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Individual variability of CYP2E1 xenobiotic oxidation in human liver
K. Nailas.Y Rabemamplenina andA. Is&la withttltt,achn,ca,
assisisnce of Mrs. A. Couvenamand A. Djald. Depanmento/ Twcolo~)! &?&I-Cenwe da Ractwche. hboi+e. &,,ce
Ether, a widelY used anaesthetk for laboratow rodents. forms an explosiw ,nixture with air For safety reasons we may be required in the future to use an akemawe, safer enaesthetic.For usa onroutine toy. icologysiudies enilurane appen to us to be the best altematiyBto ether.Enflurane is volatile. nor+Flammablsand is reportedto baoneof the safest anaesthetics availablefor humanand animaluse. The objectiveof this study was to characterizethe optimal condllions of anaesthesiawkh enfluraneand to evaluate119use in routine toximlogY studies. Young,% weeks) and old 129-30 weeks) Sprague Dawky rats and CD1 mice were used. EnSwans wes dellwed using a commerciallY waifable apparatus(Mtnewe VeterinaryEquipment). A rangeof enlluBne mncentrations (3 and 5%). carrierflow rates I1 and 3 Wmin)and time of expasura12.3 and 5 min) to he anaestheticwere tasted. Parametersevaluatedconsisted of monalitv. clinicelsigns and the depth at anaesthesia{absenceol resct~ontoorbital sinus puncture). 1. rals anaesthmiawas obtainedfollowing 3 or 5 minuteee~poswe to 5% snflurane with en ox~genflow rete of 3 Ilmin. In mice anaemhe sia we8 obtamediollowing5 minutesto exposureto Slenllurane and oxygenflow aateof 3 limin. In both species. there were no deaths. but dwpnoea occ~::w :n some mice. There were no experimentallysignilicantdifferencesin the response of Youngand old rats to enflurane. On the basis of the depth of aneesthea obtained.the good toleration of anaesthesiewe concludethat entluraneis suitable for use I” 1oufinotoximkxgystudies. Keywords: anaesthesia; rodents; enfturane: toxicity studies, blood eampll”g
Analysis of Urinary Perphyrln9 In Aluminum Exposed Rlr M. Nasiadek.J. Chmielnicka.J. Hryzajemka. E. Lewandowska-iyndul. Depamnenfof Txicolagic~l Chemistry. Medical ,,~&,a-~ L6d.f. Polend As a result of saveral human and animal studies parenterelexpaure lo aluminum has been recognizedes a risk factor of “dislyois ewefalopathy” 111.osteomalacia121and microcyw anemia(31 The mechanism bY which art excess aluminum induces anemia remaineto be clarified.It may be causedbYaluminum overloadresulting a reversible block in hame syntheae due elher to a defect in porphyrin eynthesis or impairediron utilization. In our studies. alummumchloridewee orallY administeredto female ‘Wiotar’ mts in dose* of 100 mg Allkg Ior a penod of 35 days with e wthout F&O4 (4 mg F&g). Control rats were treated with 0.9% NaCl or with Fe (4 mgfig). The dwwmir-. of urine porphyrins@-. 7.. &, 5.4-oarbow porpryrinjwas dofew ,,ned by HPLC on 7th. 14th. Zlst. 2&h and -5th day. Free er,qb;. 71s pn toporphyrw {FEPI ~15 estimsled in the blood in the earn.. Q!,ne.The measurementswere performedboth in exposedand contml rats. The resuiis of the experiment indicatethat examinedaluminum I”. ducadstatistalk increasein percentageof uropotphyr~n end decreasein copmporphiwnin urine (cummulativedose 2lOa mg ml. An increasaof FEP in the blood. 8150observedduring the first wsekofexpo~urelcummulativedoee1400mglkgl,meYpro~etobesn early indicator ofaluminum exposure. Iron administeredtogetherwith aluminum dlminishsd described changes in porphyrin metabolism. causedby aluminum itself.
signrlicant
The eflect of oral administration of rlobedins (ST%) on the activities of the Cosomal enzymes acid phosphatsse (APhI and N.aceM.8 glucosaminaaseINAGAI and on the lwet of protems m pregnant ICR mice spleen. kidney and liver and I” lo&al liver was studied after cyclophosphamideICPI administration ST6 weeadministeredfrom gee. tat10naldaY11unt~ldaY1?inthedo,ses0~23.6and70.7mglLg:!hprior to CPllO mgRglrntraperitoneal injection.CP induceddamagewasp. rociated with an Increasein maternal acwilies of APH and NAGA in liver and soleen but not in kidnev and wdh an incream in toeta! APH liver activity. ST6 pretreatment&as able to prevent partially the CPinducedbiochemicalchanges. Rewords: slobadine; cwlophosphamids; lysosomal enzymes: mice
We investigated the expression of CYPZEi in 13 human liver microsomsl samples by immumdeteotion in Western blots and catalYthiceetiyitiestowardseevBraIspBc~c CYPZEI substrates(chlorzoxazone.4.nkrophenol. dimeth$fonnamide. aniline) m vitro. Moreover. the metabolism ol benzeneIEl to water soluble (SM) and covakm binding(CB) metabaliteswee wrmlated with these data and with the Western blots and specific catalfihic activities of other forms of P450 IlAl. th2,3A4and X9/10). A4foklvariabiliin CYPZEl contentcor. relatedwith lhe me18boksmof all the CVPZEl specificsubstratesand correspondentvariability in benzenemetabolism to SM and CB uyae observed.The studies on ename kinetics of benzenemetebalism re. vealedthe existenceof human livers metaboliiing benzeneto SM with dlfferentKmandVm~xdespiteoftheirsimilarCYPPE1 content.Thevariable Km values for benzeneoxidation m different humanmicrosomes were probablyrwt caused by catal>& of other P450 enzymes. since the activity well correlatedwith 2El levels in immumblots. was effectively inhlbited by diethVldithlocarbamata and correlatedwith the oxidationrates of sevwal CYPZEl substrates. Rather.they can bedue to a microheterogenihlof CYP2El. to phenoh/piccounterpartof Ihe reportedgeneticpolymorphism01CYF’2Et in humane
atiivitv, but mixtures of &ticideswd various tormulations oi ihem ere estimated by the data91 their individualcomponents.II is rather a non+alidatedapproachtothematter.bBc~~seitisev~dent.lhatacom. binedactionot a number of ingredients is ableto modify tumorogenx proCeES. It is necessewto test eachof such substancefor the potential c-rcinogenicactiviw It is impossible to do in the course of traditional chmnicexpnments. becausesuch experimentsarewry long and sw phlsticated.To solve this problem is necessaryto use express-tests. We hew chosenthe genotoxictests “in ti”: the alkaline urwnd
assisisnce of Mrs. A. Couvenamand A. Djald. Depanmento/ Twcolo~)! &?&I-Cenwe da Ractwche. hboi+e. &,,ce
Ether, a widelY used anaesthetk for laboratow rodents. forms an explosiw ,nixture with air For safety reasons we may be required in the future to use an akemawe, safer enaesthetic.For usa onroutine toy. icologysiudies enilurane appen to us to be the best altematiyBto ether.Enflurane is volatile. nor+Flammablsand is reportedto baoneof the safest anaesthetics availablefor humanand animaluse. The objectiveof this study was to characterizethe optimal condllions of anaesthesiawkh enfluraneand to evaluate119use in routine toximlogY studies. Young,% weeks) and old 129-30 weeks) Sprague Dawky rats and CD1 mice were used. EnSwans wes dellwed using a commerciallY waifable apparatus(Mtnewe VeterinaryEquipment). A rangeof enlluBne mncentrations (3 and 5%). carrierflow rates I1 and 3 Wmin)and time of expasura12.3 and 5 min) to he anaestheticwere tasted. Parametersevaluatedconsisted of monalitv. clinicelsigns and the depth at anaesthesia{absenceol resct~ontoorbital sinus puncture). 1. rals anaesthmiawas obtainedfollowing 3 or 5 minuteee~poswe to 5% snflurane with en ox~genflow rete of 3 Ilmin. In mice anaemhe sia we8 obtamediollowing5 minutesto exposureto Slenllurane and oxygenflow aateof 3 limin. In both species. there were no deaths. but dwpnoea occ~::w :n some mice. There were no experimentallysignilicantdifferencesin the response of Youngand old rats to enflurane. On the basis of the depth of aneesthea obtained.the good toleration of anaesthesiewe concludethat entluraneis suitable for use I” 1oufinotoximkxgystudies. Keywords: anaesthesia; rodents; enfturane: toxicity studies, blood eampll”g
Analysis of Urinary Perphyrln9 In Aluminum Exposed Rlr M. Nasiadek.J. Chmielnicka.J. Hryzajemka. E. Lewandowska-iyndul. Depamnenfof Txicolagic~l Chemistry. Medical ,,~&,a-~ L6d.f. Polend As a result of saveral human and animal studies parenterelexpaure lo aluminum has been recognizedes a risk factor of “dislyois ewefalopathy” 111.osteomalacia121and microcyw anemia(31 The mechanism bY which art excess aluminum induces anemia remaineto be clarified.It may be causedbYaluminum overloadresulting a reversible block in hame syntheae due elher to a defect in porphyrin eynthesis or impairediron utilization. In our studies. alummumchloridewee orallY administeredto female ‘Wiotar’ mts in dose* of 100 mg Allkg Ior a penod of 35 days with e wthout F&O4 (4 mg F&g). Control rats were treated with 0.9% NaCl or with Fe (4 mgfig). The dwwmir-. of urine porphyrins@-. 7.. &, 5.4-oarbow porpryrinjwas dofew ,,ned by HPLC on 7th. 14th. Zlst. 2&h and -5th day. Free er,qb;. 71s pn toporphyrw {FEPI ~15 estimsled in the blood in the earn.. Q!,ne.The measurementswere performedboth in exposedand contml rats. The resuiis of the experiment indicatethat examinedaluminum I”. ducadstatistalk increasein percentageof uropotphyr~n end decreasein copmporphiwnin urine (cummulativedose 2lOa mg ml. An increasaof FEP in the blood. 8150observedduring the first wsekofexpo~urelcummulativedoee1400mglkgl,meYpro~etobesn early indicator ofaluminum exposure. Iron administeredtogetherwith aluminum dlminishsd described changes in porphyrin metabolism. causedby aluminum itself.
signrlicant
The eflect of oral administration of rlobedins (ST%) on the activities of the Cosomal enzymes acid phosphatsse (APhI and N.aceM.8 glucosaminaaseINAGAI and on the lwet of protems m pregnant ICR mice spleen. kidney and liver and I” lo&al liver was studied after cyclophosphamideICPI administration ST6 weeadministeredfrom gee. tat10naldaY11unt~ldaY1?inthedo,ses0~23.6and70.7mglLg:!hprior to CPllO mgRglrntraperitoneal injection.CP induceddamagewasp. rociated with an Increasein maternal acwilies of APH and NAGA in liver and soleen but not in kidnev and wdh an incream in toeta! APH liver activity. ST6 pretreatment&as able to prevent partially the CPinducedbiochemicalchanges. Rewords: slobadine; cwlophosphamids; lysosomal enzymes: mice
We investigated the expression of CYPZEi in 13 human liver microsomsl samples by immumdeteotion in Western blots and catalYthiceetiyitiestowardseevBraIspBc~c CYPZEI substrates(chlorzoxazone.4.nkrophenol. dimeth$fonnamide. aniline) m vitro. Moreover. the metabolism ol benzeneIEl to water soluble (SM) and covakm binding(CB) metabaliteswee wrmlated with these data and with the Western blots and specific catalfihic activities of other forms of P450 IlAl. th2,3A4and X9/10). A4foklvariabiliin CYPZEl contentcor. relatedwith lhe me18boksmof all the CVPZEl specificsubstratesand correspondentvariability in benzenemetabolism to SM and CB uyae observed.The studies on ename kinetics of benzenemetebalism re. vealedthe existenceof human livers metaboliiing benzeneto SM with dlfferentKmandVm~xdespiteoftheirsimilarCYPPE1 content.Thevariable Km values for benzeneoxidation m different humanmicrosomes were probablyrwt caused by catal>& of other P450 enzymes. since the activity well correlatedwith 2El levels in immumblots. was effectively inhlbited by diethVldithlocarbamata and correlatedwith the oxidationrates of sevwal CYPZEl substrates. Rather.they can bedue to a microheterogenihlof CYP2El. to phenoh/piccounterpartof Ihe reportedgeneticpolymorphism01CYF’2Et in humane
atiivitv, but mixtures of &ticideswd various tormulations oi ihem ere estimated by the data91 their individualcomponents.II is rather a non+alidatedapproachtothematter.bBc~~seitisev~dent.lhatacom. binedactionot a number of ingredients is ableto modify tumorogenx proCeES. It is necessewto test eachof such substancefor the potential c-rcinogenicactiviw It is impossible to do in the course of traditional chmnicexpnments. becausesuch experimentsarewry long and sw phlsticated.To solve this problem is necessaryto use express-tests. We hew chosenthe genotoxictests “in ti”: the alkaline urwnd