Document LJpVBqabMevGe33RVxMNGQZw

a - ARIG-Oles kbw-2398 _ Effect of N-Alkyl Perfluorooctylsulfonamides on Mitochondrial ~~ Bioenergetics In Vitro i Kendall B. Wallace, Ph.D. Department of Biochemistry& Molecular Biology University of Minnesota School of Medicine - Executive Summary - The following report summarizes research conducted the past 7 months concerning the effects of 6 different perfluorooctanonyl compounds (PFC's; . suppliedbyThe 3M Company) on the bioenergetic characteristics of isolated rat liver mitochondria in vitro. For purposes of confidentiality, the trade names and chemical structures were not disclosed to those conducting the research and analyzing the results. Consequently, the identities of the individual compounds are codified throughout the report. They are: PFI0; PFI0H; PF12L; PFI2M; PF95; PF143; FC10 the carboxylic acid of FC10 linear FX12 mixed FX12 FC9% FC143 [Results for the sulfonamide and the N-acetic acid of the sulfonamide are not summarized in this report] AilnlcrseiaxsePdFC'thseexapamsisnievdetopdroattoenexhlieabki,tedrsesoumlteinegffecitnonamisttoicmhuolnadtrioinal boifoensertgeetic4s. 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Kendall B. Wallace, Ph.D, Cnivensity of Minnesota School of Medicine Department of Biochemistry & Molecular Biology The initial perfluorachemicals phase of this investigation revealed that all of (PC's) examined were capable of interfering with the the vbiitoroe.nergHeotwiecveprro,petrhteiepsrecofisefrmesehclhyanisioslmatebdy iwnthaiccthcaetaclhivePrRCmiitnotecrhaocntderdiawitihn the mitochondrial membranes protonophoric uncoupling of differed, the activities ranging from aspecific oxidative phosphorylation to a full-blown detergent-like disorganization of the membranes. molecular mechanism, the results indicate Regardless of thespecific that sufficiently high concentrations of any phosphorylation and of the PEC's have ATP synthesis. It the potential of is presumed that inhibiting at the cell oxidative or tissue tlheveelr,egtuhleatrieosnulatnidngidnetfeigcriattioofnAoTfPthweilvlabreiomuasniefneesrtgeyd-daespeanndienntterifnetreenrcmeewdiiatrhy metabolic pathways, cell signaling, and cell balance between ATP-dependent apoptosis cycle control points, including and cell proliferation. the Al potential this point, all that has bien established is that the to interfere with mitochondrial bioenergetics and PFCs have presumably the cell number Whether and such cfounnccetniotnr,atipornosviardeedachtiheevecdonicnenretarlaitstiiocn exipsossuurfeficsiceenntalryioshihgahs. not yet been established. Accordingly, 1) Is the inhibition of mitochondrial two pressing bioenergetics questions in vitro that remain are. by the various PFCs manifosted as a bioenergetic deficit and interference with metabolic : ) 04142 09/15 `98 11551 IDBischemtolBio FAX:218-726-3014 PE 5 May 7. 1998 regulation at higher levels of biological organization, such as in cell cultures, intact tissues. or whole organisms? and 2) Are the nominally effective : concentrations within (he range to warrant concern for human exposures? Inherent in this latter question is the suitability of isolated rat liver mitochondria to predict adverse human health outcomes in vivo We propose to extend the investigation to address these questions by assessing the response of primary cultures of isolated hepatocytes to selected PRC's in vitro (Gay ct al., 1983). The specific design is to assess the effects of exposing cells to individual PRC's on various indicators of cell bioenergetics, including changes in oxygen consumption, mitochondrial membrane potential, ATP phosphorylation potential, and cell viability. Tn addition, we intend to incorporate into these cell studies measures designed to assess the effects of PEC's on the expression of genes believed to be responsible for mediating the effects of related chemicals on peroxisome proliferation, lipid metabolism and cell proliferation. The entire suite of genes tobe analyzed is : believed to be under the control of the "peroxisome proliferator response elements" (PRE) and include peroxisomal fatty acyl CoA oxidase, cytosolic fatty acid binding protein (FABP), miccosomal CYP,,, mitochondrial HMG- CoA synthase, activating adipocyte protein (aP2), and apoproteins Al and Cll (Latruffe and Vamecg, 1997; Gonzalez, 1997). All of these proteins have been implicated to be responsible for the effects of peroxisome proliferators on altered lipid metabolism in vioo and may prove to be valuable markers of the biological response to PFC exposures in the corresponding species. Finally, since there are strong arguments questioning the validity of data from rats and mice to predict the proliferation of peroxisomes and Promotion of tumorigenesis in humans, we propose to include in the study design the opportunity to compare and contrast the metabolic and genctic response of rat and human hepatocyte cell cultures to PFC exposure in vitro. There is substantial evidence that exposuces of non-human primates to related compounds does not induce the proliferation of peroxisomes, . hepatomegaly, and tumorigenesis as is characteristic of rats and mice. 2 04143 uss WE ALI2 1D:BioTchohleBino FAX3218-726-3014 PGE 5 ; May 7. 1998 pEopoirdesmuirozlooggaitceasl feovritdheencheucmoanncurrsespwointshe.theBsausgegdesotnionpubtlhaitshreadts eaxnpdermiimceentaarle evidence, our representative original of the suggestion was human response that guinea bo exposure pigs are more to peroxisome prolifecators. We Pig hepatocytes in intend to tost this the cell exposure by including experiments. primary cultures of guinea The intent is to compare and contrast exposures to the the response individual of rat, PFC. guinea pig and human hepatocytes to Comparisons will be quantitative and based on metabolic dose-related changes in bioenergetic response to PFC exposure. and genetic indicators of a : EXPERIMENTAL DLSIGN 2 isolated rat, guinea common vendor in pig, and order to human hepatocytes will be purchased from minimize the possibility of artifactual differences among the different coll types The cells will be suspended in Hopes-based due to cell isolation procedures, minimum essential media at 37C and distributed among wells at a density of approximately in suspension willbe exposed to the individual PFC' for 10 cells/ml, up to 24 hr. Colle The concentration of each PFC range of concentrations for will cach be varied end-point in order to establish (bioenergetic or gene an effective expression) for cach PEC. "this cange quantitative. comparisons ofeffective amongst concentrations the individual will serve as the basis for PC'S and for assessing edxifpfoesruecnecewsiiln bseendsiictitvaitteidesbyoftthheeki3nestpieccsieosf-stpheecirfeiscpcoenlsletaynpeds.itsThreeladtuiroantshiiopn of to indications for gross changes in cell viability. The exposures bioencrgetic parameters to be include oxygen consumption, assessed during mitochondeial the course membrane of the cell potential, ATP phosphocylation potential, consumption will be monitored in and cell a manner viability. similar to Whole cll oxygen that employed for the : 3 04144 0915 '98 11:52 1D:Biochentio 1Bio FAX:218-725-8014 PRE 7 May 7. 1998 isolated mitochondria. Cells will be suspended in a stirred, closed chamber into which is inserted a Clark-type oxygen electrode metered to a continuous strip-chart recorder. The cell suspending medium will be supplemented with glucose and insulin to provide the requisite metabolic fuels. Once an equilibrium is established, sequential additions of the selected IC will be made and any change in the rate of oxygen consumption recorded. In order to confirm and normalize any effect on mitochondrial respiration, all reactions will be terminated Oxygen consumption in the by first presence adding antimycin A followed by FCCP. of antimycin A will serve to reflect non- N mitochondrial respiration, as one might expect if the agent in question undergoes cytochrome P4sO-dependent oxygenation or redox cycling, for example. The rate of oxygen consumption in the presence of the uncoupler, FCCP, will provide a measure of any direct effect of the individual PFC on the electron transport chain. The potential of effect of the cells the individual in culture will be PFC's on monitored mitochondrial continuously on membrane the basis of the distribution of the cationic fluorometric dye, rhodamine 123 (R123) as wwiethhaRvhe12p3u,blfioslhleodwepdrbevyiowuasslhyin(Pga,lmaenidrathetenala.ll1o9c96a)t.ed Cteollisndwiivllidbuaelpreex-ploosaudreed wells. Again, increasing concentrations of sequentially and the fluorescence recorded. the The respective reactions PFC will will be added be terminated by adding FCCP to achieve a followed by digitonin to assure full depolarization of membrane potential, complete dissolution of cell membranes and release of non-specific latent fluorescence. Cell membrane viability potential will using be the recorded in fluorometric parallel indicator with mitochondrial propidium iodide (Pl) according Sequential to standard additions of procedures in the individual our laboratory (Palmeira PRC's and termination of et al. 199). the reaction with [CCP and digitonin will be performed just as described for Rh123 fluorescence. Unfortunately, PL apparently quenches some of the Rh123 fluorescence, prohibiting the concurrent measurement of both membrane s 04145 Uo 98 11352 IDiBiochendiolBio Fix:218-726-3014 PAGE 3 May 7, 1998 potential and conducted in cell viability in the same cells. Instead, parallel using the cells from the same the experiments willbe culture sample for both measures. by The concentrations of the various adenine HPLC (Jones, 1981). Isolated hepatocytes from nucleotides cach of the will be assessed 3 species will be . eexxppuossuerdesinwiclulltbueresttooppveadrbyiyngalkcaolnicneinztirnagtitohnes roefacttihoenimndeidviiduumal wiPtFhC'SK.OIThteo stabilize Samples the adenine nucleotides in the from cach exposure well will respective phosphorylation state, be centrifuged to remove debris, netealized nucleotides and injected are eluted at onto a Waters Radial 1 ml/min in 100 mM Pak C18 column. Individual potassium phosphate (pH 6) followed by a detected at 260 gradient to 5% methanol. Individual nm and quantified by integrating the peak nucleotides will area as compared be to known standards. displayAcutsiivnagtiosntanodfaPrPdARm-orleescpuolnasrivbeiogloegnyestewcilhlnibqeueasss(eWsasendg;byanddiffMeroernatiisa,l 1997). The method corresponding IC. consists of exposing all 3 specics of hepatocytes to the At pre-determined times, the reactions will be stopped in liquid genes nitrogen encoding and for analyzed for peroxisomal the enhanced expression of a and mitochondrial proteins. select group of This entails isolation of for selected total RNA mRNA's. followed by Probes have gel separation been prepared and Northernhybridization by PCR for the tuo "house- keeping" genes Practin and glyceraldehyde phosphate dehydrogenase (GAPDH), Rene copy which are supposedly number (cell number). un-inducible and thus valid The PRC-induced activation measures for of all other oABfueantneottsiatlwaitglielvnebolemyingcoaruDmgNaelAi.tzheoddetogrtehee ocfosptyimnuulmabtieorn offorgtehneeseextpwreossgieonnesonin tohredebrasitso basis Fxposuretelated of the stimulated prolifecation of peroxisomes expression of the following will be estimated on the genes: peroxisomal fatty . s 04146 wwID U8 11193 ID:Biochemtio IBio FRX:218-726-8014 PAGE 9 May 7. 1998 acyl CoA oxidase, cytosolic fatty acid binding protein (FABP), microsomal CPra, mitochundrial HMG-CoA synthase. activating adipocyte protein (ap2), and apoproteins Al and CII (Latruffe and Vamecq, 1997; Gonzalez, 1997). : Mitochondrial expression of proliferation will citrate synthase be assessed on the basis of theenhanced and succinate dehydrogenase (SDH). Amplification of the mitochondrial genome (MIDNA per mitochondrion as opposed to an increase an in increase in mitochondrial number per cell) will gene copy number for mitochondrial transcript of be assessedby cytochrome b (CYTb) and experiments NDI relative (Boultwood 10 et SDH (a nuclear encoded gene) using gene dosing al, 1996). "P-labeled probes have been generated by PCR for all 3 genes. Increased expression of CYTb and NDI relative to SDH has been used as an indicator of an increase in MIDNA copy number in cardiac tissue following acute intoxication of rats with adriamycin (unpublished observation). The stimulation of both peroxisomal and mitochondrialproliferation is ascribed to altered transcriptional control. To verify this, we will substantiate our results of the gene dosing and differential display experiments by measuring the corresponding enzyme activitics following, each of the various cell treatment paradigms. The exposure incubations will be stopped and the cells recoveredby centrifugation Fatty acyl CoA oxidase activity will be estimated from the cyanide insensitive oxidation ofpalmitoyl CoA according to the spectrophotometric Succinate dehydrogenase (SDH), citrate technique synthase of Lazarow * (1981). and cytochrome oxidase(COX) are measured by the corcesponding spectrophotometric methods (Pennington, 1961; Trounce ct al, 1996; Lash and Jones,1993). 6 04147 wots ws 11:83 ID:BiochentiolBio FAX:218-726-3014 PAGE 10 SUMMARY May 7. 1998 fhe proposed investigation is designed to take our current findings regarding the effects of PEC's on the bioenergetic properties of isolated mitochondrial membranes to the next level of biological integration. The obiective is two-fold: to determine whether the effects of PFC's an isolated mitochondria is manifested as bioenergetic dysfunction al the level of whole cell and to compare and contrast the response of hepatocytes from different species to PEC exposure in culture. The metabolic and genetic studies will yield valuable understanding into the mechanism(s) of biological activity as well as reveal potentially important biomarkers for assessing PFC exposure in vive. The comparisons among species will further test the question regarding the suitability of the rat as an experimental model forpredicting adverse health outcomes in humans exposed to peroxisome proliferators and whether the guinea pig may prove to be a more suitable surrogate. These data eavreideesnsceentiloalprteodiecxttrnaopoelfafteictngledvoesles-arnedspmoanrsgeirnesloaftisoanfsehtiypsfofrrpoomtenetxipaelrihmuemntaanl exposures, especially that isolated hepatic in light of our mitochondria preliminary from guinea evidence pigs is which indicates 36 times more mreistioscthanotndtroiaPF(Cu-nipnudbulciesdhedintoebrsfeorrveanticoensw)i.th bioenergetics than are rat liver 7 : 04148 wats WS 11583 1DBischentio Bis FAX:218-726-3014 PAGE 11 May 7. 1998 REFERENCES Birche-svMoklae.clheit2na;l, MMmi.ut.soccJlhaeocnkmdsirotino,aclh$o.nDKdyersirfa.ulnRcdtSyi.sofnuannc(dtLiaoTsnuh.rnabunInld: l,MJeoDnteMhs.o,dse(1di9e9n3))T.oAxSitcuodlyogyo,f Press cad Boultwood. Gale, RJ.E.FilLdienrc,h,C.,DMCi,llsL,itKtIl,ewoFordo,dsThJa,m.MoPssM,.P.KuAsHe.c, anR.d, Gaiger, A., Wainscoat, 1lSe.ukem(1i9a9.6).BritA.mpJ.liHfaiecmaattiooln. o9f5:m4i2t6o-c4h3o1ndrial DNA in acute myeloid Gonzale7z9:,13F9J.-14(41.997). Recent update on the PPARa-null mouse. Biochimie Gray. 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