Document wDjk6wQ2L4BpBgep9o080MEjV

A R I I M g kbw-2/3/98 Effect of N -Alkyl Perfluorooctylsulfonamides on M itochondrial Bioenergetics In Vitro Kendall B. Wallace, Ph.D. Department of Biochemistry & Molecular Biology University of Minnesota School of Medicine - Executive Summary - The following report summarizes research conducted the past 7 m onths concerning the effects of 6 different perfluorooctanonyl compounds (PFC's; supplied by The 3M Company) on the bioenergetic characteristics of isolated rat liver mitochondria in vitro. For purposes of confidentiality, the trade names and chemical structures were not disclosed to those conducting the research and analyzing the results. Consequently, the identities of the individual compounds are codified throughout the report. They are: PF10; PF10H; PF12L; PF12M; PF95; PF143; FC10 the carboxylic acid of FC10 linear FX12 mixed FX12 FC95 FC143 [Results for the sulfonamide and the N-acetic acid of the sulfonamide are not summarized in this report.] All six PFC's examined to date exhibited some effect on mitochondrial bioenergetics. FC10 increased the passive proton leak, resulting in a stimulation of state 4 (nonphosphorylating) respiration without uncoupling oxidative phosphorylation. The acid of FC10 increased membrane permeability to ion conductance, resulting in membrane depolarization and release of cytochrome c to inhibit respiration. Both linear and mixed FX12 were potent protonophore uncouplers of oxidative phosphorylation. Both FC95 and FC143 exhibited a general detergent-like effect on mitochondrial membranes as reflected by a decrease in respiratory control without uncoupling oxidative phosphorylation. In conclusion, although all PFC's interfered with mitochondrial bioenergetics, they differed in both potency and mechanism. Regardless, the ultimate effect is the same; inhibition of ATP synthesis. At the cellular level this may well manifest itself as failure of assorted energy-dependent metabolic or cell signaling pathways leading to disruption of metabolic regulation and cell cycle control. Whether and by what mechanism this may relate to the alleged peroxisome proliferating activity and tumorigenic activity of these compounds remains to be defined. 004140 1 kbw-2/4/98 CH2CH3 C 8F 1 7 -SO2 -N ^ CH2CH2OH / IFC10 increases passive proton leak @ 60 /iM V c h 2c h 3 C8Fi7-S02- N < H / c h 2c h 3 C8F17- S 0 2 - N < CH2COOH FX12 protonophore uncoupler @ 6 /iM (mixed and linear are comparable) PF10-H increased permeability to ion conductance @ 6 /iM 1r H C 8F 1 7 -SO 2 -N < CH2COOH Cmpd Vili C gF i 7 - S 0 2 -NH2 C8F17-SO3" FC95 weak increase in membrane fluidity @ 10 /iM ( >-5- C7F15-C oe H FC143 detergent @ 100 /iM 004141 11 ' J i iu '-t i ochem/Do IBio FOX : 2 1 8 - 7 2 6 - 8 0 U ATTACHMENT A PAGE 4 May 7, |WJ Biochemical and Molecular Mechanistic Studies of N-alkyl Perfluorosulfonamidcs Kendall R. Wallace, Ph.D. University of Minnesota School of Medicine Department of Biochemistry & Molecular Biology T h e initial phase of this investigation revealed that ail of th e perfluorochemicals (PFC's) examined wore capable of interfering with the bioenergctic properties of freshly isolated intact rat liver mitochondria in vitru. However, the precise mechanism by which each PFC interacted w ith the mitochondrial membranes differed, the activities ranging from a specific protonophoric uncoupling of oxidative phosphorylation to a full-blow n detergent-like disorganization of the membranes. Regardless of the specific m olecular mechanism, the results indicate that sufficiently high concentrations of any of the PFC's have the potential of inhibiting oxidative phosphorylation and ATP synthesis, it is presumed that at the cell or tissue level, the resulting deficit of ATP will be manifested as an interference w ith the regulation and integration of the various energy-dependent intermediary metabolic pathways, cell signaling, and cell cycle control points, including the balance between ATP-dcpendent apoptosis and cell proliferation. At this point, all that has been established is that the PFC's have the potential to interfere with mitochondrial bioenergetics and presumably cell num ber and function, provided the concentration is sufficiently high. W hether such concentrations are achieved in realistic exposvire scenarios has not yet been established. Accordingly, two pressing questions that remain are: 1) Is the inhibition of mitochondrial bioenergetics in v i t r o by the v a rio u s PFC's manifested as a bioenergetic deficit and interference with m etabolic 004142 1 9/15 '98 11:51 ID:Biochem/flo IBio FAX=218-726-3014 PAGE 5 May 7. 1998 regulation at higher levels of biological organization, such as in cell cultures, intact tissues, or whole organisms? and 2) Are the nominally effective concentrations within the range to warrant concern for human e x p o s u r e s ' Inherent in this latter question is the suitability of isolated rat liv e r m itochondria to predict adverse human health outcomes in vivo. W e propose to extend the investigation to address these questions by assessing the response of primary cultures of isolated hepatocytes to selected P F C 's in vitro (Gray et al., 1983). The specific design is to assess the effects o f exposing cells to individual PFC's on various indicators of cell bioenergetics, including changes in oxygen consumption, mitochondrial m e m b ra n e potential, ATP phosphorylation potential, and cell viability. In addition, w c intend to incorporate into these cell studios measures designed to assess the effects of PFC's on the expression of genes believed to be responsible for m ediating the effects of related chemicals on peroxisome proliferation, lipid m etabolism and cel! proliferation. The entire suite of genes to be analyzed is believed to be under the control of the "peroxisome proliferator response e le m e n ts" (PPRE) and include peroxisomal fatty acyl CoA oxidase, cytosolic fatty acid binding protein (FABP), microsomal CYP,VA, mitochondrial H M G C oA synthase, activating adipocyte protein (aP2), and apoproteins AI and CLII (Latruffe and Vamecq, 1997; Gonzalez, 1997). All of these proteins have been implicated to be responsible for the effects of peroxisome proliferators on altered lipid metabolism in vivo and may prove to be valuable markers of the biological response to PFC exposures in the corresponding species. Finally, since there are strong arguments questioning the validity of data from rats and mice to predict the proliferation of peroxisomes and p ro m o tio n of tumorigenesis in humans, we propose to include in the study design the opportunity to compare and contrast the metabolic and geneticresponse of rat and human hepatocyte cell cultures to PFC exposure in v i t r o . There is substantial evidence that exposures of non-human primates to related compounds does not induce the proliferation of peroxisomes, hepatom egaly, and tumorigenesis as is characteristic of rats and m ice. -> 004143 'jy/j.3 y 11:52 ID:Biochem/Flo !Bio FAX:218-726-801d PAGE 6 May 7. 1998 Epidem iological evidence concurs with the suggestion that rats and mice are poor sxirrogates for the human response. Based on published ex p e rim en ta l eviden ce, our original suggestion was that guinea pigs are m o re representative of the human response to exposure to peroxisom e proliferators. We intend to test this by including primary cultures of guinea pig hepatocytes in the cell exposure experiments. The intent is to co m p are and contrast the response of rat, guinea pig and human hepatocytes to exposures to the individual PFC's. Comparisons will be quantitative and based on dose-related changes in bioenergetic and genetic indicators of a m etabolic response to PFC exposure. EXPERIMENTAL DESIGN Isolated rat, guinea pig, and human hepatocytes will be purchased from a co m m o n vendor in order to minimize the possibility of artifactual differences am on g the different cell types due to cell isolation procedures. The cells wilt be suspended in 1-lepes-based minimum essential media at 37"C and distributed among wells at a density of approximately ItT cells/m). Cells in su spension will be exposed to the individual PFC's for up to 24 hr. T h e con cen tration o f each PFC will be varied in order to establish an effective range of concentrations for each end-point (bioenergetic or gene expression) for each PFC. This range of effective concentrations will serve as the basis for quantitative comparisons amongst the individual PFC'S and for assessing differences in sensitivities of the 3 species-specific cell types. The duration of exposure will be dictated by the kinetics of the response and its relationship to indications for gross changes in cell viability. The bioenergetic parameters to be assessed during the course of the cell exposures include oxygen consumption, mitochondrial membrane potential, ATP phosphorylation potential, and cell viability. Whole cell oxygen consu m ption will be monitored in a manner similar to that employed for the 3 004144 09/15 '98 11=52 ID-Biochem/hoIB io FAX:218-726-8014 PAGE 7 May 7. 1098 isolated m itochondria. Cells will be suspended in a stirred, closed ch a m b er into which is inserted a Clark-type oxygen electrode metered lo a c o n tin u o u s strip-chart recorder. The cell suspending medium will be supplemented w ith glucose and insulin to provide the requisite metabolic fuels. Once an equilibrium is established, sequential additions of the selected Pl;C will be made and any change in the rate of oxygen consumption recorded. In order to confirm and normalize any effect on mitochondrial respiration, all reactions will be terminated by first adding antimycin A followed by FCCP. Oxygen consumption in the presence of antimycin A will serve to reflect nunm itochondrial respiration, as one might expect if the agent in qu estion undergoes cytochrome P450-dependent oxygenation or redox cycling, for example. The rate of oxygen consumption in the presence of the nncoupler, FCCP, will provide a measure of any direct effect of the individual PFC on the electron transport chain. The effect of the individual PFC's on mitochondrial m em brane potential of the cells in culture will be monitored continuously on the basis of the distribution of the cationic fluorometric dye, rhodamine 123 (Rh 123) as we have published previously (Palmeira et al., 1996). Cells will be pre-loaded with R hl23, followed by washing, and then allocated to individual exposure wells. Again, increasing concentrations of the respective PFC will be added sequentially and the fluorescence recorded. The reactions will be term inated by adding FCCP to achieve a full depolarization of membrane potential, followed by digitonin to assure complete dissolution of cell membranes and release of non-specific latent fluorescence. Cell viability will be recorded in parallel with m itoch on d rial m em brane potential using the fluorometric indicator propidium iodide (PI) according to standard procedures in our laboratory (Palmeira et al., 1996). Sequential additions of the individual PFC's and termination of the reaction with FCCP and digitonin will be performed just as described for R h l 2 3 fluorescence. Unfortunately, PI apparently quenches some of the R h l2 3 fluorescence, prohibiting the concurrent measurement of both m em brane 4 004145 oy/lb '98 IT-52 iD`Biochem/MoIB io FAX:21S-726-301J PAGE 8 May 7, 1998 potential and cell viability in the same cells. Instead, the experiments will be conducted in parallel using the cells from the same culture sample for both measures. The concentrations of the various adenine nucleotides will be assessed by HPLC (Jones, 1981). Isolated hepatocytes from each of the 3 species will bo exposed in culture to varying concentrations of the individual PFC'S. T h e exposures will be stopped by alkalinizing the reaction medium with KOI I to stabilize the adenine nucleotides in the respective phosphorylation state. Sam p les from each exposure well will be centrifuged to remove debris, neutralized and injected onto a Waters Radial Pak 0 8 column. Individual nucleotides are eluted at 1 m l/m in in 100 mM potassium phosphate (pH 6) followed by a gradient to 5% methanol. Individual nucleotides will be detected at 260 nm and quantified by integrating the peak area as compared to known standards. Activation of PPAR-responsive genes will be assessed by differential display using standard molecular biology techniques (Wang and Morais, 1997). The method consists of exposing all 3 species of hepatocytes to the corresponding l 'PC. At pre-determined times, the reactions will be stopped in liquid nitrogen and analyzed for the enhanced expression of a select group of genes encoding for peroxisomal and mitochondrial proteins. This entails isolation of total RNA followed by gel separation and Northern hybridization for selected mKNA's. Probes have been prepared by PCR for the two "h o u s e keeping'' genes p-actin and glyceraldehyde 3-phosphate dehydrogenase; (GAPDH), which are supposedly un-inducible and thus valid measures for gene copy num ber (cell number). The PFC-induced activation of all o th e r genes will be normalized to the copy number for these two genes in order to quantitatively gauge the degree of stimulation of gene expression on the basis of total genomic DMA. F.xposure-related proliferation of peroxisomes will be estimated on the basis of the stimulated expression of the following genes: peroxisomal fatty 004146 5 u y/io ay li:b 3 [D:Biochem/f1olBio FAX:218-726-8014 PAGE 9 May 7. 1998 acyl Co A oxidase, cytosolic fatty acid binding protein (FABP), m icro so m a l C Y P [Va, mitochondrial IlMC.-CoA synthase, activating adipocyte protein (aP2), and apoproteins Al and CIO (Latruffe and Vamecq, 1997; Gonzalez, 1997). Mitochondrial proliferation will be assessed on the basis of the enhaneed expression of citrate synthase and succinate dehydrogenase (SDH). Amplification of the mitochondrial genome (mtDNA per mitochondrion as opposed to an increase in mitochondrial number per cell) will be assessed by an increase in gene copy number for mitochondria] transcript of cytochrome b (CYTb) and ND1 relative lo SDH (a nuclear encoded gene) using gene dosing experim ents (BouJtwood et al., 1996). uP-bbclod probes have been generated by FCR for all 3 genes. Increased expression of CYTb and ND1 relative to SDH has been used as an indicator of an increase in mtDNA copy num ber in cardiac tissue following acute intoxication of rats with adriamycin (unpublished observation). The stimulation of both peroxisomal and mitochondrial proliferation is ascribed to altered transcriptional control. To verify this, we will substantiate our results of the gene dosing and differential display experim ents by measuring the corresponding enzyme activities follow ing each of the various cell treatment paradigms. The exposure incubations will be stopped and the cells recovered by centrifugation. Fatty acyl CoA oxidase activity will be estimated from the cyanide insensitive oxidation of palmitoyi CoA according to the spectrophotometric technique of Lazarow (1981). Succinate dehydrogenase (SDH), citrate synthase and cytochrome oxidnse(COX) are measured by the corresponding spectrophotometric methods (Pennington, 1961; Trounce et al., 1996; Lash and Jones, 1993). 6 004147 'y /15 yy 11:53 ID:Biochem/flo IBio SUMMARY FAX :2 1 8 -7 2 6 -8 0 1 4 PAGE 10 May 7. 1998 The proposed investigation is designed to take our current findings regarding the effects of PFC's on the bioenergetic properties of isolated m itochondrial membranes to the next level of biological integration. T h e objective is two-fold: to determine whether the effects of PFC's on isolated m itochondria is manifested as bioenergetic dysfunction at the level of w h o le cell and to compare and contrast the response of hepatocytes from different species to PfC exposure in culture. The metabolic and genetic studies will yield valuable understanding into the mechanism(s) of biological activity as well as reveal potentially important biomarkers for assessing PFC exposure in v i v o . The comparisons among species will further test the question regarding the suitability of the rat as an experimental model for predicting adverse health outcomes in humans exposed to peroxisome proliferators and w hether the guinea pig may prove to be a more suitable surrogate. These data are essential to extrapolating dose-response relationships from experim ental evidence to predict no effect levels and margins of safety for potential h u m a n exposures, especially in light of our preliminary evidence which indicates that isolated hepatic mitochondria from guinea pigs is 3-6 times m o re resistant to PFC-indured interference with bioenergetics than are rat liver mitochondria (unpublished observations). 004148 7 y/l 'yb 1 1 ;53 ID:Biochem/flo IBio FAX'.218-726-8014 PAGE 11 May 7, 1998 REFERENCES Birch-M achin, M., Jackson, S. Kler, R.S-, and Turnbull, D .M (1993). Study o f skeletal muscle mitochondrial dysfunction. In: Methods in Toxicology, vol. 2; M itochondrial Dysfunction (Lash and Jones, cds.) A cadem ic Press. Boultwnod, J., Filder, C , Mills, K.I., Frodsham, P.M., Kusec, R., Gaigor, A., Gale, R.E., Linch, D.C., Littlewood, T.J., Moss, P.A.H., and W ain scoat, J.S. (1996). Amplification of mitochondrial DNA in acute myeloid leukemia. Brit. J. 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