chloride Cl 40 570 550 Calcium Ca 00003 10 10 Magnesium Mg 10 55 54 Data from SchmldterelSen 1997 on Squid glarit ax When stimuli are received either electrical stimuli from other nerves or peptide chemical transmitters from neurosecretory cells the membrane begins to depolarize Proteins in the cell membrane change their conformation and allow free passage of sodium ions into the axoplasm This rush of positively charged Na causes the membrane potential to change in the positive direction The Na channel is called a voltagegated channel because it is triggered open by electrical signal The opening ofthe sodium channels to influx of Na along the length ofthe nerve axon give rise to the action potential However there is a threshold of membrane depolarization that must be reached for the action potential to be generated Once the action potential is generated its amplitude is always the same The action potential propagates along the axon without any loss in amplitude The change in electrical potential causes the appearance of the action potential Modmed from Panda et An allornone response al 2002 Eclltert s Animal Physlology MechanismS amp Ad t t 5th Ed Na gates open 59 Na gates close Adopted from Randall et ai 2002 Eclltert 5 Animal hysiology M n N H 5th Ed my maim m f iui N M Kiwi inn in v39 4ar ic 0 1quot a 1 w Mm in l quotL VIquot 39 aaiiicia Shortly alter the sodium channel opens up a potassium channel also opens 7 Note that the potasslum channel Works to allow K back ll lto the cells but at a much lowerrate than the ll l LlX of Na lOl lS V thin a millisecond alter the Na hannel op ns the membrane potential continues past 0 mV to the positive side of 40 mV N c lmonmscm a Ionic Comm Action potential is transmitted along tne lengtn o e axon in one irection tnrougn a mecnanism called tne local circuit current As tne membrane in one area goes tnrougn depolarization and tnen hyperpolal lzatlol l tne adiacent area also begins to depolarlze triggering tne action potential 7 Thus tne action potential moves llKE a Wave along tne lengtn ortne axon until it reaches tne synapse Na Gate Opens N a Gate ses Adiacent Memorane Area starts to Depolarize inside o c A i ExlvacellulavFluld Ii Threshold Reached Na Gate Opens quot quot inside oiCell Exlvacellulal39Fluld ll Artertne action potential passes a speciric i m gate Potassium efflux slows out it takes several milliseconds longer oerore its cnannel closes and efflux sto s Meanwhlle tne membrane potential ralls back tnrougn zero until it reacnes tne initial resting state The actlorl potehtlal stops at the synapse Where there ls a small gap etweeh the axon and the den hte of the Next heme ceH The actlorl potehtlal stlmulates the release of the neurotransmltterchemlcal acetylchohl le stored lh veslcles tn the pre ynaptlc ceH axoplasm ACh dlttuses across the gap to the post Synaptlc membrane 0 CH8 Acetylchullne HEC amp0CH2CH2N CH3 CH 31quot lufwi w Mp Adamad m Randall eta zuuz Ewen Ahhal thsalagv Memanlsmsa Adaptatlarls slh Ed 7 synaplic cletl 4 J Receptor Pastsynaplh a e n msmnlane I HM II l ar r39lnwn l Adapted mm Randall el al zuuz E men s Arlmlal Neurolmnsml sl blndlng sne Ach phus dlrectlytu receptuh upeth up l Adapted mm Randall 21 at zuuz Em en s Ahhal phvsmlagv Meehamsms a Adammmnsquot slh Ed thsalagv Extracellular space will Receplol cvnwlllsm E rn edded tn the post Synapttc membrane are protetn receptors ACh otnos to the ACh receptors caustng a conformatton change tn the protetn and tncrease tn permeabmty to otorn Fast ece C 03 h recepmr ts a tgandrgated hannet ootorn moves tnto the axoptaamt eventuaHy trtggertng anotheractton poten at However the response ts graded as opposed to what happens tn the pre syna ttc axon 7 There IS a mreshold for number ofACh molecules that mus blnd to the receptor SmaH vestctes t meow Adamo want RandaH et at 2on2 mew physotoev sAmmat 5th Ed mutton Mutton The Acn onty stays bound to the receptor a shortttrne t dtSaSSoctateS from the receptoran d then bmd 0 otherreceptors on the membrane ttmtot T V However t e ts a so rtch tn an Iquot t enzyme caHed acetytchottnesterase tha 3721173 tatmettotatmmv tonehanttet Hydrotyzes the AC 7 AC has abuut an nee or otnotng wtth the receptor as tt ooes thh the Enzyme nn Ecken sAmmat e Amtylcholt39neslemse V 0 CH3 Acyt Functtonat Group chottne kl k2 k EAXlt 39 EAX gt EAX gt EA rl E Acetylcholinesterase Enzyme AX Acetylcholine X Choline Leaving Group A Acyl group In addition to the stimulatory signal of acetylcholine the pre synaptic axon can also release an inhibitory neurotransmitter GABA diffuses across the synapse and binds with GABA receptors Causes protein to change configuration and permit the in ux of chlon39 e ions Chloride ions cause hyperpolarization of the membrane in the negative direction making it more difficult to evoke an action potential when stimulated Ho g CHZCWNCHZNHZ Nerve physiology is universally similar among all vertebrates and invertebrates Thus pesticides with pharmacodynamic actions on nerve tissue is likely to be toxic to a Wide range ofanimals The nervous system is vulnerable to toxicosis at two levels Ion Channels Synapse Disrupt functioning of sodium channel on the axon Prevent sodium channel from closing Thus threshold for generation ofnew action potentials is lowered The inhibitory activity is inversely related to temperature Acute toxicity of DDT and pyrethroids to maIs is moderate to low ie LD50 s in the hundreds or greater Pyrethroids are synthetic versions of pyrethrins the cons ituents ofthe botanical extract pyrethrum from enum cinnaeri o ium 7 They are stableto uv catalyzed photodegradatlon Pyrethroids are used at low application rates of01 lbacre Older insecticides are used at rates of at least 1 lbacre Pyrethroids have become important urban use insecticides as the older organophosphorus insecticides have been removed form all urban uses indoor amp outdoor Water monitoring studies in California are n startin to show the presence of pyrethroid residues but the greatest masses will be bound to sediment owing to very low water solubility Two classes of pyrethroids amp Characterized by how long sodium channel is held open Bind to different regions of the channel ie different protein binding regions tehametmm auemvm o 700 0 Paymelmm Charactensuc Swgns ufTuxmg savere netremu re ex hyperexmtabmty sympatheu neweus syste am annn we MP W n marked m Nmeme Warm N mneuuna emup N O F F a U N N o O we 0 w a F a U EWWVW denametmn Pram Waterysahvatmn cuarse mtabmty mesa Charactenstm 5 ns urtuxm se tremur mcreased Extensurtune muderat re ex hyperex sympathe e actwatmn hureuametusws sewzures pares Cuntml Acliun Potennar U mV rm mV Ymesnnm 5mm Pymhmid Made uf Acliun Slowly decaying mum to 0 my resting pnlemial due 0 open Na39 gate 37 mV 2 3 t 4 We mnlhsecands ESRP 531 Fundamentals of Environmental Toxicology Fall 2003 December 1 2003 Lecture 22 Biomarkers Environmental and Tissue Contaminant Residues I Biomarkers A Biomarkers are biochemical physiological or histological indicators of either exposure to or effects of contaminants or even natural products at the suborganismal or organismal level of organization 1 Biomarkers would be indicative of a contaminant effect or an exposure at the individual level 2 However biomarker quantification can be used to compare populations living in polluted vs pristine sites to gain insight into potential exposure to toxic substances among populations B Biomarkers are generally measurements of sublethal effects in that living organisms are collected and assayed However some biomarkers can be reasonably hypothesized to be indicative of a type of contaminant and thus biomarkers can be more specific measures of exposure One example of specificity is the use of acetylcholinesterase AChE activity which can be inhibited by insecticides of the organophosphorus OP and carbamate CB classes a AChE can be measured in live or dead organisms assuming they haven t been so long that the tissue and therefore the enzymes has decomposed b However caution must be used because other compounds can inhibit AChE including natural products eg certain alkaloids and heavy metals e g copper Ci Types of biomarkers 1 Enzymes a AChEimay be indicative of exposure to OP and CB insecticides b ALAD deltaaminolevulinic acid dehydratase indicative of lead exposure c ATPase 1 Plant enzymes peroxidase RUBISCO 2 Energetics a Adenylate energy charge bi Energy reserves c Whole body Calorimetry d Enzymes of intermediary metabolism e Growth 3 Endocrine a Hormone levels 1 Corticosteroidscatecholamines 2 Thyroid hormone 3 EstrogenTestosterone 4 InsulinGlucagon 5 Growth hormone b Protein synthesis under endocrine control 1 Vitellogenin levels in male fish ESRP531 Lect 22 Biomar71doc Page 1 0f 23 Fundamentals of Environmental Toxicology Fall 2003 ESRP 531 4 Blood chemrstry 5 Growth Rate RNA and prote1n synthesis D erltauons of bromarkers a Laek of speer crty 2 Natural endogenous varrab lty of response or varrab lty of response as 1 related to other environmental stres sors a n tal m arraumry allltathmne rsr transferase GST and acetylchohnesterase vaned by factors of 1847 and 1817fold respectively In Chxmrwmux larvae held under ambrent eondruons of 13 uncontamlnated srtesquot see Frgure 1 n m 351 z 4 Sin Frgure 1 Mean GST and AChE mMLmIng proteln 1n Whole body of Chilanumus Environ Toxlcol Chem 201725 1732 UK Olsen et al 2001 ha been 11 Analysis of Contaminant Residues Issues in Environmental Analytical Chemistry A exposed What an B Page 2 of23 ESRP531 Lea 22 Biomax7ldoc ESRP 531 Fundamentals of Environmental Toxicology Fall 2003 estimate exposure Thus environmental analytical chemistry is the tool of choice for exposure estimation 1 In addition to their importance in estimating exposure residues of contaminants are monitored for compliance with various numerical standards a For example under the Clean Water Act EPA can promulgate guidelines known as ambient water quality criteria for the protection of aquatic biota 1 These guidelines are not enforceable on the Federal level but states can adopt similar guidelines that may be enforceable standards 2 In some cases the standards criteria or guidelines for protection of aquatic biota are as low as 1 part per trillion ie 1 pptapplies to DDT residues 2 Thus knowing how residue numbers are produced and some of the issues related to detection and quantitation makes us a bit more skeptical and perhaps appreciative of the tremendous amount of residue monitoring that is presented in technical reports and the refereed scientific literature C The Desperately Seeking Nothing Syndrome ll Capability of analyzing contaminants to levels of ppt and even below has evolved without a corresponding understanding of biological effects at these levels Figure 2 100 ppth gL 1950 1960 1970 1980 1990 2000 Year Figure 2 Evolution of analytical capability a Bear in mind that requirements for toxicological testing only demand comparatively high dose testing ie the doses where a biological effect is likely to be observed although many contaminants will not produce a biological effect in chronic toxicity testing unless high doses relative to environmental concentrations are administered 2 What does it mean to analyze something to the level of ppt a Significance of substance purity by percentage 1 99 9999 pure a 0 0001 impurities or 1 ppm of impurities 2 99 9999999 pure a 0 0000001 impurities or 1 ppb of impurities 3 99 9999999999 pure a 0 0000000001 impurities or 1 ppt of impurities ESRP531 Lect 22 Biomar71doc Page 3 0f 23 ESRP 531 Fundamentals of Environmental Toxicology Fall 2003 b On the other hand 1 ppb of a pesticide with molecular weight of 300 contains 2 x 1015 molecules per Liter and 1 ppt in water contains 2 x 1012 molecules 1 It s a lot of molecules but is it enough to react with an enzyme or other biological receptor to cause a measurable reaction a Remember the kinetic parameter Km used in enzyme biochemistry to indicate the affinity of a substrate and enzyme Km 2 the concentration at which the reaction velocity is 50 of maximal velocity 3 Consequences of Increased Analytical Capabilities my opinion a Contaminants seem to occur in places that we had never seen them before 1 For ex based on the Koc of DDT we would not have predicted it would leach to ground water yet it has been found in ground water albeit at levels of ppt b We have the notion that synthetic chemicals are everywhere in our environment 11 Isn t that what we would expect if compounds used as pesticides are spread across hundreds of acres at a time or if the public consumes billions of pounds of refined chemical products each year but discharges some as waste into wastewater treatment plants that are less than the ideal of 100 biodegradation efficiency 2 In the Midwest aquifers are perceived to be frequently contaminated with the herbicide atrazine this perception has been enhanced by the increasingly lower detection limits for atrazine a A study by Kolpin et all 1995 J Environ Quality v1 24 shows that the frequency of detection of atrazine in shallow aquifers increases significantly as the reporting limit of detection decreases Figure 3 Frequency 45 40 35 30 25 20 15 10 5 21001 001 01 1 10 Atrazine Reporting Limit pgL Figure 3 Effect of atrazine reporting limit on percentage of wells in shallow aquifers with detected residues Kolpin et all 1995 b Another example of how detection limit or reporting limits influences perspective of just how contaminated water is can be seen in Hoffman et all 2000 Comparison of pesticides in eight ESRP531 Lect 22 Biomar71doc Page 4 0f 23 ESRP 531 Fundamentals of Environmental Toxicology Fall 2003 US urban streams Environ Toxicol Chem l9922492258l Table 1 Table 1 Detection frequency of pesticides in an urban stream study as influenced by limits 2 200 et all 7 177 140 Note that the USGS in its technical report release for various watersheds uses reporting limit for many pesticide that are 0 001 14 gL When their scientists publish in the peer reviewed literature however their reporting limits are set much higher cl Tendency to lower regulatory limits i e criteria for permissible concentrations of chemicals in water or other media to increasingly unrealistic levels 1 Regulations that limit chemicals to levels that are at or below analytical detection capabilities become impossible to reliably regulate 4 Consider that we are regulating the concentrations of contaminants to levels of purity greater than the solvents we use to extract these chemicals from water the concept of analytical damage a Methylene chloride used to extract pesticides from water is only about 999 pure yet we may be trying to extract a contaminant that has a relative purity in water of 1 ppb 99 9999999 equivalent purity The amounts of chemical reagents and solvents used in analysis and released into the environment is 10 million times greater than the contaminants to be analyzed The people who report on the significance of the residue numbers or regulate the contaminants are different from those who generate the numbers 6 Thus it is important to understand what a residue number really is D The objectives of analytical detection a QualificationWhat is the identity of an unknown material bl QuantificationHow much is there E The first step is to identify the chemical 1 Another way of looking at this process is one of making a decision as to whether or not an analyte ie the compound you are looking for or trying to determine in a matrix is really present or not 0quot U1 ESRP531 Lect 22 Biomar71doc Page 5 0f 23
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