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/Utt!6'OI$3 01/ 21/2000 Perfluorooctane Sulfonic Add Induced HMG-CoA Reductase Inhibition in Pregnant Rats and Rat P ups. oA-T Deanna J. Luebker, M.S., Advanced Research Toxicologist Corporate Toxicology, 3M Medical Department 3M Center, Building 220-2E-02, Saint Paul, MN 55144 Phone (651) 737-1373 FAX (651) 736-2285 D iluebker@ m m m .com PREFACE This project is designed to serve both as a 3M research project and as a project to fulfill the requirements of a Ph.D. thesis in Toxicology through the University of Minnesota Twin Cities. This proposal is being submitted with the understanding that publication of the results will be subject to a first-right-of-review by 3M. PROPOSED PRO JECT AND ITS PURPOSE The objective of this study is to gain knowledge into the mechanism of action by which perfluorooctane sulfonic acid (PFOS) induces adverse effects on peri-/postnatal development in rats. The hypothesis that PFOS acts via inhibition of 3-hydroxy-3methylglutaryl-CoA reductase (HMG-CoA reductase), the rate-limiting enzyme of cholesterol synthesis, will be examined. An attempt will be made to prevent PFOS toxicity by co-administrating mevalonate, the immediate product of HMG-CoA reductase. HMG-CoA reductase activity will be compared between dams and their pups and to age matched positive and negative controls. Assays will be performed to determine if PFOS acts directly on HMG-CoA reductase, indirectly via activation of AMP-activated protein kinase (AMP-PK) or through inducing an increase in AMP levels. Other parameters, which will be followed where possible, include body weight, liver weight, liver arid serum PFOS, liver and serum lipids and miocardial fiber injury and/or inflammation. IN TR O D U C TIO N Perfluorooctane sulfonic acid (PFOS) is the ultimate metabolite of perfluoroalkyl acids and their derivatives, a class of chemicals produced by 3M and broadly utilized across multiple markets. The large production volume and broad-based utilization of this class of chemicals justifies the need for a comprehensive understanding of the potential adverse health effects they may pose. Because PFOS is the breakdown product of these chemicals, research focusing on its effects and mechanism(s) of action is of primary 004441 D. J. Luebker Page 1 o f 16 01/21/2000 concern. This proposal is aimed at gaining knowledge into the mechanism(s) by which PFOS induces effects on peri-/postnatal development in rats. Figure 1 : Chemical Structure of PFOS CF3---- (CF2>6-- CF2 jj O O General effects seen in 3M PFOS reproduction studies in rats are reduced maternal body weight gain, increased percent stillborn and decreased pup survival (Case, presentation, 1999). The primary target organ identified from adult rat studies is the liver. Effects of PFOS treatment in adult rats include decreased body weight gain, increased liver weight, decreased serum cholesterol, triglycerides and bilirubin and mild peroxisome proliferation. The first detectable biological effect seen in adult rat PFOS studies is reduced serum cholesterol At high doses of PFOS in rats, toxicity response curves become very steep and an apparent wasting syndrome occurs which culminates in death (3M Corporate Toxicology, unpublished, 1999). Haughom and Spydevold (1992) investigated the mechanism underlying the hypolipemic effect of PFOS in rats. They found PFOS to down regulate 3-hydroxy-3-methylglutarylCoA reductase (HMG-CoA reductase) activity to 35% of control HMG-CoA reductase is a microsomal enzyme which controls the rate-limiting step in cholesterol synthesis, the conversion of HMG-CoA to mevalonate (HMG-CoA + 2 NADPH + 2 H+-* mevalonate + 2NADPH++ CoA). The amount of HMG-CoA reductase present in a cell is controlled via negative feedback regulation through repressed gene transcription (Edwards et at., 1983), inhibition of mRNA translation (Nakanishi et al., 1988) and stimulation of the rate of degradation (Gill et al., 1985). The primary mechanism by which this enzyme is controlled acutely is reversible phosphorylation (Gibson, 1985; Beg et al., 1987; Hardie and Carling, 1997) with phosphorylation inactivating the enzyme. Other control mechanisms include reversible thiol-disulfide formation (Roitelman and Shechter, 1984a), allosteric activation by NAD(P)H (Roitelman and Shechter, 1984b) and alterations in membrane fluidity (Mitropoulos and Venkatesan, 1985). AMP-activated protein kinase (AMP-PK) is the major HMG-CoA reductase kinase in rat liver (Carling et al., 1989). It is controlled by reversible phosphorylation, with phosphorylation activating the kinase. AMP-activate protein kinase kinase (AMP-PKK) is the upstream component in this highly conserved kinase cascade (Hardie and Carling, 1997; Hardie, Carling and Carlson, 1998). Elevation of AMP can activate AMP-PK by allosterically activating AMP-PKK, by binding to AMP-PK and making it a poorer substrate for protein phosphatases, by binding to AMP-PK and making it a better substrate for the AMP-PKK and by allosterically activating AMP-PK (Corton et al., 1995; Hawley et al., 1995; Davies et al., 1995). The AMP-PK cascade is therefore 0 0 4 : 4 4 2 activated under conditions where AMP is elevated and ATP is depressed. The D. J. Luebker Page 2 of 16 01/21/2000 consequences of AMP-PK activation include inactivation of ATP-consuming anabolic pathways (e.g., fatty acid synthesis via phosphorylation of acetyl CoA carboxylase) and sterol synthesis, via phosphorylation of HMG-CoA reductase (Corton et a l, 1995; Henin et a l, 1995). This cascade also activates the ATP-generating catabolic pathway of fatty acid oxidation via depression of malonyl-CoA levels (Merrill et a l, 1997; Hayashi et a l, 1998). AMP-PK could thus act to balance the supply of ATP to the demand of the nucleotide (Hardie et a l, 1999). This may be important during periods of cellular stress associated with ATP depletion, such as treatment with metabolic poisons in the hepatocyte (Corton, Gillespie and Hardie, 1994). Figure 2: Reversible Phosphorylation of HMG-CoA Reductase Solid line = activation, dotted line = inactivation Inhibition of HMG-CoA reductase activity increases the cellular demand for cholesterol. This results in an increase in hepatic low density lipoprotein (LDL) receptors and an increase in clearance of plasma LDL by the liver (Kovanen et a l, 1981). Because LDL carries most of the cholesterol in humans, inhibition of HMG-CoA reductase results in a decrease in plasma cholesterol. Cholesterol, however, is an important precursor to hormones needed for the maintenance of pregnancy, parturition, lactation and maternal behavior (Numan, 1988; Freeman, 1988; Hodgen & Itskovitz, 1988) and is required for normal in utero and postnatal development in mammals (Belknap & Dietschy, 1988; Yount & McNamara, 1991). Inhibition of HMG-CoA reductase also leads to a deficiency of mevalonate and isoprenoids such as dolichol, isopentyl adenine, ubiquinone, haem A and many proteins bound to famesyl and geranylgeranyl residues (Soma et a l, 1992; Brewer et al., 1993; Corsini et a l, 1995). These isoprenoids are involved in membrane synthesis, DNA replication, cellular growth and metabolism and protein glycosylation (Quesney-Huneeus et al., 1983; Surani et a l, 1983; Farnsworth et a l, 1987; Brewer e ta l, 1993). 004443 D. J. Luebker Page 3 o f 16 Figure 3: HMG-CoA Reductase Inhibition 01/21/2000 Statins, a class of compounds used to treat hyperlipidemia, act as competitive inhibitors of HMG-CoA reductase (Stryer, 1995). Some statins, for example lovastatin (Mevinolin, MEVACOR) and simvastatin (synvinolin, ZOCOR), exist as inactive "prodrugs" which must be enzymatically or chemically opened to their respective carboxylate forms to elicit inhibitory activity against HMG-CoA reductase (Corsini et al., 1995). Other statins, for example pravastatin (eptastatin) and fluvastatin (LESCOL), are dosed in their active, open ring form (Corsini et a l, 1995). The active open-acid structures resemble that of HMG-CoA, the normal substrate for the reductase. Statins bind to the reductase, prevent it from binding to HMG-CoA and thus block the production of mevalonate. 004444 D. J. Luebker Page 4 of 16 01/21/2000 Figure 4 : Chemical Structures - Statins & HMG-CoA Example Statins: Lovastatin (Mevinolin, MEVACOR) Inactive Prodrag Mevinolinic Acid Active Inhibitor Pravastatin Active Inhibitor HMG-CoA: Normal HMG-CoA reductase substrate The majority of statins are known to be developmental^ toxic to rats (Henck et al., 1998). Effects include increased malformations (Minsker et al., 1983); decreased postweaning survival and delayed development of reflexes and behavior (FDA, 1987); reduced fetal body weight (Wise et al., 1990a); reduced fetal and offspring body weights (Wise et al, 1990b); reduced offspring body weight and increased swim maze errors (Minsker et al., 1990); reduced offspring body weight and decrease neonatal survival (Hrab et al., 1994); and retarded skeletal development and reduced fetal body weights (FDA, 1993). Studies have found that co-administration of mevalonate, the immediate product of HMG-CoA reductase, prevents or antagonizes various organ toxicities resulting from HMG-CoA reductase inhibition (MacDonald et al., 1988; Kombrast et al., 1989; Minsker et al., 1983; Hrab et a l, 1994). Minsker et al. (1983) found that co administration of cholesterol, however, had no effect on the teratogenicity of lovastatin (Mevinolin, MEVACOR). Some statins are also known to produce myocardial effects. D. J. Luebker Page 5 o f 16 01/21/2000 Hrab et a l (1994) found significant cardiac myopathy in pregnant rats dosed with 24mg/kg/dy of fluvastatin (LESCOL). All animals dying in this dose group had cardiac lesions which Were considered treatment related and believed to be associated to the cause of death (Hrab et al, 1994). The authors concluded that inhibition of mevalonate and cholesterol synthesis was a significant factor contributing to the myocardial damage and/or death of the fluvastatin treated rats. Co-administration of mevalonate complete blocked and/or ameliorated the mortality, cardiac myopathy and other adverse effects seen with fluvastatin (Hrab et a l, 1994). In-vitro studies by Guijarro et a l (1998, 1999) found the lipophilic statins atorvastatin (LIPITOR), simvastatin (synvinolin, ZOCOR) and lovastatin (mevinolin, MEVACOR) to produce apoptosis of rat and human vascular smooth muscle cells (VSMC). Mevalonate administration completely prevented this apoptosis (Guijarro et a l, 1998, 1999). Several mevalonate metabolites were evaluated to determine which were involved in the statin-induced apoptosis. Cholesterol, isopentenyl adenine, ubiquinone (coenzyme Q10) and squalene failed to prevent the statin induced effects while the isoprenoids farnesyl-pyrophosphate (FPP) and geranylgeranyl-pyrophosphate (GGPP) fully reversed the effects (Guijarro et al., 1998, 1999). These studies suggest the adverse effects seen with HMG-CoA reductase inhibitors may be more a result of decreased synthesis of certain isoprenoids than of decrease cholesterol synthesis. 004446 D. J. Luebker Page 6 o f 16 01/21/2000 Figure 5: Isoprenoid Biosynthetic Pathway Key. PP = pyrophosphate = shown to prevent statin induced VSM C apoptosis in-vitro1 it = shown NOT to prevent statin induced VSMC apoptosis in-vitro1 # = shown to prevent other statin induced adverse effects in-vivo and/or in-vitro2 0 = shown NOT to prevent other statin induced adverse effects in-vivo and/or in-vitro2 1 Guijarro et a /.,1998 & 1999 2 Minsker et al., 1983; Surani et al., 1983; Carson & Lennarz, 1979: Hrab et al., 1994 MacDonald et al., 1988; Kombrust et al., 1989, etc...) 004L4 4LV D. J. Luebker Page 7 o f 16 01/21/2000 Smith, Lear and Erickson (1995) found no sex related differences in HMG-CoA reductase enzyme activity in rats less than or equal to 28 days old. However, significantly higher activity levels are found in female rats as compared to males following puberty (Carlson, Mitchell and Goldfarb, 1978). Levin et al. (1989) investigated the developmental changes in expression of genes involved in cholesterol biosynthesis and lipid transport in human and rat fetal and neonatal livers. Levels of HMG-CoA reductase mRNA undergo large fluctuations during rat liver ontogeny (McNamara, Quackenbush and Rodwell, 1972; Leoni et a l, 1984), being high prior to birth, declining to low levels during suckling (days 1-13 postnatal) and transiently increasing at weaning (day 19-21 postnatal). In humans, however, these mRNAs appear at an earlier phase of fetal life (by week 8) and undergo only minimal (< 3 fold) changes in concentration during the rest of the intrauterine as well as postnatal life (Levin et al, 1989). Fetal and early postnatal rats may thus be very sensitive to inhibitors of HMGCoA reductase activity while fetal and early postnatal humans may not. The study outlined in this proposal is designed to help determine how PFOS administration in pregnant rats results in reduced maternal body weight gain, increased percent stillborn pups and decreased pup survival after birth. The hypothesis to be tested is that PFOS acts to inhibit HMG-CoA reductase activity in pregnant rats and/or their offspring and thus can behave as a developmental toxicant at higher dose levels. The results of this study, along with what is already known about human HMG-CoA reductase activity, will help determine the potential risk PFOS holds to be a developmental toxicant in humans. Figure 6: Hypothesis inhib ition o f HM G-CoA R eductase in Dam s in h ib ition o f HM G-CoA R eductase in pups RESEARCH PLAN OBJECTIVES 1. Determine whether hepatic HMG-CoA reductase activity is inhibited in PFOS treated pregnant rats. 2. Determine whether hepatic HMG-CoA reductase activity is inhibited in rat pups from PFOS treated dams. 00444,, 8_ D. J. Luebker Page 8 of 16 01/21/2000 3. Investigate the mechanism by which PFOS acts to inhibit HMG-CoA reductase activity. a) Determine if PFOS acts directly on HMG-CoA reductase. b) Determine if PFOS acts on AMP-PK. c) Determine if PFOS induces an increase in cellular AMP levels. EXPERIMENTAL APPROACH All results will be compared between dams and their pups to identify any relationship between dam effect and pup effect. Body weight and total liver weight will be recorded for each dam and litter as indices of general health and hepatomegally. Each group of dams and pups will be compared to age matched positive controls (dosed with a statin), negative controls (dosed with vehicle) and mevalonate challenged controls. Other parameters that will be followed where possible include liver and serum PFOS levels, liver and serum lipid levels and myocardial fiber injury and inflammation. As deemed necessary, portions of the study may be sent to contract labs for completion. Table 1: Dose Groups PFOS Groups Positive Control Groups Group 1 2 3 4 5 6 Compound(s) PFOS PFOS + mevalonate PFOS vehicle control statin statin + mevalonate statin vehicle control In-Vitro Phase HMG-CoA reductase The ability of PFOS to directly inhibit HMG-CoA reductase will be examined using rat liver microsomes. Microsomes will be prepared from untreated female rats with and without sodium fluoride (NaF) in all buffers and assay media. NaF inhibits phosphoprotein phosphatase activity (Kato and Bishop, 1972) and thus, inclusion of NaF at all stages of isolation and purification severely inhibits the conversion of inactive (phosphorylated) reductase to the active (unphosphorylated) form (Nordstrom, Rodwell and Mitchelen, 1977). Microsomes will be dosed as listed in table 1 and, following an incubation period, assayed for total and active HMG-CoA reductase by omission and inclusion, respectively, of NaF. Activity will be calculated as the rate (picomoles/minute) of formation of [3H]mevalonate from [3H]HMG-CoA per milligram of microsomal protein. Lipid levels of the various dosed microsomes will be measured using commercially available kits and/or HPLC. Cholesterol production in cultured hepatocytes, a biomarker of HMG-CoA reductase activity, may be used as a secondary measure of in-vitro HMG-CoA reductase inhibition. 0_ 0_ 4. 4. 4. 9~ D. J. Luebker Page 9 o f 16 01/21/2000 AMP levels To determine whether PFOS induces an increase in cellular AMP in-vitro, AMP levels of the microsomes will be quantified using HPLC. AMP-PK To assess the ability of PFOS to act directly to activate AMP-PK in-vitro, a peptide assay developed by Davies, Carling and Hardie (1989) which uses a synthetic peptide substrate, SAMS peptide, will be used. SAMS peptide was developed based on the amino acid sequence (Ser79) which is phosphorylated exclusively by AMP-PK. AMP-PK specimens will be prepared by partially purifying the cytosolic protein fraction of control rat liver as described by Carling et al. (1989). These specimens will be treated as outlined in table 1. Following an incubation period, AMP-PK activity will be measured based on the incorporation of radioactivity from [y-32P]ATP into the SAMS peptide (Davies et al., 1989). In-Vivo Phase HMG-CoA Reductase Activity in Pregnant Rats and Rat Pups Isolation of cellular subfractions to directly evaluate HMG-CoA reductase activity results in dissociation of the drug from the enzyme, which returns catalytically active (Corsini et al., 1995). The most favorable method to detect in-vivo inhibition of HMG-CoA reductase is by determining the in-vivo rate of incorporation of precursors, for example acetate, into cholesterol (Corsini et al., 1995). The potency of PFOS to inhibit the conversion of 14C-acetate into 14C-cholesterol in-vivo, therefore, will be used as a biomarker to determine HMG-CoA reductase in dams. These results should parallel the inhibition of HMG-CoA reductase inhibition found in the in-vitro assays. Other biomarkers which have been used successfully include urinary and plasma mevalonate levels (Corsini et al, 1995). Pregnant rats will be repeat dosed during gestation according to the dose groups listed in table 1. Prior to euthanization, dams will be dosed with 14C-acetate. Half of all dams will be allowed to come to term and half will be euthanized the morning of day 21 of gestation and pups will be removed. If PFOS is a HMG-CoA reductase inhibitor, the reduced maternal weight gain, increased stillborn and decreased pup survival seen in previous studies should be eliminated or ameliorated in the mevalonate supplemented dose group. Plasma and/or urine may be collected and analyzed for mevalonate to use as additional biomarkers of HMG-CoA reductase activity in the dams. Plasma mevalonate levels will serve as the biomarker to determine the degree of HMG-CoA reductase inhibition in each litter. Liver (from each dam and pooled for each litter) will be used for AMP-PK activity determination, liver lipid analysis and AMP determination. Sera will be collected from each dam and pooled for each litter for PFOS determination and serum 0 0 4 4 5 0 D. J. Luebker Page 10 o f 16 01/21/2000 lipid analysis. Liver and serum high density lipoprotein (HDL), low density lipoprotein (LDL) and total cholesterol will be determined using commercially available kits and/or HPLC. Liver and serum PFOS determination will be performed by the 3M Environmental Laboratory Fluorine Analytical Chemistry Team (FACT) headed by Kris Hansen, Ph.D. Myocardial tissue will be examined by electron microscopy (EM) or other appropriate method for fiber injury and/or inflammation. Liver AMP Levels in Pregnant Rats and Pups To determine whether PFOS induces an increase in cellular AMP levels in-vivo, liver AMP levels will be determined using HPLC. AMP-PK Activity in Pregnant Rats and Pups AMP-PK specimens will be prepared by partially purifying the cytosolic protein fraction as described by Carling et al. (1989). AMP-PK activity will be measured based on the incorporation of radioactivity from [y-32P]ATP into the SAMS peptide (Davies et al., 1989). SUMMARY The study outlined in this proposal is aimed at gaining knowledge into the mechanism(s) by which PFOS leads to reduced maternal body weight gain, increased percent stillborn and decreased pup survival in rats. The hypothesis that PFOS acts via inhibition of HMG- CoA reductase, the rate-limiting enzyme of cholesterol synthesis, will be examined. PFOS has previously been shown to down regulate HMG-CoA reductase activity in rats (Haughom and Spydevold, 1992). Several HMG-CoA reductase inhibitors (statins) have been shown to induce developmental effects similar to those seen with PFOS (Minsker et al., 1983; FDA, 1987; Wise et al., 1990a; Wise et al., 1990b; Minsker et al., 1990; Hrab et al., 1994; FDA, 1993). Studies have found that co-administration of mevalonate, the immediate product of HMG-CoA reductase, prevents or antagonizes various organ toxicities resulting from HMG-CoA reductase inhibition in rats and rabbits (MacDonald et al., 1988; Kombrust et a l, 1989; Minsker et al., 1983; Hrab et a l, 1994). An attempt will be made to prevent PFOS toxicity by co-administrating mevalonate. HMG-CoA reductase activity will be compared between dams and their pups and to age matched positive and negative controls. Specimens will be examined to determine if PFOS acts directly on HMG-CoA reductase, indirectly via activation of AMP-activated protein kinase (AMP-PK) or through inducing an increase in AMP levels. Other parameters, which will be followed where possible, include body weight, liver weight, liver and serum PFOS, liver and serum lipids and myocardial fiber injury and inflammation. Due to normal developmental differences in HMG-CoA reductase activity, fetal and early postnatal rats may be much more sensitive to HMG-CoA reductase inhibition than are fetal and early postnatal humans (Levin et a l, 1989). 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