Document NGRm39MjOpmYxaJmO3jZO4rjp

AR226 _ 0652 PFOS Pharmacokinetic - | 000113 AN INVESTIGATIONFAOTFTTYHAECEIFDF-EBCITNDSIONFG FPRLOUTOERIONC.ARBONS ON LIVER DEANNA J. NABBEFELD Masters Thesis Key FCI: Perfluorooctane Sulfonic Acid (PFOS) FC2: Ammonium Perfluorooctonate (APFO) FC3: N-cthylperfluorooctane Sulfonamide Ethanol (N-E{FOSE) FC4: N-ethylperfluorooctane Sulfonamide (N-Et FOSE Amide; FX-12) 000114 AN INVESTIGATION OF THE EFFECTS OF FLUOROCARBONS ON LIVER FATTY ACID-BINDING PROTEIN. A THESIS SUBMITTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF MINNESOTA BY DEANNA J. NABBEFELD IN FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE/ENVIRONMENTAL HEALTH APRIL, 1998 000115 AN INVESTIGATION OF THE EFFECTS OF FLUOROCARBONS ON LIVER FATTY ACID-BINDING PROTEIN. ABSTRACT `The objectiveof this study was to investigate thehypothesisthatcertainFluorocarbons (FCs) bind liver fatty acid-binding protein (L-FABP) and displace endogenous fatty acids (FAs) as an initial event leading to peroxisome proliferation. The goalsof the study were to assess the effect of FCs on L-FABP function as evaluated by the ability ofthe fluorescent FA analogue 11 - (S-dimethylaminonapthalenesulphonyl) - undecanoic acid (DAUDA to bind to L-FABP isolated fromratsandguineapigstreated and not treated with FCI in vivo,andtoasstheepotsenscy ofFC1, FC2, FC3and FC4 for bindtionLgFABP. Resultsshow adecreased maximumbindingcapacityofL-FABPfromFCItreatedrats without an increase in Kd. The most potent L-FABPbinderwas FC, followed by FC4 and (with equal IC1os) FC3 and FC2. Results forguineapig L-FABPsampleswere inconclusive. This may be because guinea pigsampleswereonly partially purified; thus resulting in a high degree of interference from remaining cellular debris and a lower concentrationofL-FABP, as & proportionoftotal protein, as compared to rat samples INTRODUCTION STUDY OBJECTIVES "This study was designed to investigate the hypothesis that certain fluorocarbons (FCs) bind to liver fatty acid-binding protein (L-FABP) and displace endogenous fatty acids (FAS)asan initial event leading to peroxisome proliferation. To examine this hypothesis, the kineticsof FAanFdC binding to L-FABP were investigated with an in vitro binding assay using the fluorescent FA analogue 11 - (5-dimethylaminonzpthalenesulphonyl) undecanoic acid (DAUDA). FCI and FC2, known peroxisome proliferators, and FC3 and FC4, suspect peroxisome proliferators, were examined. Wyeth-14,643 (WY), a well known peroxisome proliferator, was used as the positive control and methanol as the negative control. Oleic acid, a FA known to bind to L-FABP with avery high affinity, was used to measure the maximum L-FABP binding. (Figure 1 - structures). L-FABP from 000116 male rats (considered to be strong responders to peroxisome proliferators) and male guinea pigs (considered to be weak or non-responders to peroxisome proliferators) (Svoboda, Grady and Azamoff, 1967; Orton efal, 1984; Lake and Gray, 1985; Elcombe and Mitchell, 1986), treated or not treated with FC1 in vivo, were examined. `Thegoalsofthe study were as follows: 1)to asstheeefsfesct ofFCs on L-FABP function,asevaluated by the ability ofDAUDA 10bindto L-FABP isolatedfromrats andguineapigs; and 2)toassessthepotencyofthevarious FCsforbindingto L-FABP. "The first goal was accomplishedasfollows: a L-FABP from rats and guinea pigs,ireatedand not treated with FCI, was isolated; b.themaximumbindingcapacityorreceptornumber(Bmax) ofeach L-FABP sample and the dissociation constant or affinity (Kd) ofDAUDA for each L-FABP sample were calculated; and . the concentrationofoleic acid which inhibited 50%ofspecific DAUDA binding to isolated L-FABP samples, the oleic acid ICs for each sample, wasmeasured `The second goal was achieved by calculating the ICs,ofeach FC for the binding of DAUDA to the isolated control rat L-FABP sample 2 000117 ECL FC2 EC3 Ct Wyeth-14.643 cl MCeHth--anOolH wl Hy Hy ~sencoon DAUDA N(CH: CHy--(CH;)rO--lCeHi=cACcHi--d(CH2 COOH. on 3 000113 BACKGROUND LIVER FATTY ACID-BINDING PROTEIN. `The exact role of L-FABP, a memberofthe intracellular lipid-binding protein (LBP) family, is unclear (Bass, Kaikaus and Ockner, 1993). It is found predominatelyinthe liver, although it is also present in the smal intestinal and colonic enterocytes, gastric brush border, and enteroendocrceilnles (Bass, 1985; Bass, 1988; Sweetser, Heuckeroth andGordon, 1987; VincentandMuller-Eberhard, 1985; Chanetal., 1985; Gordetoaln., 1982; Sorofand Custer, 1987). Accepted functionsofL-FABP include binding and transporting FAs within the cell, regulating lipid metabolism, and protecting the cell by `maintaining the concentrationoffree fatty acids(FFAS)belowtoxic levels (Baetsals, 1993). L-FABP is unique to the iLBP family in that it has a larger binding cavity (Thompson et al, 1997); broader ligand specificity (binds multiple hydrophobic `compounds such as heme, certain eicosanoids, bilirubin, thyroxine, steroids, specific carcinogens and peroxisome proliferators as wellas FAs) (Kaikaus, Bass and Ockner, 1990; Ockneretal, 1972; Roelralf, 1995; Thumser, VoyansdWieltoyn, 1994;Khan `and Sorof, 1990; Levi, Gatmaitanand Area, 1969); andtheability to bind two molecules per protein while other iLBP bind only one (Thompson ef al, 1997) The crystal structoufraetL-FABP (Thompson ef al, 1997) reveals two short antiparallel achelices positioned over oneendofan 11-stranded antiparallel p-barrel. Thisdiffers from other iLBPs, which are 10-stranded, butdoes not significantly ater the conformation of theprotein. Acavityisformedwithinthe B-barrelthatservesasanintemalized ligand 000119 binding site with polar and nonpolar residues and bound water. In addition to a normal apbetween the two B-strands, L-FABP has a second gap formed by missing hydrogen bonds. The functionofthis gap is unknown, but the missing hydrogenbondsincrease the rangeofmotion in L-FABP compared to other iLBPs. This localized conformational flexibility may contribute to the broad ligand specificity exhibited by L-FABP. `Two L-FABP bindingsitesexist, and interact allosterically. Crystal structuresofthe proteinpreparedwitholeicacidhavecharacterizedtheprimarybindingsiteby an internalized carboxylate and a U-shaped hydrocarbon chain. Fatty acids boundin the primary binding site interact with Arg, a conserved resiidnuaellLBP; and are: surrounded by protein atoms, structural water and nearby atomsofthe second bound FA. `Theoleicacidintheprimarybindingsite isinvolvedinhydrogenbondinteractionsat the `carboxyl group with Ser", Arg'TM and SerTM. The secondary binding site is characterized by having the carboxylate ofthe second oleic acid near the surface, and the hydrocarbon tail inserted toward the centerofthe molecule and between the U-shaped hydrocarbon chain intheprimary binding site. Thecarboxylate at this ite is solvent-accessible, but still involved in anetworkofhydrogen bonds with residues forming the entrance to the primary binding cavity. The two ligands are in physical contact and it is believed that they influence each others relative affinities. Structural data suggest the second site may not existuntiltheprismiteaisfrileyd, orthatthepriorpresenceofa FAin theprimarysite mayberequiredforanythinglargerthat a Ci FAto bindthesecondarysite (Thompsonef al, 1997) 5 000120 FLUOROCARBONS Fluorocarbons (FCs) are compounds structurally analogous to hydrocarbons with the hydrogens replaced with fluorines. The FCs under investigation resemble long chain FAs, havinag hydrophobic tal and apolar head group. Thetails of FCs aremore rigid in structure than the tailsofFAs, however, and thus the conformational flexibilityof FCs is `more restricted than thatof FAs (Zisman, 1964). FCs have unique chemical and physical `propertiessuchasbeingveryheatstable, inertandchemically andelectricallynonreactive (Bryce, 1964;Bankes, 1970; GeaondrAndegrsoen,1986;Gilliland, 1992;Clarketal, 1973). Such charactermiaskettihcesm idealforuse in many consumer products and industrial procedures (Bryce, 1964). FCsarecomponentsofproducts including household cleaners, leather treatments, insecticides and fire-extinguishing foams; used as surfactants intheaqueous polymerizationoffluorinated monomers; and used in industrial processes such as insulating, cooling, wetting, and corrosion inhibition (Bryce, 1964; Bakes, 1970; George and Anderson, 1986; Gilliland, 1992; Clark ef al., 1973). Despite the usefulness of these chemicals, some are knowntocause mitochondrial inhibition, cholestasis, `peroxisome proliferation andtumorformation in rodents (Gililand and Mandel, 1996; Langely, 1990; Ikeefdala. 1985; Pastefoa.r, 1987; Harriseifaoln, 1988;Abdellatifer al, 1991). Permadi etal (1993) suggest chain lengthofFCs influences the severity ofeffect, finding the greatest significance extibited by Cs compounds followed closely by Cio compounds, andincreasinglyless severe consequences exhibited with shorter chained molecules. The . workof FellerandIntrasuksti (1993)agreedwiththatofPermadi ef a (1993),andadded 6 000121 that a carboxylic function was important for the stimulationofperoxisome prioliferation. Similar results were reported by Kennedyefal. (1998), who analyzed FCs ranging in lengthfrom4-9carbonsfortheeffectofstructureon toxicity.TheyfoundCs FCs to produce effects at a 10-fold lowerdosethan C, FCs, and short chained FCs to be the least toxic. Althtoheueffgechts seenin rodentshavenotbeenseeninhumans,thepotentifaolr cumulative and long-term human toxicity resulting from continuous exposure to low concentrationsof FCs isofconcern (Gilliland and Mandel, 1996;Gilliland, 1992). PEROXISOMES & PEROXISOMEPROLIFERATION. According to Small (1993), peroxisomes (also called microbodies or, in plants, glyoxysomeasr)esinglemembrane-limited cytoplasmic organelles present in most eukaryotic cells. The major functionofperoxisomes is the B-oxidation of FAs and FA derivatives (Mannaerts and Van Veldhoven, 1993). Peroxisomes contain no DNA; rather, their proteins are synthesized on free polyribosomes in the cell cytosol and imported into pre-existing peroxisomes post-translationally. It is believed that new peroxisomes form by fission from existing peroxisomes. Peroxisomes have been shown to proliferate following exposure to adiverse class of chemicals referred to as peroxisome proliferators (Green, Issemann and Tugwood, 1993) `The mechabnyiwhsicmh this occurs is unclear. Peroxisome proliferation is thought to be `mediated by peroxisome proliferator activated receptors (PPARs), nuclear hormone receptors which, upon binding ligand, recognize specific DNAsequencemotifs located upstreamof the peroxisomeproliferatortarget genes (peroxisome proliferator response 7 000122 elements (PPRES)), and activate specific gene transcription (1sseman and Green, 1990; Dryeretal., 1993). Due to the diversityofperoxisome proliferators shown to activate PPAR (Green et al., 1993), speculation exists over a direct modulation ofPPAR by peroxisome proliferators, and an indirect mechanism is suggested. Inaddition to chemical `and xenobiotic peroxisome proliferators, natural factors suchas a high fat diet, starvation and diabetes (Flatmark ef a., 1988; Ishi efl., 1980; Ishii, Horieand Suga, 1980; Horie, FukuamndoSrugia, 1991;Gottlicher, WidanmdGuastarfsskon,1992)havebeenshown to cause peroxisome proliferation. This correlation between peroxisome proliferation and FA metabolism suggests that PPAR serves an important role in lipid homeostasis (Vanden Heuval, 1996). It is probable, thus, that PPAR activation represents a physiological response to a biological stimulus, likely a factor involved in FA metabolism (Green et a., 1993). Possible stimuli/PPAR ligands include steroids, FAs and derivatives ofFA `metabolism, and cholesterol metabolites (Green ef al, 1993). Target genes include those for acyl-CoA oxidase (Tugwood ef al, 1992; Feller and Intrasuksti, 1993), L-FABP (Issemaneral, 1992) and P450ra genes (Greeneta., 1993). Significant interest surrounds the issueofperoxisome proliferation because some peroxisome proliferators have been shown to cause hepatocellular carcinomas in laboratory rodents (Moody ef al, 1991; Vanden Heuval, 1996). The mechanism by which peroxisomeproliferatorscausecancerinrodentsisunknown. Theyare classifaisead novel classofepigenic chemical carcinogen (Vanden Heuvel, 1996), are nonmutagenic in the Ames assay and do not appear to bind DNA (Conway efal, 1989;Cohenand Grasso, 1981; Reddy and Lalwani, 1983, Stott, 1988;Reddyand Rao, 1989;Lakeet al, 1990). 8 000123 Multiple mechanisms have been proposed to explain peroxisome proliferator-induced liver tumor formation, including oxidativestress(Reddy and Rao, 1989), enhanced cell replication (Marsman ef al., 1988) and promotionofspontaneously formed lesions (Schulte-Hermann et al., 1989). Greenefa. (1993) propose peroxisome proliferators are "complete carcinogens" which exhibit a combinationofinitiation (oxidative radical production) and promotion (liver mitogenesis), possibly leading to sustained DNA replicationdependingonthecompoundanddose. Doubtabout acausalrelationship between peroxisome proliferationandcarcinogenesis inrodentsexists, however, and the relevance to human health isunclear (Tucker and Orton, 1993). `Mammalian species diffc in their response to peroxisome proliferators (Lake and Gray, 1985;RodranidTcurnkbulsl, 1987). Ratsareconsideredstrongresponadndegruisne,a pigs and nonhuman primates low to non-responders (Svoboda ef al, 1967; Orton et al, 1984; Lake and Gray, 1985; Elcombe and Mitchell, 1986). Slight to no increase in peroxisomes were found in human patients treated with colfibrate (Hanefeld, Kemmerand Kadner, 1983) and fenofibrate (Blumecfkale, 1983),drugsusedinthe treatment of hypercholesterolemia and known peroxisome proliferators in rodents. Many hypothesize that iaf causal relationship does exist between peroxisomeproliferationand hepatocarcinogenesis, it i specific to rodents and not a risk to man (Tucker and Orton, 1993) Other well documented effectsofperoxisome proliferatoirns rodentsinclude inhibition of ` mitochondria B-oxidation (Elcombe and Mitchel, 1986; Eacho and Foxworthy, 1988; 9 000124 Foxworthy and Eacho, 1988; Lock, Mitchell and Elcombe, 1989; Wallace, 1998), inductionofperoxisomal f-oxidationanda-oxidation in the ER (Reddy and Lalwari, 1983; Hawkins efal, 1987), inductionofL-FABP expression (Bass, Manning and Ockner, 1985, Das, Gourisankar and Mukherjea, 1989; Fleiscehfnae.r, 1975), cholestasis (Elcombe and Mitchell, 1986; Foxworthyand Eacho, 1988; Lock ef al, 1989; Van Rafeighem efal, 1988) and hepatomegaly (Moodyefal, 1991). FATTY ACID CATABOLISINMTHE HEPATOCYTE Free faty acids (FFAS), formed by the breakdownoftriacylglycerol stored in adipocytes, arecarried in the blood by serum albumin and transported into hepatocbywtheast is thought to be a plasma membrane bound fatty acid-binding protein (FABPpm) (Stremmel, Strohmeyaendr Berk, 1986; Stremmelefal, 1985). Once in the cell, FFAs are picked up by L-FABP and, under routine conditions, the majority are transported to the mitochondria for f-oxidation, a process by which FAs are degraded to acetyl-CoA by the sequential removaloftwo carbon segments (Moran and Scrimgeour, 1994). Mitochondrial B-oxidation is coupled to the generationofhigh energy phosphate bonds via oxidative phosphorylation, and results in the synthesisof ATP and ketone bodies. In order to gain entry into the mitochondria, FA must first be converted to acyl-CoA estersby acyl-CoA synthetases, FA specific enzymes located in the mitochondrial outer membrane (Singh, Derwas and Poulos, 1987). The rate of FA entry into the mitochondria is regulated by camitine acyltransferase I, a second enzyme located in the outer membraneofthe mitochondria,whichconverts acyl-0Aestersto acylcarnitines (Murthy and Pande, 1987). Onceinsidethe mitochondria, acylcarnitianreesconvertedbacktoby acyl-CoA 10 000125 esters by camitine acyltransferase IT, and degraded by mitochondrial B-oxidation to acetylCoA (McGarry and Foster, 1980; Bieber, 1988). Acetyl-CoA is shuttled into the cytosol by the citrate transport system for cholesterol and lipid synthesis (Stryer, 1994). The key factor regulating the rateofmitochondrial B-oxidation is the amountofFA entering the `mitochondria which, as stated above, is controlled by carnitine acyltransferaseI. The activityofcamitine acyl-transferase |iscontrolledbythe abundance ofmalonyl-CoA, the first committed intermediaitne FAsynthesis(McGarry and Foster, 1980). According to Bass ef al. (1993), under conditionsofincreased FA biosynthesis, malonyl-CoA production is increased. Malonyl-CoA isproduced from acetyl-CoA in a reaction catalyzed by acetyl-CoA carboxylase, an enzyme controlled by reversible phosphorylation responding to hormone signals and the presence offatty acyl-CoA (Moran and Scrimgeour, 1994). When fatty acyl-CoA levels are low, acetyl-CoA carboxylase activity is high. Thisenhances the conversionofacetyl-CoA to malonyl-CoA. When malonylCoA is plentiful, the activity ofcamitine-acyltransferase I is limited. This causes the rate of mitochondrial p-oxidation to decrease; FFAS to accumulate; acyl-CoA production and hence cholesterol synthesis to slow; and the ratesofaltemate routesof FA catabolism, peroxisomal p-oxidation and -oxidatioin the endoplasmic reticutum (ER), to increase (Lock et l., 1989). Peroxisomal B-oxidation is normally responsibleforcatabolizing most,ifnot all,of the very longchain fatty acids broughtintothehepatocyte (Singh eral., 1981; Singh etal, 1984;Lazoetal., 1990; JaakndoWanbderss, 1991). This sysitsaelsmocapableof oxidizing medium andlongchain FAsand previously activated CoA estersofmediumand n 000126 tong chain dicarboxylic acids. Under normal conditions, however, mitochondrial Boxidation is the dominant route ofcatabolism for such substrates (Singh er al., 1987). Peroxisomal B-oxidation proceeds through similar steps as does mitochondrial Boxidation, however, important differences exist. First, theenzymesusedineach process aredifferent proteins (Hashimoto, 1987). Secondly, peroxisomal B-oxidationdoes not degrade FAsto their two carbon fragmentsas does mitochondrial B-oxidation; rather, peroxisomal B-oxidationstopsafter afew cycles, onlyshorteningthe carbon chain (Lazarow, 1978; Thomas et al,, 1980). Thirdly, peroxisomal B-oxidation is not coupled 10 an electron transportchainand oxidative phosphorylationas is mitochondrial Boxidation (Lazarow and de Duve, 1976; Mannaertsetal., 1979). Thus, while mitochondrial B-oxidation produces ATP and ketone bodies, peroxisomal B-oxidation produces hydrogen peroxide and heat. The rateofperoxisomal B-oxidation is thought to be controlled by substrate supply, specifically the activity ofacyl-CoA oxidase, which reduces molecular oxygen to hydrogen peroxide in the first stepofperoxisomal `oxidation (Mannaerts ef al, 1979; Miyazawa ef al. 1983). Like substrates for `mitochondrial B-oxidation, substratesforperoxisomal B-oxidation must be esterified to their acyl-CoA derivatives; however, peroxisomal B-oxidation is not dependent on camitine acyl transferase I, as is mitochondrial B-oxidation (Mannaerts and Van Veldhoven, 1993), 0-Oxidation in the ER, a P450 vay mediated process, i responsibleforconverting . monocarboxylic acids to dicarboxylic acids. Dicarboxylic acids are activated in the ER by 2 000127 dicarboxylyl-CoA synthetase, an enzyme absent inmitochondriaand peroxisomes. CoA estersofdicarboxylic acarie adlmosst entirely dependent on mitochondrial B-oxidation for catabolism (Suzuki efal., 1989). TheER also oxidizes bile acid intermediates and is able to esterify very longchainfattyacids (Singhand Poulos, 1988;Lazo efal., 1990). A prerequisiteofesterification is activationof FAS to their CoA derivatives (Mannaerts and VanVeldhoven, 1993). `@-Oxidation intheER isenhanced incasesof FA overload(eg. uncontrolled diabetes) or inhibitionofmitochondrial B-oxidation (Mortensen and Gregersen, 1981; Golden and Kean, 1984; Mortensen, 1986; Vianey -Liaud efal., 1987); and like peroxisomal B-oxidation, @-oxidatisinoont dependent on camitine acyl transferase | (Mannaeratnds Van Veldhoven, 1993). Hyromizsis As stated above,exposure to FCs leads to mitochondrial inhibition, cholestasis, peroxisome proliferation, and tumor formation in rodents. Threeofthese endpoints - `mitochondrial inhibition, cholestasis and peroxisome proliferation - are directly linked to FAmetabolism. Thisstudy wasdesignedto testthe hypothesisthataninitialstep in FC- induced peroxisome proliferation is displacement of FAs from L-FABPby FCs. This `hypothesis is supported by thefact that L-FABP has been shown to bind nongenotoxic peroxisome proliferators, includingcertain FCs, in vitro (Vanden Heuvel, 1996; Issemann etal, 1992), withrelativestrengthsofbinding that paralleltheirability to elicit peroxisomeproliferation(Brandeser al., 1990; Kandaetal,, 1990; Cannon and Eacho, 1991). According to thetheoryunder question, upon displacementof FAs fromL-FABP, ' theintracellularlevelsoffattyacyl-CoA woulddecrease.Thiswouldincrtheeaactsiviety 5 000128 of acetyl-CoA carboxylase and enhance the conversionofacetyl-CoA to malonyl-CoA. An increase in the level of malonyl-CoA would repress the activity ofcamitine acyltransferase 1, and inhibit mitochondrial B-oxidation. @-Oxidatinitohne ER would be enhanced,increasingthe productionofdicarboxylic acids. PPARs wouldbeactivated, by the bindingof FAs or metabolicintermediatessuch as dicarboxylic acids, and specific gene transcriptionofacyl-CoA oxidase, L-FABP and P450 wai wouldbe induced. A positive. relationship between the amountofL-FABP and the rateofperoxisomal p-oxidation has `been found (Appelkvist and Dallner, 1980), and the level ofacyl-CoAoxidase is thought to determine the rateofperoxisomal B-oxidation (Manneafaelr,1t97s9;Miyazeafwala., 1983). Thus, increased transcriptionofacyl-CoA oxidase and L-FABP would increase atesofperoxisomal B-oxidation and elicit peroxisome proliferation. InductionofP4S0as `genes would further increase the rateof o-oxidation in the ER. Cholesterol synthesis `would eventually cease in response to mitochondrial inhibition and lackofacetyl-CoA production, and decreased esterificationby the ER due to decreased acyl-CoA. This `would lead tocholestasis. Themechanismsby whichcarcinogenesiscouldbeinducedor `promoted will not be discussed 1 000129 MATERIALS AND METHODS MATERIALS `Wyeth-14,643 (WY)wasobtained from ChemSyn Science Laboratories, Lexena, KS; FCs `wereprovided by 3M Speciality Chemicals Division, St. Paul, MN; Optifluor LSCcocktail was obtainedfrom the Packard Instrument Company, Meriden, CT; AMICON 'YM-5 membrane was purchased from Amicon Corporation, Lexington, MA; BCA Protein Assay was obtained from Pierce Chemical Company, Rockford, IL; and 11-(5- Dimethylaminonapthalenesulphonyl)-undecanoic acid (DAUDA) was purchased from Molecular Probes, Eugene, OR. All other chemicals wereobtainedfrom VWR Scientific, West Chester, PA. ANIANMDTRAEALTMESNT Male rats and guinea pigs, 6-8 weeofkagse, weighing between 150 and 250grams were purchased from Charles River Labs, Wilmington, MA. Following an adaptation period of `oneweekafter arivaalt 3M,aniwemreaweilghesd, ear-tanadgexgpoesedd. The treatment groups consistedofthe following: 1. Guinea Pig Vehicle Control - Tween 80, 2% (n = 4); 2. Rat Vehicle Control - Tween 80, 2% (n =4); 3. GuineaPig FC1 - FCIin Tween80,2%(n = 4);and, 4. Rat FCI-FCIin Tw80e ,2%e (n=n 4). All treatments were administered by intraperitoneal (ip) injection. Thevehicle control groupswere dosed at Smi/ kg body weight 2% Tween 80. The FC1 groupswere dosed at 1s 000130 5ml /kgbody weightwith asuspensoifo3n2mM FCIin Twee2n%(-868mg0F,C1 / kg body weight). All animals were housed individually in controlled environments and. observed for mortality and clinical signsof toxicity during the first four hours after dosing, at 24 hours, and daily thereafter forthedurationofthe study. Animals were sacrificed with CO; 12 days after dosing. Body weights and selected organ weights (liver, kidneys, testes) were recorded at necropsy.Organtissues and body fluids were stored frozen at - 70C for biochemical analyosriisn 10%buffered formaldehydeforsubsequent histological analysis. Selected liverswereperfusedwithand storedingluteraldehyde for future histological analysis by light microscopy. PURIFICOAFLT-IFAOBNP Protein purification was performed at the Universityof San Francisco, CA (UCSF) Liver Research Center. Three frozen livers (rat FC1, guinea pig vehicle control, and guinea pig. FC1) were shipped in dry ice from 3M to UCSF. These livers and one fresh liver, froma. non-treated rat (control) sacrificed at UCSF, were purified. Frozen livers (approximately 10geach)were thawed and weighed. The fresh liver (approximately 10g) was isolated and perfused with isotonic saline. Eachliverwas homogenized 30% (w/v) in ice-cold 10mM potassium phosphate buffer, pH 7.4, using a `Teflon-glass Potter-Elvehjem tissue homogenizer. The homogenates were centrifuged for 20 minutes at 10,000g in a Sorvall superspeed RC2-B centrifuge maintained at 4C. The `supernatantswere removed and subsequently centrifugedforone hour at 38-40,000 rpm (4C) in a Beckman L7 Ultracentrifuge. The resulting supernatant (cytosol) waslabeled ' `with0.54Ci of1-ColeatetotracetheL-FABPduringpurification. 16 000131 Purification steps were performed in a cold room at 4C. The cytosol was loaded on a Sephadex G50 M column (5 x 60 cm) equilibrated with 10mMpotassium phosphate buffer, pH 7.4. Proteinwaseluted from the column at a flow rateofapproximately 1.4 `mi/minute. One hundred fractions were collected (approximately 14.5 mUfraction). Optifluor LSC-cockail (5 ml)was added to a 20 aliquotofeach fraction and a Packard Tri-carb 4530 scintillation cowuasnusetdtoeassress L-FABP activity. Fractions with LFABPactivitywerepooledandconcentratedtoapproximately Smusing avacuumfilter apparatus fitted with an AMICON YM-S membrane. Guineapig samples (control and FCItreated)wereconceanndftrozrenaattthisepoidnt.Ratsamples (controland FC1 treated) were further purified as follows. `Theconcentratedsolutionwas loaded on a Sephadex GS0 (fine) gel filtration column (2.5 x45 em) equilibrated with 10mM potassium phosphate buffer, pH 7.4. The flow rate was. `approximately 0.9 m/minute. Sixty fractions were collected at a volume of 3.48mUfraction. The fractions containing L-FABP activity were pooled and concentrated to approximately Sml. The concentrated cytosol was dialyzed overnight at 4C against 30mM Tris-HCL, pH 9, using a Spectrapore Membrane MWCO 3,500. The sample was then applied to a DEAE-cellulose column (Whatman DE-S2, 1.25 x 15cm or 2.5 x 15cm) previouslyequilibratedwith 30mM Tris-HCL, pH 9, (degassed). The column was eluted with 30mM Tris-HCI, pH 9, (degassed) followed by alineargradientof NaCl (0-0.2M) in 30mM Tris-HCL pH9. The flow rate was approximatly Imlminute. Twenty fractions, 7.8 ml each, were collected. 1" 000132 Homogeneity of the final rat control and rat FC fractions was assessed using sodium dodecyl sulfate/ polyacrylamide-gel electrophoresis (SDS PAGE) analysis, and confirmed by theappearanceof adominantproteinbandatmolecularweight (MW) 14,000Daltons (Da. (Figure 4). Protein concentrationofallsampleswas determined using the bicinchoric acid (BCA) protein assay by Pierce with BSAasthe standard (Table 1) Noneofthe samples were delipidated. FIGURE 3 SDS PAGE ANALYSIS OF FRACTIONS FOLLOWING PURFICATION OF L-FABH FMROMaRAMT,rvLaIlVuk EeRsaCrYeeTsOhSorOwLn.. RAT CONTROL L-FABP -- Ce -- 100 x --o _ -- -------- = --n RATECI L-FABP Nm --te -- - ` . bt mm... i fot T--w --s --a =--a3 | =n . I. i----wa----] --e 1 000133 TPArBoLteEin|c-onPcReOnTtrEaItNiCoOnwNaCsENdTeRteArTmIiOnNe.d using the BCA protein assay with BSA as the standard_Valuesare a mean + standard deviationof 2 trials FRACTIONS, TOTAL PROTEIN CONCENTRATION (ug/ml) PRaUtRCIoFnItrEoDl Rat FCI 123.1% 137 3088+ 14 PARTIALLY PURIFIED Guinea Pig Vehicle Control Guinea Pig FC1 1757+ 101 1021021297 FLUORESCENCEMEASUREMENTS Fluorescence measurements were based on the workofWilkinson and Wilton (1986). All assayswerecarriedoutatroomtemperatureusing a litwithof Snmin a SPEX 1681 0.22m spectrometer, SPEX Industries, Incorporated. A stock solution ofDAUDA, 0.1mM, was prepared by slowly adding SOmM potassium phosphate (KE,POL) buffer, pH 72,0 1mM DAUDAin methanol. Al further dilutions ofDAUDA were in SOmM KH;PO, pH 7.4. All dilutions of L-FABP sampleswerein S0mM KH;POL, pH 7.4. All FCs, WY andoleicacidweredissolved in methanol. All measurements weremadeafter binding had reached equilibrium. FLUORESCENCE CHARACTERIZATION `Themaximumemissionandexcitationwavelengths (nm)and averagemaximum fluorescence inte(nFTs)(ictpmy) were determined for 1yM DAUDA binding to each L- 19 000134 FABP sample. L-FABP, from original undiluted stock, was added to 2ml [uM DAUDA in aliquots of 1.6 -114yl (depending on the concentrationofprotein) until no further change in emission or excitation wavelength or FI was detected. The rangeofprotein concentrations analyzed for each L-FABP sample was 0.1uM-3uM. Excitation scans, from 250-400nm using an emission wavelength of 500mm, and emission scans, from 350600nm upon excitation at 350mm, were performed with each addition. FI (Em. 50am, Ex, 350m) was measured folleoacwhadidintigonof L-FABP. CurvesofFI versus concentrationofL-FABP, represeating an average standard deviation of3 trials, were constructed for each L-FABP sample. The three highest FI values for each curve were averaged to determine the average maximum FI for DAUDA binding to each sample. Specific DAUDABinding Total binodfDiAnUDgA (0-8) to each L-FABP sample was determined by adding 220ul aliquots of 0.1mM DAUDtAo 2m! 1M L-FABP. Increased FI (Em. 500mm, Ex., 350mm) due to the binding ofDAUDA to protein was measured. Nonspecificbindingwas assessed by saturating L-FABP binding sites with oleic acid and performing the same titration. Aliquots, 2-20ul, of 0.1mM DAUDA wereaddedto a 2ml solution of 13M LFABP and 1004 oleic acid. Specific binding ofDAUDA to each L-FABP sample was determined by subtracting nonspecific binding from total binding. ANALYSISOF THEEFOF F FCE SOFC L-FT ABP CalculationofDAUDABinding Constants(KyBau) Kdand Bmaxvaluesweredeterminedforeachprotein.Specificbindingwastransformed tounitsofboundDAUDA (iM)bydividingthespecificFI (cpm)by themaximumFIper 20 000135 14M DAUDA (cpm) for each L-FABP sample. Computer assisted nonlinear regression (GraFit Version 3, Erithacus Software Limited) was used o construct curves of specific bound DAUDA versus free DAUDA representing an average of 3-6 rials. The following equation was used, Bound = (L] x Bmax}/(Kd + [L]). `CalcofuOlleiac Atciid IoC'ns "The concentration of oleic acid which inhibits S0%of specific DAUDA binding, the ICs was calculated for each combinationofoleic acid and L-FABP sample. Cuvettes contained 2m 1uM L-FABP and 14M DAUDA. Oleic acid, IM in 10% methanol, was added in 0.4-20 pl aliquots. FI (cpm) (Em. 500mm, Ex. 350nm) due (0 the binding of DAUDA to protein following each addition was measured. Curves of percent inhibition of specific DAUDA binding versus oleic acid concentration, representing an average + standard deviation of 3-6 trials corrected for the effect of methanol, were constructed. ANALYSIS OF THEPOTENCY OF VARIOUS FCS FOR BINDING 70L-FABP FR i (Ica Cuvettes contained 2 ml 1M rat control L-FABP and 1uM DAUDA. Ligands, ImM (FCs and WY in 100% methanol,andmethanol in S0mM KEH:PO, pH 7.2), were added in 0.420 ul aliquots. FI (cpm) (Em. 500nm, Ex. 350nm) due the binding of DAUDA to protein following each addition was measured. Curves of percent inhibition of specific DAUDA binding versus competitor concentration, representing an average + standard deviation of 3-6 tials corrected for the effect of methanol were constructed. The concentrationofeach competitor which inhibited 50% of specific DAUDA binding, the . ICao, was calculated. 2 00013 RESULTS & DISCUSSION FLUORESCENCEMEASUREMENTS FLUORESCENCE CHARACTERIZATION Emi ands Excitsatioin Maoximnum `The emission and excitation spectraofeach protein binding DAUDA was analyzed to determine the optimal conditionsforthe study. The maximum emission and excitation wavelengths for all proteins were approximately 500 and 350nm respectively (Table 2). Values for guinea pig L-FABP samples were slightly higher than those for rat samples. `This difference is likely because guinea pig samples were only partially purified; thus, more cellular debris remained to potentially interfere with DAUDA binding, and lessL-FABPas a proportion of total protein was present. A "blueshift"in both emission and excitation wavelength occurred upon the additionofeach L-FABP samplteoDAUDA. Excitation `wavelength shifted from approximately 330 to 340nm (Figure 4), and emission wavelength shifted from approximately 550 to S00nm (Figure 5). This shifting of wavelength is characteristic of DAUDA binding to a nonpolar ste on L-FABP (Wilkinson and Wilton, 1986). According to Thumser efa. (19942), DAUDA only binds one of the two oleatebinding sitesofL-FABP. Due to the stringent conformational requirements of primary site binding (intemalized carboxylate/polar group and a U-shaped hydrocarbon/hydrophobic chain), DAUDA most likely bindsthesecondary site. Similar shifting of fluorescence wavelengthandemissionandexcitation maxima were found by Wilkinson and Wilton (1986)andThumser, Vosey and Wilton (1996). 2 000137 `FTlAuBoLrEes2ce-nEcMeIeSmSiIsOsiNoAnNmDaxEXiCmIaT(AnTmI)OwNeMrAeXmIeMaAsu.red upon excitation at 350nm. Excitation tm0ax1i1mMa D(nAmU)DwAeurneimielansourfuerdtuhseirncghaanngeemiinsseimoinsswiaovneolrenegxctihtoafti5o0n0wmamv.elLe-nFgAthBPwawsas added detected. Eachvalueisan averg standarddeviationof 3trials. SAMPLE EMISSION EXCITATION MAXIMUM (am) MAXIMUM (am) DAUDA only 55133153 33.00% 1.00 Rat Control L-FABP 502.67 4.62 34433208 Rat FCI L-FABP 502.33 4.04 339.67:6.66 (GuineaPigVehicle Control LFABP 511+.07.081 340672513 GuiPnigeFCa1 L-FABP 511.00 +7.81 333.67 Ls FFlIuGoUrReEs4ce-ncEeXeCxIcTitAaTtIiOonNMmAaxXiImMaA.(um) were measured using an emission wavelength of w50a0nmv. eL-lFwAeaBsnPdwegatescttaedhdd.eEdactoh c2umrlve1i4sMreDpArUeDseAnutnattiloifvn3oefturritalhser(c1huaMngDeAiUn DexAc,ita3tuioMnLF FABPA).dKB=ReatPC:aon=t,GruoilnLeFaPAiBPg.FCIL-FABP;b= GuioeaPig VehicleControl LFABP; = Rat FCI L- a b a Wavelength(vm) _%--0 350 "<x 2 000135 EIGURE 5- EMISSIONMAXIMA, wFlausoraedsdceedncteoe2mmilss1io4nMmaDxAiUmaDA(numn)tiwlenrofeumretahseurrcehdanugpeoninexecmiitsastiioonn awtav3e5l0ennmg.thLw-aFsABP K0Dd2e.AtY1Ue,cD2MtA-eL.d0F.3AED4BaAMPcU:LhD-cdAF=uA.rBv1OPe4.LiMFsDArASeUP.DpASr,0em0Ms5Ke4FMnPoLtOfaA.3tBbti:r=ival1es=.M1D3AUDDAAU,DA0.LF1A4BMFL: A=B:1M=DLAuUbD.A. RATCONTROLL-FABP RATECILFABP e. 4 d "c = c: = . . --_-- |= GUINEA PIGFCLL-FABP tr . ac 'a . d " c Zl b. pn 000139 AvFerlagu e Maoxir mumescenIntcenseity The average maximum FI (pm) of Ium DAUDAbindingto L-FABP from FCI treated `and non-treated animals did not substantially differ. The average maximum FI was approximately 850,000 cpmforrat L-FABP samples, and 660,000 cpm for guinea pig LFABP samples (Table 3). Maximum FI was reachedfollowingthe addition of 15M rat control L-FABP, 2.54M rat FCI L-FABP, 2.5uM guinea pig vehicle control L-FABP and 24M guinea pig FCI L-FABP (Figure 6). Thelowerthe affinityofreceptor for probe, the higher the concentrationofreceptor needed for binding, and viceversa(Matthews, 1993). `Thus,thesedatasuggestthat theratFC1 L-saF mplehA adaB decreP asedaffinityfor DAUDAascompared tothe rat control L-FABP sample, and that the guinea pig FC1 sample had an increased affinity for DAUDAas compared to the guinea pig vehicle control L-FABP sample. The average maximum FI for guinea pig samples was 77.6% Tower than that for rat samples. Thismaybe due to the impuroiftthye guinea pig samples resulting in a high degreeofinterference in DAUDA binding and a lower concentration of L-FABP as a proportionoftotal protein. TABLE3-MAXIMUMFIOF1uM DAUDA. Cuvettes containing 2 ml 14M DAUDA were titrated with rat andguineapig L-FABP samples (0. 1mM) to determine the maximum FIof1um DAUDA binding to each sample. Three trials per protein were performed. Valuesare the average + standard deviation of the 3 highest average FI values per protein. LFABP SAMPLE MAXIMUM FI (cpm) Rat Control 848,093 13,639 Rat FC 852,438 24776 Guinea Pig Vehicle Control 655,348 + 63,812 Guinea Pig FC1 669,632 + 33,619 2 000140 EIGURE6-AVERAGEMAXIMUMFLOF uMDAUDA.FTVS[L-FABPL Cuvettes conning 2 ml 1M DAUDA were titrated with rat and guinea pig L-FABP samples (0.1mM) to determine the maximumFT of 1m DAUDA binding to each sample. FL due to the binding of DAUDA ( protein, was measured after cach addition. Each curve is an averageof 3 rials. s00000 s7eonocooo = 0000 sooo $ dao Er] ------ 220000000000| + aGuinea PgV.Con |ecui Pg 0s Toc o - o1 oa os 1 1s 2 25 3 tram a0) SpecificDAUDABinding. Because of its well documented high affinity for L-FABP (Thumser et al, 1994 ab; Thumser and Wilton, 1994; Thumser ef al, 1996: Thumserand Wilon, 1995), oleic acid was chosen as the displacing ligandtodetermine nonspecific binding of DAUDA to LFABP. Excess oleic acid was added to occupy all L-FABP binding sites and prevent the specific binding of DAUDAto L-FABP. DAUDA was titrated into the assay and FI, due 10 nonspecific binding, was observed. Total binding was determined by performing the assay in the absence of oleic acid; specific binding was calculated by subtracting nonspecific binding from total binding. Absolute FI values are given in Table 4. The breakdown of total binding into percent specific and nonspecific binding is shown in Figure 7 2 000141 |TToataLlEbin-diLnugMofDDAAUUDDAABtIoNeDaIcNhGLT-OFLAUBMPLs-amFpAlBePw.as determined by adding aliquots of 0.1mM DAUDA to 2ral 1uM L-FABP. IncreasedFI due to the binding of DAUDAto apcriodt.eiSnpewcaisfimcebaisnudriendg.oNfoDnsApeUcDifAictbienadcihngL-wFasABmPeassaumrpeldeiwnatshedeptreersmeinnceedofby1s0u0butrMacotlienigc nonspecific trials (cpm). binding from total binding. Values are an average + standard deviation of 3-6 L-FABP SAMPLE TOTAL NONSPECIFIC _ SPECIFIC Rat Control 318.0002 [24,000 78.000 % 26,000 740,000 = 124,000 Rat FCI 302000+ 81000 Guinea Pig V. Control 219,000 138,000 9600021,000 206000% 81,000 92,0004 23,000 127,000 138,000 Guinea Pig FC1 162000+ 15000 9400065000 68,000 15000 Ed 000142 [EToItGaUlRbEi7nd-iDngAoUfDDAABUIDNADtINoGe.ach L-FABPsample was determined by adding aliquots of 0pr.o1temiMn wDaAsUmeDaAsur1e0d2.mlNon1supMecLi-fiFcABbPi.ndiInngcrweaassemdFeaIsduurcedtointthheebpirnedsiengncoefoDfA1U0D0AM tooleic nacoinds.peScpiefciicfibcibnidnidnignfgroofmDtoAtaUl DbiAndi1n0ge.acVhalLu-esFAaBrePasnaamvpelreagweasofde3-t6eramlisne.d by subtracting] RAT CONTROL LABP Nonaopexctic Spoecxht. RAT Feo. LFABP NonsEpYoctic Spocti_ `GUINEA PIG LVEFHaIBCPLE CONTROL. CFNorapactc a GUINEA PIG FCT LFABP =Soucti Ey 2 000143 Specific binding ofDAUDA to rat control L-FABP represented approximately 90% of total binding, while specific bindingof DAUDtAo rat FCI L-FABP accounted for only 68% oftotal binding. When a ligandhas lower affinity for a receptor, a larger proportion oftotal binding isnonspecific(Matthews, 1993); thus, these data suggest the rat FCI LFABP sample had a three fold loweraffinity for DAUDAthan the rat control L-FABP sample. This is consistentwiththe resultsofthe average maximum FI analysisof the rat L-FABP samples, which also suggests the affinityofL-FABP for DAUDA was decressed inthe FCIsample.Lessof a differencewasseen betweenthetwoguineapig L-FABP samples. Specific bindingof 11M DAUDA to 14M L-FABP represented 58%oftotal binding intheguinea pig vehicle control L-FABP sample, and 42%oftotal bindingin the guipnigeFCa1 sample. ThedatasuggesttheguineapigFC1 L-FABPsamplehadlower affinityfor DAtU hanthDeguA inea pig vehicle control L-FABP sample. This is not consistent withetresuhltsofthe average maximum FI analysis, which suggest the relative affinityofthe guinea pig FC1 L-FABP sample was higher than that for the guinea pig `vehicle control L-FABP sample. This inconsistency may be due to the crudenessofthe guinea pig samples giving rise to a high degreeofinterference in the binding ofDAUDA to L-FABP. The overall rudeness ofthe guinea pig L-FABP samples, as compared to the rat L-FABP samples, is reflected in a lower absolute total DAUDA binding (cpm), a higher percent nonspecific binding and a lower percent specific binding. . 2 000144 ANALYSOIFS THEEFFOEFCFCTs ONL-FABP Calocf DuAUl DABaindtini gConostan nts DAUDA binding constants were calculated foreach L-FABP sample. The purpose of this analysis was to assess the effect of FCI on the functionality of L-FABP; and to confirm the results shown thus far, which suggest the affinity of L-FABP for DAUDA was decreased in FCI treated rat L-FABP samples, and that guinea pig L-FABP samples were 100impureto accurately analyze. `The maximum binding capacity or receptor number (Bmax)ofeach L-FABP sample and the dissociation constant or affinity (Kd) of each L-FABP sample for DAUDA were: determined using computer assisted nonlinear regression (Table 5 and Figure 9). Specific binding of DAUDA to each L-FABP sample was converted to uM by dividing the FI (cpm) due to specific DAUDA binding (Table 4) by the average maximum FI (cpm) per 1uM DAUDA for each L-FABP sample (Table 3). `The Bmaxofrat control L-FABPwasnearly 1M. This agrees with the work of Thumser etal. (1994 ab), Thumser and Wilton (1994) and Thumser ef al. (1996) who all found DAUDA to bindto one of the oleic acid binding sites on L-FABP. The Bmax for the rat FCI L-FABP sample was approximately 0.351M, suggesting the capacity of rat FCI LFABP to bind DAUDA was nearly one third that of rat control L-FABP. One possible explanation for this decreased capacityi that FC! is bound to L-FABPin the FCI sample, rendering fewer available binding sites and allowing less DAUDtAo bind. The rigid tail of 0 000145 FCI (Zisman, 1964) and strict conformational requirements for primary site binding to LFABP(Thompson et al., 1997) suggest FC binding would occur inthesecondary binding site. Another possible explanation is that the vehicle (Tween 80, 2%) somehow affected the binding of DAUDA to L-FABP in the FC1 weated sample. This is unlikely, however, since Tween 80 is a mild, non-ionic detergent, designedtoallow solubilized proteins to retain their native structure (Sigma, 1998); and because a low concentrationofTween 80 used (seemethods).This does point out, however, the importance of including a vehicle control in the experiment. The Kds for the rat control L-FABPand rat FC1 L-FABP samples were not notably differentand were comparable 1 the Kdof0.38 + 0.0241M found by Thumser et al. (1996). Thus, althoughthedata for average maximum FI and percent specific binding sugagdeecresasted affinity of rat FCI L-FABP for DAUDA as compared to rat control L-FABP, the Kd and Bmax valuessuggestthe maximum binding capacity/number of binding sites rather than the binding affinity was affected. The Bmax ofthe guinea pig vehicle control L-FABP sample was much lower than expected, 0.2811M as compared to approximately WM for the rat control L-FABP sample. The Kd for the guinea pig vehicle control L-FABP sample was higher than expected, 0.48uM as compared to approximately 0.3uM for the rat control L-FABP. One possible explanation for thesedifferences in results is that rat L-FABP and guinea pig L-FABP are different. Althoughthe crystal structure of guinea pig L-FABP has not been deduced, sequence identity for FABPsofthe same tissue type from different species is 3 000146 approximately 82-92% (Richieri, Ogata and Kleinfeld, 1994). Onecan, therefore, expect the binding constants of control guinea pig and rat L-FABP to be comparable. Other possible explanationsforthisdifference arethatthe vehicle (Tween 80, 2%) had some effect on the L-FABP, orthatthe crudenessofthe guinea pig sample caused a greatdeal of interference with the binodfDiAUnDgA to L-FABP. As mentioned above, the affect of Tween 80 is presumably minimal; thus,thecrudenessofthesample is likely to be to blame forthe unexpected binding constants. As discussed previously, the results for specific binding of DAUDA to guinea pig L-FABP suggest the guinea pig FC L-FABP sample had lower affinity for DAUDA than the guinea pig vehicle control L-FABP sample. This suggestion was counter to the results for average maximum Fl, which indicate the guinea pig FCI sample had anincreasedaffinity for DAUDA as compared to the guinea pig vehicle control L-FABP sample. These: contradictory data, when combined to calculate a Kd and Bmax for cach guinea pig LFABP sample, resulted in a Kd for the guinea pig FC1 L-FABP sample that was about 4.5 times thatofthe vehicle control sample, and acapacity of the guinea pig FC1 L-FABP sample that was about twice thatofthe guinea pig vehicle control L-FABP sample. These results areinconsistentand inconclusive. likely due to the impurity of the guinea pig samples, as discussed above. 2 000147 TABLE5-DAUDABINDINGCONSTANTS, Values are an average + standard deviationof 3 trials as calculated by computer assisted nonlinear regression. Bound = ([L] x Bmax)/(Kd + [L)). L-FABP SAMPLE Bmax(uM) Kd ) Rat Control 0.987 + 0.016 0.278 0.020 Rat FC1 0.3451 0.012 0.260 + 0.063 Guinea Pig Vehicle Control 0.281 + 0.043 0.480 0.391 |GuineaPig FC1 0.413 0.060 2240+0.852 |FCIoGmUpRuEter-aSsPsEiCstIeFdICnoBnOliUnNeDarvrseFgrResEsEioDnAwUasDAus.ed to construct curvesofspecific bound DAUDA versus free average of 3-6 trials. DAUDA (Bound = ([L] The reduced chi square x Bmax)/(Kd + [L])). Each curveisan valuefor each regression was as follows: rat control FABP, L-FABP, 6.12-15; 3.83-16; rat FC1 L-FABP, 7.83-16; guinea and guinea pig FC1 L-FABP, 1.67e-15. pig vehicle control L- RAT CONTROL L-FABP RAT 3 L.FABP L=2,yri; aioEpseEsE,] BBzEg.ahtCreri aaoTse eE=T 5 08: = 1 02s =7 Lee `GUINEA PIG CONTROL L-FABP f=) 0] Awe 9 HJox=lSg aaem=aezmdess,t GUINEA PIG FC L-FABP En 030 FyoeS inttRa g T E5e E= " 000148 . fic AIC An ICso was calculated for each combinationofoleic acid and L-FABP sampletao ssess the functionality of L-FABP from FC!l-treated and non-treated rats and guinea pigs. Oleic acid, ImM in 10% methanol, was titrated into 2 ml 1uM L-FABP and 1uM DAUDA. FI (cpm), due the binding of DAUDA to protein, was measured following cach addition. Curves of percent inhibition of specific DAUDA binding versus oleic acid concentration were constructed (Figure 9). Ta6bsholwstehe percent inhibitionof specific DAUDA binding by 2M oleicacid andthe ICs valuesforeachL-FABPsample. As discussed in the introduction, oleic acid is capable of binding both primary and secondary L-FABP binding sites; and once a ligand has bound the primary sit, binding to the secondary sit is facilitated (Thompson et a., 1997). This relatively unconstrained binding of oleic acid to L-FABPis reflected in asharp decrease in FI (cpm)andan increase in DAUDA-specific binding inhibition upon the addition of micro-molar quantities of oleic acidto solutions of L-FABP and DAUDA. Ninety one percent of specific DAUDA binding to rat control L-FABP was inhibited by 2uM oleic acid; and the ICsoof oleic acid for the rat control L-FABP sample was 0.01uM. `This percent inhibition washigherthan that found by Thumser ef al. (1994b), Thumser er al. (1996) and Thumser and Wilton (1995), of 80.5%, 76% and 80% respectively, under similar conditions. The percent inhibition was much lower (52%) and ICso much higher (0.51M) for the rat FC1 L-FABP sample as compared to the rat control L-FABP sample. This suggeststheability of L-FABP from the FC1-reated ratt bind oleic acid was decreased. One explanation for this decrease in functionality, is that FC1 was bound to L- El 000149 FABP in the FCI treated sample, rendering fewer sites available to bind oleic acid and requiring more oleic acid to achieve 50% inhibition. This agrees with the results presented for the analysisofaverage maximum FL, specific DAUDA bindingand DAUDA binding constants in rat L-FABP samples. Thepercentinhibitionof specific DAUDA bindintog the guinea pig vehicle control LFABP sample upon addition of 2M oleic acidwas 93%; that for the guinea pig FC1 sample was 63%. This is similar to te results for the rat samples; however, the curves of percent inhibition versus oleic acid concentration for rat and guinea pig samples were very different (Figure 9). The curves for rat samples were hyperbolic whilethe curves for guinea pig samples were more linear and scattered. The resulting ICso values for both guinea pig L-FABP samples were the same, 0.24M oleic acid. The data are ambiguous, and ae likely due tothecrudenessofthe guinea pig samples. iTnhe10I%Csmoe0tfhoalneoilc,actiodafsoorlcuatcihonLo-fF1AuBMP Lsa-mFpAlBePwaansdca1lcuuMlaDteAdUbDyAa.ddiVnagluoelseiacraecIidC,s'IsomfM oleic 24M acid for cach oleic acid. L-FABP sample and percent inhibition of specific DAUDA binding by L-FABP Sample Oleic AcidICs (UM) __% Inibitiobny 3M oleic acid Rat Control 001 91 Rat FCI 0s 52 Guinea Pig Vehicle Control 02 93 Guinea Pig FCI 02 6 3s 000150 |EIGURE9-%DAUDAINHIBITIONVS[OLEICACID]. Oleic acid, 1mM DDAUDA. Curves in 10% methanol, was added to a represent an average of 3-6 trials solution of 1uM L-FABPand uM correctedfortheeffect of methanol. a w-o o- eWwe * a-xpartoem amen raL o-n i= == m : i-|d A - af om pea a ANALYSIS OF THE POTENCY OF FCS FOR -- BINDINGTOL-FABP AnICio of each FC, WY and methanol was calculated for DAUDA binding to the rat control L-FABP sample. Each ligand (mM, in 100% methanol or SomM KH;PO4, pH. . 7.2) was titrated into 2m! 1uMrat control L-FABP and 1uM DAUDA. The addition of 000151 competitor to L-FABP and DAUDA induced a shiftofemission wavelength back towards the curve ofDAUDA with no L-FABP (Figure 10). This is indicative of DAUDA being displaced from a nonpolar binding site on L-FABP,suchas the secondary binding site. To Varying degrees, similar shifting and decreases in FI occurred upon additionofeach competitor. FI (cpm),duethe binding ofDAUDA to protein, was measured following `each additionofcompetitor; and curvesofpercent inhibitionofspecific DAUDA binding versusligandconcencotnsrtruactted(iFiogunre 11).Table 8givesthepercentinhibition ofspecific DAUDAbinding by 101Mcompetitorandthe ICso valuesforeachcompetitor. FCI wasthestrongest inhibitorofspecific DAUDA binding, with 69% inkibition uponthe addition of 101M and an ICsoof 4.9uM. FCA was the next strongest, with 51%inkibition upontheaddition of 101M and an ICsof 9.7uM. Wyeth followed FC4, with 50% inkibition upon the addition of 104M, and an ICsoof 104M. FC3 and FC2 both inhibited 43%ofspecific DAUDAbindingupon the additionof 104M and had IC's greater than 100M Thumserand Wilton (1996) suggest tha ligands for L-FABPneedto have both a hydrophobic and a hydrophilic domain. Each FC and WY fis this description to varying degrees.Duetotherigidtai oftheFCsandthebulkystructureofWY,itislikelythat bindingofeachofthese competitotros L-FABP occurs in the secondary binding site on L-FABP. Itisconceivablethat the FCs bind with their rigid hydrophobic CF tails, `analogousto theflexible CHtailofoleicacid,inthesecondarybindingsiteandthatthe ' `polar headgroupsof FCs aresolventexposedasithecarboxylategrouponoleicacid 37 000152 To reason by structure, the varying potenciesofeach FC to binding L-FABP is difficult `Thompsonetal. (1997) suggest that anything larger than a Cs molecule may require prior binding in the primary site. Aswas mentioned in the introduction, Cs and Cio FCs are the most potent peroxisome proliferators. Itmaybethecasethat Cy and Cio molecules bind most readily to the secondary binding siteon L-FABP, not requiring a molecule to be boundin thepribmindainrg syite.Allofthe FCsexaminedinthepresentstudywereCs molecules;thus,ananalysisofchainleagthcannotbeundertakenatthistime. The disparatepotenciesofbindingL-FABPbetween FCsdonotappeartobe due to differencesinpolarityandhydrogenbondingability. Forexample, FC2, havaivnergy `polar carboxylate head group, and capableofextensive hydrogen bonding, has a higher IC1othan FC4,whichhas alesspolarheadgroupand lessabilitytohydrogenbond(see. Figure 1). `The binding ofWY to the secondary binding site on L-FABP is difficult to reason. In place ofthe hydrophobic tail, presoenFnCts andoleicacid,WY has two aeromatic rings. `The conformational flexibilityof L-FABP may accommodate this bulky structure internally, and position the carboxylate externally, ina solvent exposed manner able to participate in hydrogenbonding. Thompsefoanl. (1997) also speculate that a third binding sie, located completely on the exterior ofL-FABP, may exist. Onlythefirst few carbonsof amoleculebindingtosuchasitewouldbeboundinanorganizedfashion,the remainingportionoftheligandwouldbedisordered.This,althoughhighly speculative, maybethesiteofWYbinding to L-FABP. 38 000153 Methanolwas chosenasthenegativecontrolbecause anon-peroxisomeproliferator, similarinstructureto theFCsbeing researched,hasnotbeen identified.Uponthe addition of 104Mmethanol, 21%ofspecific DAUDAbindingwasinhibited.The ICs0of `methanolwasmuchgreaterthan 104M. Itireasonabletosuggestmethanoliscapableof `weaklybindingthesecondary L-FABPbindingsite,withtheCH groupinternally located andthe OHgroupexposedtotheexterioroftheprotein. Thestruocftmeuthraneol rendersitanunlikelycandidatefor loopinginto a U-shapeandparticipatinginprimarysite binding. ( 39 000154 ECIGHURRE1O0M-ATOGRAM. The additionofcompetitor to L-FABP and DAUDA inducedashiftofemission wavelength back towardsthecurve of DAUDA with no L-FABP. This is indicative of DAUDA beingdisplacedfrom anonpolar binding site on L-FABP. To varying degrees, `similarshiftinganddecreasesinFIoccurreduponadditionofeachcompetitor. The BeEFxAYamBapTlAaellSsL yrhonawdnsDBA,isUfaSDoorAsFmoCebo2eboLri-dnF.dAiDnBAgSDrpaAt=,cWoWnMtMrDEoAlFULD-AF.AABPJ1.ALMFBAABPPC,.EI05MFJCMY;D=A1T4MADAU0 DA g, 2 ! 5 We MA -- f e 5 we %|[Pw R =em: " 000155 Each competitor, ImM, was added to 1uM L-FABP and 1M DAUDA. Measurements ofFl were made upon each addition. Curves represent an average of 3-6 trials corrected for the effect of methanol. RAT CONTROL L-FASP pest oat eJoaos soon wi *Pr2000 rott =Zl -- So1o z 34r 56o 78z 5 {competion (44) wz --[--wrrr [ers v-ea-Freaa ARLES IC's OF FCS AND WY & PERCENT INHIBITION OF SPECIFIC DAUDA BIND The ICsoof each competitor for the rat L-FABP sample was calculated by adding competitor (1M) to 14M L-FABP and 1M DAUDA. Values are the ICso'sofeachcompetitorand % inhibitionof specific DAUDA binding by 10uM of each competitor [COMPETITOR Co (um) % INHIBITION FCI 49 [3 FCa 97 51 wy 10 50 FC3 >10 a3 FC2 >10 43 METHANOL >10 21 a 000156 CONCLUSIONS & FUTURE DIRECTIONS `This study was designed to investigate the hypothesis that certain Fluorocarbons (FCs) bind to liver faty acid-binding protein (L-FABP) and displace endogenous fay acids (FAS) as an initial event in peroxisome proliferation. To examine this hypothesis, the kineticsof FA and FC binding to L-FABP were investigated using DAUDA, a fluorescently labeled octanoic acid analogue. L-FABP from male rats (considered to be strong responders to peroxisome proliferators) and male guinea pigs (considered to be. `weak or non-responders to peroxisome proliferators) (Svobodaefal, 1967; Orton et al., 1984; LakeandGray, 1985 Elcombe and Mitchell, 1986) were examined. `The first goal was to assess the effect of FCs on L-FABP functionas evaluated by the ability of DAUDA to bind to L-FABP isolated from rats and guinea pigs treated and not treated with FCL in vivo. Results sh adeocreawsed maximum binding capacity of LFABP from FCl-treated rats withouant increase in Kd. This was demonstrated by a lower Bmax, higher oleic acid ICsoand unchanged Kd in L-FABPfrom rats treated with FCI as compared to samples from control rats. These results are likely due to FC1 binding to the secondary binding site on L-FABP; thus preventing the binding of DAUDA to L-FABP. Results for guinea pig L-FABP samples were inconclusive. This is presumably because. guinea pig samples were only partially purified, resulting in a high degree of interference from remaining cellular debris and a lower concentration of L-FABP, as a proportion of total protein,as compared to the more highly purified rat samples. A second attempt will be made t0 analyzethedifference in response to FCs seen in rodent and guinea pig LFABP. This analysis will be performed on fully purified L-FABP samples. 2 000157 The second goalofthestudy was to assess the potency of the various FCs for binding to L-FABP, as suggested by IC values. Results indicate the most potent L-FABP binder is FCI, followed by FC4,WY and (with equal ICsos) FC3 and FC2. BindingofFCs to LFABP likely occurs in the secondary binding ste; and the variance in potency is likely due 0 structural differences. Future work will foonccorruelatsingnewdatawith the resultsofthis study, and relating the effects seen in rodents and guinea pigs to the relevance they pose to human health. The possibilityofcompleting a new set of kinetics assays using a probe capableofbinding both L-FABP bindingsiteswill be looked into. This would help characterize the interaction of DAUDA with L-FABP and create a new set of results for analysis. Human L-FABP will be examined, and the relevance to human healthofFC induced peroxisome proliferation in rodentswill be investigated. FCs ofdiferent chain lengthwillbe investigated, and the effect of chain length on ability 0 bind L-FABPanalyzed. The abilityofeach FC to induce peroxisome proliferation in rodent L-FABP will be assessed using the methodofLazarow and de Duve (1976). Anatemptwillbe madetocomrelat this capacity with the relative: potencyofeach FC to bind L-FABP, as determined inthe current study. Electro-Spray Mass Spectrometry analysis onthesame L-FABPsamples as analyzed in this study is currently underway. The objective isto quantify FC1 bound to L-FABP from FC1 treated animals, correlate this with the decrease in L-FABP capacity observed in this study and with the ability of FC1 to induce peroxisome proliferation in rodents. The long range goal { is 1 refinethecorrelation between the amount of FC bound to L-FABP with peroxisome. a 000158 proliferation, and develoap biomarker for peroxisome proliferation based on bound FC levels. This biomarker would be used as a methodofscreening FCs for their ability to induce peroxisome proliferation. " 000159 REFERENCES AbdellcaariAcf.inoPgreenastisV.b,yTpaeprefrluHo.roaoncdtaRnoobiecrafcriodi,daMp.er(o19x9i1s)o.meThperolmiofdeuraltaotri.oTnoxoifcoralt. iAvpeprl. Pharmacol.. 111: 530-7. AppelkviinstpeEr. oaxnidsoDmaalliB-noexGri.da(t1i9o8n0)o.f Pfoasttsyibalceidis.nvBoilovcehmiemn.toBfiofpahtytys.acAicdt-ab,i6n1d7in:g 1p5r6o-t6e0i.n Bankes R. (1970). Scieniific. Fluoroancd thaeirc Debriovatnivess. London. 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