Aquatic Toxicology 65(2003)337–360
Fish tolerance to organophosphate-induced oxidative stress is
dependent on the glutathione metabolism and
enhanced by N -acetylcysteine
Samuel Pe?a-Llopis a ,?,M.Dolores Ferrando b ,Juan B.Pe?a a
a Institute of Aquaculture Torre de la Sal (CSIC),E-12595Ribera de Cabanes,Castellón,Spain b
Department of Animal Biology (Animal Physiology),Faculty of Biology,University of Valencia,
Dr.Moliner-50,E-46100Burjassot,Valencia,Spain Received 24October 2002;received in revised form 5June 2003;accepted 7June 2003
Abstract
Dichlorvos (2,2-dichlorovinyl dimethyl phosphate,DDVP)is an organophosphorus (OP)insecticide and acaricide extensively used to treat external parasitic infections of farmed ?sh.In previous studies we have demonstrated the importance of the glutathione (GSH)metabolism in the resistance of the European eel (Anguilla anguilla L.)to thiocarbamate herbicides.The present work studied the effects of the antioxidant and glutathione pro-drug N -acetyl-l -cysteine (NAC)on the survival of a natural population of A.anguilla exposed to a lethal concentration of dichlorvos,focusing on the glutathione metabolism and the enzyme activities of acetylcholinesterase (AChE)and caspase-3as biomarkers of neurotoxicity and induction of apoptosis,respectively.Fish pre-treated with NAC (1mmol kg ?1,i.p.)and exposed to 1.5mg l ?1(the 96-h LC 85)of dichlorvos for 96h in a static-renewal system achieved an increase of the GSH content,GSH/GSSG ratio,hepatic glutathione reductase (GR),glutathione S -transferase (GST),glutamate:cysteine ligase (GCL),and ?-glutamyl transferase (?GT)activities,which ameliorated the glutathione loss and oxidation,and enzyme inactivation,caused by the OP pesticide.Although NAC-treated ?sh presented a higher survival and were two-fold less likely to die within the study period of 96h,Cox proportional hazard models showed that hepatic GSH/GSSG ratio was the best explanatory variable related to survival.Hence,tolerance to a lethal concentration of dichlorvos can be explained by the individual capacity to maintain and improve the hepatic glutathione redox status.Impairment of the GSH/GSSG ratio can lead to excessive oxidative stress and inhibition of caspase-3-like activity,inducing cell death by necrosis,and,ultimately,resulting in the death of the organism.We therefore propose a reconsideration of the individual effective dose or individual tolerance concept postulated by Gaddum 50years ago for the log-normal dose–response relationship.In addition,as NAC increased the tolerance to dichlorvos,it could be a potential antidote for OP poisoning,complementary to current treatments.?2003Elsevier B.V .All rights reserved.
Keywords:Dichlorvos;Organophosphorus pesticide;Tolerance;Necrosis;Glutathione redox status;Biomarkers
Abbreviations:AChE,acetylcholinesterase;EAAs,excitatory amino acids;GCL,glutamate:cysteine ligase;GPx,glutathione peroxidase;GR,glutathione reductase;GSH,reduced glutathione;GSSG,oxidised glutathione or glutathione disulphide;GST,glutathione S -transferase;?GT,?-glutamyl transferase;NAC,N -acetyl-l -cysteine;NMDA,N -methyl-d -aspartate;OP,organophosphate;ROS,reactive oxygen species;TTD,time-to-death
?Corresponding author.Tel.:+34-964-319500;fax:+34-964-319509.E-mail address:samuel@iats.csic.es (S.Pe?a-Llopis).0166-445X/$–see front matter ?2003Elsevier B.V .All rights reserved.doi:10.1016/S0166-445X(03)00148-6
338S.Pe?a-Llopis et al./Aquatic Toxicology65(2003)337–360
1.Introduction
Dichlorvos(2,2-dichlorovinyl dimethyl phosphate; DDVP)is a relatively non-persistent organophosphate (OP)compound that undergoes fast and complete hy-drolysis in most environmental compartments and is rapidly degraded by mammalian metabolism(WHO, 1989).These characteristics made it attractive for worldwide use to control insects on crops,household, stored products,and treat external parasitic infections of farmed?sh,livestock,and domestic animals.In fact,dichlorvos has extensively been used to treat sea lice infestations(by the copepod parasites Lep-eophtheirus salmonis and Caligus elongatus)in the Atlantic salmon(Salmo salar)culture.
The primary effect of dichlorvos and other OPs on vertebrate and invertebrate organisms is the inhibition of the enzyme acetylcholinesterase(AChE),which is responsible for terminating the transmission of the nerve impulse.OPs block the hydrolysis of the neu-rotransmitter acetylcholine(ACh)at the central and peripheral neuronal synapses,leading to excessive ac-cumulation of ACh and activation of ACh receptors. The overstimulation of cholinergic neurones initiates a process of hyperexcitation and convulsive activity that progresses rapidly to status epilecticus,leading to profound structural brain damage,respiratory dis-tress,coma,and ultimately the death of the organism if the muscarinic ACh receptor antagonist atropine is not rapidly administered(Shih and McDonough, 1997).Until recently,the toxic effects of OPs were believed to be largely due to the hyperactivity of the cholinergic system as a result of the accumulation of ACh at the synaptic cleft.However,recent stud-ies have highlighted the role of glutamate receptors in the propagation and maintenance of OP-induced seizures,as well as the role of glutamate in mediat-ing neuronal death after OP poisoning(Solberg and Belkin,1997).A few minutes after the beginning of OP-induced seizures,other neurotransmitter systems become progressively more disrupted,releasing ini-tially catecholamines and afterwards excitatory amino acids(EAAs),such as glutamate and aspartate,which prolong the convulsive activity.After a certain dura-tion of convulsions(about40min in rats exposed to soman)the atropine treatment becomes ineffective be-cause the seizure activity can be sustained in absence of the initial cholinergic drive(Shih and McDonough,1997).The high extracellular concentrations of EAA are neurotoxic,because they are able to activate the N-methyl-d-aspartate(NMDA)receptor,leading to intracellular in?ux of Ca2+,which triggers the acti-vation of proteolytic enzymes,nitric oxide synthase, and the generation of free radicals(Beal,1995).Re-active oxygen species(ROS)such as hydrogen perox-ide(H2O2)and the free radicals superoxide(O2??) and hydroxyl radical(HO?)can react with biologi-cal macromolecules(especially the hydroxyl radical) and produce enzyme inactivation,lipid peroxidation, and DNA damage,resulting in oxidative stress.The degree of this oxidative stress is determined by the balance between ROS production and antioxidant de-fences.Pesticides are recently known to be able to induce in vitro and in vivo generation of ROS(Bagchi et al.,1995).In previous studies we demonstrated that thiocarbamate herbicides induced oxidative stress in the European eel(Anguilla anguilla L.)(Pe?a et al., 2000;Pe?a-Llopis et al.,2001).Oxidative stress ef-fects have also been observed in the carp(Cyprinus carpio)and cat?sh(Ictalurus nebulosus)intoxicated with dichlorvos(Hai et al.,1997).
OPs are also capable to induce programmed cell death(apoptosis)by multifunctional pathways (Carlson et al.,2000).Apoptosis is a complex pro-cess characterised by a cell shrinkage,chromatin condensation,and internucleosomal DNA fragmenta-tion that allows unwanted or useless cell removal by phagocytosis,preventing an in?ammatory response to the intracellular components.Caspases are a fam-ily of cysteine proteases that are present in cytosol as inactive pro-enzymes but become activated when apoptosis is initiated,playing an essential role at var-ious stages of it(Cohen,1997).Caspase-3is one of the key executioners of apoptosis,being responsible either partially or totally for the proteolytic cleavage of many structural and regulatory proteins.However, at conditions of higher stress,the cellular impairment is so high that apoptosis is suppressed.This leads to cell death by necrosis,which causes further tissue damage and an intense in?ammatory response. Dichlorvos is metabolised in the rat liver mainly via two enzymatic pathways:one,producing desmethyl-dichlorvos,is glutathione(GSH)dependent,while the other,resulting in dimethyl phosphate and dichloroac-etaldehyde,is glutathione independent(Dicowsky and Morello,1971).Hence,GSH availability can
S.Pe?a-Llopis et al./Aquatic Toxicology65(2003)337–360339
result in a limiting factor for dichlorvos elimination.
Glutathione is a ubiquitous thiol-containing tripeptide
that is involved in numerous processes that are essen-
tial for normal biological function,such as DNA and
protein synthesis(Meister and Anderson,1983).It
is predominantly present in cells in its reduced form
(GSH),which is the active state.Among the several
important functions of GSH,it contributes to the re-
moval of reactive electrophiles(such as many metabo-
lites formed by the cytochrome P-450system)through
conjugation by means of glutathione S-transferases
(GSTs).GSH also scavenges ROS directly or in a
reaction catalysed by glutathione peroxidase(GPx)
through the oxidation of two molecules of GSH to a
molecule of glutathione disulphide(GSSG).The re-
lationship between the reduced and oxidised state of
glutathione,the GSH/GSSG ratio or glutathione redox
status,is then considered as an index of the cellular
redox status and a biomarker of oxidative damage,
because glutathione maintains the thiol-disulphide
status of proteins,acting as a redox buffer.
Glutathione levels are regulated by several en-
zymes(Meister and Anderson,1983),but mainly
depend on the balance between GSH synthesis rate
(by glutamate:cysteine ligase,GCL),conjugation rate
(by GSTs),oxidation rate(non-enzymatically or by
GPx),and GSSG reduction to GSH(by glutathione
reductase,GR).GCL is an enzyme also known as ?-glutamylcysteine synthetase,which catalyses the rate limiting step of GSH biosynthesis in which
the amino acid l-cysteine is linked to l-glutamate.
GR reduces GSSG to GSH at expenses of oxidising
NADPH to NADP+,which is recycled by the pentose
phosphate pathway.In extrahepatic tissues,high GSH
concentrations are also maintained by?-glutamyl
transferase(?GT,traditionally known as?-glutamyl
transpeptidase),which is the only protease that can
cleave intact GSH and GSH-conjugates(Curthoys and
Hughey,1979).?GT is a membrane-bound enzyme
with its active site orientated on the outer surface of the
cell membrane that enables resorption of extracellular
GSH catabolites from plasma(Horiuchi et al.,1978).
We found previously that eels showing a higher sur-
vival upon herbicide exposure had enhanced GR ac-
tivity and increased GSH and GSH/GSSG ratio in the
liver(Pe?a-Llopis et al.,2001).Hence,a drug that
could increase the GSH content and act as a reductant
could improve the survival of OP-poisoned?sh.We used in this study the well-known antioxidant and free radical scavenger N-acetyl-l-cysteine(NAC),which can easily be deacetylated to l-cysteine,the limiting amino acid for glutathione biosynthesis.NAC is used clinically to treat several diseases related to oxida-tive stress and/or glutathione de?ciency such as parac-etamol(acetaminophen)overdose,VIH infection,and lung and heart diseases(Prescott et al.,1977;Gillissen and Nowak,1998;De Rosa et al.,2000;Sochman, 2002).It has also been proven to be useful in the treatment of acute paraquat and heavy metal poison-ing(Hoffer et al.,1996;Ballatori et al.,1998;Gurer and Ercal,2000).
So far,studies of tolerance to pollutants and/or ox-idative stress have principally been focused on the role of genetic variations in natural populations(e.g. Sullivan and Lydy,1999)or antioxidant defences of different species(Hasspieler et al.,1994;Hansen et al., 2001)and strains(Mathews and Leiter,1999),but not on the effect of antioxidant defences on the survival of a natural population exposed lethally to a pollutant. At this point,we try to?ll this gap by studying the effect of the antioxidant NAC on dichlorvos survival of a genetically diverse population of European eels by analysing endpoints of the glutathione metabolism, in addition to the use of AChE and caspase-3activ-ities as biomarkers of neurotoxicity and induction of apoptosis,respectively.
2.Materials and methods
2.1.Animals
Sexually undifferentiated yellow eels of the species A.anguilla(5–15g)were used to avoid the effects of sex variation and minimise hormonal interactions in toxicity assays.These European eels were captured on the coast of Portugal(averaging0.33g)and cultured for about6months in a?sh farm(Valenciana de Acui-cultura S.A.,Spain)free of any disease.Acclimation and selection of?sh for acute toxicity tests were car-ried out according to OECD guidelines(1992).Be-fore starting the experiments,animals were kept for2 weeks in aerated and?ltered dechlorinated freshwater (total hardness:192±5mg l?1as CaCO3;pH7.5±0.1; dissolved oxygen:7.2±0.1mg l?1)at24.0±0.5?C, and with a12-h photoperiod.
340S.Pe?a-Llopis et al./Aquatic Toxicology65(2003)337–360
2.2.Chemicals
Hexipra Solucion?,an emulsi?able concentrate containing40%of dichlorvos,8%of emulgators,and 47%of non-toxic solvents,composed principally by 2-propanol,was obtained from Laboratorios Hipra S.A.(Girona,Spain).2-Vinylpyridine was acquired from Aldrich.NADPH was purchased from Ap-plichem(Darmstadt,Germany).NAC and all other reagents were obtained from Sigma Chemical Co. (St.Louis,MO,USA)unless mentioned otherwise.
2.3.N-Acetylcysteine supplementation assay
Fish received a single intraperitoneal(i.p.)injection of either1mmol kg?1NAC or its vehicle(physiolog-ical saline).This amount of NAC was used in order to induce GSH synthesis beyond physiological levels. Five animals were removed from the water at3,12,24, 48,72,and96h after the injection,and anaesthetised in ice instead of using a chemical anaesthesia to prevent interfere with the glutathione metabolism(Brigelius et al.,1982).They were then weighed,the lengths measured,and were euthanised by decapitation.The livers and muscles were excised,weighed and stored frozen at?80?C until biochemical determinations.
2.4.Time-to-death(TTD)static-renewal tests
Mortality within96h was the main end point of this study.In order to ensure a low percentage of sur-vivors at96h in the TTD tests,preliminary acute tox-icity tests were performed in accordance with OECD guidelines(1992)to estimate the lethal concentration that causes85%mortality at96h(96-h LC85).Fish were exposed to different nominal concentrations of dichlorvos at24.0±0.5?C in a static-renewal system,where water and pesticide were completely replaced every24h in40-l glass aquaria.These concentration–effect experiments indicated that the median lethal concentration at96h(96-h LC50)for dichlorvos in the European eel was0.852mg l?1(95% con?dence interval(CI),0.735–0.957),and the96-h LC85was1.498mg l?1(95%CI,1.378–1.774).This latter concentration(1.5mg l?1)was then used in the TTD tests to expect a mortality of85%.This nominal concentration of dichlorvos includes 1.7mg l?1of 2-propanol.Although this aliphatic alcohol can poten-tiate the toxicity of carbon tetrachloride(Traiger and Plaa,1971),the96-h LC50of this solvent for fresh-water?sh ranged from4200to11,130mg l?1(WHO, 1990).Therefore,the toxicity of Hexipra Solucion?observed was virtually due exclusively to dichlorvos. One hundred randomly selected eels were separated into two groups.Fifty ice-anaesthetised?sh were in-jected i.p.with1mmol kg?1NAC,whereas the other 50were only injected with the same amount of saline and were assigned to four40-l tanks,receiving25?sh each.Fish were allowed to recover in clean water for 3h because the injection time lasted10min from the ?rst animal injected to the last.After that,?sh were exposed to1.5mg l?1of dichlorvos for96h under semi-static conditions as mentioned before,where water and pesticide were completely replaced once a day.Water temperature was recorded every3h and maintained at24.0±0.5?C in all tanks during the experiment.Fish were continually inspected at3-h intervals,but during the?rst24h they were checked every90min because a higher mortality was expected. Dead animals were immediately removed,the TTD noted,weighed,the length measured and the livers and muscles were excised,weighed and stored frozen at?80?C.At96h,survivors were anaesthetised with ice and processed as previously described.The same TTD experiment was replicated again in order to have 100NAC-treated and100non-treated?sh,and then gain statistical power.
2.5.Glutathione determination
Tissue samples were homogenised with5volumes of ice-cold5%5-sulfosalicylic acid per gram of wet weight tissue,and further processed by sonica-tion(Vibra-Cell,Sonics&Materials Inc.,Danbury, CT,USA).Homogenates were then centrifuged at 20,000×g for20min at4?C.Total glutathione con-tent(tGSx)and oxidised glutathione(GSSG)were de-termined in supernatant fractions with a sensitive and speci?c assay using a recycling reaction of GSH with 5,5 -dithiobis(2-nitrobenzoic acid)(DTNB)in the presence of excess GR according to Baker et al.(1990) in a microplate reader(Model3550,Bio-Rad Labora-tories,Richmond,CA,USA)as previously described (Pe?a-Llopis et al.,2001).Glutathione concentrations were expressed as nmol of GSH equivalents(GSx) per mg of protein(GSx=[GSH]+2×[GSSG]).
S.Pe?a-Llopis et al./Aquatic Toxicology65(2003)337–360341 GSH was calculated by subtracting GSSG levels from
the tGSx levels determined.GSH/GSSG ratio was
expressed as number of molecules but not moles:
GSH GSSG =tGSx?GSSG
GSSG/2
.
2.6.Kinetic enzyme assays
Liver and muscle tissues were homogenised with 5and4volumes,respectively,of Henriksson stabil-ising medium(Henriksson et al.,1986),which con-tained50%glycerol,20mM phosphate buffer pH7.4, 0.5mM EDTA,and0.02%defatted bovine serum al-bumin.?-Mercaptoethanol was not included because it interferes with the GR assay.Homogenates were centrifuged at20,000×g for20min at4?C,and the resulting supernatants were diluted5-or10-fold with buffer and assayed rapidly for enzyme activities.
2.6.1.AChE(EC
3.1.1.7)activity
AChE activity was determined at415nm with acetylthiocholine as substrate in accordance to an adaptation of the Ellman method(Ellman et al., 1961)to microtiter plates by Doctor et al.(1987), but with0.1M phosphate buffer,pH7.27and1mM EDTA as recommended by Riddles et al.(1979).Eel cholinesterase activity detected in muscle was con-sidered as true AChE as was previously characterised (Lundin,1962;Ferenczy et al.,1997).
2.6.2.GR(EC1.6.4.2)activity
The method of Cribb et al.(1989)was used to assay the GR activity through the increase of absorbance at 415nm with reference wavelength at595nm.The?nal concentrations of0.075mM DTNB,0.1mM NADPH, and1mM GSSG were used in accordance to Smith et al.(1988).
2.6.
3.GST(EC2.5.1.18)activity
GST activity was measured through the conjugation of GSH with1-chloro-2,4-dinitrobenzene(CDNB) according to Habig et al.(1974).The assay mixture contained100mM potassium phosphate buffer,pH 6.5,1mM CDNB in ethanol,and1mM GSH.The formation of the adduct of CDNB,S-2,4-dinitrophenyl glutathione,was monitored by measuring the rate of increase in absorbance at340nm with a Multi-skan Ascent microplate reader(Thermo Labsystems, Helsinki,Finland).
2.6.4.γGT(EC2.
3.2.2)activity
?GT activity was determined by the method of Silber et al.(1986).The rate of the substrate ana-logue?-glutamyl-p-nitroanilide cleavage to form p-nitroaniline(pNA)by transfer of a glutamyl moiety to glycylglycine was monitored at405nm for at least 10min.
2.6.5.GCL(EC6.
3.2.2)activity
GCL activity assay was adapted to microtiter plates from the indirect method of Seeling and Meister (1985),which utilises the coupled reaction of pyruvate kinase(PK)and lactate dehydrogenase(LDH)to de-termine the rate of formation of ADP by GCL through the oxidation of NADH.Each well contained0.1M Tris–HCl buffer,pH8,150mM KCl,2mM EDTA, 20mM MgCl2,5mM ATP,2mM phosphoenolpyru-vate,10mM l-glutamate,10mM l-?-aminobutyrate, 0.2mM NADH,7U ml?1PK,and10U ml?1LDH. Enzyme activity was evaluated by following the de-crease in the absorbance of NADH at340nm at25?C with the Multiskan Ascent microplate reader.
A calibration curve of known activities of puri?ed enzymes was used on every96-well plate to avoid mis-calculations resulting from an ill-de?ned path length. AChE(type V)from electric eel,GR(type III)from baker’s yeast,GST from equine liver,and?GT(type I)from bovine kidney were used as standards,whose activities were determined in quartz cuvettes using a Hitachi U-2001UV-Vis spectrophotometer(Hitachi Instruments Inc.,USA).A molar absorption coef?-cient at412nm(ε412)of14.150was used for the di-anion of DTNB(TNB2?)as Riddles et al.(1979)de-termined.As no puri?ed GCL enzyme was available, several samples were used as standards and their activ-ity were validated spectrophotometrically.Reliability of AChE and?GT assays was veri?ed with the stan-dard ACCUTROL TM Normal.Speci?c enzyme activi-ties were expressed as nmoles of substrate hydrolysed per min per milligram of protein(mU mg?1prot).
2.7.Caspase-3assay
Caspase-3activity was measured in96-well plates using the Sigma caspase-3colorimetric assay kit
342S.Pe?a-Llopis et al./Aquatic Toxicology65(2003)337–360
according to the manufacturer’s instructions.The
hydrolysis of the peptide substrate acetyl-Asp-Glu-
Val-Asp p-nitroanilide(Ac-DEVD-pNA)to release
pNA was monitored at405nm and calculated using
a pNA calibration curve,whose concentrations were
determined with a spectrophotometer.Sealed and
light preserved microplates were incubated at25?C
for several days in order to detect extremely low
enzyme activities.Pseudo-zero-order kinetics was
ensured by plotting absorbance against time on every
well.Recombinant human caspase-3was used as a
positive control to validate results.Speci?c enzyme
activity was expressed as pmol of Ac-DEVD-pNA
hydrolysed per min per mg protein(?U mg?1prot).
The DEVDase activity measured was considered as
caspase-3-like because caspase-7is another key ex-
ecutioner of apoptosis that has similar function and
substrate speci?city to caspase-3(Fernandes-Alnemri
et al.,1995).
2.8.Protein determination
Protein content was determined by the Bio-Rad Pro-
tein Assay kit(Bio-Rad Laboratories GmbH,Munich,
Germany)based on the Bradford dye-binding proce-
dure,using bovine serum albumin as standard.
2.9.Statistics
The96-h lethal concentrations(LC50and LC85)
were determined with the Probit Analysis procedure
using the SPSS10.0statistical software package
(SPSS Inc.,Chicago,IL,USA),which was used for
all other statistical analyses.Survival curves were
constructed using the Kaplan–Meier method(Kaplan
and Meier,1958)and compared by the log-rank χ2statistics.Two-factor ANOV A with the type III sum-of-squares method was used to investigate the
effect of pre-treatment and pesticide exposure and
their interaction on studied variables.The time de-
pendence of variables after NAC injection in controls
was also tested by the two-way ANOV A.A priori
contrasts between selected single levels of factors
were made to compare means.Variables with hetero-
geneity of variances,according to the Levene test,
were properly transformed.Pearson correlation coef-
?cients were calculated among all studied parameters
of dichlorvos-exposed eels in order to measure the
strength of a linear association between two variables.These relationships were also tested after removing the effect of TTD by means of partial correlations. These variables were checked for normality with the Kolmogorov–Smirnov test with Lilliefors signi?cance correction,and data not normally distributed were appropriately transformed.Sequential Bonferroni cor-rection was applied to multiple signi?cance tests to avoid spurious signi?cant differences(Rice,1989). As standard ANOV A-type and common multivari-ate regression methods cannot be used for survival data because of the presence of censored observa-tions and skewing of the data(Piegorsch and Bailer, 1997),the Cox proportional hazards regression model (Cox,1972)was used to determine the relationship between dichlorvos mortality and studied variables. Unadjusted hazard ratios were obtained from univari-ate Cox proportional hazard models.Adjusted hazard ratios were obtained from signi?cant explanatory variables determined using a multivariate stepwise forward selection procedure from all covariates based on conditional parameter estimates.The P≤0.05 and P>0.10were set,respectively,as limits for variable inclusion and exclusion.These covariates were then adjusted for the effect of length and weight. The assumption of proportional hazards was ensured by visual inspection of the smoothed plots of scaled Schoenfeld residuals(Schoenfeld,1982)versus sur-vival time,in accordance with Hess(1995),and the plots of martingale residuals against the covariates.
3.Results
3.1.Dichlorvos mortality
Mortality observed upon exposure to the96-h LC85 of dichlorvos was91%for eels pre-treated with saline (92and90%in the?rst and second replicate of the ex-periment,respectively),whereas it was of85%in NAC pre-treated?sh(86and84%in the?rst and second replicate,respectively).Replicates showed no different survival curves when compared stratifying for treat-ment(log-rankχ2=0.6,P=0.43).Aquaria also did not affect survival in saline-treated?sh(log-rankχ2= 0.6,P=0.90)nor NAC-treated?sh(log-rankχ2=
1.8,P=0.60),thus data of replicates and aquaria were pooled.Then,only nine of the100?sh injected with the vehicle survived within the96h of the study
S.Pe?a-Llopis et al./Aquatic Toxicology65(2003)337–360
343 Fig.1.Kaplan–Meier estimates of survival of eels i.p.injected either with1mmol kg?1NAC or its vehicle(saline)and,after3h,were exposed to1.5mg l?1(the96-h LC85)of dichlorvos.Censored observations at the end of the observation time were represented by crosses.
period,with a mean survival of25h(95%CI,20–30), while15of the100?sh pre-treated with NAC sur-vived,with a mean survival of34h(95%CI,28–41). Therefore,eels pre-treated with1mmol kg?1of NAC presented a66.7%higher survival than non-treated ?sh(log-rankχ2=7.8,P<0.005;Fig.1),which was more evident within the?rst24h.
3.2.Effect of NAC and/or dichlorvos on biochemical parameters
Basically,?sh exposure to the96-h LC85of dichlorvos resulted in a decrease of the hepatic and muscular GSH levels(P<0.001;Table1),but a muscular GSSG increase(P<0.01)that lowered the GSH/GSSG ratio in the muscle(P<0.001).The glutathione redox status was also decreased in the liver(P<0.001).The activities of hepatic GR and GST,hepatic and muscular?GT,hepatic GCL and caspase-3-like,and especially muscular AChE were also diminished(P<0.001),whereas GST activity increased in the muscle(P<0.001).Conversely, NAC treatment achieved an increase of the GSH con-tent and GSH/GSSG ratio in the liver(P<0.001) and muscle(P<0.001and0.01,respectively),in ad-dition to an enhancement of hepatic GR,GST,GCL (P<0.001),and?GT(P<0.01)activities,and mus-cular GST(P<0.05).Interactions of treatment and dichlorvos exposure were only found on the hepatic and muscular?GT activities(P<0.05).
The single i.p.injection of1mmol kg?1NAC in-creased the levels of hepatic and muscular GSH by 39and14%(P<0.001and0.05,respectively),hep-atic GSH/GSSG ratio by53%(P<0.001),hepatic GR activity by12%,hepatic and muscular GST activ-ity by18and16%(P<0.01and0.05,respectively), and hepatic GCL activity by31%(P<0.001).How-ever,GSH content in the liver was time-dependent (Fig.2B).Three hours after the injection,GSH rose (P<0.05)and reached a two-fold increase at12h (P<0.001),that returned to baseline after48h.The 12h after the injection corresponded with the9h after dichlorvos exposure,and the beginning of?sh mortali-ties(Fig.1).The administration of NAC also enhanced the hepatic GSH/GSSG ratio by134%(Fig.3B)and GR activity by26%(Fig.3D)at the?rst3h(P< 0.001and0.01,respectively),but were not different afterwards,except for GR activity at96h(P<0.01). The decrease of glutathione levels and enzyme ac-tivities found in dichlorvos-exposed eels were amelio-rated with NAC pre-treatment(Table1).Hepatic and muscular GSH levels of NAC-treated animals were59 and16%higher(P<0.001and0.01,respectively), as can be observed in Figs.2A and4A,which resulted
344
S.Pe?a-Llopis et al./Aquatic Toxicology 65(2003)337–360
(A)
(B)
H e p a t i c G S H (n m o l G S x m g -1
p r o t )
20
40
60
80
100
120
Time After ip Injection (h)
H e p a t i c G S H (n m o l G S x m g -1
p r o t )
Fig.2.Effect of NAC treatment on hepatic GSH levels.(A)Eels received a single i.p.injection of 1mmol kg ?1NAC,or its vehicle,3h before that were exposed to dichlorvos (1.5mg l ?1,the 96-h LC 85).Data represent individual GSH levels at mortality time or at the end of the experiment (96h).(B)Fish were given a single dose of NAC (1mmol kg ?1i.p.),or saline,and were maintained in clean water.Data are means ±S .E .(n =5).?P <0.05,???P <0.001,respectively,compared to controls at the same post-injection time.Vertical dotted lines indicate overlapping times for i.p.injection.
in 29and 9%lower GSH diminution than non-treated ?sh compared to non-exposed animals.The GSSG increase in muscle as a consequence of dichlorvos ex-posure was 13%lower in NAC-treated ?sh.This fact elevated the GSH/GSSG ratio by 37%(P <0.001)in the muscle,which was an 18%less oxidised than controls.Glutathione redox status in the liver of NAC-treated ?sh (Fig.3A )was also 41%superior.Activities of hepatic GR (Fig.3C ),GST (Fig.5A ),?GT (Fig.5B ),GCL (Fig.5C ),and caspase-3-like (Fig.5D )were increased by 24%(P <0.001),8%(P <0.01),33%(P <0.001),44%(P <0.001),and 18%(P <0.01),respectively,than non-treated ?sh.When compared to non-exposed ?sh,these activities were 16,6,22,29,and 8%,respectively,less inhibited than saline-treated animals.However,no signi ?cant differences of enzyme activities were found in the skeletal muscle between treatments (Fig.4B –D ).
S.Pe?a-Llopis et al./Aquatic Toxicology65(2003)337–360349
As glutathione levels and enzyme activities were measured in dead?sh,the time between the?sh death to when its tissues were excised and frozen at?80?C might have allowed the autooxidation of GSH,cleav-age of GSH and GSSG by?GT(Anderson,1985),and protein degradation(Gallenkamp et al.,1981).How-ever,although eel liver and muscle were not espe-cially rich in?GT compared to other tissues(Tate and Meister,1981)or species(Sulakhe and Lautt,1985), and intervals of time were inferior to3h,we performed an extra experiment in order to test the effect of mor-tality on possible glutathione loss and oxidation and protein degradation.We compared the glutathione lev-els and enzyme activities in the liver and muscle of ?ve eels excised immediately after being killed by a blow on the head with other?sh kept in clean water at 24?C for1.5,3,and6h after being euthanised.There were no signi?cant differences between?sh sampled immediately and those sampled1.5and3h after death, but GSH was slightly diminished and oxidised and GR inhibited in the liver and muscle of?sh maintained for 6h in water after death(Dunnet’s test,P<0.05;data not shown).Therefore,the effect of postmortem de-lay on glutathione levels and enzyme activities in this study seemed to be negligible.
3.3.Bivariate and partial correlations
Tables2and3show correlations among parame-ters in?sh exposed to dichlorvos.Hepatic GSH con-tent was correlated with the length and weight of non-treated eels(r=0.40and0.35,respectively). Caspase-3-like activity was linearly correlated with the hepatic GSH/GSSG ratio of saline-and NAC-treated eels(r=0.52and0.55,respectively).Furthermore, the pattern of individual caspase-3-like activity against TTD(Fig.5D)was very similar to that of hepatic GSH/GSSG ratio(Fig.3A),which suggests that the caspase-3-like activity may be regulated by the glu-tathione redox status as previously stated by Ueda et al. (1998).Caspase-3-like activity was also related to hep-atic GSH(r=0.46),GR(r=0.50),and GST(r= 0.50)activity of saline-treated eels.GCL activity was correlated with hepatic GSH(r=0.43),GSH/GSSG ratio(r=0.54),?GT activity(r=0.50),and hep-atic and muscular GR activity(r=0.61and0.37, respectively).Other signi?cant correlations found in saline-treated eels were between GST activity and GSH in the liver(r=0.39),between the?GT activity in the liver and muscle(r=0.36),and between mus-cular AChE and GR(r=0.36),and GST(r=0.36) activities.However,many relationships between two variables were non-signi?cant when the linear effect of the TTD was removed,because both variables were re-lated to the TTD.Conversely,after adjusting for TTD, the hepatic and muscular GSH/GSSG ratios were cor-related in saline-and NAC-treated eels(r=0.31and 0.31,respectively,P<0.01;data not shown).
3.4.Cox proportional hazards models
The Cox proportional hazards model is a semi-parametric multivariate regression method used to estimate the effects of several explanatory variables (covariates)on survival.Although this model does not assume a particular mathematical distribution for the survival times,it assumes that the effects of the different covariates on survival do not change over time.The hazard ratio(HR)evaluates the percentage change in risk with each unit change in the covariate. When HR is higher than1,the risk of death rises when the covariate increases;when HR is lower than1,the risk of death decreases when the covariate increases. Univariate Cox models(Table4)show that hep-atic GSH,GSH/GSSG ratio,GR,GST,GCL,and caspase-3-like activities were signi?cantly associated with dichlorvos survival in saline-treated?sh when each covariate was considered solely on the model (P<0.001,except for hepatic GST;P=0.0014). The risk of death of these?sh would decrease by 4.3%on every increase of one unit in the GSH con-tent(one nmol of GSx mg?1prot).Each increase in one GSH/GSSG unit would reduce independently the hazard of death by3.6%.The risk of death of saline-treated?sh would decrease independently by 5.3,0.13,0.8,or14%on every unit increase of the ac-tivity of the enzymes GR,GST,GCL,and caspase-3, respectively.On the contrary,muscular GSH,hep-atic and muscular GSSG,and muscular AChE ac-tivity were negatively associated with survival(P< 0.001).In the case of NAC-treated?sh,the hepatic glutathione redox status and the GR,GST,?GT,GCL, and caspase-3-like activities were independently and positively associated with survival(P<0.001,ex-cept for hepatic GST;P=0.008).The hepatic and muscular GSH,hepatic GSSG,and muscular AChE
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Table4
Unadjusted hazard ratios relating survival under dichlorvos exposure to studied variables of?sh pre-treated with saline or1mmol kg?1NAC Covariates Saline NAC
HR(95%CI)P HR(95%CI)P Length0.925(0.856–1.009)0.0470.882(0.797–0.977)0.016 Weight0.951(0.902–1.006)0.070.918(0.858–0.983)0.014 Liver
GSH0.957(0.938–0.983)2×10?5 1.023(1.012–1.034)5×10?5 GSSG 1.320(1.181–1.299)9×10?7 1.342(1.227–1.468)10?10 GSH/GSSG0.964(0.953–0.983)8×10?90.965(0.954–0.977)2×10?9 GR0.947(0.922–0.973)5×10?50.942(0.910–0.976)0.0008 GST0.999(0.998–0.999)0.00140.999(0.998–1.000)0.008
?GT0.979(0.929–1.032)0.430.913(0.869–0.958)0.0002 GCL0.992(0.987–0.996)0.00050.987(0.984–0.991)10?10 Caspase-30.864(0.813–0.934)3×10?60.856(0.805–0.910)6×10?7 Muscle
GSH 1.271(1.177–1.372)10?9 1.072(1.023–1.122)0.003 GSSG 1.593(1.230–2.063)0.00040.974(0.787–1.205)0.81 GSH/GSSG 1.011(0.982–1.040)0.46 1.010(0.988–1.033)0.37
GR0.881(0.778–1.016)0.0440.947(0.804–1.116)0.51
GST0.994(0.976–1.012)0.500.989(0.973–1.006)0.21
?GT 1.070(0.669–1.287)0.78 1.163(0.661–2.049)0.60 AChE 1.123(1.065–1.124)2×10?5 1.158(1.091–1.230)10?6 Italic P values indicate signi?cant ones atα=0.05after sequential Bonferroni correction for multiple tests.HR:hazard ratio,CI:con?dence interval.
activity were negatively and independently associated with survival when each covariate was considered solely on the model(P<0.001).The paradoxical negative association of GSH contents and AChE ac-tivity with survival does not mean that having less levels will decrease the risk of death.Simply,mus-cular GSH content and AChE activity was lower in resistant?sh because the exposure time was greater. Therefore,more GSH consumption and AChE inhi-bition was observed.
Dichlorvos mortality can be explained as a conse-quence of the effects of several covariates(Table5). The best explanatory variables associated with survival in non-treated eels were muscular GSH(P= 8×10?7),hepatic GSH/GSSG ratio(P=10?5), muscular AChE(P=2×10?5),and hepatic and mus-cular GR activity(P=0.033and0.049,respectively). Hence,?sh with higher hepatic glutathione redox sta-tus or hepatic or muscular GR activity are expected to have2.8,3.2,or14%less probability of dying, respectively,on each unit change of these parameters, after adjustment for the other variables in the model. When?sh were pre-treated with NAC,survival was much more dependent on the availability of GSH(P= 2×10?5)and maintenance of the glutathione redox status(P=3×10?5)than on the AChE activity (P=0.006).In addition,in excess of glutathione,the activities of hepatic GCL(P=0.0002)and GR(P= 0.048),and muscular GST(P=0.019)were decisive for prolonged survival.
The Cox regression analysis was also carried out in all animals exposed to the pesticide to determine the net weight of the treatment after adjusting for the other explanatory variables in the model.As a result, pre-treatment with NAC decreased the risk of death by51%(P=0.0008).Hepatic glutathione redox sta-tus(P=4×10?11),GCL activity(P=0.0007), and muscular GR activity(P=0.0002)were inde-pendently related to higher survival,whereas muscu-lar(P=0.00018)and hepatic(P=0.0006)GSH, and AChE activity(P=4×10?8)were negatively associated with survival.
Despite the fact that the caspase-3-like activity was highly related to survival when considered solely in the model,it was not among the best explanatory vari-ables when adjusted to the other covariates.This is
S.Pe?a-Llopis et al./Aquatic Toxicology65(2003)337–360353
Table5
Adjusted hazard ratios of the best explanatory variables related to dichlorvos mortality of European eels pre-treated with saline or 1mmol kg?1NAC
Covariates HR(95%CI)P
Saline-treated?sh
Hepatic GSH/GSSG0.972(0.959–0.984)10?5 Hepatic GR0.968(0.939–0.997)0.033 Muscular GSH 1.266(1.153–1.390)8×10?7 Muscular GR0.860(0.739–0.999)0.049 Muscular AChE 1.134(1.071–1.202)2×10?5 NAC-treated?sh
Hepatic GSH 1.031(1.017-1.045)2×10?5 Hepatic GSH/GSSG0.972(0.959–0.985)3×10?5 Hepatic GR0.960(0.923–1.000)0.048 Hepatic GCL0.992(0.988–0.996)0.0002 Muscular GST0.977(0.957–0.996)0.019 Muscular AChE 1.115(1.033–1.205)0.006
All the individuals
Treatment0.493(0.326–0.746)0.0008 Hepatic GSH 1.020(1.009–1.032)0.0006 Hepatic GSH/GSSG0.967(0.957–0.977)4×10?11 Hepatic GCL0.994(0.991–0.998)0.0007 Muscular GSH 1.085(1.040–1.132)0.00018 Muscular GR0.815(0.730–0.909)0.0002 Muscular AChE 1.133(1.084–1.185)4×10?8 A stepwise forward selection procedure from all covariates was used on each model to obtain those covariates more associated with survival.Length and weight were added to these models in order to adjust for them.HR:hazard ratio,CI:con?dence interval. because the contribution of each variable to the regres-sion model is determined in the context of the contri-bution of all the other variables in the model.Then,as caspase-3-like activity was strongly correlated to the GSH/GSSG ratio(Tables2and3),the latter entered ?rst in the equation excluding the caspase-3-like ac-tivity.If the model were constructed without the glu-tathione redox status,the caspase-3-like activity could be among the best explanatory variables(data not shown).Hence,the caspase-3-like activity can also be considered as signi?cant related to survival.
4.Discussion
The present work demonstrates the relevance of the glutathione metabolism in the tolerance to dichlorvos exposure.A lethal concentration of this OP pesticide (1.5mg l?1,the96-h LC85)decreased and oxidised glutathione levels and inhibited enzyme activities in a higher degree than that observed in?sh exposed to a sublethal concentration of dichlorvos(Pe?a-Llopis et al.,2003).The oxidation of GSH and inhibition of GR indicated the presence of oxidative processes, and was in agreement with Hai et al.(1997),who found a GSH diminution in the carp liver and mus-cle,and AChE inhibition after being exposed to1 and5mg l?1of dichlorvos for24h.Dichlorvos also decreased glutathione levels and inhibited AChE and GPx activities in several tissues of rats(Julka et al., 1992).
This study also demonstrates that NAC injected i.p.improved eel survival upon dichlorvos exposure. The single i.p.injection of this drug enhanced the GCL activity and increased transiently the hepatic GSH lev-els two-fold,although de novo synthesis of glutathione by GCL is regulated by feedback inhibition of GSH (Richman and Meister,1975).NAC also increased the muscular GSH and hepatic glutathione redox status, in addition to the activities of GR in the liver and GST in the liver and muscle.Therefore,in general,and es-pecially during the?rst24h,NAC-treated?sh pre-sented higher GSH levels and GSH/GSSG ratios in the liver and muscle,and less inhibited GR,GST,?GT, and GCL activities in the liver.These features allowed them to maintain GSH levels,detoxify dichlorvos,and remove the ROS generated by it more ef?ciently than saline-treated animals,and for that reason,improve tolerance.
Mortality in non-treated?sh was dependent pri-marily on muscular GSH,hepatic GSH/GSSG ratio, muscular AChE activity,and hepatic and muscular GR activity.This was in agreement with previous re-sults of A.anguilla exposed to the herbicide moli-nate(Pe?a-Llopis et al.,2001).On the other hand, in NAC-treated?sh survival relied principally on the individual capacity to maintain and enhance the hep-atic GSH/GSSG ratio and synthesise GSH by GCL, because GSH was in excess during the?rst24h of dichlorvos exposure.The survival in this group was less dependent on muscular AChE and GST activities and hepatic GR activity.It is remarkable that hepatic GST activity was not among the best explanatory vari-ables of dichlorvos mortality,although this enzyme is directly linked to dichlorvos detoxi?cation and was re-lated to TTD of treated and non-treated?sh when con-sidered solely in the model.This suggests that the role
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of glutathione in extending survival would be more related with the removal of free radicals than with the detoxi?cation of dichlorvos.
Although NAC-treated?sh were two times less likely to die within the study period of96h,survival to dichlorvos exposure was mainly dependent on hep-atic glutathione redox status.As this dependence was greater than that of the muscular AChE activity,the glutathione redox status represents a better biomarker of effect and individual susceptibility than the mus-cular AChE activity.In addition,the individual dif-ferences in glutathione levels and enzyme activities were more important than the effect of the treatment to explain survival.Then,those?sh that were able to preserve or increase the GSH/GSSG ratio were expected to live longer upon dichlorvos exposure than those that lost redox homeostasis.Similarly,we found that mortality of two marine bivalves exposed to the OP insecticide fenitrothion was also associated with impaired glutathione redox status(Pe?a-Llopis et al.,2002).Raising the GSH/GSSG ratio by reduc-ing GSSG to two molecules of GSH is energetically less costly than synthesising GSH de novo.Despite the relevance of GR in the tolerance to oxidative stress(Pe?a-Llopis et al.,2001),its activity also de-pends on NADPH availability.Then,the individual capacity to enhance enzyme activities of gluconeo-genesis and the pentose phosphate pathway could also determine the tolerance to the OP.In fact,resis-tance to oxidative stress and GSH levels were found to be modulated by several of these enzymes,such as glucose-6-phosphate dehydrogenase(Salvemini et al.,1999),transaldolase(Banki et al.,1996), and cytosolic NADP+-dependent isocitrate dehy-drogenase(Lee et al.,2002).In addition,Godon et al.(1998),using a proteomic approach,established that over100proteins whose levels changed after ox-idative stress in yeast(Saccharomyces cerevisiae),a quarter were involved in the repression of glucolysis and the tricarboxylic acid cycle,and redirection of the carbohydrate metabolism towards the regenera-tion of NADPH at expense of ATP.V oehringer et al. (2000),using DNA microarrays,found that cells re-sistant to apoptosis preserved high intracellular pools of GSH by enhancing pathways for establishment and maintenance of high intracellular redox potential. Glutathione depletion is considered a biomarker of environmental stress as it was observed in?sh stressed either by chemical or natural pollutants (e.g.Chatterjee and Bhattacharya,1983;Allen et al., 1988;Gallagher et al.,1992;Hasspieler et al.,1994; Almar et al.,1998;Vaglio and Landriscina,1999; Pe?a-Llopis et al.,2001).Nevertheless,hepatic GSH synthesis can be induced as a consequence of the pollutant interaction with GCL,and then increase GSH beyond control levels,as it was observed in sev-eral?sh studies(Stein et al.,1992;Hasspieler et al., 1994;Pe?a et al.,2000;Pe?a-Llopis et al.,2001). Conversely,diminution of muscular GSH content seems to be an intrinsic factor of pesticide exposure, because the longer the exposure,the lower its values. Glutathione levels in muscle depend largely on GSH capture from plasma by?GT(Grif?th and Meister, 1979).It is known that the liver is the main source of plasma GSH(Kaplowitz et al.,1985).Therefore, if there was hepatic GSH depletion,less glutathione could be exported to the plasma,and less would be available for extrahepatic tissues,such as muscle and brain.This would conduce to the exhaustion of muscular GSH.Thus,as GSH replenishment in the muscle is more dif?cult than in the liver,depletion of GSH in muscle could be a better biochemical marker of pollutant exposure than in liver.
Domenicotti et al.(2000)found an apoptotic path-way dependent on glutathione depletion.A moder-ate perturbation of the cellular redox state can be suf?cient to activate protein kinase C novel iso-forms and induce the nuclear binding of the activator protein-1(AP-1),leading to apoptosis.In fact,many cellular signalling molecules(such as protein tyro-sine and serine/threonine kinases and phosphatases, Ca2+channels and transporters,and transcription factors like NF-?B,p53and AP-1)are regulated by the redox state of cells(Kamata and Hirata,1999). Therefore,an important oxidation of the hepatic glu-tathione redox status of?sh by dichlorvos can alter transcriptional responses to induce programmed cell death(Esteve et al.,1999).Dichlorvos-exposed?sh showed marked inhibition of the caspase-3-like activ-ity compared to non-exposed animals.Caspases have cysteine residues in the catalytic domain(Wilson et al.,1994)that are prone to oxidation or thiol alky-lation,and therefore,are regulated by the oxidative stress and intracellular redox state(Ueda et al.,1998). Many studies have demonstrated that oxidant-induced inhibition of caspase-3-like activity is suf?cient to
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switch apoptosis to necrosis (e.g.Lemaire et al.,1998;Samali et al.,1999).Hence,our results suggest that ?sh individual differences in the homeostasis of the hepatic glutathione redox status can determine the degree of dichlorvos-induced oxidative stress and caspase-3-like inactivation,resulting in hepatic cell death by necrosis.Schafer and Buettner (2001)found that the glutathione redox status can be indicative of the biological status of the cell.A decrease in the GSH/GSSG ratio may shift cells through different bi-ological stages,such as proliferation,differentiation,apoptosis and,at very low values,necrosis.In our study,NAC treatment diminished caspase-3-like in-hibition,allowing higher ?sh tolerance to dichlorvos.Evidence indicates that NAC can decrease
apoptosis Fig.6.Proposal of the involvement of the glutathione metabolism and NAC in the tolerance to dichlorvos mortality.The OP inhibits the AChE activity,leading to excessive accumulation of ACh and overstimulation of muscarinic receptors.This process produces neuronal hyperexcitation and seizures that allow the release of EAA,such as Glu and Asp,which activate the NMDA receptor.Excessive activation of NMDA receptors triggers the in ?ux of large amounts of extracellular Ca 2+,which activates proteolytic enzymes,nitric oxide,and leads to formation of free radicals.Then,GSH oxidises to GSSG to remove ROS,catalysed by GPx or non-enzymatically.GSSG is subsequently reduced to GSH by GR at expense of oxidising NADPH to NADP +,which is recycled by the pentose phosphate pathway.In addition,GSH can be conjugated to dichlorvos by GST allowing the detoxi ?cation of the pesticide.The antioxidant NAC acts as a reductant and free radical scavenger that can also be deacetylated to Cys,the limiting amino acid for glutathione synthesis,which is regulated by GCL.Extracellular GSH is degraded to its catabolites by ?GT and dipeptidase,enabling intracellular GSH synthesis.Excessive ROS can lead to high oxidative stress,which activates caspases and induces cell death by apoptosis.However,when oxidative stress is very high —like in this study —caspases are inhibited leading to cell death by necrosis.Ultimately,these tissular damages can cause the death of the organism.
and necrosis in numerous systems (e.g.Atkins et al.,2000;Rojas et al.,2002).
According to the impairment-disability concept of Depledge (1989),an initial perturbation caused by a toxic exposure will generate responses in order to com-pensate the cellular and tissular homeostasis of an or-ganism.This compensatory phase will be established with higher stress-induced disturbance,but when fur-ther repair and/or compensation are impossible,the organism dies.Tolerance to pollutants generally fol-lows a log-normal distribution (as is assumed in the Probit model for dose –response relationships).Two hypotheses have been proposed to explain it.Gaddum (1953)introduced the concept of individual tolerance or individual effective dose,where every individual
356S.Pe?a-Llopis et al./Aquatic Toxicology65(2003)337–360
has an innate background to tolerate a pollutant until it receives a dose equal to or greater than its individ-ual effective dose,and then dies.Conversely,mortal-ity can be the result of stochastic processes occurring within all individuals with the chance of death be-ing essentially the same for all individuals receiving a dose.Although the?rst hypothesis remained univer-sally accepted,Newman and McCloskey(2000)found that the tolerance to a lethal concentration of a toxicant is neither due to innate qualities of individuals nor stochastic processes.Therefore,according to informa-tion provided in this study,we propose an intermediate hypothesis to explain tolerance to the OP dichlorvos: tolerance depends on maintaining and increasing the hepatic glutathione redox status.Therefore,individ-ual regulations to synthesise GSH,enhance GR ac-tivity or other redox systems,or derive carbohydrate metabolism towards NADPH regeneration,will mod-ulate intracellular redox potentials.When glutathione metabolism is impaired and is not rapidly restored, oxidative stress generated by the OP prevails and may ?nally lead to cell death by necrosis,resulting in tis-sular dysfunction,and eventually?sh death(Fig.6). As mentioned before,glutathione depletion in the liver can allow the exhaustion of glutathione levels in ex-trahepatic tissues,such as brain and muscle.These tissues are more susceptible than the liver to oxida-tive damage because antioxidant defences are more limited(Facchinetti et al.,1998).We suggest that ex-cessive dichlorvos-induced oxidative stress might lead to suf?cient brain and muscle necrosis,causing cen-tral nervous system and muscular dysfunction(Santos et al.,2002),which results in respiratory distress and, at last,the death of the organism.
As OP pesticides are extensively used in widespread applications,poisoning by these compounds repre-sents a serious public health problem,especially since OPs are used as agents of chemical warfare(Goozner et al.,2002).Although the standard treatment against OP poisoning consists of reactivation of the inhibited AChE with an oxime and reversal of the biochemical effects of acetylcholine with atropine(Kwong,2002), the present study demonstrates that NAC may be a complementary antidote for OP poisoning,acting at different target sites.NAC characteristics,such as its low toxicity,low price,and high solubility makes it suitable for treatment of OP intoxications in?sh,es-pecially prophylactically before antiparasitic actions.In fact,bath treatments with NAC improve the recov-ery of OP-intoxicated eels(Pe?a-Llopis et al.,2003).
5.Conclusions
1.Tolerance to the OP dichlorvos can be explained
as the individual capacity of maintaining and in-creasing the hepatic GSH/GSSG ratio through glutathione metabolism.An impairment of the glutathione redox status could lead to excessive oxidative stress,necrosis,and eventually the death of the organism.
2.NAC improves?sh survival to a lethal concentra-
tion of dichlorvos by enhancing the glutathione metabolism and decreasing the glutathione loss and oxidation,and enzyme inactivation caused by the OP pesticide.Therefore,NAC increases the tolerance to dichlorvos-induced oxidative stress and necrosis,being a potential antidote for OP poi-soning and complementary to current treatments.
3.Muscular GSH depletion was a better biomarker
of pollutant exposure than the liver.Hepatic glu-tathione redox status,GR,GCL,and caspase-3-like activities,in addition to muscular AChE activity, can be used as biochemical markers of effect and individual susceptibility to dichlorvos mortality or other pollutants that induce oxidative stress Acknowledgements
This work was supported by the CICYT-FEDER Grant1FD1997-0943-CO2-02.Samuel Pe?a-Llopis was recipient of a research grant from the Spanish Council for Scienti?c Research(CSIC).The authors thank Germán Ca?avate for his help in checking?sh mortality and performing some biochemical deter-minations;Dr.Guido van den Thillart and Richard van Heeswijk for providing the Multiskan Ascent microplate reader;and Dr.Carlos Saavedra for re-viewing the manuscript.The?sh farm Valenciana de Acuicultura S.A.is also greatly acknowledged for kindly providing European eels.
Appendix A.General comments
The GSH levels and AChE activity were measured spectrophotometrically by kinetic enzyme assays.
、 毕业设计（论文）外 译文题目：轴承的摩擦与润滑 2010年 10 月 15 日 外文文献原文： Friction , Lubrication of Bearing In many of the problem thus far , the student has been asked to disregard or neglect friction . Actually , friction is present to some degree whenever two parts are in contact and move on each other. The term friction refers to the resistance of two or more parts to movement. Friction is harmful or valuable depending upon where it occurs. friction is necessary for fastening devices such as screws and rivets which depend upon friction to hold the fastener and the parts together. Belt drivers, brakes, and tires are additional applications where friction is necessary. The friction of moving parts in a machine is harmful because it reduces the mechanical advantage of the device. The heat produced by friction is lost energy because no work takes place. Also , greater power is required to overcome the increased friction. Heat is destructive in that it causes expansion. Expansion may cause a bearing or sliding surface to fit tighter. If a great enough pressure builds up because made from low temperature materials may melt. There are three types of friction which must be overcome in moving parts: (1)starting, (2)sliding, and(3)rolling. Starting friction is the friction between two solids that tend to resist movement. When two parts are at a state of rest, the surface irregularities of both parts tend to interlock and form a wedging action.
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外文翻译 专业机械设计制造及其自动化学生姓名刘链柱 班级机制111 学号1110101102 指导教师葛友华
外文资料名称： Design and performance evaluation of vacuum cleaners using cyclone technology 外文资料出处：Korean J. Chem. Eng., 23(6), (用外文写) 925-930 (2006) 附件： 1.外文资料翻译译文 2.外文原文
应用旋风技术真空吸尘器的设计和性能介绍 吉尔泰金，洪城铱昌，宰瑾李， 刘链柱译 摘要：旋风型分离器技术用于真空吸尘器 - 轴向进流旋风和切向进气道流旋风有效地收集粉尘和降低压力降已被实验研究。优化设计等因素作为集尘效率，压降，并切成尺寸被粒度对应于分级收集的50％的效率进行了研究。颗粒切成大小降低入口面积，体直径，减小涡取景器直径的旋风。切向入口的双流量气旋具有良好的性能考虑的350毫米汞柱的低压降和为1.5μm的质量中位直径在1米3的流量的截止尺寸。一使用切向入口的双流量旋风吸尘器示出了势是一种有效的方法，用于收集在家庭中产生的粉尘。 摘要及关键词：吸尘器; 粉尘; 旋风分离器 引言 我们这个时代的很大一部分都花在了房子，工作场所，或其他建筑，因此，室内空间应该是既舒适情绪和卫生。但室内空气中含有超过室外空气因气密性的二次污染物，毒物，食品气味。这是通过使用产生在建筑中的新材料和设备。真空吸尘器为代表的家电去除有害物质从地板到地毯所用的商用真空吸尘器房子由纸过滤，预过滤器和排气过滤器通过洁净的空气排放到大气中。虽然真空吸尘器是方便在使用中，吸入压力下降说唱空转成比例地清洗的时间，以及纸过滤器也应定期更换，由于压力下降，气味和细菌通过纸过滤器内的残留粉尘。 图1示出了大气气溶胶的粒度分布通常是双峰形，在粗颗粒（>2.0微米）模式为主要的外部来源，如风吹尘，海盐喷雾，火山，从工厂直接排放和车辆废气排放，以及那些在细颗粒模式包括燃烧或光化学反应。表1显示模式，典型的大气航空的直径和质量浓度溶胶被许多研究者测量。精细模式在0.18?0.36 在5.7到25微米尺寸范围微米尺寸范围。质量浓度为2?205微克，可直接在大气气溶胶和 3.85至36.3μg/m3柴油气溶胶。
Optimum blank design of an automobile sub-frame Jong-Yop Kim a ,Naksoo Kim a,*,Man-Sung Huh b a Department of Mechanical Engineering,Sogang University,Shinsu-dong 1,Mapo-ku,Seoul 121-742,South Korea b Hwa-shin Corporation,Young-chun,Kyung-buk,770-140,South Korea Received 17July 1998 Abstract A roll-back method is proposed to predict the optimum initial blank shape in the sheet metal forming process.The method takes the difference between the ?nal deformed shape and the target contour shape into account.Based on the method,a computer program composed of a blank design module,an FE-analysis program and a mesh generation module is developed.The roll-back method is applied to the drawing of a square cup with the ˉange of uniform size around its periphery,to con?rm its validity.Good agreement is recognized between the numerical results and the published results for initial blank shape and thickness strain distribution.The optimum blank shapes for two parts of an automobile sub-frame are designed.Both the thickness distribution and the level of punch load are improved with the designed blank.Also,the method is applied to design the weld line in a tailor-welded blank.It is concluded that the roll-back method is an effective and convenient method for an optimum blank shape design.#2000Elsevier Science S.A.All rights reserved. Keywords:Blank design;Sheet metal forming;Finite element method;Roll-back method
A Case Study of Pattern-based Software Framework to Improve the Quality of Software Development Chih-Hung Chang, Chih-Wei Lu Dept. of Information Management, Hsiuping Institute of Technology No.11, Gongye Rd., Dali City, Taichung County, Taiwan(R.O.C.) 886-4-24961123 ext 3112 {chchang,cwlu}@ https://www.doczj.com/doc/242837454.html,.tw William C. Chu Dept. of Computer Science and Information Engineering, Tunghai University No.181, Sec. 3, Taichung Port Rd.,Taichung City, Taiwan (R.O.C.) 886-4-23508983 cchu@https://www.doczj.com/doc/242837454.html,.tw Nien-Lin Hsueh Dept. of Information Engineering and Computer Science, Feng Chia University No. 100 Wenhwa Rd., Taichung, Taiwan (R.O.C.) 886-4- 24517250 ext 3773 nlhsueh@https://www.doczj.com/doc/242837454.html,.tw Chorng-Shiuh Koong Dept. of Computer and Information Science, Taichung University No.140, Ming-Sheng Rd., Taichung City, Taiwan (R.O.C.) 886-4-22183804 csko@https://www.doczj.com/doc/242837454.html,.tw ABSTRACT In recent years, development of the software industry and demand for software systems have increased rapidly, but developers often does not know whose suggestion to follow regarding methodologies of software engineering. One reason for that is the difficulty in applying new software engineering technologies. Developers take a long time to train. Another reason is the difficulty in integrating CASE toolsets. So many indeterminate factors make the development process more and more complex. On the other hand, software development is too customized, and software reuse is difficult. T he reasons above are the cause for software development and maintenance to become more complex and difficult to control. In this paper we explore the importation of a software pattern-based framework, and the development of an ERP/support chain system. Based on software patterns, developers can separate development and business so as to reduce problems caused by the developer’s lack of business experience. T he quality of the product can thus be enhanced, software development costs be reduced, and software maintenance be improved. Keywords Design Pattern, Framework, Software Development Process, XML 1.INTRODUCTION In Object-Oriented T echnology, the property of inheritance allows software components to be reused, which can obviously reduce the cost of software development. For this reason, to produce a highly reusable software component is an important goal of software engineering. However, programmers are usually focused on code reuse while ignoring design reuse. Design patterns provide a clear concept of design structure by describing the relationships of inheritance and reference between components of the system. Design patterns are a series of familiar usages and constructions utilized throughout system design. Design patterns allow rapid coding of certain components by following certain patterns of steps. T his can improve the documentation and maintenance of existing systems by providing an explicit specification of class, object interactions and their underlying intents. One of the main purposes of design patterns is to help software engineers to understand the common characteristics of software objects/components in specialized domain. In recent years, due to the development and maturation of WWW and Java [14] technologies, many applications are now web applications or leaning in that direction. Many software concepts are utilized for the web as well, such as Design Patterns and Frameworks. The Apache Struts [12] and Spring Framework [13] are both open source frameworks used to address and reduce the complexity of developing an enterprise application. T he advantage of using a framework is the layered architecture it provides. Layered architecture allowed users to choose the component desired, while also providing the integration framework when developing application using J2EE. T hese developing web concepts can facilitate the development of web applications. However, these very useful tools and concepts lack a systematic organization. We hope to use these open source software technologies to develop a software framework which can be applied to web application. T his should solve the problem of web applications lacking a good structure, while through applying these open source software technologies, software development costs will be reduced. Furthermore, a guideline for programmers who wants to use these open source technologies will be provided. This paper is organized as follows: In the next section, we discuss works related to our project; in section 3, the open source technologies used in the paper and the system implementation will be described; Section 4 is a sample experiment. T he conclusion is given in section 5.
A comparison of tool±repair methods using CO2laser surfacing and arc surfacing J.Grum*,J.M.Slabe Faculty of Mechanical Engineering,University of Ljubljana,As?kerc?eva6,1000Ljubljana,Slovenia Abstract The life of loaded machine elements and the vital parts of tools can be successfully extended by systematic maintenance and the timely repair of damaged surfaces.It has been proved that with the regular maintenance of tool parts the cost of the tool in the price of a?nished product can be considerably reduced.It is a very economical practise to manufacture certain parts from low-cost,tough structural steel on which a layer of wear-resistant alloy has been surfaced.In such a case the volume fraction of the surfaced layer is usually much lower than10%of the total volume of the tool or the machine element. In this paper,we report some of our latest results involving comparative studies of repair surfacing on maraging steel and the cladding of common structural steel with a Ni±Co±Mo alloy similar to the maraging steel using a laser process and submerged-arc surfacing.The results are based on micro-structural and micro-chemical analyses of the surfaced layer and are supported by analyses of the micro-hardness and the residual stresses,carried out on suitably adaptedˉat specimens. #2002Elsevier Science B.V.All rights reserved. Keywords:Laser cladding;Submerged-arc surfacing;Maraging steel 1.Introduction Precipitation-hardened maraging steels are suitable for the manufacture of machine elements and tool parts that are subjected to high thermo-mechanical loads as a result of their exceptionally favourable mechanical properties,particularly their excellent fracture toughness combined with their high tensile strength and hardness[1,2].Grum and Zupanc?ic?[3,4] have investigated the inˉuence of various temperature/ time conditions of precipitation hardening on the mechanical properties of these steels.Tool manufac-turers have been successfully introducing maraging steels in the manufacture of die-casting tools for aluminium and magnesium alloys,for which a high resistance to cyclic thermo-mechanical loads is required.An exceptionally good weldability of mara-ging steels in all states,i.e.prior to and after precipita-tion annealing[1],permits the successful repair of damage to tools in order to extend the operating life of the tools and preserve the quality of the die-cast parts. Gehricke[5]proved that the weld or the surfacing weld did not show inclusions and pores,which guar-antees the high quality of a product.Precipitation annealing of the weld and the heat-affected zone ensures that the nominal hardness of the material recommended for the operation of the tools can be achieved. Laser cladding,which involves the preliminary deposition of a powder of a certain chemical composi-tion and grain size on the workpiece surface,is the simplest way of improving the surface properties of a, usually cheap,parent metal.The selected method of Applied Surface Science208±209(2003)424±431 *Corresponding author.Tel.: 386-1-4771-203; fax: 386-1-2518-567. E-mail address:janez.grum@fs.uni-lj.si(J.Grum). 0169-4332/02/$±see front matter#2002Elsevier Science B.V.All rights reserved. doi:10.1016/S0169-4332(02)01427-7
外文翻译 原文 Foreign T rade o f China Material Source:W anfang Database Author:Hitomi Iizaka 1.Introduction On December11,2001,China officially joined the World T rade Organization(WTO)and be c a me its143rd member.China’s presence in the worl d economy will continue to grow and deepen.The foreign trade sector plays an important andmultifaceted role in China’s economic development.At the same time, China’s expanded role in the world economy is beneficial t o all its trading partners. Regions that trade with China benefit from cheaper and mor e varieties of imported consumer goods,raw materials and intermediate products.China is also a large and growing export market.While the entry of any major trading nation in the global trading system can create a process of adjustment,the o u t c o me is fundamentally a win-win situation.In this p aper we would like t o provide a survey of the various institutions,laws and characteristics of China’s trade.Among some of the findings, we can highlight thefollowing: ?In2001,total trade to gross domestic pr oduct(GDP)ratio in China is44% ?In2001,47%of Chinese trade is processed trade1 ?In2001,51%of Chinese trade is conduct ed by foreign firms in China2 ?In2001,36%of Chinese exports originate from Gu an gdon g province ?In2001,39%of China’s exports go through Hong Kong to be re-exported elsewhere 2.Evolution of China’s Trade Regime Equally remarkable are the changes in the commodity composition of China’s exports and imports.Table2a shows China’s annu al export volumes of primary goods and manufactured goods over time.In1980,primary goods accounted for 50.3%of China’s exports and manufactured goods accounted for49.7%.Although the share of primary good declines slightly during the first half of1980’s,it remains at50.6%in1985.Since then,exports of manufactured goods have grown at a much
204/JOURNAL OF BRIDGE ENGINEERING/AUGUST1999
JOURNAL OF BRIDGE ENGINEERING /AUGUST 1999/205 ends.The stress state in each cylindrical strip was determined from the total potential energy of a nonlinear arch model using the Rayleigh-Ritz method. It was emphasized that the membrane stresses in the com-pression region of the curved models were less than those predicted by linear theory and that there was an accompanying increase in ?ange resultant force.The maximum web bending stress was shown to occur at 0.20h from the compression ?ange for the simple support stiffness condition and 0.24h for the ?xed condition,where h is the height of the analytical panel.It was noted that 0.20h would be the optimum position for longitudinal stiffeners in curved girders,which is the same as for straight girders based on stability requirements.From the ?xed condition cases it was determined that there was no signi?cant change in the membrane stresses (from free to ?xed)but that there was a signi?cant effect on the web bend-ing stresses.Numerical results were generated for the reduc-tion in effective moment required to produce initial yield in the ?anges based on curvature and web slenderness for a panel aspect ratio of 1.0and a web-to-?ange area ratio of 2.0.From the results,a maximum reduction of about 13%was noted for a /R =0.167and about 8%for a /R =0.10(h /t w =150),both of which would correspond to extreme curvature,where a is the length of the analytical panel (modeling the distance be-tween transverse stiffeners)and R is the radius of curvature.To apply the parametric results to developing design criteria for practical curved girders,the de?ections and web bending stresses that would occur for girders with a curvature corre-sponding to the initial imperfection out-of-?atness limit of D /120was used.It was noted that,for a panel with an aspect ratio of 1.0,this would correspond to a curvature of a /R =0.067.The values of moment reduction using this approach were compared with those presented by Basler (Basler and Thurlimann 1961;Vincent 1969).Numerical results based on this limit were generated,and the following web-slenderness requirement was derived: 2 D 36,500a a =1?8.6?34 (1) ? ??? t R R F w ?y where D =unsupported distance between ?anges;and F y =yield stress in psi. An extension of this work was published a year later,when Culver et al.(1973)checked the accuracy of the isolated elas-tically supported cylindrical strips by treating the panel as a unit two-way shell rather than as individual strips.The ?ange/web boundaries were modeled as ?xed,and the boundaries at the transverse stiffeners were modeled as ?xed and simple.Longitudinal stiffeners were modeled with moments of inertias as multiples of the AASHO (Standard 1969)values for straight https://www.doczj.com/doc/242837454.html,ing analytical results obtained for the slenderness required to limit the plate bending stresses in the curved panel to those of a ?at panel with the maximum allowed out-of-?atness (a /R =0.067)and with D /t w =330,the following equa-tion was developed for curved plate girder web slenderness with one longitudinal stiffener: D 46,000a a =1?2.9 ?2.2 (2) ? ? ? t R f R w ?b where the calculated bending stress,f b ,is in psi.It was further concluded that if longitudinal stiffeners are located in both the tension and compression regions,the reduction in D /t w will not be required.For the case of two stiffeners,web bending in both regions is reduced and the web slenderness could be de-signed as a straight girder panel.Eq.(1)is currently used in the ‘‘Load Factor Design’’portion of the Guide Speci?cations ,and (2)is used in the ‘‘Allowable Stress Design’’portion for girders stiffened with one longitudinal stiffener.This work was continued by Mariani et al.(1973),where the optimum trans-verse stiffener rigidity was determined analytically. During almost the same time,Abdel-Sayed (1973)studied the prebuckling and elastic buckling behavior of curved web panels and proposed approximate conservative equations for estimating the critical load under pure normal loading (stress),pure shear,and combined normal and shear loading.The linear theory of shells was used.The panel was simply supported along all four edges with no torsional rigidity of the ?anges provided.The transverse stiffeners were therefore assumed to be rigid in their directions (no strains could be developed along the edges of the panels).The Galerkin method was used to solve the governing differential equations,and minimum eigenvalues of the critical load were calculated and presented for a wide range of loading conditions (bedding,shear,and combined),aspect ratios,and curvatures.For all cases,it was demonstrated that the critical load is higher for curved panels over the comparable ?at panel and increases with an increase in curvature. In 1980,Daniels et al.summarized the Lehigh University ?ve-year experimental research program on the fatigue behav-ior of horizontally curved bridges and concluded that the slen-derness limits suggested by Culver were too severe.Equations for ‘‘Load Factor Design’’and for ‘‘Allowable Stress Design’’were developed (respectively)as D 36,500a =1?4?192(3)? ?t R F w ?y D 23,000a =1?4 ?170 (4) ? ? t R f w ?b The latter equation is currently used in the ‘‘Allowable Stress Design’’portion of the Guide Speci?cations for girders not stiffened longitudinally. Numerous analytical and experimental works on the subject have also been published by Japanese researchers since the end of the CURT project.Mikami and colleagues presented work in Japanese journals (Mikami et al.1980;Mikami and Furunishi 1981)and later in the ASCE Journal of Engineering Mechanics (Mikami and Furunishi 1984)on the nonlinear be-havior of cylindrical web panels under bending and combined bending and shear.They analyzed the cylindrical panels based on Washizu’s (1975)nonlinear theory of shells.The governing nonlinear differential equations were solved numerically by the ?nite-difference method.Simple support boundary condi-tions were assumed along the curved boundaries (top and bot-tom at the ?ange locations)and both simple and ?xed support conditions were used at the straight (vertical)boundaries.The large displacement behavior was demonstrated by Mi-kami and Furunishi for a range of geometric properties.Nu-merical values of the load,de?ection,membrane stress,bend-ing stress,and torsional stress were obtained,but no equations for design use were presented.Signi?cant conclusions include that:(1)the compressive membrane stress in the circumfer-ential direction decreases with an increase in curvature;(2)the panel under combined bending and shear exhibits a lower level of the circumferential membrane stress as compared with the panel under pure bending,and as a result,the bending moment carried by the web panel is reduced;and (3)the plate bending stress under combined bending and shear is larger than that under pure bending.No formulations or recommendations for direct design use were made. Kuranishi and Hiwatashi (1981,1983)used the ?nite-ele-ment method to demonstrate the elastic ?nite displacement be-havior of curved I-girder webs under bending using models with and without ?ange rigidities.Rotation was not allowed (?xed condition)about the vertical axis at the ends of the panel (transverse stiffener locations).Again,the nonlinear distribu-