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Key Proteins Of Oxidative Stress And Addiction Potential Induced With Tramadol By Proteomics And Related Analytical Technologies In Zebrafish Brain

Posted on:2010-12-10Degree:DoctorType:Dissertation
Country:ChinaCandidate:H Q ZhuoFull Text:PDF
GTID:1100360275988083Subject:Biochemistry and Molecular Biology
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Tramadol hydrochloride(TH),an atypical centrally acting opioid analgesic,hasbecome the most prescribed drug worldwide.Previous reports predominantly havefocused on the numerous positive response of TH in vertebrates,including analgesiafor moderate to severe pain;antidepressant,anti-inflammatory,andimmunostimulatory effects,micturition control;an ability to lower glucose indiabetics and so on.However,the data on the increase in TH related fatalities,adversereactions,and the drug addiction presented in recent years have been obtained frompostmarketing surveillance and case reports,unfortunately,cellular and molecularmechanisms involved in neurotoxicity,dysfunction and addiction potential afterchronic and abused TH administration are not well documented.These issueshighlight the need to assess the addiction potential and neurotoxicity of TH abuse atthe cellular and molecular levels.In this thesis,zebrafish(Danio rerio) was chosen as experimental object.Theresults,from the perspective of behavior observation and statistical,showed thatoxygen demand and preference of oxygen-rich surface water layer were heightened infish,thus significantly increased the mean distribution density of fish in surface layer,by a TH stimulus.Compared with those in pethidine hydrochloride(more effectiveanalgesic) positive control group,the abnormal behavior was even more significant,which was observed to emerge much faster,and last much longer(the whole processup to 6 h).Moreover,it was also accompanied by a significant reduction in activity.The phenomenon was belongs to TH specific abnormal behavior and physiologicalresponse in zebrafish.Body and brain mean weight loss in zebrafish,after long-termreceiving food containing TH.Specific physiological responses to TH exposure,makes zebrafish a favorable animal model for experimental studies of the mechanismsof TH toxicity and drug addiction.With the similar prooxidant properties with other opioids,TH was shown to inducea decrease in the activity of SOD,and an increase in lipoperoxide.Based on the regulation ofantioxidant enzyme activity,zebrafish had developed certain adaptabilityand tolerance to the oxidative toxicity during the chronic TH administration.Aftersingle IM injection,Gas Chromatography-Mass Spectrometry(GC/MS) trackinganalysis was used to examine the diffusion and elimination of the parent compound in7 kinds of tissue,including brain,eyes,gill,heart,liver,gut,and muscle.Regulationof TH content vs.time demonstrated a rapid drug diffusion and longer eliminationprocess in fish brain.Two principal metabolites O-desmethyl(M1) and N-desmethyl(M2) TH were detected in the brain tissue,and content of M2 was significantly higherthan that of M1,as the main desmethyl metabolite in zebrafish.Methods of IMinjection and GC/MS tracking analysis,could be well introduced to the study on THabsorption,distribution,clearance,metabolism and drug effect,especially themechanism of neurotoxicity in zebrafish,and their combination with the assays ofSOD enzyme activity and MDA content,could be used to assessment of degree ofoxidative stress in brain,but also to offer scientific reference values for theappropriate doses selection and experimental samples preparation for the follow-upscreening and identification of the key proteins by proteomics technologies.Proteomics has recently become one of the best analytical techniques to screen andidentify the differential proteins in tissues and organs.One practical research strategyhad been set up to screen and identify differential proteins in zebrafish brain byproteomics,especially in the establishment of semi-quantitative 2D-PAGE andintroduction of two strategies for sample preparation for peptide mass fingerprintinganalysis,namely usages of protein intensifiers(including human serum transferrin,albumin,ferritin),and mineral oil-,glycerol-,Vaseline-coated targets.All of thesestrategies led to effective improvement of identification of differential proteins insensitivity and reliability.A total of 30 TH induced differential protein spots wereidentified,and classifed into 13 categories of proteins,in which various subtypes of14-3-3 proteins,creatine kinase,ATP synthase(H+ transporting mitochondrial F1complex beta polypeptide),and tubulin were identified at the separated location onthe gel for 3,3,4,and l l times respectively.The TH responsive proteins identifiedwere involved in cytoskeletal system,energy metabolism,signal transduction, synaptic function,protein modification or degradation,and calcium signal regulator.Most of them have functions in oxidative stress or oxidative stress related diseasespathogenesis,including 14-3-3 proteins,creatine kinase BB(CK BB),ubiquitincarboxy-terminal hydrolase L-1(UCHL-1),ATP synthase,synaptosome-associatedprotein(SNAP),tubulin,actin.Furthermore,proteins including aldolase C,ATPsynthase,CK BB,pyruvate dehydrogenase kinase,UCHL-1,14-3-3 proteins,dynamin 1,SNAP,tubulin and actin,also differentially expressed in proteomics ofknown drug addiction.Meanwhile some other identified proteins have not yet beenreported,such as calcium signal regulation related protein—novel protein similar tovertebrate EF hand calcium binding protein 2,cytoskeletal related protein—novelintermediate filament protein similar to centrosomal protein 110 kDa.Similar toknown addiction drugs,TH induced neurotoxicity in zebrafish brain by three paths,namely energy metabolism,oxidative stress and protein modification or degradation.Western blotting and enzyme activity assay confirmed the results that chronic THinduced up-regulation of 14-3-3 epsilon peptide,and inhibition of aldolase andcreatine kinase enzyme activity,the two important enzymes in energy metabolism.All effects were still significant after two weeks of TH cessation.Furthermore,allabove results verified the reliability of screening and identification of the differentialproteins by proteomics technologies.Chronic TH treatment induced thedown-regulation of some important proteins involved in oxidative stress and energymetabolism,which directly or indirectly led to the changes of mitochondrial structureand function.Ultrastructureal observation on mitochondria showed series ofmorphological changes in the case of TH exposure,for example,distribution disorder,numeral density decreased significantly,the mitochondrial matrix was cleared,electron density decreased,swelling and vacuolation was observable,meanwhile themitochondria cristae show significant reduction,breakdown and in some cases wereabsent.Combining multiple methods,including behavior observation,drug metabolismdetection,western blotting,enzymes activity assay,and ultrastructural observation,with proteomics to screen and identify TH-associated key proteins,results suggested the involvement of neurotoxicity evaluated by oxidative stress,mitochondrialdysfunctions,and protein modification or degradation,moreover imbalances ofenergy metabolism,damages of cytoskeletal system in the effects of long-tern THabuse,at the cellular and molecular level.Furthermore,the addiction potential is mostlikely to accompany with chronic and long period exposure to TH.The results of thisthesis also provide meaningful evidence for the safety of TH,including neurotoxicityand addiction potential,as it is widely used throughout the world,and proteomicstechnologies based on a variety of other research methods and the identified keyproteins,also have application value in assessment the neurotoxicity and addictionpotential of other drugs.
Keywords/Search Tags:tramadol neurotoxicity, proteomics, addiction potential
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