Document zzYNZEK4LQLzL0zqmR0LxMy2z

AR2%6-0161 ors 3M ME- DSTI4C3:ALStaDndEaPrdAPRroTceMdEurNeTfo,r LCivOerRSPubOceRluAlaTrEFraTctOioXnaItCioOn L- OGY Study Objective: 0R-S Perfluorochemical compounds suchas perfluorooctane sulfonate (PFOS) have. been linked with reduction in serum cholesterol levels and liver has been identified as the primary target site. The exact cellular mechanism of how perfluorochemical `compounds interact with liver tissues is unknown and it is necessatroy look into the sub-cellular fractions. `The purposeofthis study is therefore to (1) isolateliver sub-cellular fractions and (2) to quantitatethe PFOS contents in each fraction. The correlation between subcellular PFOS contents and serum biochemical markers willbe further investigated. "This protocoils partofan ongoing study in the 3M Strategic Toxicology Laboratory designed to understand the effectsofperfluorooctanesulfonamides and their metabolites, including PFOS, on intracellular fatty acid transport and `metabolism, mitochondrial function, and cholesterol synthesis. This protocol provides a meansof combining our resources and focus analytical capabilities on the same set of tissues. Study Location: 3M Strategic Toxicology Laboratory 3M Center, Building 270-SB-314 Saint Paul, MN 55144-1000 Sponsor: 3M Specialty Chemicals 3M Center, Building 236 Saint Paul MN 55144-1000 Study Director: Andrew M. Seacat Ph.D. 3M Medical Dept. Corporate Toxicology 3M Center Building 220-2-02 Saint Paul, MN 55144-1000 Ph.: 651-575-3161, FAX: 651-733-1773 Study Toxicologist: Sue Chang, M.S. Advanced Research Toxicologist M Medical Dept./Corporate Toxicology 3M Center Building 220-2E-02 Saint Paul, MN 55144-1000 Ph: 651-736-2212, FAX: 651-733-1773 Methods & Materials: 04429 boris Regulatory Compliance: "This is an exploratory in nature and thus classified as non-GLP as explained in TOX SOP 0950, Strategic Toxicology Lab GLP Program Procedure. Test Material: "The spansor has provided samplesofPFOS to the investigators. Analytical `documentationofthe starting material will be the responsibilityofthe sponsor. A chemical composition specification sheet will be kept on fil. Animals: Species: Rat Strain: Sprague Dawley Source: Harlan Laboratories, Inc. Age at initiationoftreatment: 10-12 weeks old Weight at initiation of treatment: approximately 250-300g Animal Handling Cares and Specimen Collection: Please see Study No. DT-15 `Specimen Handling / Processing: For perfluorosulfonamide metabolite analysis, liver samples wil be fractionized according to the procedures described below. The isolated sub-cellular fractions willbe packed in dry ice and shipped to Dr. Kris Hansen at 3M Environmental Lab. Kris Hansen, Ph.D. 3M Environmental Technology and Safety Services 935 Bush Avenue St. Paul, MN 55133-3331 Ph: 651-778-6081, Fax: 651-778-6176. Procedures: Buffer needed: 0.25M sucrose in 0.05M Tris-HCL, pH 7.4 Equipment needed: Tissue grinder Centrifuge: Polypropylene centrifuge tubes (1.5 ml and 50 mi) Animal: Rat livers from both control and PFOS-treated groups. 04430 TIS (NOTE - Allprocedures should be carried out at 0 - C. ) 1. Suspend and homogenize the liver in 0.25 M Sucrose in 0.05 MTris-HCI buffer (pH 7.4) using 20% weight/volume ratio, or approximately volumesofthe liver weights. The homogenization should be carried out on ice at all ime to prevent the enzymes and tissue proteins from degradation. Homogenize on ice at low speed using a 50ml Tenebrock tissue grinder with the hollow handleofthe pestle filledwithcrushed ice. Homogenize at least 10 strokes, or until the suspension is visibly homogencous. ORbetcaoirndttwhoe t1o.t5almviomliucmreocaenndtrtihfeugceortruebsepsoannddinlgabweelitghhetsmo"f1t"h.eAhloimqougoetn1atmelso.fthe homogenate into cach tube. Store the tubes frozen for further analysis. Transfer the remaining of the homogenates into two 50-ml centrifuge tubes. Note: For this step and steps belowfollowing the centrifugation, make sure to `measure and record the volumes and weightsfor each supernatant fraction. 2. Spin the whole homogenate at 700 x g for 10 minutes at 4C. `Supematants: Decant supernatant fluid into new centrifuge tubes on ice. Weigh and record the total volume. Make two 1 ml aliquotofthe supematants into two 1.5 ml microcentrifuge tubes labeled "2". Store frozen. Pellet: `The resulting pellet contains debris and unbroken cells. Add 1 ml of buffer per 1 gram of original sample, re-suspend the pelletin 0.25 M Sucrosein 0.05 MTris-HCI buffer (pH 7.4) and label it as "Plasma membrane". Make two 1 ml aliquot ofthe suspension into two 1.5 ml microcentrifuge tubes labeled "3". Store frozen. 3. Spinthe supernatants at 2500 xg for 10 minutes at 4C. `Supernatants: Decant supernatant fluid into new centrifuge tubes on ice. Weigh and record the total volume. Make two 1 ml aliquotofthe: supernatants into two 1.5 ml microcentrifuge tubes labeled "4". Store frozen. Pellet `The resulting pellet contains nuclei and unbroken cells. Add 1 ml of buffer per 1 gramoforiginal sample, re-suspend the pelleitn 0.25 M Sucrosein0.05 M Tris-HCI buffer (pH 7.4). Label it as 004431 bis "nuclei". Make two 1mlaliquotofthe suspension into two 1.5 ml microcentrifuge tubes labeled "5". Store frozen. 4. Centrifuge the supernatant fluid obtained from step 3 at 10,000 x g for 15 minutes at dC. Supernatant: Decant supernatant fluid into a new centirfuge tube. Weigh and record the total volume. Make two 1 ml aliquotofthe supernatants into two 1.5 ml microcentrifuge tubes labeled "6". Store frozen. Pellet: `The resulting pellet contains mitochondria. Add 1 ml of buffer per 1 gram of original sample, re-suspend the pellet in 0.25 M Sucrose in 0.05 M Tris-HCI buffer (pH 7.4). Label it as "mitochondria". Make two 1 mi aliquotof the mitochondrial suspension. into two 1.5 ml microcentrifuge tubes labeled "7". Store frozen. 5. Centrifugethe supernatant fluid obtained from step 4 at 30,000 x g for 10minutes adc. Supernatants: Decant supernatant fluid into new centrifuge tubes on ice. Weigh and record the total volume. Make two 1 ml aliquotofthe. supernatants into two 1.5 ml microcentrifuge tubes labeled "8". Store frozen. Pellet: The resulting pellet contains lysosomes. Add 1 mlofbuffer per 1 `gramoforiginal sample, re-suspend the pelleint 0.25M Sucrose in 0.05 M Tris-HCI buffer (pH 7.4) and labeli as "lysosome". Make two 1 mi aliquot of the lysosome suspension into two 1.5 ml microcentrifuge tubes labeled "9". Store frozen. 6. Centrifugethe supernatant fluid obtained from step 5 above at 105,000 x gfor60 minutes at 4C. `Supernatants: Decant supernatant fluid intonewcentrifuge tubes on ice. Weigh and record the total volume. Labelthe supernatant as "cytosol. Make two 1 ml aliquotofthe Supematants into two 1.5 ml microcentrifuge tubes labeled "10". Store frozen. Pellet: `The resulting pellet contains microsomes. Add 1 mlofbuffer per 1 gramoforiginal sample, re-suspend the pelletin 0.25 M Sucrose in 0.05 M Tris-HCI buffer (pH 7.4) and labeli as "microsome'" Make two 1 ml aliquotofthe microsomal suspension into two 1.5 `ml microcentrifuge tubes labeled "11". Store frozen. 04432 ois Data: 3M Study Number = __ + Date Animal #, sex, and strain Dosegroup - = =_ I * Weight of liver sample (2) |-- Volumeof sucrose-Tris HCl added (mL) = Fraction Fraction| (Minus) weight | amount (Minus) amout before | saved for| saved for # homogenate aliquot 8 LC-MS (mL) gel electr. (mL) -- bmw aor]| membre sup. | su Pl Rae EeA A pel0l0etRTEE ER e E a a]a me ee a aTieRmad 2 N RNce lR igcelR a isn2 aael5 in asHo0 xnXy g g0 ae len sho A N e aetE lE atE E e SlR biE ns ie vA eav `Mitochondrial RR ER ETEEEE oo June] |]|] EER a pe SR ee I Issa a ar RRR Responsibilities: Andrew Seacat and Sue Chang will be responsible for performing the experiment and sending specimens for analysis. 604433 pris Signatures: Andrew M. Seacat, Ph.D. Date Toxicology Specialist Study Director Sue Chang, M.S. Date Advanced Research Toxicologist Study Toxicologist Sponsor Representative Date 004434