(EL AO. C I . 1.75.7‘ ‘ . V . V 8/th , pan» xr .4. Mb 5.. T s . b. a? . .....s. «V n “me ‘ am... 3,; ._ ,V , V V .. V . V iww a, rut . . . . , ., . . , . .. . .. . . 1.1.4.1. ..JVu . Its? . _.; V. .. . .. h . . x V . _ V . “3V, : Tr. ,‘ . .. ‘ . . u . o. u . A .v , , ‘ . , V , . . s. . A, c p! ., V . . ,. . V .V V. V V V . , . V. . . .. . fiWw-fltgxnmte. . .. A .. V V .. V. ....V . .. . V . . . , 2 V V. 9%. at. ...énwfiaWi . XI a... . ... V? l: 1 1 {.5 80%;? rfiv .....P . .. . . .3 3:31.93. . . V v?- .v A: . 19:13. 11-. W5, ., V ., ‘ .firhfllehn V £549.29! kw“ ..l’ ......I. .._. U. r vim-19‘ .... ? 3:111? . :51 I: -.- $1.\%I.Vr.V. .1 i x k I: set-.... 1‘ It i ... $3.! 4 in... . {fibrfinuflyiliii}... 1 ~ . . .v 2(’ ‘i‘ .v ... .. . :ufkw . . .54: . .... . (J's?! ., ..J.., ulv‘.4 ‘O.1L III)».- . Inll‘b‘ i"! It, . . -, \Vl, I.d.;...LV.‘V....w:| 4 c , . . It .l.§!,.,.1uwfl....‘t>.tmm\fiunn.i|{; 5... 5.5V uaadimisivlv 1 , ... : x . V ‘ V . ‘HfififlusznnmwwngflnV .-.. . q Justus; m 1 IN 2000 SITY LIBRARIES “lillmmmumll ll [ll \llllllllllll 3 1293 02080 6133 # ”—4— This is to certify that the thesis entitled In Vitro Studies of the Biochemical Toxicity of Perfluorooctane Sulfonic Acid and its possible Interaction with 2,3,7,8- Tetrachlorodibenzo-p-dioxin presented by Wen Yue Hu has been accepted towards fulfillment of the requirements for M.S. Zoology and degree in Institute of Environmental Toxicology / Major professor I)ate ‘é(/22‘i9496) 7/ ’/ 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution PLACE IN RETURN Box to remove this checkout from your record. TO AVOID FINE return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 11/00 cJCIRCJDmDuepss-pu IN VITRO STUDIES OF THE BIOCHEMICAL TOXICITY OF PERFLUOROOCTANE SULFONIC ACID AND ITS POSSIBLE INTERACTION WITH 2,3,7,8 — TETRACHLORODIBENZO-P-DIOXIN By Wen Yue Hu A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Zoology Institute of Environmental Toxicology 2000 ABSTRACT IN VITRO STUDIES OF THE BIOCHEMICAL TOXICITY OF PERFLUOROOCTANE SULFONIC ACID AND ITS POSSIBLE INTERACTION WITH 2,3,7,8 — TETRACHLORODIBENZO-P-DIOXIN By Wen Yue Hu In the current study, three aspects of the biochemical toxicity of perfluorooctane sulfonic acid (PFOS) were investigated using in vitro cell culture systems. The effects of PF OS on aryl hydrocarbon receptor (AhR) mediated cytochrome P4501A1 (CYP1A1) activity were tested using in vitro cell bioassays. The results showed that PFOS had neither adverse effect on cell viability nor direct effect on CYP1A1 activity within the dose range tested. However when cells were dosed with PFOS and TCDD in combination, interactive effects on both CYP1A1 induction and AhR activation were observed at environmentally relevant concentrations, in which PFOS increased the effects of TCDD by 30-40 %. It was further tested with time course experiment and transcription inhibition experiment that this interactive effect possibly occurred at transcriptional level. In addition, the effects of PF OS on gap junctional intercellular communication (GJIC) were tested. PFOS inhibited GJIC in a dose-dependent manner, and the inhibitory effect occurred in a very short time period. It was proven that this inhibitory effect was neither species-specific nor tissue-specific. Finally aromatase assay was conducted, results from which indicated that PF OS at a concentration of 50 mg/L could induce aromatase activity in vitro by 1.5 fold for 24 hrs exposure, and by 1.7 fold for 48 hrs exposure for PFOS concentration at 10 mg/L and 50 mg/L. To my dear parents Hua Hu and Xiao Ru Liu, for their never stopped love and encouragement. iii ACKNOWLEDGMENTS I Sincerely thank my academic advisors Dr. John Giesy and Dr. Paul Jones for their continuous support and tremendous help. I really appreciate the cooperative tradition in the aquatic toxicology laboratory at Michigan State University, only with the friendly supports from all the personnel in this laboratory, could I make through all the difficulties during the process of doing this project and writing my thesis. I am honored to be able to have Dr. Don Hall, Dr. Will Kopachik and Dr. Robert Roth on my graduate committee, and to receive invaluable advises and comments from them. Special thanks were owed to Dr. James Trosko and Dr. Brat Upham at Michigan State University for their kindly providing experimental instrument and instructions that made part of this thesis possible. I am thankful to Dr. Wim Decoen at Antwerp University, Belgium for his advises on cell bioassays; Dr. Thomas Sanderson and Marjoke from Utrecht University at Netherlands for their help on NCI-H295R cell culture and aromatase assay. Debts are owed to Dr. Heather Eisthen for her support during the first year and a half in my graduate studies, and especially for her understanding on my switching program. Thanks are owed to 3M company for their funding of this project, and the aquatic toxicology laboratory at Michigan State University for providing me the graduate assistantship. TABLE OF CONTENTS LIST OF TABLES ............................................................................ vii LIST OF FIGURES ............................................................................ viii LIST OF SYMBOLS OR ABBREVIATIONS ............................................. xi INTRODUCTION ................................................................................. 1 Acute Toxicity ............................................................................ 2 Tissue Distribution, Metabolism and Excretion ..................................... 3 Induction of Oxidative Stress and Lipid Metabolizing Enzyme Activities ...... 4 Effect on Hepatic Microsomal Cytochrome P450 Enzyme Activity .............. 4 Non-genotoxic Tumor Promoter ...................................................... 5 Chapter 1. CELL BIOASSAY BACKGROUND ................................................................................ 7 MATERIALS AND METHODS ............................................................... 8 Chemicals ................................................................................. 8 Cell Culture ............................................................................... 8 Bioassay Procedure ..................................... ' ................................ 9 Plating Cells ..................................................................... 9 Dosing Cells .................................................................... 10 Cell Viability Assay ........................................................... ll EROD Assay ................................................................... 1 1 Luciferase assay ............................................................... 13 Cell Bioassay Data Analysis .......................................................... 13 RESULT .......................................................................................... 14 Cell Viability Assay ..................................................................... 14 Direct Effect of PFOS on P450 1A1 Activity ...................................... 14 Interactive Effects ofPFOS and TCDD in EROD Assay 17 Interactive Effects of PF OS and TCDD in Luciferase Assay ..................... 20 Time Course Experiment ............................................................... 20 Transcription Inhibition Experiment .................................................. 23 DISCUSSION ...................................................................................... 28 Chapter 2. GJIC ASSAY BACKGROUND ................................................................................ 34 MATERIALS AND METHODS ............................................................... 34 Chemicals ................................................................................ 34 Cell Culture .............................................................................. 35 GJIC Assay .............................................................................. 35 Cell Plating ..................................................................... 35 GJIC Measuring ............................................................... 36 RESULT ............................................................................................ 37 Dose Response Experiment ............................................................. 37 Time Course Experiment ................................................................ 40 DISCUSSION ....................................................................................... 40 Chapter 3. AROMATASE ASSAY BACKGROUND .................................................................................. 45 MATERIALS AND METHODS ............................................................... 46 Chemicals ................................................................................. 46 Cell Culture ............................................................................... 46 Aromatase Assay ........................................................................ 47 Plating Cells ..................................................................... 47 Dosing Cells ..................................................................... 47 Assay Procedure ................................................................ 48 F luorescamine Protein Assay ................................................ 49 RESULT .......................................................................................... 49 DISCUSSION ..................................................................................... 52 CONCLUSION .................................................................................. 55 APPENDIX A ..................................................................................... 60 APPENDIX B ..................................................................................... 61 REFERENCES ................................................................................... 64 vi LIST OF TABLES Table A1. Culture Medium for NCI-H295R Cells. (page 60) Table A2. Culture Medium for CDK Cells. (page 60) Table B1. F luorescamine Protein Assay Data for BSA Standard. (page 61) vii LIST OF FIGURES Fig. 1 Effects of PFOS on H4IIE-luc cell viability in the absence or presence of TCDD. A) cells were dosed with PFOS only; B) cells were dosed with different concentrations of PFOS and TCDD in combination. PFOS concentration in mg/L, TCDD concentration in lug/L. (page 15) Fig. 2 Direct effects of PFOS on H4IIE-luc cell and PLHC-l cell EROD activity and on H4IIE-luc cell luciferase activity, compared with the effects of TCDD. A) EROD activity of H4IIE-luc cells dosed with PFOS or TCDD; B) EROD activity of PLHC-l cells dosed with PFOS or TCDD; C) Luciferase activity of H4IIE-luc cells dosed with PF OS or TCDD. (page 16) Fig. 3 Interactive effects of PFOS and TCDD on H4IIE-luc cell EROD activity. A) H4IIE-luc cells were exposed to PFOS at concentrations of 0.001 mg/L, 0.1 mg/L and 10 mg/L in the presence of TCDD; B) H4IIE-luc cells were exposed to PFOS at wider concentration range, from 10 to 0.0001 mg/L with 10 fold dilution, in the presence of TCDD. (page 18) Fig. 4 3-D plot of interactive effects of PFOS and TCDD on H4IIE-luc cell EROD activity (with the same data as in Fig. 3 B). X-axis represents PFOS concentration in mg/L; Y-axis represents TCDD concentration in rig/L; Z-axis represents relative EROD activity which is resorufin fluorescence divided by protein concentration. General linear model pairwise comparisons were conducted (* P<0.05, ** P<0.01). (page 19) Fig. 5 Interactive effects of PF OS and TCDD on PLH C-l cell EROD activity. A) PLH C-l cells were dosed with different concentrations of PFOS (mg/L) and TCDD (pg/L) in combination; B) a set of data in A where TCDD concentration equals to 0.2 ug/L were plotted in a histogram, general linear model pairwise comparisons were conducted (* p<0.05; ** p<0.01). (page 21) Fig. 6 Interactive effects of PF OS and TCDD on H4IIE-luc cell luciferase activity. A) H4IIE-luc cells were dosed with different concentrations of PF OS (mg/L) and TCDD (pg/L) in combination; B) 3-D graph with same data from A, X-axis represents PFOS concentration in mg/L, Y-axis represents TCDD concentration in pg/L, Z-axis represents luciferase light production. General linear model pairwise comparisons were conducted (* p<0.05, ** p<0.01). (page 22) viii Fig. 7 Time Course experiment of interactive effects of PFOS and TCDD on PLHC-l cell EROD activity. A) PLH 01 cells were dosed with TCDD and PF OS on day 2 (72 hrs before running the assay). B) Cells were dosed with TCDD only on day 2, and with PFOS on day 5 (5 min before running the assay). C) Cells were dosed with TCDD only on day 2, and with PFOS on day 5 (20 min before running the assay). D) Cells were closed with TCDD only on day 2, and with PF OS on day 5 (1 hr before running the assay). (page 24) Fig. 8 Time Course experiment of interactive effects of PF OS and TCDD on H4IIE-luc cell EROD activity. A) Cells were dosed with TCDD and PFOS on day 2 (72 hrs before running the assay). B) Cells were dosed with TCDD only on day 2, and with PFOS on day 5 (5 min before rumiing the assay). C) Cells were dosed with TCDD only on day 2, and with PFOS on day 5 (10 min before running the assay). D) Cells were dosed with TCDD only on day 2, and with PFOS on day 5 (30 min before running the assay). (page 25) Fig. 9 Transcription inhibition assay on interactive effects of PFOS and TCDD on H4IIE- luc cell luciferase activity. A) Cells were dosed with TCDD, TCDD in the presence of 10 uM amanitin and TCDD in the presence of 10 pM amanitin plus 0.1 mg/L PFOS. B) Cells were dosed with TCDD, TCDD in the presence of 100 uM amanitin and TCDD in the presence of 100 pM amanitin plus 0.1 mg/L PFOS. C) Cells were dosed with TCDD and TCDD in the presence of 0.1 mg/L PFOS; no inhibitor was added. (page 27) Fig. 10 Diagram Showing the mechanisms for EROD assay and Luciferase. (page 29) Fig. 11 Dose Range Box showing the environmental relevance of data. X-axis shows PFOS concentration in mg/L, and Y-axis shows TCDD concentration in ug/L. The two dash lines indicate the maximum concentrations of these two chemicals found in wildlife, 2mg/L and 500 ug/L for PFOS and TCDD respectively. The lower left area of the box indicates the concentration range of environmental concern. The interactive dose range based on cell bioassay results is 0.01 mg/L to 0.1 mg/L for PFOS and 200 ug/L to 1000 ug/L for TCDD, shown as a cross. (page 33) Fig. 12 Image of WB F-344 cells dosed with different concentration of PFOS / PFOSA. (page 38) Fig. 13 Dose response effects of PFOS and PFOSA on WB F-344 cells Gap Junctional Intercellular Communication (with exposure time of 30 min). (page 39) Fig. 14 Dose response effect of PFOS on CDK cells Gap Junctional Intercellular Communication (with exposure time of 30 min). (page 39) Fig. 15 Image of WB F-344 cells exposed to PFOS / PFOSA (50 mg/L) for different time period. (page 41) ix Fig. 16 Time course of PFOS and PFOSA (at concentration of 50 mg/L) induced inhibition of Gap Junctional Intercellular Communication in WB F-344 cells. (page 42) Fig. 17 Effect of PFOS on NCI-H295R cell aromatase activity. A) Cells dosed with 8-Br CAMP as positive control; B) Cells exposed to PFOS for 24 hrs; C)Cells exposed to PFOS for 48 hrs; D) Cells dosed with 50mg/L PFOS at different time interval before running the assay. Aromatase activity was expressed as picomolar substrate aromatized per mg protein per hr. (page 50, page 51) Fig. 18 Diagram shows the environmental relevance of the data. The PFOS concentration that caused interactive effects with TCDD in cell bioassays ranges from 0.01 mg/L to 0.1 mg/L; the PFOS concentration that caused significant inhibitory effects on GJIC ranges from 12.5 mg/L to 200 mg/L; the PFOS concentration that caused significant effects on aromatase activity ranges from 10 mg/L to 50 mg/L. Dashed line indicates the maximum concentration of PF OS found in wildlife” indicates range of concentration determined at both end; H indicates range of concentration determined at only one end. (page 56) Fig. Bl BSA protein standard curve. (page 62) AhR ATCC CDK CYP 1 9 CYP1A1 DMEM DRES ECSO EROD F BS GJIC H4IIE-luc MEM NADPH NCI-H295R PBS PF DA PFFAS PFOA PFOS PF OSA PKC PLHC- l PPAR PPRE TCDD WB-F 344 LIST OF SYMBOLS AND ABBREVIATIONS --- aryl hydrocarbon receptor --- American Type Culture Collection --- Carvan dolphin kidney cell line, isolated from a prematurely born female-bottle-nose dolphin --- cytochrome P450 19 (aromatase) --— cytochrome P4501Al --- Dulbecco’s Modified Eagle Medium --- dioxin-responsive elements --- concentration of 50% effectiveness --- ethoxyresorufin-o—deethylase --- fetal bovine serum --- gap junctional intercellular communication --- rat hepatoma cell line, which was stably transfected with firefly luciferase reporter gene --- Minimum Essential Medium Eagle --- B-nicotinamide adenine dinucleotide phosphate (reduced form) --- human adrenocortical carcinoma cells --- phosphate buffer saline --- perfluorodecanoic acid --- perfluorinated fatty acids --- perfluorooctanoic acid --- perfluorooctane sulfonic acid --- perfluorooctanoic sulfonamide --- protein kinase C --- hepatocellular carcinoma cells derived from desert topminnow (Poeciliopsis lucida). --- peroxisome proliferator activated receptor --- peroxisome proliferator responsive element --- 2,3,7,8 — tetrachlorodibenzo-p-dioxin --- rat liver epithelial cells xi INTRODUCTION Perfluorinated fatty acids (PFFAS) are commonly used in industrial materials such as wetting agents, lubricants, corrosion inhibitors, stain resistants for leather, paper and clothing, as well as in foam fire extinguishers (Sohlenius, et al. 1994) due to their low surface tension, stable physical and chemical properties. PFFAS also possess unique biological characteristics that make them suitable for red blood cell substitutes and hepatic drugs (Ravis, et al., 1991). Because of their growing list of applications and increasing potential for exposure to humans and wildlife, toxicologists are now assessing the potential toxicity of PF F AS at environmentally relevant concentrations. Based on the fact that PFFAS are chemically stabilized by strong covalent bonds between carbons and fluorines, they were historically considered to be metabolically inert and non-toxic (Sargent et al. 1970). However, only recently, has it been found that they are biologically active and can induce effects on peroxisomal proliferation, lipid metabolizing enzyme activity, xenobiotics metabolizing enzyme activity, and other important biochemical processes in exposed organisms (Obourn, et al., 1997; Sohlenius, et al., 1994). The major target organ of PFFAS is the liver, but this does not exclude other possible target organs such as the pancreas, testis and kidney (Olson, et al., 1983). Acute Toxicity The most well studied compounds in the PF PA family are perfluorooctanoic acid (PFOA) and perfluorodecanoic acid (PFDA). The acute toxicities of these two compounds were evaluated in male Fisher rats, and the LD50/30 days for PFOA was found to be 189 mg/kg-body weight and 41 mg/kg-body weight for PFDA (Olson and Andersen, 1983). Rats treated with a lethal dose of PFOA exhibited incipient death within the first five days; however, those exposed to PFDA showed a delayed lethality after two weeks (Olson and Andersen, 1983). This difference is probably due to their different rate of accumulation and elimination in male rats. Tissue Distribution, Metabolism and Excretion PFFAS are completely ionized in hydrophilic environments, and their basic hydrocarbon backbone is similar to fatty acids, except that all of their carbon atoms are covalently bound with fluorine atoms, which is also responsible for their chemical and metabolically inertness. When Wistar rats were treated with a single intraperitoneal dose (20 mg/kg- body weight) of PF DA, approximately 15% of the administered PF F As were found in the serum, with more than 99% bound to the serum proteins. In the liver, 5% of PF FAS were found to be either in the free anionic form or bound to lipid portion (Ylinen and Aurivla, 1990). Most of the PFFAS administered via the diet were unaffected by metabolic enzymes. Elimination of PFFAs was primarily through urinary excretion, with little bilary and fecal excretion, and the rate of elimination was sex-related (Ylinen, et al., 1989; Hanhijarvi, et al. 1987). The renal elimination rate of PFOA in female Wistar rats was ten-fold greater than in male rats. It was suggested that estradiol played a considerable role in controlling PFOA excretion (Ylinen, et al., 1989). Induction of Oxidative Stress and Lipid Metabolizing Enzyme Activities Although the mechanism by which PFFAS elicit their toxic effects is unknown, the one consistent conclusion drawn by most researchers is that they acted as peroxisome proliferators. Peroxisome proliferators include a number of structurally diverse compounds. Regardless of their dissimilarities in structure, these compounds all have one thing in common: they all induce the proliferation of peroxisomes, and result in an increase in both the number of peroxisomes and their corresponding enzyme activities (Kawashima, et al. 1989). PFFAs can interfere with lipid metabolism by increasing peroxisomal fatty acid B-oxidation, and induce several hepatic enzyme activities (Sohlenius and Reinfeldt, 1996). Both in vivo and in vitro exposures to PFFAS result in increased activities of peroxisomal Acyl-COA oxidase, which is known to catalyze the first and rate-limiting step in fatty acid oxidation (Sohlenius, et al., 1994). Fatty acid oxidation is also a process that can produce hydrogen peroxide, a oxidative radical, which can cause oxidative stress and result in DNA damage (Sohlenius, et al., 1994). The peroxisome proliferator activated receptor (PPAR), a member of steroid hormone receptor family, can be activated by peroxisome proliferators and bind to the peroxisome proliferator responsive element (PPRE). Previous studies identified several PPRES which located upstream from the structural gene for Acyl-CoA oxidase (Sohlenius and Reinfeldt, 1996; Braissant, et al., 1996). A good correlation had been observed between PPAR activation and peroxisome proliferation potency (Green, 1992). PFFAS have been shown to be involved in regulating tissue fatty acid composition and content. PFFAS can reduce cholesterol and triacylglycerol level in serum, increase liver triacylglycerol concentration, and reduce hepatic lipid output (Haughom and Spydevold, 1992). It has also been found that treatment with PFFAS can inhibit Acyl-COA synthetase activity and result in an increase in the level of free fatty acids (Rec, et al., 1996). Free fatty acids are known to be able to activate protein kinase C (PKC), which leads to a signaling cascade that is important for normal cell function, cell proliferation and gene expression. Effect on Hepatic Microsomal Cytochrome P45 0 Enzyme Activity Cytochrome P450 enzymes (CYP) are a group of primary oxidative enzymes involved in phase I metabolism, a process that detoxifies xenobiotics by making them more polar so that they can be conjugated and excreted easily. Microsomal cytochrome P450 enzymes were induced in rats treated with PFFAS (Perrnadi, et al., 1992). This induction was sex- related and organ—specific, based on the fact that male rats were more sensitive than female rats, and liver was the major target organ compared to the kidney. For example, administration of PFOA to male rats induced CYP4A1 enzyme activity by 6.8 fold in liver and 2.1 fold in kidney (Diaz, et al., 1994). The CYP4A sub-family is a group of nine enzymes that are specific for fatty acid (o-hydroxylation. Other CYP enzymes may be induced depending on the administration pathways and duration of exposures. Effect on Leydig Cell Function PFFAS can affect Leydig cell function and produce Leydig cell adenomas (Liu, Hahn, and Hurtt, 1996). So far most information available is for the effects of ammonium perfluorooctanoate (C8) on Leydig cells of adult male rats. Three levels of effects have been observed: 1) overall depression of Leydig cell function in vitro (Cook, et al., 1992); 2) decreased testosterone release and increased estradiol level in vivo (Biegel, et al., 1995); 3) elevation of aromatase (CYP 19) activity by 16 fold in vivo (Liu, et al., 1996). Non-genotoxic Tumor Promoter Treatment with PF FAs has been associated with the induction of hepatic necrosis, hepatocyte carcinomas, Leydig cell adenomas, and pancreatic tumors (Oboum, et al., 1997). It has been postulated that the increase in oxidative stress and alteration in protein kinase C level are responsible for the possible carcinogenic property of PFFAS (Rec, et al., 1996). Recently the alternative hypothesis has been suggested that these effects may be non—genotoxic and caused by the disruption of hormone regulation (Cook, et al., 1992) or blocking of intercellular communication (Upham, et al., 1998). Perfluorooctane sulfonic acid (PFOS) appears to be the end metabolite of a number of perfluorinated compounds used extensively in commercial applications. The amount of PF OS produced is much greater than any other PFFAS (3M internal report). However so far most studies have been conducted on PFOA instead of PFOS. Whether PFOS can cause similar adverse biological effects as PFOA and other PFFAS is still under investigation, and its possible mechanism of action remains to be elucidated. In this study three aspects of biochemical toxicity of PFOS were investigated using in vitro cell culture systems. The effect of PFOS on aryl hydrocarbon receptor (AhR) mediated cytochrome P4501A1 activity and its possible interaction with 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD) were tested using EROD and Luciferase bioassays. In addition, the effects of PFOS on gap junctional intercellular communication were tested using the scrape/loading dye technique. Finally, in vitro aromatase assays were conducted on human adrenocorticol cells in order to determine the possible effects of PFOS on steroid hormone metabolism. Together these studies investigate possible modes of action of PF OS based on known toxicological end-points. F F F FF F OH FCF\/ FCFV \CF/ FCF\/ F \/ \/ \/ |_ F:\C\/c F\C99.9%. a-Amanitin was purchased from Molecular Probe (A 6920). Cell Culture H4IIE-luc cells are rat hepatoma cells that were stablely transfected with firefly luciferase reporter gene under direct control of dioxin-responsive elements (DRES) (Sanderson, et al. 1998). Due to this unique feature, the H4IIE-luc cell line can be used for both luciferase assay and ethoxyresorufin-O-deethylase (EROD) assay. PLHC-l cells are derived from a hepatocellular carcinoma of desert topminnow (Poeciliopsis lucida). Previous studies have indicated the presence of AhR and inducible cytochrome P450 1A1 activity (Hahn, 1993). H4IIE-luc and PLHC-l cells were cultured in 100 mm disposable tissue culture dishes (Corning, 25020). All cells were grown under sterile conditions (pH=7.4) in a humidified 5/95% COz/air incubator (Forrna Scientific, Model 8173). H4IIE-luc cells were cultured at 37 C°, and the PLHC-l cells were grown at 30 C°. H4IIE-luc cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Sigma D-2902), supplemented with 10% fetal bovine serum (FBS, Hyclone). PLHC-l cells were cultured in Minimum Essential Medium Eagle (MEM, Sigma M3024) supplemented with 292 mg/L L-glutamine (Life Technologies), and 10% FBS (Hyclone). All cells were passaged when cells became confluent, and new cultures were started from frozen stocks after 30 passages. Bioassay Procedure Day I Plating cells When cells reached 80-100% confluence, they were trypsinized from tissue culture dish using 1x trypsin—EDTA (Sigma), and resuspended in cell culture media. The concentration of stock cell solution was estimated using a hemocytometer. H4IIE-luc cells were diluted to a concentration of approximately 7.5 x 105 cells / m1, and PLHC-l cells were diluted to a concentration of approximately 1.25 x 10‘5 cells / m1. Cells were seeded into the 60 interior wells of the 96 well flat bottom micro-plate (view plates, Packard 600-5181) using Eppendorf repeater pipette (Brinkmann Instruments, NY). 250 pl of cell solution was seeded into each well. The 36 exterior wells were filled with 250 pl culture medium to avoid marginal effects. Cells were incubated for 24 hrs before dosing to allow for cell attachment. Day 2 Dosing cells TCDD was dissolved in isooctane, and the solvent for PFOS was methanol. Each standard consisted of six concentrations prepared by 5 fold and 10 fold serial dilutions for TCDD and PFOS, respectively. The concentration range for TCDD was determined based on previous studies, which showed the whole range of dose-response; where as PFOS concentration centered at the environmental average concentration 0.1mg/L: TCDD: 1 pig/L, 0.2 jig/L, 0.04 pig/L, 0.008 pg/L, 0.0016 jig/L, 0.00032 pg/L; PFOS: 10 mg/L, 1 mg/L, 0.1 mg/L, 0.01 mg/L, 0.001 mg/L, 0.0001 mg/L. Before dosing, cells were briefly inspected under microscope, checking for contamination and even cell distribution. Control wells and treatment wells were dosed with 2.5 pl of appropriate solvent and chemicals. Blank wells received no dose (see the spread Sheet below for example). Each treatment was tested in triplicate. All exposures except for those in the time course experiment were 72 hrs. TCDD alone TCDD lug/L + TCDD lug/L + TCDD lug/L + lug/L PFOS PFOS 0.1mg/L PFOS lOmg/L 0.001mg/L 0.2ug/L 0.2ug/L+ 0.2ug/L + 0.2ug/L + 0.001mg/L . 0.1mg/L lOmg/L 0.04ug/L 0.04ug/L + 0.04ug/L + 0.04ug/L + 0.001mg/L 0.1mg/L 10mg/L 0.008ug/L 0.008ug/L + 0.008ug/L + 0.008ug/L + 0.001mg/L 0.1mg/L lOmg/L 0.0016ug/L 0.0016ug/L + 0.0016ug/L + 0.0016ug/L + 0.001mg/L 0.1mg/L 10mg/L 0.00032ug/L 0.00032ug/L + 0.00032ug/L + 0.00032ug/L + 0.001mg/L 0.1mg/L lOmg/L Day 5 Cell Viability Assay Cytotoxicity was measured using the live/dead viability kit (Molecular Probes L-3224). The kit comprises two probes: calcein AM and ethidium homodimer. Calcein AM is a fluorogenic esterase substrate that when hydrolyzed produces green fluorescence. Thus, green fluorescence is an indicator of living cells that have esterase activity as well as intact cell membranes. Ethidium homodimer is a red fluorescent nucleic acid stain that is only able to pass through the broken membranes of dead cells. Therefore by measuring the ratio of these two fluorescent emissions at two different wavelength, an estimation of the ratio of live to dead cells can be obtained. Preparation of the viability assay reagent was done by diluting the appropriate amounts of calcein and ethidium with the appropriate volume of media without FBS. Plates were removed from the incubator and media was aspirated, then cells were rinsed twice with phosphate buffer saline (PBS). 50 pl of PBS with calcium and magnesium and 50 pl of viability assay reagent were added to cell containing wells using 8-channel pipette. Plates were incubated at 30 C° for 10 min and then scanned in the Cytofluor 2300 Fluorescence Measurement System (Millipore, Bedford, MA) at 500 nm and 600 nm wavelength for calcein and ethidium respectively. Day 5 EROD Assay The ethoxyresorufin-O—deethylase (EROD) assay is a useful tool for identification of dioxin-like compounds which can induce P450 1A1 (CYP 1A1) activity. EROD assay 11 with H4IIE-luc and PLHC-l cells were performed following a modified version of the EROD assay procedure (Sanderson and Giesy 1998). On the day of assay, exposed cells were briefly inspected under microscope, checking for degree of confluence, homogeneity among wells, and any Sign of contamination and cytotoxicity. Cell culture medium was aspirated by vacuum aspirator, and cells were rinsed three times with PBS. 30 pl of distilled water was added to each cell-containing well, and cells were lyzed by freezing and thawing. Cells were treated with 70 pl of Hepes-dicoumarol buffer (Sigma, M1390) and 50 pl of 20 pM 7-ethoxyresorufin (Molecular Probes, Eugene, OR), and incubated at 30 C° for 20 min to ensure temperature uniformity. Reactions were initiated by adding 50 p1 of 1.25 mM NADPH (Sigma N-6505) in Hepes, and plates were incubated exactly for 1 hr. at 30 C°. Reactions were stopped by adding 50 pl of 1.08mM fluorescamine (Sigma, F-9015) in acetonitrile, and plates were incubated for another 15 min. Fluorescamine is intrinsically non- fluorescent, but reacts with amine groups on protein to yield a fluorogenic derivative; thus it was used to determine the protein concentration of cell lysate (Udenfriend, et al., 1972). Resorufin was measured. using a Cytofluor 2300 Fluorescence Measurement System (Millipore) at 1. ex =530 nm and 1. cm =590 nm, and fluorescamine was measured at A 3,, = 400 nm and 7t em = 460 nm. P4501Al induction was expressed as relative EROD activity, which was calculated as resorufin illumination divided by protein concentration. 12 Day 5 Luciferase Assay Luciferase assay is an in vitro technique using a genetically modified system to identify Ah receptor-active compounds. H4IIE-luc cells were stablely transfected with an AhR controlled luciferase reporter gene, and these cells express firefly luciferase in response to Ah receptor agonists. On the day of assay, exposed cells were briefly inspected visually for signs of contamination and abnormal cell growth. Luciferase Reporter Gene Assay Kit (Packard, 6016916) were reconstituted freshly right before performing the aSSay. One bottle of lyophilized substrate was dissolved with 10ml buffer, and agitated gently until a homogeneous solution was formed. Cell culture media was aspirated, and cells were rinsed three times with PBS. The bottom of the view plates was sealed with self-adhesive Topseal-A (Packard, 6005185). 75 pl of PBS including calcium and magnesium was added to 60 interior wells using 8-channel pipette. Under subdued light condition, 75 pl per well of reconstituted substrate solution was added and agitated, and the plates were incubated for 10 min at 30 C°. Luminescence was measured on a plate-reading luminometer (Microlite ML3000, Dynatech). Cell Bioassay Data Analysis All cell bioassay data were collected electronically and converted into Spreadsheet for analysis. Dose response curves were drawn using microsofi EXCEL 98, and statistical tests were conducted using SYSTAT 8.0. 13 RESULTS Cell Viability Assay H4IIE-luc cells exposed to PFOS and TCDD showed no Sign of cytotoxicity or abnormal cell grth within the concentration range tested (Fig. 1A and 1B). Live/dead ratios of cells treated with PF OS alone up to 10 mg/L were not significantly different from that of the control (Fig. 1A). When cells were treated with PFOS and TCDD in combination, the live/dead ratios for treated cells were not Significantly less than that of the control (Fig. 18). In fact, at relatively high dose of TCDD and PFOS, the live/dead ratios of treated cells were greater than that of the control, even though the difference was not significant. This could be explained by excessive cell grth observed with visual assessment which was possibly due to the inhibition of cell-cell communication (see GJIC assay results) and the block of contact inhibition. Direct Effect of PFOS on EROD activity and luciferase activity Two cell lines H4IIE—luc and PLHC-l were used to assess any direct effects of PF OS on EROD activity and luciferase activity. Cells were exposed to PFOS or TCDD for the purpose of comparison. PFOS alone did not induce Cytochrome P450 1A1 (CYP1A1) activity compared to that of the control (Fig. 2A). EROD activity of H4IIE-luc cells dosed with PFOS was Similar to that of the control, whereas TCDD induced EROD activity in a dose-dependent manner, with the greatest induction being 17 fold. In order to check whether this effect is Species-Specific, the same experiment was conducted using fish PLHC-l cells (Fig. 2B). Results were very Similar to those observed for H4IIE-luc l4 200 l 80 160 140 120 100 80 60 40 20 % of Control HH 0.0001 0.001 0.01 PFOS concentration (ppm) 0.1 1 60 140 1 20 1 00 80 60 40 20 0 % of control W W 0.0001 0.001 0.01 0.1 1 TCDD concentration (ppb) 10 —o— rcoo + TCDD&PFOS0.001 + TCDD&PFOS0.01 —x—— TCDD&FFOSO.1 —x— TCDD&PFOS1 + TCDD&FF0610 Fig. 1 Effects of PFOS on H4IIE-luc cell viability in the absence or presence of TCDD. A) cells were dosed with PFOS only; B) cells were dosed with different concentrations of PFOS and TCDD in combination. PFOS concentration in mg/L, TCDD concentration in pg/L. Cell viability was measured as live/dead ratio, and expressed as % of control, which was cells dosed with solvent (methanol). Error bars represent standard deviation of three measurements. 15 2000 1800 A 1600 1400 1200 1000 800 600 400 200 + 0 «I ——- --~ — +- 0.0001 0.001 0.01 0.1 1 10 dose of PFOS (ppm) TCDD (ppb) + TCDD + PFOS % of control 1200 1000 B 800 600 l-O—TCDD ‘ + PFOS °/o of control 400 200 0 . V 0.0001 0.001 0.01 0.1 1 10 concentration of PFOS(ppm) TCDD (ppb) 0.01 0.009 C 0.008 0.007 I i 0.006 1 0°05 / + PFOS 0.004 0.003 + TCDD 0.002 0.0014;— ¢ 1' m 0 luc 0.0001 0.001 0.01 0.1 1 10 concentration of PFOS (ppm) TCDD (ppb) Fig. 2 Direct effects of PFOS on H4IIE-luc cell and PLH C-l cell EROD activity, and on H4IIE-luc cell luciferase activity compared with the effects of TCDD. A) EROD activity of H4IIE-luc cells dosed with PFOS or TCDD; B) EROD activity of PLH 01 cells dosed with PFOS or TCDD; C) luciferase activity of H4IIE-luc cells dosed with PFOS or TCDD. EROD activity was expressed as % of control, luciferase activity was expressed as luciferase light production. Control cells were dosed with 0.1% (v/v) solvent (methanol) only. Error bars represent standard deviation of three measurements. 16 cells: PF OS exhibited no detectable effect on CYP1A1 induction. Luciferase assay was conducted on H4IIE-luc cells dosed with PFOS or TCDD. PFOS when dosed alone did not induce AhR-mediated luciferase activity relative to that of the control. In contrast, TCDD induced luciferase activity in a dose-dependent manner (Fig. 2C). Interactive Effects of PF OS and TCDD in EROD Assay In order to assess the possible interaction between TCDD and PFOS, cells were exposed to these two chemicals in combination. Cells were dosed with TCDD standard alone, and TCDD in combination with 0.001, 0.1 or 10 mg/L of PFOS (Fig. 3A). Co-exposure of cells to PFOS and TCDD increased the CYP1A1 activity induced by TCDD. Compared to the TCDD standard dose-response curve, the addition of PFOS increased both the slope of the curve and the magnitude of maximum response, with the medium concentration of PF OS (0.1 mg/L) having the most Significant interaction with TCDD. In order to confirm the interactive relationship between these two chemicals, more PFOS concentrations were tested. Cells were exposed to PFOS with concentrations ranging from 10mg/L to 0.0001mg/L in a serial dilution of 10 fold, in combination with the TCDD standard concentration. A similar interactive relationship between PFOS and TCDD was observed (Fig. 3B). To permit visual assessment, the same data was plotted as a 3-D graph and General Linear Model (GLM) pairwise comparison was conducted (Fig. 4). Significant interactive effects were observed at 0.2 pg/L TCDD plus 0.1 mg/L PFOS (p<0.05), 1 pg/L TCDD plus 0.01 mg/L PFOS (p<0.05), and 1 pg/L TCDD plus 0.1mg/L 17 4.5 4A .2- €3.5— co 3. +TOIdee D 825 +TCImFF®m1 I: 2 +mn=oso1 > 515 +mr=r=cs10 a: 1 (I 0.5 0 , 0.0001 0.001 0.01 0.1 1 10 TGDconoertraimmg’L) 6 5 +1000 4 +TCDD&PFOS0.0001 +TCDD&PFOS0.00I - X” TCDD&PFO$0.01 +TCDD&PF080.I +TCDD8IPF081 -—+—TCDD&PFO$10 relative EROD activity 00 0.0001 0.001 0.01 0.1 1 1O dose of TCDD (ug/L) Fig. 3 Interactive effects of PFOS and TCDD on H4IIE-luc cell EROD activity. A) H4IIE-luc cells were exposed to PFOS at concentrations of 0.001 mg/L, 0.1 mg/L and 10 mg/L in the presence of TCDD; B) H4IIE-luc cells were exposed to PFOS at wider concentration range, from 10 to 0.0001 mg/L with 10 fold dilution, in the presence of TCDD. EROD activity was expressed as relative EROD which equals to resorufin fluorescence divided by protein concentration. Error bars represent standard deviation of three measurements. 18 Fig. 4 3-D plot of interactive effects of PFOS and TCDD on H4IIE-luc cell EROD activity (with the same data as in Fig. 3B. X-axis represents PFOS concentration in mg/L; Y-axis represents TCDD concentration in pg/L; Z-axis represents relative EROD activity which is resorufin fluorescence divided by protein concentration. General linear model pairwise comparison was conducted (* P<0.05, ** P<0.01). PFOS (p<0.01). In the last combination, the addition of PFOS increased the effect of TCDD by 40%. To compare responses between species, the same experiment was conducted using PLHC-l cells (Fig. 5A). For PLHC-l cells the standard TCDD dose-response curve had a slightly different shape compared to that of the H4IIE-luc cells, however the general trend of interactive effects was similar to that of the H4IIE-luc cells. The most significant interactive effects were observed at a TCDD concentration of 0.2 pg/L, therefore, this set of data was plotted in a histogram (Fig. SB). The maximum induction was observed at a PF OS concentration of 0.1 mg/L (p<0.01), which increased the effect of TCDD by 40%. Interactive Effects of PF OS and TCDD on Luciferase Expression The luciferase assay was conducted using H4IIE-luc cells dosed with TCDD and PFOS in combination, at the same concentrations used as in the interactive EROD assay (Fig. 6A). The same data was plotted as a 3-D histogram for better visual assessment (Fig. 6B), and general linear model (GLM) pairwise comparison was conducted. Exposure to l pg/L TCDD plus 0.1 mg/L PFOS (p<0.05), and 0.2 pg/L TCDD plus 0.1 mg/L PFOS (p<0.05), significantly increased induction over TCDD alone, with the maximum of increase by 40%. Time-course Experiment To determine whether the interactive effects on EROD induction was due to interaction between PFOS and CYP 1A1 enzyme or EROD reaction substrate, a time course 20 4000 3500 3000 .E‘ —o—TCDD .2 2500 ~5- +TCDD&PFOS0.001 (U o 2000 +TCDD&PFOS0.01 8 1500 —)(—TCDD&PFOSO.1 UJ 1000 +Tcooar=i=os1 500 +TCDD&PFOS10 o Log [TCDD concentration ug/L] 8 7— *,; é? at M E 6+- t? é? R 7 t? D 5 g Z / / A / 4 / / 3 a a 0.0001 0.001 0.01 0.1 1 10 PFOS(mg/L) Fig. 5 Interactive effects of PFOS and TCDD on PLH C-l cell EROD activity. A) PLH C-l cells were dosed with different concentrations of PFOS (mg/L) and TCDD (pg/L) in combination; B)a set of data in A where TCDD concentration equals to 0.2pg/L were plotted in a histogram, general linear model pairwise comparisons were conducted (* p<0.05; ** p<0.01). 21 0.01 A 0.009 I 0.008 T I 92‘ 0.007 > +rwo “3 0.006 (U +mifl1 to 0.005 (I) +TmPFCm01 E 0004 .93 Toomprosm — 0.002 10 0.001 0 0.0001 0.001 0.01 0.1 1 10 Concentration ofTCDD (ugIL) Fig. 6 Interactive effects of PFOS and TCDD on H4IIE-luc cell luciferase activity. A) H4IIE-luc cells were dosed with different concentrations of PFOS and TCDD (pg/L) in combination; B) 3-D graph with same data from A, X-axis represents PFOS concentration in mg/L, Y-axis represents TCDD concentration in pg/L, Z-axis represents luciferase light production. General linear model pairwise comparisons were conducted (* p<0.05, ** p<0.01). 22 experiment was conducted. Instead of dosing cells with both TCDD and PFOS on day 2, which is 72 hrs before performing the assay, PLHC-l cells were dosed with TCDD standard only. On the same day of the assay, cells were then dosed with PFOS at 5 min, 20 min, and 60 min before running the EROD assay (Fig. 7B, 7C, 7D). A PFOS concentration of 0.1 mg/L was used in this experiment, because based on previous results, this concentration of PFOS caused the greatest interaction between TCDD and PFOS on both EROD and luciferase induction. In all three assays, there was no significant difference between cells dosed with TCDD alone and TCDD in the presence of PFOS added at different time intervals before the assay. In contrast, when cells were exposed to TCDD and PFOS at 72 hrs before assay, a significant interaction was observed (Fig. 7A). Thus, it can be concluded that the Significant interactive effects occurred only in the long-term exposure (72hrs), and it was not due to the direct interaction between PFOS and P450 enzyme or the EROD reaction substrate, which should have happened much more quickly. Species-specificity was studied by conducting the same experiment with H4IIE-luc cells. The result was very similar to that of the PLHC-l cells, with interactive effects observed only in long—term exposure (Fig. 8). Transcription Inhibition Experiment a-Amanitin is an inhibitor of eukaryotic polymerase II (pol II) enzyme, which is responsible for mRNA transcription. This experiment was conducted to determine whether PF OS could still elicit its interactive effects with TCDD after pol II transcription was inhibited. Cells were seeded into the 96 well plate on day 1 as described in materials 23 .338 00 mo cougcoocoo “a mOmmv 9580532: 025 00 502.60 03053 832%.. ES Sum .383 05 meg 20.80 E S m .30 no mo"; 5.3 28 .N .80 no baa SOUP 5S, 0800 803 £00 a ”98mm 05 mean: 20.03 58 0N0 m .30 no mOmm .23 05 .N .30 co bee CODE 55 0080 303 £3 6 ”98mm 05 ms? 283 ES 3 m .30 :0 m9: 503 0:“ .N .30 8 .25 once as, 858 253 £8 a ”95.8 55 a: 8&3 an S N be 8 won: as once as, 85% 253 £8 To m: 2 55:8 comm :8 To an .5 coup Ea 8.3 05 Boots 5588355 058590 50:8 58:. A .wE @3883 age 8880p 0F _. V0 «0.0 50.0 wgd 0.. _. V0 _.0.0 50.0 500.0 . o . o F F W m m N m... .Boglql N m. . a m 38.599 _ ... a PEI-I v w 0851-1 v m 0 99+ m @191 m M o W o m... A R M. a A U m 3388 08» . . Livosunbc. 2 a 3 so Soc 59:. 2 . 2. a... s: as... 0 1 o w J _ m w W . N a N m. FEII n W. F. MESH; m w rglul . m PEI-I v m REIT M m + B o .m. a 0.. m— s < a 24 .33:: .0 mo 5080:8000 we mOnEv 8:082:80:— ooufi 00 0008.60 0.805% 88808 8.3 EEO .988 20 mafia 283 :0: 09 m 80 co mpg .005 05 .N .30 co baa OOU... £05 0080 20? 230 O ”03me 20 @005: 283 SE 03 m .30 :o mOmm £05 05 .N .30 no baa OOUO. £05 0800 0.203 38 G ”gamma 20 mag 0.503 58 9 m .30 co mOmm 50$ 05 .N .30 so 300 OOU... 505 0800 803 m=8 Am ”983 20 mam—EB 883 8: 8 N .30 no mOmm 05w OOUO. 505 0800 82> £3 2 .0585 8% =8 878:: 8 oooe 8a mots E 885 2,8825 .3 858890 50:8 2:0. w .wa 280 coegc8c8 coo» 380 coa§=8=8 89. 2 F 3 5o .88 .83 2 v to So .88 .83 o o 0.0 0.0 P m m 8&31‘1 W égk‘l w W m; OOOHII M. Ell M. 0.? 9 u a a o m on 0 N a n 3 O on U m 238 :3 8389889 2 F 3 5.9 88 588 9 p 3 5o 88 .82. o O . no mo W P F an... Ell 3 w 800868101 3 m BIT N M. 9.9+ N E a mN 0 mu 3 m G a 0 an e m. m V < Q? (N m... ... 25 and methods. On day 2 cells were dosed with TCDD, and incubated at 37 C° for 36 hrs to allow for TCDD to induce luciferase activity. On day 3 cells were dosed with either amanitin or amanitin and PFOS in combination. The amanitin concentrations used were 10 pM and 100 pM, and the PFOS concentration used was 0.1 mg/L based on previous studies. Luciferase assay was conducted on day 5 as previously described. Amanitin at a concentration of 100 uM inhibited luciferase gene transcription efficiently, whereas a concentration of 10 uM had no effect (Fig. 9A and 9B). Both concentrations showed no sign of cytotoxicity based on visual observation and cell viability assay results. Cells dosed with 10 uM amanitin plus TCDD were observed in the same magnitude of luciferase induction as those dosed with TCDD only, and cells dosed with TCDD, amanitin plus PFOS had a significantly greater luciferase induction (Fig. 9A). In contrast, cells dosed with 100 uM amanitin plus TCDD showed a much lesser luciferase induction relative to cells dosed with TCDD only (Fig. 93). Cells dosed with TCDD, amanitin plus PFOS were observed in the same magnitude of luciferase induction as those dosed with TCDD and amanitin (Fig. 9B). When cells were dosed with TCDD only, TCDD and PFOS in combination (no amanitin added), PFOS increased the TCDD induced luciferase activity by 40 % (Fig. 9C), which was of the same magnitude of interaction as that observed in Fig. 9A and was consistent with results from the interactive luciferase assay (Fig. 6). This result suggested that interactive effects occurred when there was no transcription inhibitor added or the concentration of the inhibitor was not high enough to inhibit transcription. The addition of PF OS did not have any effect on 26 0.14 A 0.12 lf—fl g 0.1 <0 0.08 i +TCDD 8 E3 0'06 +TCDD+Amanitin § 0.04 - +TCDD+ 0'02 Amanitin+PFOS 0 o 0.5 1 1.5 TCDD concentration (ug/L) 0.12 B 0.1 .2 > 33 0-08 +Tcoo m 0 006 “a? +TCIDrAnanitin ,9; 0.04 g +1701} — 0.02 I Ananitin+FFOS 0 0 0.5 1 1.5 TCDD concentration(ugIL) 0.14 C 0.12 0i g. 01 > '8 ' r 5 g 0.08 8 0.06 +TCDD .9 +Tooo+n=os '6 0.04 :3 .1 0.02 0 0 0.2 0.4 0.6 0.8 1 1.2 TCDD concentration (ug/L) Fig. 9 Transcription inhibition assay on interactive effects of PF OS and TCDD on H4IIE- luc cell luciferase activity. A) cells were dosed with TCDD, TCDD in the presence of lOuM amanitin and TCDD in the presence of lOpM amanitin plus 0.1mg/L PFOS; B) cells were closed with TCDD, TCDD in the presence of 100 1.1M amanitin and TCDD in the presence of 100 uM amanitin plus 0.1mg/L PFOS; C) cells were dosed with TCDD and TCDD in the presence of 0.1mg/L PFOS, no inhibitor was added. 27 TCDD induced luciferase activity when transcription of luciferase gene in these cells had been inhibited. DISCUSSION The mechanisms of EROD assay and Luciferase expression are diagramed in Fig. 10A and 103, respectively. Induction of EROD activity is an endogenous response of cells to dioxin-like compounds. When TCDD or other dioxin-like compounds are introduced into the cell, they bind to aryl hydrocarbon receptor (AhR), and form a receptor-ligand complex facilitated by heat shock protein (Hsp70). This complex translocates to the nucleus and interacts with specific sequences on the DNA, termed dioxin-responsive elements (DRES), which is an enhancer of the CYP1A1 gene. This binding can up- regulate the transcription of CYP1A1 gene, increase the amount of CYP1A1 mRNA, and subsequently increase the amount of CYP1A1 protein. CYP1A1 is involved mainly in oxidative metabolism of exogenous chemicals, and one of its characteristic activities in vitro is to catalyze the reaction from ethoxyresorufin to resorufin. Resorufin is a fluorogenic compound. Therefore the induction of CYP 1A1 can be quantitated by fluorometric measurement of EROD activity (Sanderson, et al. 1998; Roman, et al., 1998). In contrast to the EROD assay, luciferase assay is a genetically engineered system to identify AhR agonists. H4IIE-luc cells were stably transfected with firefly luciferase reporter gene, which is under direct control of the DRE. Binding of the agonist to the 28 fl \ I CYPlAlgene TC-DD DRE n1111/ TCDD A CYP1A1 V A. EROD assay Ethoxyresorufin TCDD \ /-__L_uf::e D) T DRE TCDD AhR RNA \ mm/ Luciferas B. Luciferase assay (32,: ,. substrate ~.- ,Qgrlight Fig. 10 Diagram showing the mechanisms of EROD assay and Luciferase assay. 29 receptor results in an activated receptor-ligand complex that translocates to the nucleus. There it interacts with the cis-acting regulatory sequences DRES, which are localized 5’ upstream of the luciferase reporter gene, and increases the production of luciferase enzyme. This enzyme can cleave the luciferin substrate to produce light. Hence light production is an indicator of AhR binding affinity (Sanderson, et al., 1996). Because of the differences in the mechanism of these two in vitro bioassays, their results have different implications. First, the induction of EROD activity has biological significance. Good correlation exists between the AhR binding affinity and the EROD induction potency in vitro and toxic potency in viva. Whereas luciferase induction is only an in vitro transcription monitoring system, and it is not associated directly with any biological effects. Secondly, an alteration in EROD activity dose not necessarily mean that it is an AhR mediated effect. Since EROD activity is an endogenous response, it is regulated at transcriptional, post transcriptional and -translational levels. Compared to the EROD assay, the expression of exogenous luciferase reporter gene may be less affected by other factors. Thus, it is a more direct indicator of AhR mediated response, and it has a greater sensitivity as well (Sanderson, et al., 1996). Based on above discussion, the results of in vitro bioassays tell us that PFOS by itself has no direct effect on cytochrome P450 isoenzyme activity. Even though PFOS can cause wasting syndrome similar to that caused by TCDD, it probably elicits its effect through a different, non AhR mediated pathway. However PFOS can induce interactive effects with TCDD not only in the EROD assay, but also in the luciferase assay, this implies a more 30 complicated transcription receptor interaction rather than direct AhR binding. As shown in previous studies peroxisome proliferator activated receptor (PPAR) is involved in mediating most of the effects of peroxisome proliferators. PPAR has been qualified as a member of the nuclear transcription receptor family. It was once called orphan receptor because its exact mode of action was unknown. The tentative hypothesis here is that PPAR may have interacted with AhR itself or AhR-associated factors, such as ARNT. However so far there is no evidence on the relationship between PPAR and AhR. Further studies would need to be conducted to reveal the mechanism of the interactive effects. To eliminate the possibility that PFOS simply interacts with EROD and luciferase assay substrates, and to confirm the finding that PF OS interacts with TCDD at a transcriptional level, two additional experiments were conducted. The time course experiment was based on the theory that alterations at the transcriptional level did not occur instantaneously, but required certain amount of time to elicit effects, whereas interactions directly with the substrates would occur rapidly (5~10 min). Since interactive effects were only observed in long-term exposure (72 hrs), it can be concluded that the observed effects were not due to direct interaction with the substrates. The transcription inhibitor experiment was designed to test effects occurring at the transcriptional level using transcription inhibitor amanitin. The idea was that if PFOS interacted with TCDD at stages other than the transcriptional level, the interactive effects between PF OS and TCDD should still persist after the transcription of luciferase gene had been inhibited. The result in Fig. 9 suggests the opposite, which is that the interaction does occur at the transcriptional level since the interactive effects between PFOS and TCDD disappeared afier the transcription of 31 luciferase gene was inhibited by amanitin. Another possible explanation of the effects observed is that afier cells were exposed to fairly great amount of amanitin for 36 hrs, they were not in a physically healthy status, even with the live/dead ratio stays the same. Therefore, no interaction could be observed no matter which stage it happened. If this is true, then the transcription inhibition assay might not be a good way to determine the effect at transcription in this study. The purpose of this study was to investigate potential mechanisms of action of PFOS, that could be used to provide general information for environmental risk assessments. Thus, the environmental relevance of the data becomes an important issue. To illustrate the environmental relevance, a dose range box was developed (Fig. 11). The two dashed lines indicate the environmental extreme value for these two chemicals. Here 2mg/L and 500 ug/L were considered the maximum concentration measured in wildlife for PFOS and TCDD respectively. Thus, the lower left area of the box indicates the concentration range of environmental concern. Based on chemical analysis data obtained in ATL this is also the concentration range within which these two chemicals can be measured together out in the field. The interactive dose range based on cell bioassay results is 0.01mg/L to 0.1mg/L for PFOS and 200 ug/L to 1000 ug/L for TCDD, shown as a cross. This dose range falls into the lower left area of the environmental ranges. 32 2mglL 100000: I T IIIIIII I I IIIIIII I I IIIIIII I I IIIIIII I I IIIIIII I I HW- : l I _ I _ _ I 2 100005— : —_:_ E I E .4 r ; : 8 1000;— : —_ U 5 ———————————————— I ——————— 5‘ 500 A — I - E . I , - (pg/L) E 100g : “g E I E “' Environmental | ‘ — Relevant Region ' — 10 E I E E I E 1 l lllllllI l llllllII I 11111111 I llllllll 4| 1 IIlUlI l IIIIIIII 0.0001 0.001 0.01 0.1 1.0 10 100 PFOS (mg/L) Fig. 11 Dose Range Box showing the environmental relevance of data. X-axis shows PF OS concentration, and Y-axis shows TCDD concentration. The two dash lines indicate the maximum concentrations of these two chemicals found in wildlife, 2 mg/L and 500 ug/L for PFOS and TCDD respectively. The lower left area of the box indicates the concentration range of environmental concern. The interactive dose range based on cell bioassay results is 0.01 mg/L to 0.1 mg/L for PF OS and 200 ug/L to 1000 ug/L for TCDD, shown as a cross. 33 GAP JUNCTIONAL IN TERCELLULAR COMMUNICATION ASSAY BACKGROUND Gap junctional intercellular communication (GJIC) is a major pathway for cells to communicate with each other, thus, it plays a crucial role in the maintenance of normal cell growth and function. The down-regulation of GJIC has been linked to tumor promoting properties of many non-genotoxic carcinogens. For a wide range of chemicals, the correlation between tumor promotion and GJIC inhibition is greater than 80% (Trosko and Ruch, 1998). Previous studies have shown that some perfluorinated fatty acids can inhibit GJIC in a dose-dependent fashion. This inhibition occurs within a short time period (<1hr), and is rapidly reversed by removing the inhibitors (Upham, 1998). In order to compare the effects of PFOS on GJIC to those of other PF FAs, and to determine the species specificity of the effects, GJIC assay was conducted using WB F-344 rat liver cells and CDK dolphin kidney cells. The dolphin cell line was used here in the effort to develop a marine mammalian model for testing the effect of PFOS, because PFOS was also detected in a fairly great amount in marine mammal samples (3M internal data). MATERIALS AND METHODS Chemicals Perfluorooctane sulfonic acid (PFOS) was obtained from 3M company (St. Paul, MN) as a mixture. Based on the NMR analysis, the mixture consisted of 68% of straight chain 34 PFOS and 17% of branched chain PFOS. Perfluorooctanoic sulfonamide (PFOSA) was obtained from Sigma. Cell Culture WB-F344 cells are rat liver epithelial cells obtained from Drs. J.W. Brisham and MS. Tsao of University of North Carolina. This cell line has been well characterized for its expression of gap junctional proteins (Tsao, et al., 1984). Carvan dolphin kidney (CDK) cell line is an epithelial cell line isolated from a prematurely born female-bottle-nose dolphin. Same as WB-F344 cells CDK cells are also non-tumorigenic primary cells. WB-F344 and CDK cells were cultured in 75cm flask (Corning 430720). All cells were grown under sterile conditions (pH=7.4) at 37 C° in a humidified 5/95% C02 /air incubator (Forrna Scientific, Model 8173). WB-F344 cells were cultured in Dulbecco’s Modified Eagle Medium (Formula 78-5470-EF, Gibco), supplemented with 5% FBS (Gibco). CDK cells were cultured in Dulbecco’s Modified Eagle Medium and Ham’s F12 (Sigma D-2906), supplemented with 10% FBS (Gibco), and other nutrients (see Appendix A for detailed formula). GJIC Assay GJIC was measured by the use of the scrape loading dye transfer technique. Two mammalian cell lines (WB-F344 and CDK) were used to compare species specificity, and two chemicals (PFOS and PF OSA) were tested to compare structure specificity. 35 Cell Plating After reaching 80-lOO% confluence, cells were trypsinized with 1x trypsin-EDTA (Gibco—BRL 15400-054» and cell solution was collected. Cell number was determined using a hemocytometer. WB-F 344 cells were diluted to a concentration of approximately 1 x 106 cells / ml, and CDK cells were diluted to a concentration of approximately 1 x 105 cells / ml. 2 ml of the cell solution was then transferred to 35 mm tissue culture plates, and cells were incubated for 24 hrs before dosing to allow for cell attachment. GJIC Measuring PFOS (MW 499) and PFOSA (MW stock solutions were prepared in acetonitrile, in a 2 fold dilution series from 20 g/L to 0.3125 g/L. 20 pl of PFOS and PFOSA stock solution or solvent was added to each cell culture plate containing 2 ml cell culture medium. Following the chemical exposures, cells were rinsed three times with PBS and then approximately 1 ml of 0.05% lucifer yellow dye was added to each plate. The size of the dye molecule is large enough to keep them from entering the intact cell membrane, but is small enough that they can go through gap junction between cells. A surgical steel blade was used to make three scrapes through the monolayer of cells. So the dye can be up- taken by the broken cells, and then transferred to adjacent cells through gap junction. After three minutes incubation at room temperature, the dye was discarded, and the cells were rinsed three times with PBS, and then fixed with 0.5 ml of 4% formalin. Dye migration was observed and photographed at 200x using a Nikon epifluorescence phase contrast microscope illuminated with an Osram HBO 200W lamp and equipped with a COHU video camera. The average distance of dye migration from the scrape indicates 36 the ability of cells to communicate with each other through gap junction, and it was calculated using the Nucleotech Gel Expert program. Each treatment was tested in triplicate. The average was calculated, and plotted using Excel. Differences among clones and between compounds were determined by 2-way ANOVA, followed by Turkey’s multiple range test. In the dose response experiment, cells were treated with PFOS at concentrations ranging from 200 mg/L to 3.125 mg/L with a 2 fold dilution series, but with the same exposure time (30 min). In the time course experiment, cells were treated with the same dosage of PFOS (50 mg/L), but with different duration of exposure: 2min, 5 min, 10min, 30min, 1 hr and 24 hrs. Dosage ranges and exposure times were determined based on previous studies. RESULT Dose-response Experiment Fig. 12 showed the pictures of WB F-344 cells in GJIC dose-response experiment taken under phase-contrast fluorescent microscope. As described in methods and materials, the distance from the front of the dye to the scrape is directly pr0portional to the level of cell- cell communication. The dye migrated to the greatest distance in cells treated with solvent. PFOS inhibited dye migration in a dose-dependent manner (Fig. 12 and Fig. 13). From the dose response curve, the EC50 for GJIC inhibition was determined to be approximately 20 mg/L PF OS for 30min exposure. The maximum inhibition of GJIC was 37 .AEE om no 080 oSmonxovanE mo 0008:5008 Echofim0 £05 0800 £00 $03-95 mo owns: NO .wE mOnE ..BE 09. 9 ummoaxm :00 m>> mOuE <9: mm 9 00.893.V =8 m>> 25:00 x:m_m :00 m>> 38 120 100 80 % of control 60 + PFOS + PFOSA 40 we 20 o 0 50 100 150 200 250 Concentration of PF OS (mg/L) and PFOSA (mg/L) Fig. 13 Dose Response Effects of PF OS and PFOSA on WB cells Gap Junctional Intercellular Communication (with exposure time of 30 min). 120 100 80 60 + PFOS 40 7 + PFOSA % of control 20 0 10 20 30 40 Duration of exposure (min) Fig. 14 Effects of PF OS on CDK cells Gap Junctional Intercellular Communication (with exposure time of 30 min). 39 caused by PFOS concentration of 50 mg/L or greater. No sign of cytotoxicity was observed within the concentration range tested. To determine the structure specificity of the chemicals, PFOSA (an insecticide) was tested at the same dose range as PFOS. A similar dose-dependent inhibition of GJIC was observed. The ECSO value for PFOSA was approximately 24 mg/L, with the maximum inhibition occurred at 100 mg/L PFOSA. To determine the species specificity of this inhibitive effect, another cell line, CDK cells were tested with the same treatment (Fig. 14). A very similar dose-response curve was obtained, with an EC 50 value of approximately 14 mg/L PFOS. The maximum effect was observed at 50 mg/L PFOS or greater. Time course experiment The phase-contrast images of WB F-344 cells exposed to 50 mg/L PFOS for 2 min, 5 min, and 10 min were shown in Fig. 15. GJIC was inhibited in a short time period by the exposure to 50 mg/L PFOS. A 50% inhibition was observed after WB F-344 cells were exposed to PFOS for only 2 min, and the maximum inhibition of 90% occurred within 5 to 10 min (Fig. 16). These results were similar to those observed for PF OSA. When WB cells were exposed for 1hr and 24 hrs, no further inhibition of GJIC was observed. DISCUSSION The mechanism of the GJIC inhibition by fluorinated compounds is poorly understood, however, inhibition of GJIC does depend on the fluorinated carbon tail. Previous studies have shown that perfluorinated fatty acids (PFFAs), such as perfluorooctanoic acid 40 .808 2:: 82006 5 fine 00 wok e 8890 “=8 $8.95 0o 82: 58:80 3.23 2 .3 EE or .8 wOnE 9 00898 m=oo m>> 58 m 8.. mourn. 9. 00896 m__wo m>> EE N 02 wOuE 9. 008qu £00 m>> 60:8 2.8 m>> 41 120 100 80 60 + PF OS % of control 20 0 1O 20 30 40 Duration of exposure (min) Fig. 16 Time Course of the Inhibitory Effects of PFOS and PFOSA on WB cells Gap Junctional Intercellular Communication (with PF OS / PFOSA concentration at 50 mg/L). 42 (PFOA) and perfluorodecanoic acid (PFDA), can inhibit GJIC in a dose-dependent manner, whereas the non-fluorinated fatty acids do not have such effects (Ketcham and Klaunig, 1996). The inhibitory potency of PFFAs depends on the length of its carbon chain. PFFAs with carbon chain length less than 5 or more than 16 did not inhibit GJIC. In contrast, PFFAs with carbon chain lengths of 7, 8, 9 and 10 can completely inhibit GJIC at a concentration of 50 mg/L (Upham et al., 1998). Results from the current study are consistent with previous published result. PF OS which has an 8 carbon chain effectively inhibits GJIC, with ECSO value of 18 mg/L. PF OSA has the same carbon chain length as PFOS but with a modified functional group. PFOSA inhibits GJIC with a potency similar to that of PFOS. This indicates that the critical feature that determines the GJIC inhibition efficiency is the length of the carbon chain, but not the functional groups. This result suggests a receptor-mediated mechanism, that is only ligand of certain structure can be recognized by the receptor, and elicits its effect on GJIC consequently. Peroxisome proliferator activated receptor (PPAR) may be a potential mediator of this process, however, to date there is no direct evidence of such a relationship between GJIC and PPAR. To date most of the studies of GJIC inhibition have been conducted using the well- developed rat liver cell model. In this study dolphin kidney cells CDK was used to test species specificity. The result showed that the inhibitory effect of PFFAs on GJIC is neither species- nor tissue—specific. 43 The results of the time course experiment indicate that inhibition of GJIC occurred within a very short period of time, which is not sufficient enough for effects at transcriptional level to occur. This suggests a possible mode of action, that is the post-translational modification of gap junctional protein was involved in this effect. Even though there is a good correlation between tumor promotion and GJIC inhibition, so far no direct evidence has been provided on the carcinogenesis of PFOS. Long term in vivo exposure studies need to be conducted to provide further information on PFOS in order to reach a definite conclusion. 44 AROMATASE ASSAY BACKGROUND In recent years there has been growing concern about the endocrine disruptive potential of environmental contaminants and commercial products. Chemical disturbances of endocrine functions can be caused by either direct interaction with steroid hormone receptors, particularly the estrogen receptor, or by interference with enzymes that are involved in steroid synthesis and breakdown (Kavlock, et al., 1996). In the adrenocortical cortex of mammals, cholesterol is transformed into l7l3-estradiol in several steps by several enzymes. An estrogenic or antiestrogenic effect may occur due to interference with one or more enzymes involved in this pathway (Drenth, et al., 1998; Sanderson and Van den Berg, 1998). The aromatase enzyme complex consists of the microsomal CYP19 enzyme and the flavoprotein NADPH-reductase. It is this enzyme that catalyzes the last step in the cholesterol to estradiol pathway, which converts testosterone to estradiol (Simpson, et al., 1994). Ammonium perfluorooctanoate (C8) has been reported to induce hepatic aromatase activity by up to 16 fold in dietary exposed Fisher rats (Cook, et al., 1992; Liu, et al., 1996). Therefore, the effect of PFOS on aromatase activity in vitro was investigated using human adrenocortical cells in order to compare the effect of PFOS with that of 45 ammonium perfluorooctanoate, and to assess the effect of PFOS on hormonal regulation in vitro. MATERIALS AND METHODS Chemicals Perfluorooctane sulfonic acid (PFOS) was obtained from 3M company (St. Paul, MN) as a mixture. Based on the NMR analysis, the mixture consisted of 68% of straight chain PF OS and 17% of branch chain PFOS. [1B-3H(N)]-Androst-4-ene-3,17-dione (28.5Ci/mmol) was obtained from New England Nuclear company (N EN-926). Cell Culture NCI-H295R is a human adrenocortical carcinoma cell line, which was obtained from the American Type Culture Collection (ATCC # CRL-2128). The NCI-H295R cells have the physiological characteristics of undifferentiated human fetal adrenal cells. They have the ability to synthesize steroid hormones and to express steroidogenic cytochrome P450 enzymes including aromatase (CYP19) activity (Rainey, et al., 1993). NCI-H295R cells were cultured in 75 cm flask (Corning 430720) under sterile conditions (pH=7.4) at 37C° in a humidified 5/95% C02 /air incubator (Forma Scientific, Model 8173). NCI-H295R cells were cultured in DMEM/Ham’s F12 medium (Gibco), supplemented with other nutrients (see Appendix A1). Medium was changed twice a week. When cells reached confluence and were ready for passaging, medium was removed, cells were washed twice with 10 ml PBS, and trypsinized with 1x trypsin 46 /EDTA (Sigma). Cells were then incubated at 37C° for 5 min, and 10 ml medium was added. The cell suspension was split into two new flasks each containing 12ml medium. Aromatase Assay Plating NCI-H295R cells were grown until reached 90% confluent. Medium was aspirated and cells were washed twice with PBS. 2 ml of 1 x trypsin/EDTA was added, the flask was incubated at 37 C° for 5 min, then cells were suspended in 10 ml medium. Cell number was determined using a hemocytometer, and cell concentration was adjusted to 2~5 x 105 cells per ml. 1 ml of diluted cell suspension was added in each well of the 24 well flat bottom view plates (Coming, 25820). Cells were incubated at 37 C° for 24 hrs to allow cell attachment. Dosing Medium was changed the day after plating, and cells were dosed at 0.2% (v/v) of PFOS stock solution dissolved in Methanol. The final concentrations of PFOS were 0.01 mg/L, 0.1 mg/L, l mg/L, 10 mg/L, and 50 mg/L. Methanol was used as solvent control. Each concentration was tested in triplicate. After dosing cells were incubated at 37 C° for 24 hrs or 48 hrs exposure. 47 Assay Procedure The aromatase assay was conducted following the modified procedure of Lephart and Simpson (1991). The method measures the production of [3H-] H2O, which is formed as a result of the aromatization of the substrate [IB-3H]-androstenedione. Medium was aspirated from the 24 well plates, and cells were washed twice with PBS. A working solution of 54 nM [3H]-androstenedione in serum-free medium was prepared. A volume of 0.25 ml of working solution was added to each well and plates were incubated at 30 C° for 1.5 hrs. 50 ul of working solution was added, in duplicate, directly to a scintillation vial, as a check of the total amount of radioactivity in the working solution. 200 pl of the rest working solution were added in duplicate to enpendorf tubes, these were used as background check, and they would go through the same extraction procedure as other treatment samples. After incubation, plates were immediately placed on ice. Exactly 200 pl of medium were withdrawn from each well, and transferred to eppendorf tubes containing 500111 chloroform. Each tube was vortexed for 15 sec, and centrifuged at 11,000x g for 2 min. 100 pl of the supernatant was carefully transferred to an eppendorf tube containing lOOul dextran-coated charcoal solution. This mixture was vortexed for 15 sec, and allowed to stand for 5 min. Then it was centrifuged at 11,000x g for 15 min. 125 pl of the supernatant was transferred to a scintillation vial, to which 4 ml of scintillation cocktail were added, and the tritium isotope activity was measured in a scintillation counter. 48 F Iuorescamine Protein Assay To correct for heterogeneity in plating and in differential cell growth, a fluorescamine protein assay was carried out on the lysed cells. Fluorescamine is intrinsically nonfluorescent but reacts in milliseconds with primary amine groups on proteins to yield a fluorescent derivative. It is widely used to determine protein concentrations of aqueous solutions (Udenfriend, et al., 1972). After completing the aromatase assay, the plates were washed twice with PBS. 200 pl of distilled water was added to each well, and cells were lysed by freezing and thawing. 50 pl of 1.08 mM fluorescamine (Sigma, F -9015) in acetonitrile was added and mixed by agitation. After 10 min incubation at room temperature, fluorescence were measured using a Cytofluor 2300 Fluorescence Measurement System (Millipore) at k ,2 = 400 nm and 2. em = 460 nm. The aromatase activity was expressed as picomole reaction per hour per milligram protein (see Appendix B for aromatase activity calculation). RESULT Aromatase was expressed constitutively by NCI-H295R cells, which responded in a predictable manner to known aromatase inducers. Aromatase activity was induced by 8- Br cAMP in a dose-dependent manner, with the maximum of 5.2 fold induction occurred at 43 mg/L 8-Br cAMP (Fig. 17A). In contrast, aromatase activity was only slightly induced by PFOS (Fig. 17B and Fig. 17C). When cells were treated with PFOS for 24 hrs, only a concentration at 50 mg/L showed a significant effect (p<0.05) compared to the solvent control, with the aromatase activity increased by a factor of 1.5 (Fig. 17B). The 49 .0 a: r .o & s P N .0 c—L aromatase actIVIty (pmol/mg protein/hr) o (A 11.; 0 0.43 4.3 43 O 8-Br CAMP concentration (mg/L) p 8% .9 on .0 aromatase actIVIty (pmollhr/mg protein) 0.05 Fig. 17 Effect of PFOS on NCI-H295R cell aromatase activity. A) cells dosed with 8-Br cAMP as positive control; B) cells dosed with PFOS for 24 hrs. Aromatase activity was expressed as picomole tritiated water formed per mg protein per hr. Control wells were incubated with 0.02% (v/v) solvent (methanol). Error bars represent the standard deviation of three measurements. 50 o o _. .0 io P ' N or 0.: I3 aromatase actIVIty (pmol/hr/mg protein) 0. 5 0.1 0.05 0 0.01 0.1 1 10 50 PFOSconcentratioMng/L) is 0.35 D g 0.3 3‘ :g 80.25 0 L. g 8 0.2 0’ E E a 0.15 a a T gé 0.1 i .Lr‘ I! 0.05 ' ‘ .3 ° . " 3 o _1=__fl . . SC 5m'n 10n'in 30rrin 60m'n exposure time to PFOS Fig. 17 Effect of PFOS on NCI-H295R cell aromatase activity. C)cells dosed with PFOS for 48 hrs. D) cells dosed with 50mg/L PFOS at different time interval before running the assay. Aromatase activity was expressed as picomolar substrate aromatized per mg protein per hr. Control wells were incubated with 0.02% (v/v) solvent (methanol). Error bars represent the standard deviation of three measurements. 51 magnitude of effect of PFOS on aromatase activity was proportional to the duration of exposure. The aromatase increased in a dose-dependent manner for cells treated with PFOS for 48 hrs (Fig. 17C). PFOS at concentrations of 10 and 50 mg/L significantly increased aromatase activity (p<0.05) by a factor of 1.7. To test the hypothesis that PF 0S affected aromatase activity by changing membrane properties of cells and allowing a greater influx of substrate, a time course experiment was conducted. NCI-H295R cells were dosed with 50 mg/L of PFOS at 5min, 10min, 30min or 60min before performing the assay (Fig. 17D). During all these short terrnexposure to PFOS (< 1 hr), no significant effects on aromatase activity was observed. This indicates that the initial hypothesis was false, that is the effects of PFOS on NCI-H295R cell aromatase activity is was not simply due to change of membrane properties and increase of substrate influx. DISCUSSION The NCI-H295R cell line was established from a human adrenocortical carcinoma. Multiple pathways of steroidogenesis are expressed by NCI-H295R cells, including formation of corticosteroids, mineralocorticoids, androgens, and estrogens (Gazdar, et al., 1990). All of the major adrenocortical enzyme systems are present in NCI-H295R cells, including desmolase (P450scc), 11 B-hydroxylase (P4500118), 21 (Jr-hydroxylase (P450021), 17 a-hydroxylase (P450017), lyase and aromatase (P450019) (Gazdar, et al., 1990). The cytochrome P450 steroid hydroxylase activity can be controlled at two levels: at the level of substrate mobilization, and at the level of gene transcription (Parker and 52 Schimmer, 1995). Some trophic hormones such as ACTH, F SH and LH regulate steroidogenesis by mobilizing substrates across mitochondrial membranes to their corresponding steroidogenic cytochrome P450 enzymes. This process is dependent on the CAMP and CAMP related protein kinase A signaling cascade (Parker and Schimmer, 1995; Pon, et al., 1986). The genes encoding for cytochrome P450 steroid enzymes are regulated in a cell selective way by some nuclear receptors and growth factors. One of the candidates is a DNA binding protein termed steroidogenic factor 1 (SF -1). SF-l can bind to SF-l responsive elements that reside close to the sequences for many of the steroid hydroxylases (Parissenti, et al., 1993). The activation of SF-l and its subsequent binding to SF-l responsive elements requires the phosphorylation of SF-lby a CAMP-dependent protein kinase (Pon, et al., 1986; Parissenti, et al., 1993). This is supported by the fact that most of the steroidogenic P450 enzymes can be up-regulated in a dose-dependent fashion by 8-Br CAMP, which is an activator of the protein kinase-A pathway. The specific mechanism under which PFOS may elicit its effect on the aromatase (CYP19) activity is still under investigation. It has been found that treatment with PF OA and PFDA can affect the level of protein kinase C, which is an important signaling pathway that may interfere with steroidogenesis (Reo, et al. 1996). Another feasible possibility is that the peroxisome proliferator activated receptor (PPAR) is involved, and it interacts with the nuclear receptors and growth factors that regulate aromatase gene expression. 53 Aromatase (CYP19) enzyme is responsible for the formation of estrogens from their androgen precursors. In most vertebrate species that have been examined, aromatase expression occurs primarily in the gonads and in the brain (Simpso, et al., 1994). In some species estrogen biosynthesis in the brain has been implicated in sex determination during development and sex related behavior such as mating (Jeyasuria, et al., 1994; Antonopoulou, et al., 1995). Therefore regulating aromatase activity can have tremendous effect on estrogen biosynthesis and estrogen related physical responses. 54 CONCLUSION The results from cell bioassays, GJIC assay and aromatase assay can be summarized as following: PFOS exhibits no cytotoxicity within the concentration range tested; PF OS by itself has no significant effects on cytochrome P4501A1 isoenzyme activity; TCDD when dosed in the presence of PFOS, elicited a greater magnitude of P4501A1 induction and Aryl Hydrocarbon activation; The interactive effects of PF OS and TCDD occurred at the level of transcription; PFOS inhibited Gap Junctional Intercellular Communication in a dose-dependent manner with a EC50 value of 20 mg/L PFOS; The inhibition of GJIC by PFOS occurred within a short period of time; This inhibitory effect is neither species- nor tissue-specific; PF OS slightly induced aromatase activity in NCI-H295R cells, significant induction only occurred at a relatively great dose of PFOS (50 mg/L); Induction increased with prolonged duration of exposure from 24 hr to 48 hr; The concentration range at which significant effects were observed on AhR and CYP1A1 activity were marginally environmentally relevant, the concentration of PFOS required to affect GJIC and aromatase activity were greater than environmentally relevant concentration (Fig. 18). Results from the current study provide useful information on three aspects of the biochemical toxicity of PFOS. To estimate the environmental risk of a compound to 55 2m 0.01 - 0.1 Cell bioassay .—-. 12.5 - 200 GJIC assa y 0—> Aromatase assa 10 I 50 y O—b l I I l _ I 0.001 0.01 0.1 l 10 100 1000 PF OS concentration (mg/L) Fig. 15 Diagram shows the environmental relevance of the data. The PFOS concentration that caused interactive effects with TCDD in cell bioassays ranges from 0.01 mg/L to 0.1 mg/L; the PFOS concentration that caused significant inhibitory effects on GJIC ranges from 12.5 mg/L to 200 mg/L; the PFOS concentration that caused significant effects on aromatase activity ranges from 10 mg/L to 50 mg/L. (dashed line indicates the maximum concentration of PFOS found in wildlife); H indicates range of concentration determined at both end; H indicates range of concentration determined at only one end. 56 human and wildlife, we have to take into account of both hazard and exposure. In cell bioassays the concentration at which significant interaction between PFOS and TCDD was observed falls into the range of environmental relevance. Furthermore, this interaction only resulted in an increase of CYP1A1 induction by 30-40%. This means that when human and wildlife are exposed to moderate concentrations of PFOS and a fairly great concentrations of TCDD at the same time, it is possible that the effect of TCDD may be increased by 30-40%. The concentrations of PFOS that significantly inhibited GJIC are approximately 10 fold greater than the maximum concentration of PF OS detected in wildlife. Similarly the effective concentration of PFOS on aromatase activity also exceed the environmentally relevant concentration range of PF OS. Furthermore, the aromatase activity was only induced by a factor of 1.5-1.7 fold. All the concentrations I mentioned in this study were the concentration of PFOS in cell culture medium, eventually how much of them end up getting into the cell still remain undetermined. Based on above discussion, it is unlikely that PFOS is currently causing significant effects on any of the biochemical pathways investigated on wildlife. Of course efforts should be made to assure that concentrations at which PFOS may elicit significant adverse effects are not reached in the future. Another issue of concern is whether it is feasible to extrapolate these in vitro results to in vivo situations. This has been a point of discussion among environmental toxicologists. Predicting in vivo toxicity from in vitro effects must be done with caution. Toxicokinetics, levels of organization and functional integrations in vivo complicate the extropolation. In vitro studies are efficient tools to study the mechanisms of effects. Once 57 the mechanisms of effects are known, greater generality in extrapolating to in vivo systems can be made. Also it is only by knowing the mechanism of action that effective biomarkers and monitoring programs can be established. The mechanism of action of PFOS is still under investigation and needs to be further clarified. No definite conclusion can be drawn upon the limited information currently available. A more in depth study is currently being conducted in the aquatic toxicology laboratory at Michigan State University. Hopefully we will be able to elucidate the mechanism of action so that accurate risk assessments of the potential effects of PFOS in a wider range of organisms can be valid. 58 APPENDICES 59 Appendix A Table A1. Culture Medium for NCI-H295R Cells. Ingradient Abbreviatio Sources and catalog # Amount 11 Dulbecco’s Modified Eagle DMEM GIBCO-BRL (12400- 7.8g dissolved Medium / Ham’s F 12 016) in 440ml diH2O Insulin, Transferrin, ITS-G GIBCO-BRL (41400- 100x, 5ml Selenium 037) Bovine BSA SIGMA (A-9647) 1.25mg/ml, 5ml 1,000U GIBCO-BRL (15140- 0.5ml penicillin/streptomycin 1 14) 10% Dextran Charcoal DCC-F BS GIBCO—BRL (16000- 50ml Coated Fetal Bovine Serum 044) Table AZ. Culture Medium for CDK Cells. Abbreviation Sources and catalog # Amount Ingradient Dulbecco’s Modified Eagle Medium / Ham’s F12 DMEM/F 12 SIGMA (D-2906) 7.8g dissolve in 850ml water medium 10% Fatal Bovine Serum FBS gggCOBRL “6000- 5ml EM Science (SXO420- lOmM NaCl lOmM 1) 50mg/L Gentamicin GIBCO-BRL (15710- 064) 5ml 3x MEM amino acid SIGMA (M6725) 15ml Including L-glutamrn 3x MEM non-essential amino SIGMA (M'7145) 15ml acid 3x MEM vitamins SIGMA (M-6895 15ml 60 APPENDIX B AROMATASE CALCULATION The aromatase activity was expressed as picomole 3H-Androst-4-ene-3,l7-dione catalyzed per milligram protein, this was calculated based on following equation: Aromatase activity (pmol/mg) = (cpm/ 35.19%)*4/1.5/73964.5/ protein content per well Cpm --- count per minute (background substracted) 35.19% --- measuring and labeling efficiency 4 --- dilution 1.5 --- incubation time (hr) 73964.5 --- factor used to convert dpm to picomole Protein content measurement In order to convert fluorescence data from fluorescamine assay to protein content, a BSA protein standard curve was produced. Table Bl F luorescamine protein assay data for BSA standard. protein(mg) fluorescence 0.1 563 0.05 352 0.025 216 0.0125 144 0.00625 104 0.003125 85 61 700 600 500 400 300 200 1 00 fluorescence 0.05 0.1 protein content (mg) 0.15 Fig. B1 BSA protein standard curve. slope 5683.441 intercept 70.08333 fluorescence=5683.4*protein + 70 protein content per well (mg) = (fluorescence - 70) / 5683.4 62 REFERENCES 63 REFERENCES Antonopoulou, E., Mayer, 1., Berglund, 1., and Borg, B. (1995). Effects of aromatase inhibitors on sexual maturation in Atlantic salmon, Salmo salar, male parr. Fish Physiology and Biochemistry. 14, 15-24. Biegel, L. B., Liu, R. C. M., Hurtt, M. E., and Cook, J. C. (1995). Effects of ammonium perfluorooctanoate on Leydig cell function: in vitro, in vivo, and ex vivo studies. T oxicol. Appl. Pharmacol. 134, 18-25. Bojes, H. K., and Thurman, R. G. (1994). Peroxisomal proliferators inhibit Acyl-CoA synthetase and stimulate protein kinase C in vivo. T oxicol. Appl. Pharmacol. 126, 233- 239. Cook, J. 0, Murray, S. M., Frame, S. R., and Hurtt, M. E. (1992). Induction of Leydig cell adonomas by ammonium perfluorooctanoate: a possible endocrine-related mechanism. T oxicol. Appl. Pharmacol. 113, 209-217. Diaz, M. J., Chinje, E., Kentish, P., Jamot, B., George, M., and Gibson, G. (1994). Induction of cytochrome P450 4A by the peroxisome proliferator perfluoro-n-octanoic acid. Toxicology. 86, 109-122. Drenth, H.-J., Bouwman, C. A., Seinen, W. and Van den Berg, M. (1998). Effects of some persistent halogenated environmental contaminants on aromatase (CYP19) activity in the human choriocarcinoma cell line JEG—3. T oxicol. Appl. Pharmacol. 148, 50-55. Gazdar, A. F., Oie, H. K., Shackleton, C. H., Chen, T. R., Triche, T. J., and Myers, C. E. (1990). Establishment and characterization of a human adrenocortical carcinoma cell line that expresses multiple pathways of steroid biosynthesis. Cancer Research. 50, 5488- 5496. Green, S. (1992). Commentary: receptor-mediated mechanisms of peroxisome proliferators. Biochemical Pharmacology. 43, 393-401. Hahn, M. E., Lamb, T. M., Schultz, M. E., Smolowitz, R. M. and Stegeman, J. J. (1993). Cytochrome P4501A induction and inhibition by 3,3’,4,4’-tetrachlorobiphenyl in an Ah receptor-containing fish hepatoma cell line (PLHC-l). Aquatic Toxicology. 26, 185-208. Hanhijarvi, H., Ylinen, M., Kojo, A., and Kosma, V. M. (1987). Elimination and toxicity of perfluorooctanoic acid during subchronic administration in the Wistar rat. Pharmacol. T oxicol. 61, 66-68. 64 Haughom, B., and Spydevold, O. (1992). The mechanism underlying the hypolipemic effect of perfluorooctanoic acid (PFOA), perfluorooctane sulphonic acid (PFOSA) and clofibric acid. Biochimica et Biophysica Acta. 1128, 65-72. Jeyasuria, P., Roosenburg, W. M., and Place, A. R. (1994). Role of P450 aromatase in sex determination of the diamondback terrapin, Malaclemys terrapin. J. Experi. Zoo. 270, 95-111. Kavlock, R. J., Daston, G. P., DeRosa, C., Fenner-Crisp, P., Gray, L. E., Kaattari, S. (1996). Research needs for the risk assessment of health and environmental effects of endocrine disruptors: a report of the USEPA-sponsored workshop. Environ. Health Perspect. 104 (suppl 4), 715-740. Kawashima, Y.,Suzuki, S., Kozuka, H., Sato, M., and Suzuki, Y. (1994). Effects of prolonged administration of perfluorooctanoic acid on hepatic activities of enzymes which detoxify peroxide and xenobiotics in the rat. Toxicology. 93, 85-97. Kawashima, Y., Uy-yu, N., and Kozuka, H. (1989). Sex-related difference in the inductions by perfluoro-n-octanoic acid of peroxisomal B-oxidation, microsomal l- acylglycerophosphocholine acyltransferase and cytosolic long-chain acyl-CoA hydrolase in rat liver. Biochem. J. 261, 595-600. Ketcham, C. A., and Klaunig, J. E. (1996). Effect of protein kinase C inhibitors on hepatic gap junctional intercellular communication blockage by peroxisome proliferators. Fundament. Appl. T oxicol. 30(suppl.), 1054-1062. Lephart, E. D. and Simpson, E. R. (1991). Assay of aromatase activity. Methods Enzymol. 206, 477-483. Liu, R. C. M., Hahn, C., and Hurtt, M. E. (1996). The direct effect of hepatic peroxisome proliferators on rat Leydig cell function in vitro. T oxicol. Appl. Pharmacol. 30, 102-108. Liu, R. C. M., Hurtt, M. E., Cook, J. C., and Biegel, L. B. (1996). Effect of the peroxisome proliferator, ammonium perfluorooctanoate (C8), on hepatic aromatase activity in adult male CrlzCD BR (CD) rats. F undam. Appl. T oxicol. 30, 220-228. Obourn, J. D., Frame, 8. R., Bell, R. H., Longnecker, D. 8., Elliott, G. S., and Cook, J. C1. (1997). Mechanisms for the pancreatic oncogenic effects of the peroxisome proliferator wyeth-l4, 643. T oxicol. Appl. Pharmacol. 145, 425-436. Okey, A. B., Riddick, D. S., and Harper, P.- A. (1994). The Ah receptor: mediator of the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds. Toxicology Letters. 70, 1-22. 65 Olson, C. T., and Andersen, M. E. (1983). The acute toxicity of perfluorooctanoic and perfluorodecanoic acids in male rats and effects on tissue fatty acids. T oxicol. Appl. Pharmacol. 70, 362-372. Parissenti, A. M., Parker, K. L., and Schimmer, B. P. (1993). Identification of promoter elements in the mouse 21-hydroxylase (CYP21) gene that require a functional cyclic adenosine 3’,5’-monophosphate-dependent protein kinase. Mol. Endocrinol. 7, 283-290. Parker, K. L., and Schimmer, B. P. (1995). Transcriptional regulation of the genes encoding the cytochrome P450 steroid hydroxylases. Vitamins and Hormones. 51, 339- 370. Perrnadi, H., Lundgren, B., Anderson, K., and Depierre, J. W. (1992). Effects of perfluoro fatty acids on xenobiotic-metabolizing enzymes, enzymes which detoxify reactive forms of oxygen and lipid peroxidation in mouse liver. Biochemical Pharmacology. 44, 1 183-1 191. Pon, L. A., Hartigan, J. A., and Orme-Johnson, N. R. (1986). Acute ACTH regulation of adrenal corticosteroid biosynthesis. Rapid accumulation of a phosphoprotein. J. Biol. Chem. 261, 13309-13316. Rainey, W. B, Bird, 1. M., Sawetawan, C., Hanley, N. A., et al. (1993). Regulation of human adrenal carcinoma cell (NCI-H295) production of C19 steroids. J. Clin. Endocrinol. Metab. 77, 732-737. Ravis, W. R., Hoke, J. F., and Parsons, D. L. (1991). Perfluorochemical erythrocyte substitutes: disposition and effects on drug distribution and elimination. Drug Metabolism Reviews. 23, 375-411. Reo, N. V., Narayanan, L., Kling, K. B., and Adinehzadeh, M. (1996). Perfluorodecanoic acid, a peroxisome proliferator, activates phospholipase C, inhibits CTP: phosphocholine cytidylyltransferase, and elevates diacylglycerol in rat liver. Toxicology Letters. 86, 1-11. Roman, B. L., Pollenz, R. S., and Peterson, R. E. (1998). Responsiveness of the adult male rat reproductive tract to 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure: Ah receptor and ARNT expression, CYP1A1 induction, and Ah Receptor down-regulation. T oxicol. Appl. Pharmacol. 150, 228-239. Sanderson, J. T. et al. (1996). Comparison of Ah receptor-mediated luciferase and ethoxyresorufin-o-deethylase induction in H4IIE cells: implicaiton for their use as bioanalytical tools for the detection of polyhalogenated aromatic hydrocarbons. T oxicol. Appl. Pharmacol. 137, 316-325. Sanderson, J. T., Kennedy, S. W., and Giesy, J. P. (1998). In vitro induction of ethoxyresorufin-o-deethylase and porphyrins by halogenated aromatic hydrocarbons in avian primary hepatocytes. Environ. T oxicol. Chem. 17, 2006-2018. 66 Sanderson, J. T. and Van den Berg, M. (1998). An in vitro system for the detection of compounds that can interfere with the expression of steroidogenic cytochrome P450 (CYP) enzymes. Organohalogen Compounds. 37, 77-80. Sargent, J. and Seffl, R. (1970). Properties of perfluorinated liquids. Fed. Proc. 29, 1699- 1703. Simpson, E. R., Mahendroo, M. 8., Means, G. D., Kilgore, M. W., et al. (1994). Aromatase cytochrome P450, the enzyme responsible for estrogen biosynthesis. Endocrine Rev. 15, 342-355. Sohlenius, A. K., et. A1. (1994). Effects of perfluorooctanoic acid, a potent peroxisome proliferator in rat, on morris hepatoma 7800C1 cells, a rat cell line. Biochimica et Biophysica Acta. 1213, 63-74. Sohlenius, A. K., Reinfeldt, M., Backstrom, K., Bergstrand, A., and Depierre, J. W. (1996). Hepatic peroxisome proliferation in vitamin A deficient mice without a simultaneous increase in peroxisomal Acyl-CoA oxidase activity. Biochemical Pharmacology. 51, 821-827. Trosko, J. E., and Ruch, R.J. (1998). Cell-cell communication in carcinogenesis. Frontiers Biosci. 3, 208-236. Tsao, M. S., Smith, J. D., Nelson, K. G. and Grisham, J. W. (1984). Adiploid epithelial cell line from normal adult rat liver with phenotypic properties of oval cells. Exp. Cell Res. 154, 38-52. Udenfriend, S., Stein, S., Bohlen, P., Dairman, W., Leimgruber, W. and Weigele, M. (1972). Fluorescamine: a reagent for assay of amino acids, peptides, proteins, and primary amines in the picomole range. Science. 178, 871-872. Upham, B. L., Deocampo, N. D., Wurl, B., and Trosko, J. E. (1998). Inhibition of gap junctional intercellular communication by perfluorinated fatty acids is dependent on the chain length of the fluorinated tail. Int. J. Cancer. 78, 491-495. Witzmann, F. A., Parker, D. N., Jamot, B. M. (1994). Induction of enory-CoA hydratase by LD50 exposure to perfluorocarboxylic acids detected by two-dimensional electrophoresis. Toxicology Letters. 71, 271-277. Ylinen, M., and Auriola, S. (1990). Tissue distribution and elimination of perfluorodecanoic acid in the rat after single intraperotoneal administration. Pharmacol. Toxicol. 66, 45-48. Ylinen, M., Hanhijarvi, H., Jaakonaho, J., and Peura, P. (1989). Stimulation by oestradiol of the urinary excretion of perfluooctanoic acid in the male rat. Pharmacol. Toxicol. 65, 274-277. 67 Yoshiaki, F. K., Imataka, H., Sogawa, K., Yasumoto, K. I. And Kikuchi, Y. (1992). Regulation of CYP1A1 expression. The FASEB J. 6, 706-710. 68