Document 5byMXopxe7ZwXZNZraMGJRVnN

-4H2ll- I7S T-6295.28 Final Report T-6295.28: Analysis of Liver Enzymes, Glycogen, and Perfluorooctane Sulfonate (PFOS) Concentration in Samples from T-6295.25: One-Generation Reproduction Study of PFOS - Mevalonic Acid/Cholesterol challenge and NOEL Investigation in Rats. Study Location: Southern Research Institute (SRI), P.O. Box 55305, 2000 Ninth Ave South Birmingham, AL 35255-5305 Study Monitors: John L. Butenhoff, Ph.D., CIH, DABT, Staff Scientist 3M Medical Dept. / Corporate Toxicology & Regulatory Services 3M Center Building 220-2E-02 Saint Paul, MN 55144 Ph.: 651-733-1962, FAX: 651-733-1773 & Deanna J. Luebker, M.S., Senior Toxicologist 3M Medical Dept. / Corporate Toxicology & Regulatory Services 3M Center, Building 220-2E-02 Saint Paul, MN 55144 Ph: 651-737-1374 FAX: 651-733-1773 Sponsored by 3M Specialty Chemicals Division 3M Center Bldg 236 St. Paul, MN 55144 reoso 33C "Om r o t-h mo z< vo CD Page 1 of 13 Study Objective T-6295.28 Final Report The objective of this study was to measure the activity of glucose-6-phosphate dehydrogenase (G6PD), malic enzyme, and UDP-glucuronosyltransferase (UDPGT) and to determine the amount of glycogen and PFOS in liver specimens collected from PFOS treated rat dams and their pups. Methods Summary The in-life portion of this study (T-6295.25) was performed at Argus research laboratories and is detailed in the final report for that study. Briefly, female rats were dosed with 0.0, 0.4, 0.8, 1.0, 1.2, 1.6, or 2.0 mg/kg Perfluorooctane Sulfonic Acid, Potassium Salt (PFOS) for 42 days prior to mating with untreated breeder males, through confirmed mating (a maximum of 14 days), and gestation day (GD) 21 or lactation day (LD) 4. Select dams were sacrificed on GD 21 just prior to delivery and all remaining dams were allowed to litter, and sacrificed with their pups on LD 5. LD5 liver samples from dams and pups (pooled by litter) in the 0.0, 0.4, 1.6, and 2.0 mg/kg dose groups were analyzed in the current study. Details and results of the PFOS analysis are included in the T-6295.25 final report. Glycogen was analyzed in pup samples only by the method of Roe and Dailey (1966) using the anthrone reagent. UDPGT, malic enzyme, and G6PD activity were measured using CaCL precipitation. The two cytosolic enzymes, malic enzyme and G6PD, were assayed spectrophotometrically by standard literature methods following the conversion of NADP+ to NADPH at 340 nm. The microsomal UDPGT assay followed the reduction of 4nitrophenol at 405 nm and also used standard literature methods. All results were compared between treated and control animals and analyzed to determine statistical differences using the students T-test. Results G6PD Reductions in average enzyme activity were observed in F0 samples at all dose groups analyzed (0.4, 1.6, and 2.0 mg/kg), but these reductions were only statistically significant in the 0.4 and 1.6 mg/kg dose group samples (Table 1 and Figure 1). Reductions in average enzyme activity were observed in the FI samples at all maternal dose groups analyzed, (0.4, 1.6, and 2.0 mg/kg), but this reduction was only statically significant in the 2.0 mg/kg dose group samples (Table 1 and Figure 2). Malic Enzyme No statistically significant differences or trends in enzyme activity were observed in the F0 or FI samples (Table 1 and Figures 3 and 4). UDPGT Statistically significant increases in activity were observed in F0 0.4 and 1.6 dose group samples but activity in the 2.0 mg/kg dose group samples did not differ from control (table 1 and Figure 5). Reductions in average enzyme activity were observed in the FI samples at all maternal dose groups analyzed, (0.4, 1.6, and 2.0 mg/kg), but this reduction was only statically significant in the 1.6 and 2.0 mg/kg dose group samples (Table 1 and Figure 6). Page 2 of 13 T-6295.28 Final Report Glycogen Increases in FI liver glycogen content were observed at the 1.6 and 2.0 mg/kg dose levels. This increase was statically significant at the 2.0 mg/kg maternal dose level only (Table 1 and Figure 7). Discussion Glycogen storage disease type la (GSD-Ia), also known as von Gierke's disease, is a condition in which G6PD levels are markedly reduced or absent, impairing or destroying the liver's ability to produce free glucose. The condition leads to abnormally large amounts of glycogen in the liver, hypoglycemia, increased dependence on fat metabolism, and increased levels of lactate and pyruvate in the blood. Without a continuous, exogenous source of glucose, severe hypoglycemia and metabolic perturbations occur that can compromise fetal outcome. Observations made in animals bom with GSD-Ia include small body size and emaciation, severely enlarged pale livers, pale kidneys, and diffuse vacuolation of hepatocytes with large amounts of glycogen and small amounts of lipid. In the current study, G6PD activity and glycogen levels were analyzed to determine if a perturbation of glycogen storage similar to that which occurs with GSD-Ia may occur upon PFOS exposure. Reductions in average enzyme activity were observed in F0 and FI samples at all dose levels analyzed. Although not all reductions were statistically significant, these results suggest that PFOS may act to down regulate activity of this enzyme. Increased FI liver glycogen content was also observed in this study. Although this increase was only statistically significant at the 2.0 mg/kg dose level, a wide range on concentrations was observed at the 1.6 mg/kg level and an overall increase was observed over control. Malic enzyme and UDPGT activity were investigated to gain insight into decreases in free and total T3 and T4 levels observed in serum samples from the T-6295.25 study. Malic enzyme, a marker of T3 action on liver, was unchanged. This indicates that, although significant decreases in levels of free and total T3 have been observed, the action of T3 on the liver appears to be unaffected. UDPGT levels were investigated to determine if increased glucuronidation and excretion of T4 and T3 may occur upon PFOS exposure and be responsible for the observed decrease in thyroid hormone levels. The results of this analysis, although unclear, do not support the hypothesis that UDPGT activity is increased upon PFOS exposure. Conclusions The results of this study indicate that perturbation of glycogen storage processes may be occurring in animals exposed to PFOS. This may related to the decreased viability observed in rat and mouse pups bom to PFOS exposed dams and should be the focus of future investigations. Results also suggest T3 activity in the liver is not altered upon PFOS exposure and that increased UDPGT activity, although unclear, is most likely not the cause of decreased T3 and T4 levels observed in PFOS exposed animals. Page 3 of 13 List of Figures: Table 1: Individual Data Figure 1: FO Glucose-6-Phosphate Dehydrogenase Activity Figure 2: FI Glucose-6-Phosphate Dehydrogenase Activity Figure 3: FO Malic Enzyme Activity Figure 4: FI Malic Enzyme Activity Figure 5: FO UDP-glucuronosyltransferase Activity Figure 6: FI UDP-glucuronosyltransferase Activity Figure 7: FI Liver Glycogen T-6295.28 Final Report Page 4 of 13 Table 1: Individual Data Maternal Dose group 0.0 0.4 1.6 2.0 G-6PD Enzyme Activity (nmoles/min/g tissue) F0 F1 4910 2035 7289 4458 2107 2221 3814 1719 2409 1783 3760 1972 3876 2358 2177 1928 3408 2055 1673 1287 1341 1783 1811 1442 1745 3558 1728 2861 2154 1863 1857 2441 2422 2506 1398 1558 1288 2723 1535 2633 2087 5841 Malic Enzyme Activity (nmoles/min/g tissue) F0 F1 1459 414 1373 288 1251 290 807 276 336 145 1193 199 1351 328 627 272 1035 309 837 169 628 155 200 980 236 1667 185 1398 1170 190 214 1021 1055 1503 269 647 177 1081 289 1073 830 1602 UDPGT Enzyme Activity (nmoles/min/g tissue) F0 F1 254 369 286 309 179 338 284 419 276 433 302 217 362 404 303 255 309 273 304 361 316 266 182 413 240 297 150 356 255 335 306 279 279 241 243 256 217 230 189 255 312 218 Glycogen (mg/g liver) F1 0.40 0.58 2.61 1.63 2.89 0.66 4.60 0.49 0.27 0.51 0.33 15.18 0.83 1.99 1.17 0.15 3.42 3.84 1.85 T-6295.28 Final Report Page 5 of 13 Figure 1: FO Glucose 6-Phosphate Dehydrogenase Activity Lactation Day 5 7000 6000 #<30W3D) 5000 O) 4000 E 'w 0 1 3000 <o 2000 1000 N=6 N=5 N=6 0.0 0.4 1.6 Dose Group (mg/kg/day PFOS) * = statistically significant difference from control, P < 0.05;** P < 0.01 T-6295.28 Final Report N=6 2.0 Page 6 of 13 Figure 2: F1 Glucose 6-Phosphate Dehydrogenase Activity Lactation Day 5 T-6295.28 Final Report Dose Group (mg/kg/day PFOS) * = statistically significant difference from control, P < 0.05; ** P < 0.01 Page 7 of 13 T-6295.28 Final Report Figure 3: FO Malic Enzyme Activity Lactation Day 5 1600 0.4 1.6 Dose Group (mg/kg/day PFOS) Page 8 of 13 Figure 4: F1 Malic Enzyme Activity Lactation Day 5 T-6295.28 Final Report Dose Group (mg/kg/day PFOS) Page 9 of 13 Figure 5: FO UDPGT Activity Lactation Day 5 T-6295.28 Final Report Dose Group (mg/kg/day PFOS) * = statistically significant difference from control, P < 0.05; ** P < 0.01 Page 10 of 13 Figure 6: F1 UDPGT Activity Lactation Day 5 T-6295.28 Final Report Dose Group (mg/kg/day PFOS) * = statistically significant difference from control, P < 0.05; ** P < 0.01 Page 11 of 13 T-6295.28 Final Report Figure 7: F1 Liver Glycogen Lactation Day 5 12 ' 10 mg glycogen / g liver N=6 - 0 .0 -2 N-5 04 N -4 16 -4 Dose Group (mg/kg/day PFOS) * = statistically significant difference from control, P < 0.05; ** P < 0.01 r N -3 2 Page 12 of 13 Signatures: Prepared By: Deanna Luebker, MS Study Monitor T-6295.28 Final Report ________ o^/^<oo<y Date Reviewed By: tA ~ '. John Butenhoff, Ph.D., DABT, CIH Study Monitor OY Date Page 13 of 13