Document 6R63dv939z9z3x2GV8kEzpGn6

AR226-0(61 DT15 3M MEDICAL DEPARTMENT, CORPORATE TOXICOLOGY - ST-43: Standard Procedure fo r Liver Subcelular Fractionation - Study Objective: Perfluorochemical compounds such as 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 o f how perfluorochemical compounds interact with liver tissues is unknown and it is necessary to look into the sub-cellular fractions. The purpose o f this study is therefore to (1) isolate liver sub-cellular fractions and (2) to quantitate the PFOS contents in each fraction. The correlation between subcellular PFOS contents and serum biochemical markers will be further investigated. This protocol is part o f an ongoing study in the 3M Strategic Toxicology Laboratory designed to understand the effects of perfluorooctanesulfonamides and their metabolites, including PFOS, on intracellular fatty acid transport and metabolism, mitochondrial function, and cholesterol synthesis. This protocol provides a means of combining our resources and focus analytical capabilities on the same set o f 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 220-2E-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: 004429 DT15 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 sponsor has provided samples of PFOS to the investigators. Analytical documentation of the starting material will be the responsibility o f the sponsor. A chemical composition specification sheet will be kept on file. Animals: Spedes: Rat Strain: Sprague Dawley Source: Harlan Laboratories, Inc. Age at initiation o f treatment: 10-12 weeks old W eight at initiation o f 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 will be fractionized according to the procedures described below. The isolated sub-cellular fractions will be 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 ml) Animal: Rat livers from both control and PFOS-treated groups. 004430 DT15 (NOTE - All procedures should be carried out atO- 4C. ) Suspend and homogenize the liver in 0.25 M Sucrose in 0.05 M Tris-HCl buffer (pH 7.4) using 20% weight/volume ratio, or approximately 5 volumes of the liver weights^ The homogenization should be carried out on ice at all time to prevent the enzymes and tissue proteins from degradation. Homogenize on ice at low speed using a 50 ml Tenebrock tissue grinder with the hollow handle of the pestle filled with crushed ice. Homogenize at least 10 strokes, or until the suspension is visibly homogeneous. Record the total volume and the corresponding weights of the homogenates. Obtain two 1.5 ml microcentrifuge tubes and label them " 1" . Aliquot 1 ml o f the homogenate into each 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 below following the centrifugation, make sure to measure and record the volumes and weightsfor each supernatant fraction. Spin the whole homogenate at 700 x g 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 aliquot of the supernatants into two 1.5 ml microcentrifuge tubes labeled "2". Store frozen. Pellet: The resulting pellet contains debris and unbroken cells. Add 1 ml o f buffer per 1 gram o f original sample, re-suspend the pellet in 0.25 M Sucrose in 0.05 M Tris-HCl buffer (pH 7.4) and label it as "Plasm a m em brane". Make two 1 ml aliquot o f the suspension into two 1.5 ml microcentrifuge tubes labeled "3" . Store frozen. Spin th supernatants at 2500 x g 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 aliquot of the 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 gram o f original sample, re-suspend the pellet in 0.25 M Sucrose in 0.05 M Tris-HCl buffer (pH 7.4). Label it as 004431 DT15 "nuclei". Make two 1 ml aliquot of the 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 4C. Supernatant: Decant supernatant fluid into a new centirfuge tube. Weigh and record the total volume. Make two 1 ml aliquot of the 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-HCl buffer (pH 7.4). Label it as "m ito ch o n d ria". Make two 1 ml aliquot of the mitochondrial suspension into two 1.5 ml microcentrifuge tubes labeled "7". Store frozen. 5. Centrifuge the supernatant fluid obtained from step 4 at 30,000 x g 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 aliquot of the supernatants into two 1.5 ml microcentrifuge tubes labeled "8" . Store frozen. Pellet: The resulting pellet contains lysosomes. Add 1 ml of buffer per 1 gram o f original sample, re-suspend the pellet in 0.25 M Sucrose in 0.05 M Tris-HCl buffer (pH 7.4) and label it as "lysosome". Make two 1 ml aliquot o f the lysosome suspension into two 1.5 ml microcentrifuge tubes labeled "9" . Store frozen. 6. Centrifuge the supernatant fluid obtained from step 5 above at 105,000 x g for 60 minutes at 4C. Supernatants: Decant supernatant fluid into new centrifuge tubes on ice. Weigh and record the total volume. Label the supernatant as "cytosol". Make two 1 ml aliquot o f the supernatants into tw o 1.5 ml microcentrifuge tubes labeled "10". Store frozen. Pellet: The resulting pellet contains microsomes. Add 1 ml o f buffer per 1 gram o f original sample, re-suspend the pellet in 0.25 M Sucrose in 0.05 M Tris-HCl buffer (pH 7.4) and label it as "m icrosome". Make two 1 ml aliquot of the microsomal suspension into two 1.5 ml microcentrifuge tubes labeled "11". Store frozen. 004432 DT15 Data: 3M Study Number = Date = Animal #, sex, and strain = Dose group = Weight of liver sample (g) = _____ Volume of sucrose-Tris HC1 added (mL) = Fraction # Whole homogenate Plasma membrane sup. Plasm i membrane cells Nuclei sup. Nuclei cells Mitochondrial sup. MitoehondriaT cells Lysosomal sup. L\.v*somal cells Cytosol Mjeiosom d cells Fraction weight before aliquot (g) (Minus) amount saved for LC-MS (mL) (Minus) amout saved for gel electr. (mL) 700 x g sup ~~ 7IHJXJ i s m s pJlei --- 2500 x g sup '2500 x g ' pellet g ip p ll lOKxg sup lUh, s g pellet 30K x g sup -- 30K x g a ssp fiF sp Ip p pellet 105K x g sup_ 105K. x g pellet Note ----------- --------------- . ...- ...... Responsibilities: Andrew Seacat and Sue Chang will be responsible for performing the experiment and sending specimens for analysis. 004433 Signatures: Andrew M. Seacat, Ph.D. Toxicology Specialist Study Director Sue Chang, M.S. Advanced Research Toxicologist Study Toxicologist Sponsor Representative Date Date Date DT15 004434