Document R3yO5oQLLnwXVxoOapbypJJn

-- .. - -- .-.-.-.-.-.-.-..- .- .-.-.-..-....- - - hi,-,lhevels)fparticulatmeatter.The Environmenta IQualityBoard of Puerto izicohas statedthatCata fio(an industrializeadrea) has a historyof exceeding @atit)naAlmbient Air QualityStandards.The Hea lthDepartment of Puerto Rict)has reportedthe highestratesof respiratoriyllnessesand alltypesof ::inccrisn thismunicipalityU.sing theEPA standardsampling methodology, 1,)tasluspended particles(TSP) and particulatmeatter of lessthan 10 @Lm @P\110)were collectedinCatafioand Fajardo(controlarea)foreach season of the year.The particulatmeatterfrom the air filterasnd from reference ,tandardmaterial(SRM 1649. urban dust)Were extractedsequentiallywith threseolventosfdifferentpolaritieussing 24-hour SoxhietextractionT.he extractsobtained were testedfor cytotoxicitoyn human keratinocytesusing the NeutralRed Bioassay (NRB). The hexane extractfrom SRM 1649 was the most toxicwith a NR,o of 5 ILg/mL. Similartoxicityto the SRM1649 were found in the Catafio'shexane and dichloromethaneextractsforwinter and summer samples, respectiveiy.Particleextractsshowed NRo values bei%%cen 6 to 120 @Lg/mL forotherseasons.None of the controlextractshad Iemonstrateddetectablelevelsoftoxicityinthe winterextractsa,lthoughin other seasons the NR@ fluctuatedbetween I I to 93 @Lg/mL Most of the dichloromethaneextractsshowed non-detectabletoxicitylevels.Fractionationsand cytotoxicityteststo some hexane toxicextractshave been done and willbe presentedalong with otherfractions. Fi 9475 ANTITUMOUR-ACTIVITIES COUMARIN IN HUMAN OF COUMARIN AND 7-OHMALIGNANT CELL LINES. U S Wel,er,B Steffen,C -P Siegers.Instituteof Toxikology,Medical b'niversitoyf Liibeck,Gerniat@v. Coumarin is found in many medicinalplantsand thereforealso used in phytomedicine forthetreatmentof vcnous diseases.The metabolicpathways of coumarin inthehuman body leadtothe intermediate7-OH-coumarin and consequent -.Iucuronidatioinn the intestinaend liver.Clinicaltrialswith c(iumarinor coumarin/cimetidinecombinationsin adjuvanttumour therapy revealedbeneficiaelffectsinpatientswith malignant melanomas (Thomes, D. etat.,Eur.J.Oncol. 15,431-435, 1989) and metastatickidney carcinomas (Marshall,M.E. etal.,J.1r.Coll.Physicians.Surg.22 (Suppl.1)73, 1993). Coumarin was alsoactivein thepreventionof tumour growth inexperimental models of carcinogen-inducedtumours (Wattenberg,L.W. et at.,Cancer Res. 39, 1651-1654, 1979) or onkogen-induced tumours in transgenicinice (,Tseng,A. Jr.,J. lr.Coll.Physicians.Surg.22 (Suppi.1)4,9-50, 1993). GTowth-inhibitoryeffectsof C and 7-OH-C were also reportedon human malignantcelllinesin vitro(Marshall,M.E. et al.,J. Cancer Res. Clin. Oncol. 120 (Suppl.1)S,39-S412, 1994).We testedthe antitumouractivities or coumarin (C) and itsknown metabolites7-OH-coumarin (7-OH-C) in severalhuman tumour ccillinesinculture.C as well as 7-OH-C inhibited cellproliferatioonfa gastriccarcinoma cellline,a colon-carcinomacellline (Caco-2),a hepatoma-derivedcellline(HepG2) and a lymphoblasticcell line(CCRF CEM) ina conccntration-dependenwtay. Inequimolarconcentra- LiOnSthe [C_,rvalueswere 1.59-3.57 mM for C and 0.68-2.69 mM for 7- OH-C. The glucuronideof 7-OH-C was ineffectivien thisrespect.Besides antiproliferatiavned cytotoxiceffectscoumarin exertsalsoimmunomodulatoryactivitiesa;lleffectstogethercharacterizceoumarin asa so-called"bio- logicalresponsemodifier" and supportthechance to use coumarin-containing phytotherapeuticpreparationsinthe adjuventtreatmentof tumour diseases; the recommended daily dosage is 100-1000 mg coumarin. Fl 9461 PREDICTABILITY OF ASSESSING CATARACTOGENIC POTENTIAL OF DRUG CANDIDATES USING EXPLANT LENS CULTURE. c lmlylwivC@Can ,W P BeierschmittRL,M Shepard, M J Avery,J H Fortner, and M D Aleo.PfizerInc.,Groton,CT The 5-lipoxygenaseinhibitoCrJ12918 (250 mg/kg/d) wa associatedwith o pa'cityformationin Sprague-Dawley ratsafterI month.S-1@I. time course of toxicityand diminishingsystemic exposure to CJ12918 suggestedthat opacityformationwas linkedto extensivemetaboliteformation.The incidence of. opacityformationwas reduced 33% with the lessmetabolicallylabile structuralnalogCJ 13454,despiteincreasedsystemicexposuretotheparent drug (AUC(O-8hr): 67 vs 2 iLg-hr/mlforCJ12918) and similardrug levels inthe lens(:516vs :517 ngaens).The referenceanalogZD2 138 was without effectand was not detectedin the lens.The presentstudy utilizedexplanted lensesfrom Sprague-Dawley ratstodetermine the predictabilitaynd limitationsof assessingcataractogenipcotentialof thesedrugs in vitro.Lenses were placed inorgan cultureand exposed dailytoeithervehicle(0.2% ETOH), CJ 129 18, CJ 13454 or ZD2138 for up to 7 days. The role of metabolitc formationwas assessedusing an arachior-induceSd9 microsomlilfraclion. In contrastto in vivo results,ZD2138 was a potent cataractolenicagent in i,itr,A)f.ter2 days of exposure to 15 @Lliml.lensclarityand ATP content in thepresenceof S9 was as follows(appearance,@IrofcontrolATP): ZD2138 (53% cloudy,32 1%), CJI"918 (42% cloudy.40=3%). CJ]3454 (100% clear,101 - 12%). Lens clarityand ATP content for all3 drugs decreased proportionatelwyith increaseddru- concentration(40 @Lg/mi)or lengthof' exposure. Parent dru- levelsin t@c lens were <0.5 @Lgfor ZD2138 and ranged between 0.6-1.3 @L- forCJ 12918 and CJ 13454. Overall.these data suggest thatwith some limitationse.xplanted lensescan be used as an i)r i-itrtooolfor discriminatingcompounds with potentialto cause lenticular opacitiesH.owever, sincethe in vitromodel bypasses potentialdifferences in metabolism,pharmacokincticsand accumulation of variouscompounds intothe targettissuein vivo,the model alone cannot be used asa definitive determinant of cataractogenicrisk. Fl-94-7@ DETERMINATION METHYL ETHYL RAT BLOOD. OF THE PARTITION COEFFICIENTS OF KETOXIME IN WATER. OLIVE OIL AND, S E Janku',H Greim' W Dekant2 and J G Filser''.GSF-itistituotfe Toxicology,Neuherberg, Germany, -universiryVt*iirzbiiWrigi.r,-bLirg, Germany. Methyl ethylketoxime (MEKO-, 2-butanoneoxime) isan industriaalntioxidant and corrosioninhibitorI.n long term inhalatiosntudieswith male rodents, MEKO induced livertumours. Knowled-c of tissuedose may be an important factorin elucidatingthe carcinogenic;.-tencyof MEKO. Tissue dose can be calculatedforvariousexposure conditionsby means of a physiologically based toxicokinetimcodel. Among otherparameters,such a model requires knowledge of partitiocnoefficient(sPCs).A PC describestheratioof the concentrationosf MEKO in a liquidphase or a tissueto the concentration in the gas phase,atequilibrium. The enrichmentof MEKO in water,oliveoiland blood of Fischer344/N ratswas measured accordingto Johanson and Filser(1993; Arch Toxicol 67:151-163). Defined amounts of liquidswere incubatedwith defined amounts of gaseous MEKO at 37'C in closedall-glassystems.ConcenLration-timecdurses of MEKO in the gas phase were monitored using a gas chromatograph equipped with a FID and a stainlessteelcolumn (0.7 in) packed with Porapak Q (60-80 mesh). The oven temperaturewas 180'C. MEKO was stableinoliveoil.However, inwater and blood slow hydrolysis to 2-butanone occurred.PCs matrix:air(mean = SD [n))were for water 22300 3400 [9],foroliveoil5000 600161 and forblood 20400 3700 [6]. Assuming a body composition of 70% water and 10% fat,thethermodynamic equilibriumconstantwhole body:airiscalculatedby means of thePCs water:airand oliveoil:aitrobe 16100.The similarvaluesof the PCs water:airand blood:airindicatean uniform distributiownithinthe body, with theexception of adipose tissueF.rom thehigh valueof thePC blood:airithas tobe deduced that inhalativeuptake is limited by the alveolarventilationS.pnnsored bv AlliedSignal. DISPLACEMENT OF A FLUORESCENTLY LABELED FATTY ACID ANALOGUE FROM FATTY ACID CARRIER PROTEINS BY WYETH-14,643, AMMONIUM PERFLUOROOCTANOATE, POTASSIUM PERFLUOROOCTANE SULFONATE AND OTHER KNOWN PEROXISOME PROLIFERATORS. D Nabbefeld',J ButenhofV, N Bass'and A Seacat'.'3M, Toxicology Services,h'edicalDepartment,SaintPauf MN-2Universirv of Califonzia San Francisco,LiverResearch Center,San Francisco,CA. Itishypo&esized thatfattyaciddisplacementfrom fattyacid-carriperroteins isan initiaelventleadingto peroxisome proliferatioannd tumor formation observed in animal studies.To testthishypothesis,displacementof the fluorescentllyabeledfattyacidanalogue I1-(5-dimethylaminonapthatenesulfphonyl)-undecanoicacid(DAUDA) from thefattyacid-carrieprroteins. bovine serum albumin (BSA) and ratliverfattyacid-bindingprotein(LFABP), by some known peroxisomeproliferato(rPsP)was measured invitro as a percentlossof initiaflluorescence(excitation= 350 nm and emission = 500 nm). The known peroxisome proliferatorWsy,eth-14.643 (WY), ammonium pertluorooctanoat(eAPFO) and potassium perfluoroocianesulfonoate (PFOS), when added to solutionscontainingI@LM L-FAB P and I ILM DAUDA resultedin up to a 66% reduction(obtainedwith PFOS) in initial fluorescenceat a concentrationof 10 @LM PP. Results for BSA were ver@v similarto thoseseen with L-FABP, wherein PFOS yieldedthe maximum SOT 1998 Annual Meeting 395 decreaseininitiaflluorescenceof67%. The effectof additionalknown and SuspectPP on theseParameters iscurrentlyunder investigationT.hese results supportthehypothesisthatfattyacidsaredisplacedfrom carrierproteinsby known PP. Future work will investigatethe in vivo correlationbetween disruptionof fattyacid processingand peroxisome proliferation. EFFECT OF COUMARIN ON UNSCHEDULED D-NA SYINTHESIS IN PRECISION-CUT HUAAN LIVER SLI( J A Beamand, PT Barton.R J Priceand B G Lake. BIBRA ltitern,i CarslialionS,urrey,Englayid Coumarin (1,2-bcnzopyronc)occurs naturallyinvariousplantsand es F19479 CHARACTERIZATION SYSTEMS. OF IN VITRO HEPATOCYTE oilsand is known to exhibitmarked speciesdifferencesin both meta@ and hepatotoxicityI.n thisstudy we have examined the effectof L:OL on unscheduled DNA synthesis(UDS) in culturedhuman liverslices. 14 M Bums, T K Baker, and M A Carfagna.InvestigativTeoxicology,Lilly sion-cutliverslices(200-300 @Lm) were prepared with a Krumdieck Research Laboratories,Eli Lilly& Co.,Greenfield,IN. slicerL.iver sliceswere culturedfor 24 hr in RPMI 1640 culturem@ There arc a number of differenitn vitrohepatocytesystems availabletouse as potentialmodels of in vivo hepatotoxicityT.he abilityof invitrosystems to be usefulforunderstandinginvivo effectsisbased on a thorough understandingof cellulaprhysiologyand mechanisms of toxicityI.n thisstudy, the effectsof cadmium (a model hepatotoxicanto)n freshlyisolatedrat hepatocyteswere characterizedin cultureand suspension/cultureF.reshly isolatedrat bepatocyteswere eitherculturedfor 24 or 48 hrs. or put into suspensionfor4 hours and then transferredto culturefor24 hrs.Hepatocytes were exposed to cadmium chloridefor4 hours and cytolethalitwyas measured by lactatedehydrogenase release.Hepatocytes in suspension/cultur(eLCO = 3 nmoL/millioncells)were shown to be ffim timesmore sensitiveto cadmium cytolethalithyan in culturefor24 hrs (LCo = 9 nmol/million cells).Therefore,the firstobjectivewas to examine a mechanism which could differentiactaedmium cytolethalitiyntheseinvitrosystems.Kukongviriyapanand Stacey (J.Cell Phys. 140, 491, 1989) showed thatcadmium containing5% fetalcalfserum. ['H]thymidineand 0-5 mM coumarin. a dynamic organ culturesystem in an atmosphere of 95% 0,15% C( serve as positivecontrols,human liversliceswere alsocultured A it.' and 0.05 n-lM2-acctylaminofluoren(e2-AAF), 0.002 and 0.02mM afL B@ (AFB,) and 0.005 and 0.05 mM 2-amino-l-methyl-6-phenvi-imid3zc bjpyridine(PHIP,a heterocyclicooked food mutagen).Liver slices fixedin formalinand processedfor autoradiographicevaluationof i Compared to controlcultures,0.05-5 mM coumarin had no effecton expressed as thenet graincount or as the percentageof centrilobulahre@ cytenucleiwith >5 and > 10 netgrains.In contrasta, threeknown mut," produced a marked inductionof UDS inhuman liverslices.Net grainc values(mean of4 experiments)incontrol.0.05 mM 2-AAF, 0.02 m.%lA and 0.05 mM PHIP treatedcultureswere 0.33.4.4,7.1and 18.2,respecti% These resultsuggest thatcouman'n i'snot a genotoxic agent in human 1: (Supportedby RIFM and Rh6ne-Poulenc,Inc.). transportwas greaterin freshlyisolatedand 48 hr culturedhepatocytesthan in 24 hr cultures.We hypothesizedthatcytolethalitwyould be modulated by cadmium transportO.ur data showed that24 and 48 hr cultureshave similarLCi@ values,thereforetransportdoes notappear to be the mechanism which differentiatetshe sensitivitoyf bepatocytesystems tocadmium cytolethalityT,he second objectiveof our studieswas to determineifisolated hepatocytescould be used to examine the sex differencein cadmium toxicity seen in vivo.Female ratshave been shown to have an increasedsensitivity to cadmium toxicityI.solatedhepatocyteswere exposed to progesteronein F19572 USE OF PRECISION-CUT RAT LIVER SLICES FOR STUDIES OF XENOBIOTIC METABOLISM A@ND TOXICI- COMPARISON OF THE KRUMDIECK TISSUE SLICERS. AND BRENDEL R J Price',S E Ball2 A B Renwick', J A Beamand' and B G Lake'. 'BIBRA Internatio'n'alC,a,.,h.1t.,,S,..,,,y,England and '%@eth-Avers: Research, Drug Metabolism Division,Princeton,NJ. conjunctionwith cadmium tostudy a possiblemechanism forthe sex difference.Progesterone(300 @Lglml)significantliyncreasedcadmium (2- 7 RM) In thisstudy we have compared freshlycut and culturedliverslicesproduc from untreatedmale Sprague-Dawley ratsby theKrumdieck (K) and Brend@ cytolethalitiyn male mt hepatocytes.However, hepatocytesisolatedfrom Vitron(BV) tissueslicersN.o significandtifferenceswere observedin levt female ratswere not more sensitivethan male hcpatocytesto cadmium- ofproicin,potassium,totalglutathione(i.eG.SH + GSSG), reducedglutao induced cytolethalitTyh.ese studiesemphasize theimportance of identifying specificendpointsof toxicityand the challengesof comparing in vitrowith in vivo data. one and cytochrome P-450 and activitieosf 7-ethoxvresorufi0n-deethyla and 7-benzoxyresorufin0-debenzylase in freshlycut slicesproduced by il two tissueslicersH.owever, levelsofoxidisedglutathionewere significan greaterin liverslicesproduced by the BV tissueslicerP.recision-cultiv F, slicesproduced by both tissueslicerswere culturedinRPMI 1640 mediui to EVALUATION OF THE EFFECTS OF KUPFFER CELLS ON for0 (i.ea. I hr preincubationperiod),24 and 72 hr in a dynamic orga 9-5-01GALACTOSAMINE-INDUCED REPATOTOXICITY USING culturesystem in an atmosphere of either95% 0,/5 % CO., or 95 % air/ THE PRF-CISION-CUT RAT LIVER SLICES. % CO,. Apart from smalldifferencesinglutathionelevelsin0 and 24 @ N Masulomi, J Sugimoto, K Shimamura, and E Tanaka. Toxicology culturcdliverslicesn,o significandtifferencesinthe above parameterswer laboratory,Yokohama Research Center MitsubishiChemical Co., Tokyo, Japan, Sponsor: M Tsuchitani. observedbetween liverslicespreparedwith both tissueslicerasnd culmm, in both gas phases. With liverslicesproduced by both tissueslicers50 RN D-galactosaniine(GAIN) isone of the most extensivelystudiedhepatotoxicantsand known toinduce liverdamages viadepletionof uridinetriphosphate. In addition,recentin vivostudieshave focused upon the involvement of Kupffer cellsin the pathogenesisof GaIN-induced liverinjury,suggesting thatKupffer cellsenhance the toxicityby releasingbiologicallayctiveproductssuch as superoxideanion or tumor necrosisfactor(x.To investigattehe roleof Kupffer cellsin GaIN-induced hepalotoxicityin vitro.we used the sodium arseniteproduced a -reaterinductionof heat shock protein70 it slicesculturedfor 24 hr in a @igh oxygen than inan air atmosphere.Thi@ data suggeststhatboth the K and BV tissueslicerscan readilyproduct precision-cultiverslicesfor studiesof xenobioticmetabolism.and toxicitv However. forany given applicationof precision-cultiverslicesitisdesirabl to establishoptimal cultureconditions.(Supported by Wyeth-Ayers, Research) precision-cultiverslicesystem.Liver slicesprepared from untreatedor Kupffer cel]-inactivate(dGdCl.@-treatedr)atswere exposed to variousconcentrationsofGAIN (-20mM) and culturedfor24 hours.GAIN atconcentrations of more than 5mM caused dose-dependentloss of intracellulpaortassium and leakageof lactatedehydrogenase (LDH) from theslicesof untreated mts. Histologicaelxaminationrevealeddiffusenecrosisof hepatocyte.ne toxicitoyf GAIN, however, was markedly reduced inthe slicesfrom Kuprfer F19573 INDUCTION OF CYTOCHROME P-450 (CYP) ISOENZYMES BY RIFAMPICIN IN CULTURED PRECISION-CUT HUMAN LIVER SLICES. B G Lake',S E Ba]12.A B Renwick', J,%4Tredgerl,J Kao2, J A Beamand' and R I Price'.'BIBRA ItiternationaCla.rsitalto?Siu,rre@vE,ngland.- cell-inactivatreadts.Moreover, pre-tmatment with dexamethasone, which 'R@'eth-AyersRtesearrh,Drug Metabolism Division.PriticetonN.J and inhibittshesynthesisof inflammatorymediators,reducedthe Ga]N-induced toxicitiynslicesfrom untreatedrats.These resultisndicatethe participation 'Instituotfe LiverStudies,King @ College School ofMeiiiciiiaend DepitistrLyo,ndon, Etigiand. of Kupffer cellsin GaIN-induced hepatotoxicitiyn vitro,and suggestthat inflammatoryfactorsmay play an importantrolein Kupffcr cell-mediated In thisstudy we have examined the effectof rifampicin(RIF) - Cyp isocnzymes in culturedprecision-cuhtuman liverslices.Liversliceswere hepatotoxicitoyf GAIN. Liverslicesmay providea usefulmodel forstudying rolesof nonparenchymal cellsin chemically induced hepatotoxicity. preparedfrom samples of human liver(surplusto transplantrequirements) with a Krumdieck tissucslicerand culturedin RPM[ 1640 medium in an atmosphere of 95% 0,/5% CO, employing a dynamic organ cultureSYSIcIll Treatmentof liverslicesfor72 hr with 50 @LM RIF induced testosterone hydroxylaseactivityb,ut had no effecton 7-ethoxyresorufi0n-dcethylasc and 7-methoxyresorufln0-demerhylase activitiesA.variahilitWyas observed 396 SOT 1998 Annual Meetino