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The role of fatty acids and related analogs in mediating peroxisome proliferation in primary cultures of rat hepatocytes
Intrasuksri, Urusa, Ph.D.
The Ohio State University, 1991
UMI 300 N. Zeeb Rd. Ann Arbor, MI 48106 THE ROLE OF FATTY ACIDS AND RELATED ANALOGS
IN MEDIATING PEROXISOME PROLIFERATION IN
PRIMARY CULTURES OF RAT HEPATOCYTES
A Dissertation
Presented in Partial Fulfillment of the Requirements
for the Degree Doctor of Philosophy in the
Graduate School of The Ohio State University
By
Urusa Intrasuksri, B.S.
The Ohio State Unversity
1991
Dissertation Committee: Approved by
Dennis R. Feller, Ph.D.
Lane J. Wallace, Ph.D.
Allan M. Burkman, Ph.D. Adviser
Robert W. Brueggemeier, Ph.D. College of Pharmacy To My Parents
ii ACKNOWLEDGEMENTS
I wish to express my sincere thanks to:
My adviser, Dr. Dennis R. Feller, for his guidance, patience, and support throughout my graduate studies. His steady concern, friendship and encouragement make me feel as if I were not far away from home.
The members of my dissertation committee: Dr. Lane J. Wallace, Dr. Allan M. Burkman and Dr. Robert W. Brueggemeier, for their valuable suggestions and comments.
Dr. P.E. Kolattukudy, Julie Li, and Leena Hiremath from Biotechnology Center for their willing help and instructions on the molecular works.
John Mitchell and Erin Hooksin for their assistance on Electron microscopic studies.
My senior friends, Tim Esbenshade and Tom Kocarek, for their instructions and guidance in the hepatocyte culture system.
My American, Korean, Egyptian, Indian, Venezualian and Chinese friends, in the Division of Pharmacology, whom make the fifth floor of Parks Hall a very enjoyable and memorable place, and whom open my world to other cultures.
My Thai friends, Suparp, Vimon, Suchada, Chawewan, Sirinart, Rutaiwan, Precha, Wirot, Kasem, Pitsanu, Panida,..., for their worthwhile amity. Their presence make O.S. U. as well as Neil Hall an unforgettable place of mine.
My room-mate and my close neighbor, Kulaya and Tidawadee, for their inestimable help, support and understanding. Their assistance in and out the apartment made it possible for me to devote all my time in writing this dissertation.
My parents and my sister. I thank you for your sincere confidence in me and for your eternal support and encouragement throughout my studies.
The Thai Ministry of Public Health, Department of Medical Sciences for partial stipend support.
Finally, I would like to thank the Ohio State University and the United States of America for giving me an opportunity to pursue the Ph.D. degree. The financial support and the education which I received from here will always be appreciated. VITA
October 27, 1960 ...... Bom - Bangkok, Thailand
March, 1984 ...... B.S. Pharmacy, Mahidol University, Bangkok, Thailand
April, 1984 - Aug., 1986 ...... Medical Scientist, Ministry of Public Health, Thailand
September, 1986 - Present ...... Graduate Student, College of Pharmacy, The Ohio State University
January, 1987 - Present ...... Graduate Teaching Associate and Graduate Research Associate, The Ohio State University
PUBLICATIONS
Intrasuksri, U. and Feller, D.R., Comparison of the Effects of Selected Monocarboxylic, Dicarboxylic and Perfluorinated Fatty Acids on Peroxisome Proliferation in Primary Cultured Rat Hepatocytes. Biochem. Pharmacol. 42: 184-188, 1991.
Intrasuksri, U., and Feller, D.R., Characteristics of Peroxisome Proliferation by Perfluorinated Octanoic Acid (PFOA) in Cultured Rat Hepatocytes. FASEB Journal 5: A1571, 1991.
Miller, D.D., Hamada, A., Clark, M.T., Adejare, A., Patil, P.N., Shams, G., Romstedt, R.J., Kim, S.U., Intrasuksri, U., McKenzie, J.L., and Feller, D.R., Synthesis and ct2- ' Adrenoceptor Effects of Substituted Catecholimidazoline and Catecholimidazole Analogues in Human Platelets. Medicinal Chemistry 33: 1138-1144,1990.
Intrasuksri, U., and Feller, D.R., Comparison of Selected Mono and Dicarboxylic Acids on Induction of Peroxisome Proliferation in Cultured Rat Hepatocyte. FASEB Journal 4: A583, 1990. PUBLICATIONS (continued)
Feller, D.R., Shams, G., Romstedt, K.J., Kim, S.U., Intrasuksri, U., Fashempour, J., Tantishaiyakul, V., Hamada, A., and Miller, D.D., Structure Function Relationships of Tolazoline Analogs on Human Platelet Aggregation: a-Adrenoceptor Activities. The Pharmacologist 32: A190, 1990.
Intrasuksri, U., and Feller, D.R., Are Fatty Acids Mediators of Peroxisome Proliferation in Cultured Rat Hepatocytes ? FASEB Journal 3: A307, 1989.
Intrasuksri, U., Shams, G., Romstedt, K.J., Adejare, A., Clark, M., Hamada, A., Miller, D.D., and Feller, D.R., Effects of 2-Benzylimidazoline and 2-Benzylimidazole Analogs on a-2 Adrenoceptors in Human Platelets. The Pharmacologist 30: A185, 1988.
Feller, D.R., Adejare, A., Intrasuksri, U., Shin, Y., and Kirk, K.L., Alpha-adrenergic Agonist Properties of Ring 2- and 6-Fluorinated Analogs of Epinephrine. The Pharmacologist 30: A53, 1988.
Intrasuksri, U. and Janviriyasopak, U., Study on the Hypoglycemic Effect of Largerstroemia Speciosa Pers. Bachelor Thesis (in TTiai), Mahidol University, Thailand, 1984.
FIELD OF STUDY
Major Field: Pharmacy Studies in Pharmacology
v TABLE OF CONTENTS
DEDICATION...... iii
ACKNOWLEDGEMENTS...... iv
VITA ...... v
TABLE OF ABBREVIATIONS ...... xi
LIST OF TABLES ...... xiii
LIST OF FIGURES...... xv
CHAPTER I. INTRODUCTION...... 1 A. Overview of Peroxisome Proliferation ...... 1 B. Peroxisomes ...... 2 1. Morphology ...... 2 2. Biochemical characteristics...... 3 3. Peroxisome biogenesis ...... 4 4. Functions of peroxisomes ...... 5 5. Peroxisomal disorders ...... 7 6. Peroxisomes and lipid metabolism ...... 8 C. Peroxisome Proliferators ...... 11 1. Fibric acids ...... 12 2. Non-fibric acid hypolipidemic drugs ...... 12 3. Plasticizers and related compounds ...... 13 4. Phenoxy acid herbicides ...... 13 5. Miscellaneous d ru g s ...... 14 6. Physiological and dietary effects ...... 14 7. Fatty acids and related compounds ...... 16 D. Hepatic Peroxisome Proliferation ...... ; ...... 18 1. Hepatomegaly ...... 18 2. Induction of peroxisomal enzymes ...... 19 3. Induction of microsomal cytochrome P-450 by peroxisome proliferators ...... 21 4. Miscellaneous changes during peroxisome proliferation ...... 22 5. Peroxisome proliferation and hepatocarcinomas ...... 23
vi 5.1 The oxidative stress th e o ry ...... 24 5.2 Tumor initiation and promotion ...... 25 6. Species differences and significance to human ...... 25 7. Methods in hepatic peroxisome proliferation studies ...... 26 8. Significant of peroxisome proliferation stu d ies ...... 29 E. Proposed Mechanisms of Peroxisome Proliferation ...... 30 1. Receptor-mediated mechanism ...... 30 2. Substrate overload mechanism ...... 32 3. Proposed regulatory components in peroxisome proliferation ...... 34 3.1 CoA and the level of long-chain acyl-CoA ...... 34 3.2 Fatty acid binding protein ...... 35 3.3 Role of calcium ...... 35 F. Statement of the Problem ...... 35 1. Objective and rationale ...... 35 2. Significance ...... 40
CHAPTER II. STRUCTURAL REQUIREMENTS OF FATTY ACIDS ANALOGS; PERFLUORINATED FATTY ACIDS AND ASYMMETRIC PHENOXYCARBOXYLIC ACIDS, ON PEROXISOME PROLIFERATION IN PRIMARY CULTURES OF RAT HEPATOCYTES ...... 53 A. Introduction ...... 53 B. Specific Aims ...... 54 C. Methods ...... 55 1. Materials ...... 55 2. Animals ...... 56 3. Isolation and preparation of primary culture of rat hepatocytes ...... 56 4. Biochemical assays...... 57 5. Determination of cytotoxicity produced by the compounds ...... 59 5.1 Morphological determination via light microscopy ...... 59 5.2 Lactate dehydrogenase measurement ...... 59 6. Analysis of d ata ...... 61 D. Results ...... 61 1. Comparison of the effects of natural and metabolically stable analogs of monocarboxylic acids on protein content, FACO and LH activities ...... 61 2. Requirement of carboxylic acid function in the structure of perfluorinated fatty a c i d ...... 62 3. Relative potencies of perfluorinated fatty acid analogs of different carbon chain-length ...... 62
vii 4. Effects of the enantiomers of two phenoxycarboxylic acid analogs on protein content and FACO activity in primary cultured rat hepatocytes; comparison to clofibric acid ...... 63 E. Discussion ...... 64
CHAPTER III. NATURALLY OCCURRING FATTY ACIDS AS MEDIATORS FOR PEROXISOME PROLIFERATION: THE SIGNIFICANCE OF FUNCTIONAL GROUPS IN AN ACTIVE PROLIFERATOR STRUCTURE AND THE ROLE OF DICARBOXYLIC A C ID S ...... 87 A. Introduction ...... 87 B. Specific Aims ...... 88 C. Methods ...... 90 1. Materials...... 90 2. Isolation and preparation of primary cultures of rat hepatocytes ...... 90 3. Biochemical assays...... 91 4. Determination of cytotoxicity produced by the compounds ...... 91 5. Analysis of d ata ...... 92 D. Results ...... 92 1. Comparison of various chain length of naturally occurring monocarboxylic fatty acids ...... 92 2. Comparison between saturated and unsaturated fatty acids of the same chain length ...... 93 3. Characterization of oleic acid-increased enzyme activities: time-course study and effect of cycloheximide ...... 94 4. Role of dicarboxylic acids: effects of short-, medium-, and long-chain dicarboxylic acids and w-hydroxy fatty acids ...... 94 5. Effects of various mitochondrial fatty acid 6-oxidation blockers on the effects of short- and medium- chain mono- and di-carboxylic a c id s ...... 95 E. Discussion ...... 95
CHAPTER IV. CHARACTERIZATION OF PERFLUORINATED OCTANOIC ACID (PFOA) AS NON-METABOLIZABLE FATTY ACID PEROXISOME PROLIFERATOR IN PRIMARY CULTURES OF RAT HEPATOCYTES ...... 114 A. Introduction ...... 114 B. Specific Aims ...... 117 C. Methods ...... 118 1. Materials...... 118 2. Isolation and preparation of primary cultures of rat hepatocytes ...... 119 3. Biochemical assays...... 120 4. Assay of carnitine palmitoyltransferase activity in isolated rat liver mitochondria ...... 120 4.1 Isolation of rat liver mitochondria ...... 120 4.2 Carnitine palmitoyltransferase-I a ssay ...... 121 5. Total RNA isolation and Northern blotting ...... 121 5.1 Isolation of total m R N A ...... 121 5.2 Northern blotting and RNA transfer ...... 122 6. Electron microscopy ...... 123 7. Data analysis ...... 124 D. Results ...... 124 1. Biochemical characteristic of peroxisome proliferation by P F O A ...... 124 1.1 Concentration-response studies ...... 124 1.2 Time-courses for induction of FACO, LH and CAT activities by PFOA as compared to C P IB ...... 125 2. Drug combination studies ...... 126 2.1 Effects of drug combination on FACO activity ...... 126 2.2 Effect of PFDA on FACO induction by PFO A ...... 127 2.3 Effects of octanoic and octanedioic acids on PFOA response ...... 127 3. Effect of nicardipine on FACO induction ...... 128 4. Mitochondrial fatty acid 6-oxidation and PFOAinduction ...... 128 5. Effect of CPT-I inhibitor on FACO induction by PFOA and C IP R O ...... 129 6. Effect of PFOA, oleic acid and CIPRO on CPT-I activity in isolated rat liver mitochondria ...... 130 7. Morphological analysis of peroxisome proliferation induced by PFOA as compared to CIPRO ...... 130 8. Molecular mechanisms of PFOA as compared to CIPRO and oleic a c id ...... 131 8.1 Induction of mRNA encoding peroxisomal FACO in cultured rat hepatocytes ...... 131 8.2 Effect of cycloheximide on FACO induction by PFOA, CIPRO and oleic a c id ...... 132 E. Discussion ...... 132
CHAPTER V. SUMMARY AND CONCLUSIONS...... 167 A. Summary ...... 167 B. Conclusions ...... 172 C. Synopsis of research work ...... 173
REFERENCES ...... 176
ix TABLE OF ABBREVIATIONS
ABBREVIATION NAMES
ALD ...... Adrenoleukodystrophy
BA ...... Butanoic acid
BOA ...... 2-Bromooctanoic acid
BPA ...... 2-Bromopalmitic acid
CAT ...... Carnitine acetyltransferase
c D N A ...... Complementary deoxyribonucleic acid
CIPRO...... Ciprofibrate
C o A ...... Coenzyme A
CPIB ...... Clofibric acid
CPOA ...... 2-(4-Chlorophenoxy)octanoic acid
CPPA ...... 2-(4-Chlorophenoxy)propanoic acid
CPT ...... Carnitine palmitoyltransferase
DA ...... Decanoic acid
DAB ...... 3,3’-Diaminobenzidine
DEHA ...... Di(2-ethylhexyl)adipate
DEHP ...... Di(2-ethylhexyl)phthalate
DEHS ...... Di(2-ethylhexyl)sebacate DMSO...... Dimethylsulfoxide
DTNB ...... 5,5’-Dithiobis-(2-nitrobenzoicacid)
EDTA ...... Ethylene diamine tetraacetate
FACO ...... Fatty acyl-CoA oxidase
FAD ...... Flavin adenine dinucleotide
H202 ...... Hydrogen peroxide
LDH ...... Lactate dehydrogenase
L-FABP...... Liver fatty acid binding protein
LH ...... Laurate hydroxylase
MEDICA 1 6 ...... B,B,B’B’-Tetramethylhexanedioic acid
MPA ...... Mercaptopropionic acid
m RNA...... Messenger ribonucleic acid
NADH ...... Nicotinamide adenine dinucleotide, reduced form
NADP ...... Nicotinamide adenine dinucleotide phosphate
NADPH...... Nicotinamide adenine dinucleotide phosphate,
reduced form
OA ...... Octanoic acid
PFBA ...... Perfluoro-n-butanoic acid
PFDA ...... Perfluoro-n-decanoic acid
PFOA ...... Perfluoro-n-octanoic acid
PFOL...... 2,2,3,3,4,4,5,5,6,6,7,7,8,8,8-pentadecafluoro-l-
octanol
PFOS ...... Perfluorinated octane sulfonic acid
POCA ...... 2-[5-(4-ch!orophenyl)pentyl]-oxiran-2-carboxylate PPAR ...... Peroxisome proliferator-activated receptor
PVC ...... Polyvinyl chloride
RNA ...... Ribonucleic acid LIST OF TABLES
TABLE Page
1. Enzymes Associated with hepatic peroxisomes ...... 42
2. Classification of peroxisomal disorders ...... 43
3. Effects of selected CPIB, mono- and perfluorinated fatty acids on cell viability assessed by LDH activity after 72 hr treatm ent ...... 71
4. Effects of natural monocarboxylic acids on cellular protein content in 72 hr primary cultured rat hepatocytes ...... 72
5. Effects of perfluorinated fatty acids and clofibric acid on cellular protein content, peroxisomal FACO and microsomal LH activities in 72 hr primary cultured rat hepatocytes ...... 73
6. Effects of perfluorinated octanoic acid and perfluorinated octanol on cellular protein content, peroxisomal FACO and microsomal LH activities in 72 hr primary cultured rat hepatocytes ...... 77
7. Relative potencies and maximal effects (EmflI) for induction of fatty acyl-CoA oxidase and laurate hydroxylase in primary cultured hepatocytes by CPIB and perfluorinated fatty acids ...... 81
8. Effects of enantiomers of 2-(4-chlorophenoxy)pentanoic acid (CPPA) and clofibric acid on cellular protein content and peroxisomal FACO activity in 72 hour primary cultured rat hepatocytes ...... 82
9. Effects of enantiomers of 2-4(-chlorophenoxy)octanoic acid (CPOA) and clofibric acid on cellular protein content and peroxisomal FACO activity in 72 hour primary cultured rat hepatocytes ...... 83
10. Chemical structures and names of fatty acids and analogs used in the effect of naturally occurring fatty acids on peroxisome proliferation ...... 102
xiii TABLE Page
11. Comparison of the effects of C18 fatty acids of various degree of unsaturation on peroxisomal FACO and microsomal LH activities ...... 105
12. The effect of long-chain polyunsaturated fatty acids on fatty acyl-CoA oxidase (FACO) activity in primary cultures of rat hepatocytes ...... 107
13. Effect of cycloheximide on oleic acid- and ciprofibrate-induced FACO activity in primary cultures of rat hepatocytes ...... 110
14. Effects of dicarboxylic acids of various chain length on FACO and LH activities in primary cultures of rat hepatocytes ...... I ll
15. Effects of dicarboxylic and hydroxycarboxylic acids of C12 and C16 on FACO activity in primary cultures of rat hepatocytes ...... 112
16. Effects of the inhibition of mitochondrial fatty acid B-oxidation on natural mono- and di-carboxylic acids in primary cultures of rat hepatocytes ...... 113
17. ECjo values for the induction of FACO and LH activities by PFOA, CPIB and CIPRO ...... 146
18. Morphometric analysis of peroxisomes after the hepatocytes were treated with CIPRO (0.1 mM) and PFOA (0.1 mM) for 3 days. The peroxisome proliferation was shown as increases in number, diameter and percent of the area occupied as compare to control ...... 162
xiv LIST OF FIGURES
FIGURE Page
1. Fatty acid fl-oxidation pathway in peroxisomes and mitochondria ...... 44
2. Chemical structures of hypolipidemic (fibric acid) peroxisome proliferators ...... 45
3. Chemical structures of non-fibric acid hypolipidemic peroxisome proliferators ...... 46
4. Chemical structures of plasticizers and related compounds ...... 47
5. Chemical structures of phenoxy acid herbicides ...... 48
6. Chemical structures of miscellaneous drugs that cause peroxisome proliferation ...... 49
7. Chemical structures of fatty acid analogs with peroxisome proliferative activity ...... 50
8. A receptor-mediated mechanism for peroxisome proliferation...... 51
9. Proposed mechanism of peroxisome proliferation by DEHP; a substrate overload mechanism...... 52
10. Chemical structures, names and abbreviations of fatty acid analogs used for the studies of structural requirements of fatty acids as mediators for peroxisome proliferation ...... 69 10. Continued ...... 70
11. Comparison of monocarboxylic acids and perfluorinated fatty acids of the C4 carbon chain length on the induction of peroxisomal fatty acyl-CoA oxidase and microsomal laurate hydroxylase ...... 74
12. Comparison of monocarboxylic acids and perfluorinated fatty acids of the C8 carbon chain length on the induction of peroxisomal fatty acyl-CoA oxidase and microsomal laurate hydroxylase ...... 75
13. Comparison of monocarboxylic acids and perfluorinated fatty acids of the C10 carbon chain length on the induction of peroxisomal fatty acyl-CoA oxidase and microsomal laurate hydroxylase ...... 76
xv FIGURE Page
14. Comparison of the effect of perfluorinated octanoic acid and perfluorinated octanol on FACO and LH activities ...... 78
15. Concentration-response curves for the induction of peroxisomal fatty acyl-CoA oxidase (FACO) activity by perfluorobutanoic acid (PFBA), perfluorooctanoic acid (PFOA), perfluorodecanoic acid (PFDA) and clofibric acid (CPIB) ...... 79
16. Concentration-response curves for the induction of microsomal laurate hydroxylase (LH) activity by perfluorobutanoic acid (PFBA), perfluorooctanoic acid (PFOA), Perfluorodecanoic acid (PFDA) and clofibric acid (CPIB) ...... 80
17. Concentration-response curves for the induction of fatty acyl-CoA oxidase (FACO) activity by the enantiomers of 2-(4-chlorophenoxy)pentanoic acid [R(+)-CPPA and S(-)-CPPA] as compared to clofibric acid (C PIB ) ...... 84
18. Concentration-response curves for the induction of fatty acyl-CoA oxidase (FACO) activity by the enantiomers of 2-(4-chlorophenoxy)octanoic acid [R(+)-CPOA and S(-)-CPOA] as compared to clofibric acid (CPIB) ...... 85
19. Concentration-response curves for the induction of fatty acyl-CoA oxidase (FACO) activity by perfluorobutanoic acid (PFBA), perfluorooctanoic acid (PFOA), S(-)-2-(4-chlorophenoxy)pentanoicacid (CPPA) and S(-)-(4- chlorophenoxy)octanoic acid (CK)A ) ...... 86
20. Schematic illustration of the substrate overload hypothesis presented in Chapter III ...... 101
21. Effects of various chain length of monocarboxylic acids (C4, C8, C10, Clg and C22) as compared to clofibric acid (CPIB) and perfluorooctanoic acid (PFOA) on FACO and LH activities in cultured rat hepatocytes ...... 103
22. Concentration-dependent studies of the effects of long-chain saturated and unsaturated C18 fatty acids in increasing peroxisomal FACO and microsomal LH activities ...... 104
23. Effects of degrees of unsaturation on fatty acyl-CoA oxidase (FACO) and laurate hydroxylase (LH) activities in primary cultures of rat hepatocytes ...... 106
24. Time courses for the induction of peroxisomal fatty acyl-CoA oxidase (FACO) activity by oleic acid (1 mM) and by control (no d r u g ) ...... 108 FIGURE Page
25. Time courses for the induction of carnitine acetyltransferase (CAT) activity by oleic acid (1 mM) and by control (no d ru g ) ...... 109
26. Function of carnition in carrying long-chain fatty acyl-CoA into the mitochondrial matrix for fatty acid oxidation ...... 143
27. Concentration-response curves for the induction of fatty acyl-CoA oxidase (FACO) activity by perfluorooctanoic acid (PFOA), ciprofibrate (CIPRO) and clofibric acid (C P IB ) ...... 144
28. Concentration-response curves for the induction of microsomal laurate hydroxylase (LH) activity by perfluorooctanoic acid (PFOA) and clofibric acid (CPIB) ...... 145
29. Time courses for the induction of peroxisomal fatty acyl-CoA oxidase (FACO) activity by 0.1 mM perfluorooctanoic acid (PFOA) and 1 mM clofibric acid (CPIB) and control (no d ru g ) ...... 147
30. Time courses for the induction of microsomal laurate hydroxylase (LH) activity by 0.1 mM perfluorooctanoic acid (PFOA) and control (no drug) ...... 148
31. Time courses for the induction of carnitine acetyltransferase (CAT) activity by 0.1 mM perfluorooctanoic acid (PFOA), 1 mM ciprofibrate (CPIB) and control (no drug) ...... 149
32. Effect of various concentrations of perfluorooctanoic acid (PFOA) on ciprofibrate (CIPRO)-concentration-response curve ...... 150
33. Effect of various concentrations of clofibric acid (CPIB) on ciprofibrate (CIPRO)-concentration-response curve ...... 151
34. Effect of the combination of perfluorooctanoic acid (PFOA) and perfluorodecanoic acid (PFDA) concentrations on fatty acyl-CoA oxidase (FACO) activity in rat hepatocytes ...... 152
35. Effect of octanoic acid and octanedioic acid on fatty acyl-CoA oxidase (FACO) induction by perfluorooctanoic acid (PFOA) ...... 153
36. Effect of nicardipine on fatty acyl-CoA oxidase (FACO) induction by perfluorooctanoic acid (PFOA), ciprofibrate (CIPRO) and oleic acid in cultured rat hepatocytes ...... 154
xvii FIGURE Page
37. Effects of various inhibitors of mitochondrial fatty acid 13-oxidation on fatty acyl-CoA oxidase (FACO) induction by 0.03 mM perfluorooctanoic acid (PFOA) ...... 155
38. Effects of carnitine palmitoyltransferase-I inhibitor on fatty acyl-CoA oxidase (FACO) induction by 0.1 mM ciprofibrate (CIPRO) and 0.1 mM perfluorooctanoic acid (PFOA) ...... 156
39. Effects of 2-bromopalmitic acid and ciprofibrate on the activity of carnitine palmitoyltransferase I (CPT-I) in isolated rat liver m itochondria ...... 157
40. Effects of perfluorooctanoic acid and oleic acid on the activity of carnitine palmitoyltransferase I (CPT-I) in isolated rat liver mitochondria ...... 158
41. Electron micrographs of the control cultured rat hepatocytes stained with 3,3’-diaminobenzidine for peroxisome visualization ...... 159
42. Electron micrographs of 0.1 mM ciprofibrate-treated cultured rat hepatocytes stained with 3,3’-diaminobenzidine for peroxisome visualization ...... 160
43. Electron micrographs of 0.1 mM PFOA-treated cultured rat hepatocytes stained with 3,3’-diaminobenzidine for peroxisome visualization ...... 161
44. Northern blot analysis (20 ng total RNA/lane) of time course changes in the mRNA level encoding fatty acyl-CoA oxidase (FACO) in hepatocyte cultured in the absence (control) or presence of 0.1 mM ciprofibrate (CIPRO), 0.1 mM perfluorooctanoic acid (PFOA) or 1 mM oleic acid ...... 163
45. Northern blot analysis of time course changes in the mRNA level encoding fatty acyl-COA oxidase (FACO) in cultured hepatocytes treated with 0.1 mM perfluorooctanoic acid (PFOA) and the corresponding control ...... 164
46. Northern blot analysis of the mRNA levels of FACO after 20 hr treatment with no drug, 0.1 mM ciprofibrate, 0.1 mM perfluorooctanoic acid, and 1 mM oleic acid ...... 165
47. Effect of cycloheximide on fatty acyl-CoA oxidase (FACO) induction produced by 0.08 mM ciprofibrate (CIPRO), 0.08 mM perfluorooctanoic acid (PFOA) and 1 mM oleic acid ...... 166
xviii FIGURE
48. Participation of two proposed mechanisms of peroxisome proliferation produced by xenobiotics, non-metabolizable fatty acids and naturally occurring fatty a c id s ...... CHAPTER I
INTRODUCTION
A. Overview of Peroxisome Proliferation
Peroxisomes are small cytoplasmic organelles with a single membrane surrounding a homogenous matrix. They were discovered in 1954 by Johannes A.G. Rhodin [Rhodin, 1954;
Goldfischer and Reddy, 1984], who called these organelles microbodies. Subsequently,
DeDuve (1965) and coworkers found that these organelles contain the enzymes catalase and several hydrogen peroxide (^O^-generating oxidases; thus, they renamed microbodies
"peroxisomes." Nearly all eukaryotic cells contain peroxisomes which contribute to cell respiration, gluconeogenesis, purine catabolism and lipid metabolism [DeDuve, 1965; DeDuve and Baudhuin, 1966; DeDuve, 1983; Reddy and Lalwani, 1983].
Current interest in peroxisomes is associated with the phenomenon of "peroxisome proliferation," which is defined as an increase in the number and volume density of peroxisomes [Reddy and Lalwani, 1983]. Exposure of rodents to several structurally dissimilar chemicals and to some dietary manipulations such as high fat diets is uniformly and predictably associated with a marked peroxisome proliferation in hepatic parenchyma cells
[Paget 1963; Reddy and Krishnakantha, 1975; Neat et al., 1980; Ishii et al., 1980; Reddy and
Lalwani, 1983]. The term "peroxisome proliferator" was introduced by Reddy et al. (1975) to designate a drug or xenobiotic which increases the size and the number of peroxisomes in
1 liver cells. Clofibrate, a hypolipidemic drug used in hyperlipoproteinemic patients, was the
first drug found to cause peroxisome proliferation [Paget, 1963; Witiak et al. , 1977]. A large
number of drugs and xenobiotics is now reported to produce peroxisome proliferation [Reddy
and Lalwani, 1983; Feller and Kocarek, 1986; Moody et al., 1991]. The mechanism by
which this diverse group of chemicals causes peroxisome proliferation is not understood.
Chronic exposure to peroxisome proliferators results eventually in the development of hepatic
tumors in rodents [Cohen etal., 1981; Reddy and Lalwani, 1983; Rao and Reddy, 1987; Rao
and Reddy, 1989]. However, these agents are non-mutagenic and do not interact with or directly damage DNA [Glauert et al., 1984; Rao and Reddy, 1987; Reddy, 1990]. Thus, the
studies of peroxisome proliferation are quite significant because this phenomenon is associated with carcinogenicity, and the relevancy to human risk remains unknown.
Several research groups have been investigated the mechanisms underlying the proliferation of subcellular organelles. In order to assess the implications of hepatic peroxisome proliferation induced by xenobiotics or dietary manipulations, the knowledge of this organelle is essential. Therefore, this chapter discusses the morphological and biochemical responses of hepatic peroxisome to peroxisome proliferators and then focuses on possible mechanisms whereby these chemicals produce peroxisome proliferation.
B. Peroxisomes
1. Morphology
Peroxisomes in hepatic parenchymal cells are single-membrane-limited organelles which measure approximately 0.3 to 1.0 fim in diameter and contain a finely granular electron-dense matrix [Reddy and Lalwani, 1983; Goldfischer & Reddy, 1984]. Hepatic peroxisomes in many species contain a dense crystalloid core or nucleoid which consists of urate oxidase (uricase) monomer only [Antonenkov and Panchenko, 1978]. No crystalloid core is detected in hepatic peroxisomes of some species that lack uricase, including humans
[Goldfischer and Reddy, 1984]. Nearly all eukaryotic cells contain peroxisomes. Depending on the cell type, the number may vary from one to several-hundred per cell [Reddy and
Lalwani, 1983]. Hepatocytes may contain up to 1000 peroxisomes per cell, accounting for approximately 1.0-1.5% of the cellular protein [Tolbert, 1981].
Peroxisomes are differentiated from mitochondria by their single membrane, electron- dense, homogeneous matrix and absence of cristae; and from lysosomes by their absence of vacuoles, lipid droplets, pigments, myelin figures and ferritin [Reddy and Lalwani, 1983].
Peroxisomes are easily identified in fixed cells or tissue slices by staining the samples with
3,3’-diaminobenzidine (DAB) under alkaline conditions [Novikoff and Goldfischer, 1969].
The DAB serves as a hydrogen donor for the peroxidatic reaction of catalase, and in the process is converted to a conjugated unsaturated structure which binds osmium tetroxide.
Peroxisomes can also be identified at the ultrastructural level by immunocytochemical procedures which utilize either the peroxidase-labeled Fab fragments of the antibodies raised against peroxisomal enzyme(s) such as catalase and enoyl-CoA hydratase [Reddy MK et al.,
1981; Yokota and Fahimi,1981], or the protein A-gold method [Bendayan et al. 1982; Reddy etal., 1982].
2. Biochemical characteristics
Peroxisomes contain more than 40 enzymes, including those producing and degrading hydrogen peroxide [DeDuve, 1965; Tolbert, 1981; DeDuve, 1983]. More than half of the peroxisomal enzymes are related to lipid metabolism. Other enzymes in mammalian peroxisomes are involved in purine and amino acid catabolism and in glyoxylate metabolism (Table 1). Most peroxisomal enzymes have their counterparts in other cell organelles
[Mannaerts and Van Veldhoven, 1990], such as mitochondria and smooth endoplasmic reticulum. For example, enzymes in the peroxisomal 6-oxidation of long-chain fatty acid function as a chain-shortening mechanism that supplies shorter-chain length fatty acids for mitochondrial fatty acid 6-oxidation [Mannaerts and Van Veldhoven, 1990].
All peroxisomal enzymes are confined to the interior of the organelle, except for acyl-
CoA synthetase, which is an integral membrane protein having its catalytic site facing the cytosol [Mannaerts & Van Veldhoven, 1990]. Most peroxisomal enzymes investigated so far are either soluble matrix proteins, core proteins, or membrane proteins the catalytic sites of which face the matrix. The peroxisome membrane possesses a non-selective pore-forming protein [Van Veldhoven et al. , 1987]. The diameter of the pore (1.7 nm in rat peroxisomes) is large enough to allow the free diffusion of substrates, products, and cofactors, e.g., sucrose and inorganic ions [DeDuve and Baudhuin, 1966; Mannaerts and Van Veldhoven, 1990].
Whether the permeability of the channels is regulated is not known [Mannaerts and Van
Veldhoven, 1990].
3. Peroxisome biogenesis
Recently, the mechanisms involved in peroxisome biogenesis have been widely investigated in yeasts, plants and higher eukaryotic cells. Peroxisomes contain no DNA; thus, all of their proteins are encoded in the nucleus similar to the case of mitochondria [Lazarow and Fujuki, 1985; Small and Lewin, 1990], Although mitochondria contain DNA, the genome encodes less than 10% of mitochondrial protein. The majority of mitochondrial proteins are nuclearly encoded, many as larger precursors, and enter the organelle post- translationally, often accompanied by proteolytic cleavage of a leader peptide [Small and Lewin, 1990]. Development of peroxisomal membrane and crystalloid core proteins also requires the post-translational import of proteins that are synthesized in the free cytosolic polyribosomes into preexisting peroxisomes. However, most peroxisomal proteins are
synthesized at their mature size and lack cleavable topogenic sequences [Small and Lewin,
1990].
A topogenic signal that is capable of targeting proteins to peroxisome has been widely investigated. Recent in vivo studies reported that this signal consists of the C-terminal tripeptide, Ser-Lys-Leu (SKL) [Gould et al. , 1987; Small and Lewin, 1990]. However, some peroxisomal proteins from higher eukaryotes do not contain SKL at their C-terminus (e.g., rat thiolase), and others do not contain SKL anywhere within their sequence (e.g., rat catalase). Therefore, more than one type of signal can direct proteins to peroxisomes.
Preexisting peroxisomes are proposed to grow in size as they accumulate more protein and phospholipid components and then divide by fission into two daughter peroxisomes. The biogenesis model implies that de novo synthesis of peroxisomes never occurs. The cells must have at least one peroxisome to form new peroxisomes. This process is believed to be similar to the proliferation of mitochondria and chloroplasts [Lazarow and Fujuki, 1985].
4. Functions of peroxisomes
Peroxisomes participate in respiration, thermogenesis, gluconeogenesis, purine catabolism and lipid metabolism [DeDuve and Baudhuin, 1966; Reddy and Lalwani, 1983].
Subsequent identification of additional peroxisomal enzymes has led to further speculation on the metabolic role of these organelles [Masters and Crane, 1984]. Most of these metabolic pathways are either similar to or complement activities in other cellular compartments.
Peroxisomes play a role in both anabolic and catabolic reactions. For the anabolic 6 process, peroxisomes participate in the early steps of the biosynthesis of plasmalogen and some other ether phospholipids, such as platelet-activating factor, followed by the synthetic steps in the endoplasmic reticulum [Naidu and Moser, 1990]. Peroxisomes are also involved in bile acid synthesis [Pedersen et al, 1987].
The respiratory pathway mediated by peroxisomal oxidases leads to hydrogen peroxide
(H202) generation, which is subsequently decomposed by catalase. Peroxisome respiration accounts for about 20% of the oxygen consumption of liver [DeDuve and Baudhuin, 1965].
The formation of H202 by H202-producing flavo-protein oxidases and its degradation by catalase leads to the production of heat [Hryb, 1981]. Evidence suggests that peroxisomes in brown fat play a role in thermogenesis in rats during cold adaptation [Nedergaard et al. ,
1980]. The function of liver peroxisomes in gluconeogenesis is not clear, but it may be attributed to the formation of a-keto acids.
Purine catabolism by peroxisomes is associated with the presence of urate oxidase.
In some reptiles, uric acid is degraded to allantoin via the sequential action of hepatic peroxisomal enzymes that humans have lost in the course of evolution [Mannaerts and Van
Veldhoven, 1990]. Mammalian peroxisomes contain D-amino-acid oxidase, which oxidizes the D-isomers of neutral and basic amino acids. Peroxisomes also contain alanine glyoxylate aminotransferase. Deficiency of this transaminase causes an overproduction of oxalate, which results in calcium oxalate nephrocalcinosis and kidney destruction [Wise et al., 1987].
Peroxisomes contain all enzymes required for fatty acid 8-oxidation system. However, the enzymes of peroxisomal 8-oxidation differ from mitochondrial enzymes with respect to the catalytic and molecular properties [Reddy and Lalwani, 1983; Hawkins et a l, 1987;
Osmundsen et al., 1987; Mannaerts and Van Veldhoven, 1990; Schulz, 1991]. Since the role of peroxisomes in lipid metabolism is very important and may be directly involved in the mechanism of peroxisome proliferation, the peroxisomal B-oxidation of fatty acids will be discussed in more detail later in this chapter [see Section B.6, Peroxisomes and lipid metabolisml.
5. Peroxisomal disorders
Quite a number of disorders has recently been attributed to dysfunction in peroxisomes. Since peroxisomes participate in the synthesis of ether lipids and bile acids and in fatty acid B-oxidation, particularly those of very long-chain fatty acids, lack of this organelle is associated with severe abnormalities in many human organs [Goldfischer and
Reddy, 1984; Moser, 1987; Kaiser, 1989; Wanders et al. , 1990]. The disorders are grouped into two major classes according to the genetic determination as shown in Table 2 [Naidu and
Moser, 1990].
Class 1 disorders are the generalized peroxisomal disorders which occur frequently in newborns or infants and are inherited in an autosomal recessive manner. The disorders are associated with a defect in the biogenesis of peroxisomes and not in peroxisomal enzyme gene transcriptional or translational events. A failure of peroxisomes to form or to maintain themselves results in a functional defect in more than one of the enzymes located in peroxisomes [Naidu and Moser, 1990]. Mannaerts and Van Veldhoven (1990) suggested that in some cases the peroxisomal enzymes are synthesized at their normal rate but not imported into peroxisomes, and the non-imported enzymes such as the B-oxidation enzymes are rapidly degraded in the cytosol. Patients with generalized peroxisomal disorders show multiple anatomical and histological abnormalities, including severe neurological symptoms.
The disorders in Class 2 are deficiencies of a single peroxisomal enzyme, which may be caused by a mutation in the gene encoding the corresponding enzyme. The disorders are commonly shown in children and adults and are inherited in both X-linked recessive and autosomal recessive manners. These peroxisomal disorders do not display all possible lipid abnormalities but show defects related to the specific peroxisomal enzyme deficiencies which occur.
For the third class (rhizomelic chondrodysplasia punctata), it is not clear whether the multiple enzyme deficiencies are the result of mutations of the corresponding genes or of a partial import deficiency [Mannaerts and Van Veldhoven, 1990]. Thus, the position of these disorders in the classification is uncertain [Naidu and Moser, 1990],
Biochemical abnormalities in disorders of peroxisomal biogenesis include the following: (1) the presence of catalase in cytosol, (2) the reduction of plasmalogen synthesis,
(3) an accumulation of very-long-chain fatty acids, phytanic acid, bile acid intermediates and pipecolic acid, and (4) an increase in the excretion of dicarboxylic acids in urine [Moser,
1987; Naidu and Moser, 1990; Wanders et al., 1990].
Whereas the treatment for the Class 1 peroxisome disorders is limited by the severity of the illness present at the time of birth, the treatment for some diseases in Class 2 disorders is being actively pursued [Naidu and Moser, 1990]. Treatment of X-linked adrenoleukodystrophy (ALD) includes replacement of steroids and a dietary reduction in very- long-chain fatty acids. There is no definitive treatment for these peroxisomal disorders; however, if the biochemical abnormalities are recognized, prenatal testing and appropriate genetic counseling can be provided [Naidu and Moser, 1990].
6. Peroxisomes and lipid metabolism
Fatty acid B-oxidation had been considered an exclusive mitochondrial activity until
Lazarow and DeDuve (1976) demonstrated the existence of peroxisomal fatty acid B-oxidation. This finding was of key importance in demonstrating that peroxisomes had a role in fatty acid
metabolism, and this in turn led to the recognition that peroxisomes perform significant
functions in mammals and humans. However, the enzymes of the mitochondrial and peroxisomal systems are different proteins [Tolbert 1981; Hashimoto, 1982; Mannaerts and
Debeer, 1982; Reddy and Lalwani, 1983; Hashimoto, 1987; Moser, 1987; Mannaerts and Van
Veldhoven, 1990; Schulz, 1991]. A comparison of the fatty acid B-oxidation pathways in peroxisomes and mitochondria is shown in Figure 1 [Moser, 1987]. The differences are these:
1. In mitochondria, transport of fatty acyl-CoA through the inner membrane is
carnitine-dependent. The peroxisomal fatty acid B-oxidation pathway is independent of
carnitine.
2. Both peroxisomes and mitochondria contain their own CoA pool [Mannaerts and
Debeer, 1982]. However, unlike mitochondria, peroxisomes do not possess their own pyridine nucleotide pool. Therefore, addition of NAD+ strongly stimulates peroxisomal B- oxidation but not mitochondrial B-oxidation of fatty acids [Mannaerts and Debeer, 1982].
3. The first enzyme of mitochondrial B-oxidation is fatty acyl-CoA dehydrogenase.
This enzyme is coupled with the electron transport system and is blocked by inhibitors such as cyanide. The initial step in the peroxisomal sequence is the fatty acyl-CoA oxidase, a flavin oxidase that produces H202. Peroxisomes also have no tricarboxylic acid (TCA) cycle and electron transport system.
4. Peroxisomes contain a bifunctional enzyme which exhibits enoylhydratase and 3- hydroxy fatty acyl-CoA dehydrogenase activities whereas two separate enzymes exist in mitochondria. Recently, it was suggested that the peroxisomal bifunctional protein also exhibits A3, A2-enoyl-CoA isomerase activity and may be a trifunctional enzyme [Palosaari 10 and Hiltunen, 1990].
5. Peroxisomal thiolase is different from the two thiolases that exist in mitochondria.
6. Fatty acid 8-oxidation by mitochondria results in the production of ATP.
Peroxisomes have no electron transport system, and 8-oxidation of fatty acids in this organelle produces H202 and heat.
7. Substrate specifications for fatty acids differ in peroxisome and mitochondria.
Mitochondria oxidize short-, medium-, and long-chain fatty acids, and the carboxy side-chain of prostaglandins. Peroxisomes, on the other hand, oxidize a wider spectrum of fatty acids which includes medium-, long-, and very-long-chain fatty acids; medium- and long-chain dicarboxylic acids; and the carboxy side-chain of prostaglandins. Whereas short-chain dicarboxylic acids cannot be oxidized by peroxisomes', very-long-chain fatty acids, xenobiotics, and bile acid intermediates are good substrates only for peroxisomal oxidation
[Mannaerts and Van Veldhoven, 1990]. Schulz (1991) suggested that these unusual fatty acids are more preferential for peroxisome 8-oxidation because of the inactivity or low activity of the mitochondrial acyl-CoA dehydrogenase and/or of the carnitine-dependent mitochondrial uptake system toward these compounds. Whether long-chain dicarboxylic acids can be metabolized by mitochondria is still unknown.
8. While carnitine acetyltransferase and carnitine palmitoyltransferase are mitochondrial enzymes which function in the transportation of specific length fatty acids into the mitochondrial compartment, peroxisomes contain carnitine octanoyltransferase, which exhibits a relatively broad substrate specificity which is most active toward hexanoyl derivatives [Hashimoto, 1987]. The peroxisomal carnitine octanoyltransferase, as well as peroxisomal carnitine acetyltransferase, is believed to aid in the transfer of chain-shortening fatty acids, which are the products of the peroxisomal 6-oxidation pathway out of peroxisomes 11 for farther oxidation in mitochondria [Naidu and Moser, 1990].
Because peroxisomal B-oxidation does not degrade fatty acids completely, it is
suspected that peroxisomes function in the chain shortening of long-chain fatty acids to
produce better substrates for the mitochondrial system [Hashimoto, 1982; Hashimoto, 1987;
Mannaerts and Van Veldhoven, 1990]. The peroxisomal B-oxidation systems may play an
important role in the metabolism of very long-chain and unsaturated fatty acids [Osmundsen,
1982], prostaglandins [Diczfalusy et al., 1990], dicarboxylic acids [Kolvraa and Grefersen,
1986], xenobiotic compounds like phenyl fatty acids [Yamada et al. , 1986], and hydroxylated
5B-cholestanoic acids formed during the conversion of cholesterol to cholic acid [Kase et al.,
1986]. Mitochondrial B-oxidation, which drives oxidative phosphorylation and supplies acetyl-
CoA for ketogenesis, may be essential only during fasting, although in some animal organs
it provides a major portion of the energy under non-fasting conditions [Schulz, 1991].
C. Peroxisome Proliferators
Paget (1963) described the fine structural changes in rat hepatocytes associated with clofibrate-induced hepatomegaly. A few years later, Hess and co-workers (1965) and subsequently Svoboda and Azamoff (1966) made the first reports of peroxisome proliferation in the livers of rat and mice after clofibrate administration. The term "peroxisome proliferator" was introduced by Reddy et al. (1975) to denote the chemical that induces proliferation of the cytoplasmic organelle peroxisome in the liver cell. Recent studies have now revealed that at least 60 xenobiotics, or specific diets, can produce peroxisome proliferation and can be classified into as many as 7 groups [Moody et al., 1991]. Chemical structures of some peroxisome proliferators are shown in Figs. 2-7. 12
1. Fibric acids
Many hypolipidemic drugs structurally related to clofibrate (designated as fibric acids) appear to have hepatic peroxisome proliferative properties-i.e., hepatomegaly and the induction of peroxisomal enzymes. Clofibrate (ethyl-a-p-chlorophenoxyisobutyrate), an aryloxyalkanoic acid derivative, was discovered and introduced as a hypolipidemic compound by Thorp and Waring (1962). The ester hydrolyzed free acid form, clofibric acid, is the active lipid-lowering and peroxisome proliferative form. It was found to possess this effect in 1963 by Paget [Paget, 1963]. Clofibrate and other fibric acid-containing peroxisome proliferators, mainly differing in the substitution of position 4 of the aromatic ring, increase in potency as lipophilicity increases [see Fig. 2], Clofibrate (Atromid-S®) and some fibric acid derivatives-i.e., bezafibrate, ciprofibrate, fenofibrate, beclobrate and gemfibrozil
(Lopid®)~have been clinically used as antihyperlipidemic drugs, in particular as hypotriglyceridemic drugs [Witiak et al., 1977; Reddy and Lalwani, 1983; Feller et al.,
1989], Most of these drugs are several times more potent than clofibrate in lowering serum lipids and in inducing hepatic peroxisome proliferation [Lalwani et al., 1983; Reddy and
Lalwani, 1983]. However, in vivo [Eacho et al., 1986] and in vitro [Kocarek and Feller,
1989] reports indicate that peroxisome proliferative effects of these agents are not causally linked to their hypotriglyceridemic actions.
2. Non-fibric acid hypolipidemic drugs
Figure 3 shows hypolipidemic agents which are not structurally related to clofibrate.
Tibric acid; Wy-14,643; tiadenol; BM-15,766; DL-040 and cetaben have been shown to exert marked hypolipidemic and peroxisome proliferative effects in rodents [Reddy and
Krishnakantha, 1975; Reddy et al., 1979; Reddy and Lalwani, 1983; Hawkins et al., 1987; 13
Moody et al., 1991]. DL-040 was also shown to induce peroxisome proliferation in Rhesus
monkeys at dose levels that exceed therapeutic levels [Lalwani et al., 1985].
3. Plasticizers and related compounds
Ester plasticizers are used extensively as industrial solvents and plasticizers in the
manufacture of a wide variety of plastics including food and medical packaging [Lake et al. ,
1975]. Structures of these compounds are shown in Figure 4. Di-(2-ethylhexyl)phthalate
(DEHP) and di-(2-ethylhexyl)adipate (DEHA) are two common plasticizers used in polyvinyl
chloride (PVC) plastics. These lipophilic compounds caused toxicological hazard because they
leak from the plastics, especially pliable plastic wraps, accumulate, and are not readily
degraded from the body [Thomas and Thomas, 1984].
DEHP and related analogs cause liver hyperplasia, proliferation of peroxisomes and hepatocarcinogenesis in rodents [Reddy and Lalwani, 1983; Elcombe and Mitchell, 1986],
However, they are weak hepatocarcinogens relative to some other peroxisome proliferators.
The hepatocellular carcinomas appeared only 10% and 16% in male and female rats, respectively, after 2 years of feeding [Conway et al., 1989]. Lundgren et al. (1987b) analyzed the structural requirements for hepatic peroxisome proliferation by 2-ethylhexanoic acid analogs and found that the most effective proliferators in this group possess an ethyl group as the substituent on carbon 2 of the main chain, which consists of six carbons.
4. Phenoxv acid herbicides
Phenoxy acid herbicides have a close structural relationship to fibric acid analogs [See
Fig. 5]. They have been reported to produce peroxisome proliferation in rodents both in vivo
[Lundgren et al., 1987a] and in vitro [Lewis et al., 1987]. Structurally unrelated herbicides 14
such as the diphenyl ether lactofen (Fig. 5) also produce hepatic peroxisome proliferation in
mice in a similar fashion to nafenopin [Butler et al., 1988].
5. Miscellaneous drugs
LY-171,883, a leukotriene D4 antagonist with a tetrazole-substituted
alkoxyacetophenone, causes hepatic peroxisome proliferation in rodents (Eacho etal., 1986;
Eacho et al., 1989]. The compound is structurally distinct from most other peroxisome
proliferators since it lacks a carboxylic function. However, the acidic tetrazole is chemically
similar to the carboxylic acid. The orientation of this acidic tetrazole group with respect to
the acetophenone ring may be related to its potency [Gray et al., 1983]. The distance of the
acidic heterocycle to the phenyl ring of LY-171,883 is comparable to the distance between the
carboxyl group and phenyl ring of clofibric acid [Foxworthy et al., 1990c].
A number of clinically-used drugs is also reported to possess peroxisome proliferative
effects. These includes an antipyretic drug, acetylsalicylic acid [Ishii and Suga, 1979]; an
anticonvulsant, valproic acid [Horie and Suga, 1985]; a tranquilizer, chlorpromazine [Van den
Branden et al., 1987]; an antigout drug, benzbromarone [Butler et al., 1990] and a steroid,
dehydroepiandrosterone [Wu et al., 1989]. Structures of these compounds are shown in
Figure 6.
6. Physiological and dietary effects
In addition to chemicals which offer simple and reproducible hepatic peroxisome proliferations, certain physiological and dietary conditions have also been shown to produce morphological and enzymatical changes in hepatic peroxisomes. These conditions include cold adaptation [Nedergaard et al., 1980], high-fat diet [Ishii et al., 1980; Neat et al., 1980; 15
Osmundsen, 1982], vitamin E deficiency [Reddy et a l, 1981], starvation [Ishii et al., 1980] and diabetes [Horie et al., 1981]. However, the maximal induction of hepatic peroxisome proliferation and peroxisomal 6-oxidation by physiological and dietary is only several-fold over the control whereas the induction by chemicals and xenobiotics may be 30-fold or greater. Therefore, these dietary manipulations have been suggested to be a unique class of peroxisome proliferation inducers apart from other chemical peroxisome proliferators [Moody etal., 1991].
High-fat diets which contain very-long-chain unsaturated fatty acids, such as a partially hydrogenated fish oil diets, have been shown to result in a higher peroxisomal 8-oxidation activity in rat liver than diets that contain oils with shorter chain fatty acids [Thomassen et al.,
1982]. Thomassen et al. (1982) also suggested that the content of trans 22:1 fatty acids in those partial hydrogenated fish and rapeseed oils is responsible for the stimulation of peroxisomal fatty acid oxidation in rats fed with these oils. Diets containing C22:1 fatty acids were reported to cause a transient accumulation of triacylglycerol in the heart and other tissue but not in the liver [Bremer and Norum, 1982]. Christiansen et al. (1985) used primary cultures of rat hepatocytes to examine the effect of cis and trans 22:1 fatty acid (erucic acid and brassidic acid, respectively) and found that only trans fatty acid, which is less oxidized by mitochondria 6-oxidation, produced a slight but significant induction of peroxisomal 6- oxidation. Moreover, as found in the induction by other peroxisome proliferators, the induction of peroxisomal 6-oxidation by a high-fat diet correlated with microsomal w-oxidation of lauric acid [Nilsson et al., 1986; Nilsson et a l, 1987].
Hormones may play a role in peroxisome proliferation. Estradiol has been found to cause peroxisome proliferation and an increase in 3-hydroxy fatty acid diesters production in the uropygial glands of male and female mallards [Bohnet et al., 1991], The peroxisomes 16 proliferate in the glands of the females during mating season and in the estradiol-treated
males. Induction of peroxisomal B-oxidation has been found in guinea pig treated with
adrenocorticotropin and dexamethasone [Russo and Black, 1982]. In addition, fibric acid as
well as methyl-substituted dicarboxylic acid was recently reported to possess a thyromimetic
effect in the livers of rats. These characteristics indicate that the peroxisome proliferators may
act as transcriptional activators of thyroid-hormone-dependent genes in the rat liver [Hertz et
a l, 1991].
7. Fattv acids and related compounds
There are several fatty acid-like compounds that produce peroxisome proliferation in
rodents. These fatty acid peroxisome proliferators include sulfur-substituted fatty acid
analogues [Aarsland et a l, 1989; Berge et a l, 1989a; Berge et a l, 1989b], 6,B’-methyl
substituted dicarboxylic acid analogs (MEDICA) [Hertz et a l , 1988; Bar-Tana et a l , 1989]
and perfluorinated fatty acid analogs [Ikeda et a l, 1985]. The chemical structures of these
fatty acid analogs are shown in Figure 7.
The sulfur-substituted, 3-thia fatty acids, such as 3-thiadicarboxylic acid and alkylthio
acetic acid, have been reported to have hypolipidemic properties and to induce in a peroxisomal B-oxidation and peroxisome proliferation in vivo [Berge et a l , 1989b; Bergseth
and Bremer, 1990], The non-B- as well as non-w-oxidizable dicarboxylic acids were
apparently more potent than the alkylthio acetic acid. Interestingly, when the sulphur atom
is in the 4-position, the fatty acid changes to a weak peroxisome proliferator, but can enhance the level of hepatic triacylglycerol and is a powerful inhibitor of B-oxidation [Hovik et a l ,
1990; Skorve et a l, 1990].
B,B’-Methyl-substituteddicarboxylicacid (MEDICA) of C14-C18 chain length, has been 17
recently reported to possess hypolipidemic activity, with MEDICA 16 being the most potent
of the series [Bar-Tana et al., 1985]. MEDICA 16 caused peroxisome proliferation in vivo
and in cultured rat hepatocytes, and the induction of peroxisome proliferation in culture was prevented by mitochondrial carnitine palmitoyltransferase inhibitors [Hertz et al., 1988].
Perfluorinated fatty acids are another group of non-metabolized fatty acids that cause peroxisome proliferation in vivo [Ikeda et al., 1985]. They are widely used in industry due to their surfactant properties, chemical and thermal stabilities and antiwetting actions
[Guenthner and Victor, 1962], The analogs that are generally used are perfluoro-n-butyric
acid (PFBA), perfluoro-n-octanoicacid (PFOA), perfluoro-n-decanoicacid (PFDA), perfluoro- n-octanol (PFOL) and perfluorinated octane sulfonic acid (PFOS).
In vivo studies revealed that PFBA, PFOA, PFDA and PFOL but not perfluorinated alkanes induced peroxisome proliferation [Ikeda et al., 1985]. PFOS, although lacking the carboxylic group, produced peroxisome proliferation [Ikeda et al., 1987]. Zonal heterogeneity
studies revealed that PFOA-induced peroxisome proliferation was more prevalent in centrilobular than in periportal hepatocyte [Just et al., 1989]. In contrast to other peroxisome proliferators, PFDA increased plasma triglycerides while decreasing plasma cholesterol.
Thus, it may be classified as the first "non-hypotriglyceridemic1' peroxisome proliferator
[Borges et al., 1990]. Interestingly, although the activity of fatty acyl-CoA oxidase was induced, the overall rate of peroxisomal fatty acid 6-oxidation was decreased [Borges et al.,
1990]. It was also found that PFOA-induced peroxisome proliferation is species- and sex- dependent in vivo. PFOA caused peroxisome proliferation in the male and female mice and in the male but not female rats [Uy-Yu et al., 1990]. Moreover, PFOA, like other peroxisome proliferators, also induced stearoyl-CoA desaturase, and the effect was dependent on testosterone [Kawashima et al., 1989a]. 18
The toxic effects of perfluorinated fatty acid are decreased food intake, thymic atrophy, bone marrow depression, hepatomegaly, disruption of hepatic architecture and delayed lethality [Harrison et al. , 1988]. Among all perfluorinated fatty acid, PFDA is the most toxic compound. It was suggested that PFDA produced toxic effects in rodents similar to those caused by 2,3,7,8-tetrachlorodibenzo-p-dioxin [Harrison et al. , 1988]. Unlike dioxin toxicity, PFDA also causes severe testicular atrophy and necrosis. The disruption of the normal feedback relationship between plasma androgen and leutinizing hormone concentration which resulted in androgenic deficiency has been reported in male rats treated with PFDA
[Bookstaff et a l, 1990].
D. Hepatic Peroxisome Proliferation
The administration of peroxisome proliferators to rodents results in several characteristic changes in liver as well as kidney. However, the most pronounced effects of these peroxisome proliferators are in the liver. Hepatic peroxisome proliferation is characterized by an increase in the size and number of peroxisomes in parenchymal cells.
These changes include hepatomegaly, inductions of peroxisome, and peroxisomal- associated enzymes [Reddy and Lalwani, 1983; Hawkins et al. , 1987; Lock et al. , 1989; Moody et al.,
1991]. In contrast to the liver response, no kidney enlargement has been reported [Hawkins et al., 1987]. Only hepatic responses to peroxisome proliferators will be discussed.
1. Hepatomegaly
Hepatomegaly appears to be a characteristic of all peroxisome proliferators. The liver enlargement induced by peroxisome proliferators results from both hyperplasia, which can be seen within the first few days of compound administration, and hypertrophy of the liver. The 19 increase in liver cell size is largely due to an expansion of the peroxisomal volume of the cell.
The peroxisome volume fraction increases from 2% in a normal rat hepatocyte to as much as
25 % after hepatocytes were exposed to peroxisome proliferators [Reddy and Lalwani, 1983].
The liver enlargement is sustained for the period that the peroxisome proliferators are administered, and reverses within two weeks after discontinuation of treatment [Reddy and
Lalwani, 1983; Marsman et a l, 1988]. Chronic administration of fenofibrate, methyl clofenapate and tibric acid results in a dose-dependent increase in liver size in a greater magnitude than clofibrate-induced hepatomegaly [Orton and Parker, 1982]. pHJThymidine incorporation is increased after rodents are treated with nafenopin [Moody et al., 1977; Bieri et al. , 1984], trichloroethylene [Elcombe et al. , 1985], and Wy-14,643 [Reddy et al. , 1979].
Nafenopin also enhances hepatocyte DNA synthesis irrespective of the calcium level in cultured rat hepatocytes [Bieri et al., 1984].
2. Induction of peroxisomal enzymes
There are numerous biochemical changes in peroxisomes after treatment with peroxisome proliferators. Induction of peroxisome in the liver is associated with a 10- to 30- fold increase in the activity of peroxisomal 6-oxidation enzymes [Reddy and Krishnakantha,
1975; Lazarow and DeDuve, 1976; Lazarow, 1977; Osumi and Hashimoto, 1979; Fahimi et al., 1982; Bakke and Berge, 1984; Berge et al., 1984; Hawkins et al., 1987], enoyl-CoA hydratase [Lazarow, 1978; Osumi and Hashimoto, 1979; Reddy and Lalwani, 1983; Reddy
MK et a l, 1981; Hawkins et a l, 1987], 3-hydroxyacyl-CoA dehydrogenase [Lazarow and
DeDuve, 1976; Osumi and Hashimoto, 1979; Reddy MK et al., 1981], and 3-ketoacyl-CoA thiolase [Lazarow, 1978; Osumi and Hashimoto, 1979; Reddy MK et al., 1981]. The enzyme inductions generally parallel the increase in peroxisomal volume. The in vivo studies of 20 peroxisome proliferation also reveal the inductions of non-fatty acid B-oxidation peroxisomal enzymes such as carnitine acetyltransferase [Moody and Reddy, 1974; Lazarow, 1977; Reddy and Lalwani, 1983], carnitine octanoyltransferase [Hashimoto 1987; Chatterjee et al., 1988] and acyl-CoA: dihydroxyacetone phosphate acyltransferase [Reddy and Lalwani, 1983].
Catalase, the peroxisomal marker enzyme that is not in the peroxisomal B-oxidation cascade, is induced only 2-fold by peroxisome proliferators at the maximum effect [Reddy and
Lalwani, 1983; Hashimoto, 1987; Hawkins et al., 1987; Lock et al., 1989; Malki et al.,
1990; Moody et al., 1991]. However, the activity of urate oxidase is not induced by peroxisome proliferators [Hashimoto, 1987; Lock et al., 1989]. Long term in vivo studies showed that the induction of peroxisome proliferation-associated enzymes was sustained for
1 year, which indicates that there was no compensatory mechanism for this phenomenon
[Malki et al., 1990],
Peroxisome proliferators, di(2-ethylhexyl)phthalate, and Wy-14,643 were shown to increase the translatable mRNA levels in rat liver [Furuta et al., 1982; Chatterjee et al.,
1983]. Development of a technique using specific cDNA probes revealed a 10- to 20-fold increase in the mRNA levels of the 3 enzymes (fatty acyl CoA-oxidase, enoyl-CoA hydratase/3-hydroxyacyl-CoAdehydrogenaseand 3-ketoacyl-CoAthiolase) in the peroxisomal
B-oxidation system in rats treated with peroxisome proliferators [Osumi et al., 1984;
Watanabe et al., 1985; Reddy et al., 1986; McQuaid et al., 1987; Nemali et al., 1988].
Clofibrate, ciprofibrate, and di(2-ethylhexyl)phthalate selectively increased the rate of transcription of the B-oxidation genes, which reached maximal rates by 1 hr and persisted for
16 hr after administration of the compound. However, the transcriptional rate of catalase was not significantly altered [Reddy et al., 1986]. 21 3. Induction of microsomal cytochrome P-450 bv peroxisome proliferators
Studies of the increase in the marker enzyme activity and proliferation of subcellular
organelles in the liver revealed that peroxisomes are the most responsive organelles in
comparison to lysosomes, mitochondria, and smooth endoplasmic reticulum (SER) [Malki et
al., 1990]. However, the proliferation of SER is associated with a concomitant increase in
certain microsomal enzymes, in particular cytochrome P-450. It was not noted until 1982 that
peroxisome proliferators selectively induced liver cytochrome P-450 that carry out co-
hydroxylation of lauric acid [Gibson et al., 1982; Orton and Parker, 1982] and arachidonic
acid [Bains et al., 1985]. The increase in this activity approached an order of magnitude, and
has not been found as a response to any other set of enzyme-inducing xenobiotics [Moody et al., 1991]. This cytochrome P-450 induced by peroxisome proliferators has been isolated and purified and designated cytochrome P-452 [Tamburini et al., 1984], cytochrome P -450^
[Hardwick et al., 1987], or the more current systematic names, cytochrome P450 IVA1
[Nebert et al., 1987; Gibson et al., 1990] and CYP4A1 [Nebert et al., 1991].
The increase in this cytochrome protein is due to specific increases in mRNA transcription [Lock et al., 1989; Gibson et al., 1990; Milton et al., 1990]. Using cDNA probes analysis, it was shown that a single dose of clofibrate leads to a 3-fold increase in the mRNA encoding for cytochrome P450 IVA1 within 3 hr of administration. The increase in this mRNA closely parallels the increase in the cytochrome P450IVA1 protein and enzymatic activity for co-hydroxylation of lauric acid [Hardwick et al., 1987]. Recent studies have demonstrated a very strong correlation between the ability of peroxisome proliferators to induce both peroxisomal fatty acid fi-oxidation and microsomal laurate co-oxidation [Lake et al., 1984; Sharma et al., 1988b; Gibson et al., 1990; Makowska et al., 1990]. It has been postulated that this cytochrome P450 IVAl-dependent enzyme and peroxisomal enzyme are 22 regulated by the same mechanism which controls changes in fatty acid metabolizing enzymes in the two separated hepatic subcellular compartments [Lock et al., 1989; Gibson et al.,
1990].
4. Miscellaneous changes during peroxisome proliferation
Hypolipidemic peroxisome proliferators increase the fatty acid-binding protein level in the livers of rats [Kawashima et al., 1983]. This 14 kDa protein abundantly found in the liver has been postulated to function in the intracellular transport and utilization of long-chain fatty acids and in protecting hepatocytes from the toxic effects of high levels of fatty acids and their CoA esters [Bass, 1988; Brandes et al., 1990]. Peroxisome proliferators do not bind to liver fatty acid-binding proteins (L-FABP) [Bass, 1988]. Brandes et al. (1990) showed that the induction of peroxisomal 6-oxidation and of L-FABP by peroxisome proliferators is a temporally continuous but closely related process. The bezafibrate-induced increase in peroxisomal palmitoyl-CoA oxidation activity progressed at a greater initial rate and declined rapidly after removal of bezafibrate whereas L-FABP continued to increase [Brandes et al.,
1990]. However, it is not clear whether L-FABP is a part of a "peroxisomal gene domain"
[Watanabe et al., 1985] or whether it is induced secondary to an increase in free fatty acid flux generated by increased peroxisomal 6-oxidation [Bass, 1988].
Peroxisome proliferators solely induced cytosolic epoxide hydrolase [Hammock and
Ota, 1983]. This induction has also become the prototypical change used to classify a xenobiotic as a peroxisome proliferator [Moody et al., 1991].
The administration of peroxisome proliferator also causes an increase in the activities of microsomal enzymes participating in lipid biosynthesis, such as glycero-3-phosphate acyltransferase [Das et al., 1983], stearoyl-CoA desaturase [Kawashima et al., 1989a], and 23 palmitoyl-CoA elongenase [Kawashima and Kozuka, 1985]. An increase in lipid biosynthesis
may be necessary for the supply of lipid components for the proliferation of the intracellular
organelles [Kawashima et al., 1989b]. Four different structural unrelated peroxisome
proliferators (clofibric acid, DEHP, tiadenol, and PFOA) but not hormonal or dietary
manipulation were reported to increase markedly the microsomal 1-acylglycerophosphocholine
acyltransferase in rat liver [Kawashima et al., 1989b]. High correlations were observed
between the induction of this microsomal enzyme and peroxisomal 8-oxidation enzyme;
therefore, this enzyme was suggested to be a useful parameter responsive to peroxisome
proliferators [Kawashima et al., 1989b],
Immunoblotting analysis revealed that peroxisome proliferators highly increase the
amount of peroxisome integral membrane proteins [Bartles et al., 1990; Malki et al., 1990].
Bartles et al. (1990) and Crane et al. (1988) reported that peroxisome proliferators induced
alterations in the expression and the modification of the rat hepatocytes membrane proteins.
Van Veldhoven et al. (1987) suggested the presence of a pore-forming protein that could be
a porin-like protein. However, the functions of these integral membrane protein are not clear.
5. Peroxisome proliferation and hepatocarcinomas
Hepatic peroxisome proliferations are classified as a unique class of nongenotoxic
chemical carcinogens [Reddy et al., 1980]. Chronic exposure to peroxisome proliferators
results in the development of liver tumors in rodents [Reddy and Lalwani, 1983; Reddy and
Rao, 1986; Conway et al., 1989; Reddy, 1990]. The mechanism of carcinogenicity remain
obscure. The studies show that the latency period and the incidence of liver tumors correlate well with the effectiveness of the administered dose in inducing peroxisome proliferation.
Biochemical characterization of neoplastic development has shown that peroxisome 24 proliferators cause a unique tumor phenotype. In contrast to many carcinogens, peroxisome proliferators are not genotoxic. They did not bind to DNA or cause mutations [Warren et a l ,
1980; Glauert et a l, 1984; Gupta et a l, 1985]. The genesis of tumors in rodents by peroxisome proliferators requires continuous exposure for more than 60 to 500 days depending on their carcinogenic potencies. Once the tumor forms, its progression is not inhibited by cessation of peroxisome proliferator exposure [Reddy and Lalwani, 1983], As with other hepatocarcinogens, peroxisome proliferator-induced altered foci have decreased ATPase and glucose-6-phosphatase. However, peroxisome proliferator-induced foci lack activities of y- glutamyl transpeptidase (GGT), glutathione S-transferase P, or a-fetoprotein [Rao et a l ,
1988]. Although the mechanism of hepatocarcinogenicity caused by peroxisome proliferators remains unclear, two theories have been proposed: the oxidative stress and the tumor initiation-promotion hypotheses.
5.1 The oxidative stress theory
Reddy and Lalwani (1983) suggested that peroxisome proliferators initiate a slow and subtle process of oxidative damage which results from increased levels of H202-generating peroxisomal fatty acid 6-oxidation enzyme systems and an imbalanced increase in catalase and peroxisomal oxidase. Leakage of H202 causes reactive oxygen species which damage intracellular membranes and/or DNA, and results in carcinogenesis [Glauert et a l , 1984;
Reddy and Rao, 1989; Rao and Reddy, 1989]. Long term treatment with peroxisome proliferators also results in a persistent increase in the formation of 8-hydroxy-deoxyguanosine
[Kasai et a l , 1989] 25
5.2 Tumor initiation and promotion
Although some indirect evidence supports the oxidative stress theory, conclusive evidence for DNA damage is lacking [Cattley et al., 1990]. Single strand breaks occurred in DNA of hepatocytes exposed to H202 in vitro [Olsen, 1988], but not in hepatocytes exposed to peroxisome proliferators [Butterworth et al., 1984; Elliot and Elcombe, 1987]. Schulte-
Hermann et al. (1983) suggested that peroxisome proliferators promote spontaneously initiated foci in liver. Therefore, the mechanisms of hepatocarcinogenesis by peroxisome proliferators may be contributed to an enhancing cell replication associated with tumor promotion [Conway et al., 1989; Cattley et a l, 1990].
6. Species differences and significance to human
Studies have shown that rats and mice are the most sensitive species to the hepatic effects of peroxisome proliferators [Reddy and Lalwani, 1983; Elcombe et al., 1985; Hawkins et a l, 1987; Lock et al., 1989; Watanabe et a l, 1989]. Reddy and coworkers (1984) reported the peroxisome proliferation induced by ciprofibrate in Cynomolgus and Rhesus monkeys. However, several peroxisome proliferators such as LY-171,883 [Eacho et al.,
1986], bezafibrate [Watanabe et al., 1989] and nafenopin [Lake et al., 1989] failed to elicit peroxisome proliferation in some other species including dogs, marmosets, Rhesus monkeys and guinea pigs. Hamsters show a hepatic response similar to that of rats but smaller in magnitude [Eacho et al., 1986]. All studies clearly demonstrated that there is a marked species difference in sensitivity to peroxisome proliferators. Rat and mice are the most sensitive, followed by hamsters, whereas guinea pigs and primates are insensitive or nonresponsive.
The question was asked whether the data from rodent studies are relevant to humans. 26 Examination of human liver biopsies, obtained from patients receiving hypolipidemic peroxisome proliferators such as clofibrate, gemfibrozil, or fenofibrate, has demonstrated marginal or no increase in peroxisomal volume densities or numbers [Lock et al., 1989].
Studies using primary cultures of human hepatocytes [Butterworth et al., 1989] revealed that whereas thein vitro rat hepatocyte DNA repair assay is a valid model for predicting potential genotoxic effects in human, rodent hepatocytes may not be appropriate for assessing the potential of nongenotoxic chemicals, including peroxisome proliferators. Trichloracetic acid was found to induce peroxisomal 6-oxidation in hepatocytes isolated from rats and mice, but not from humans [Elcombe et al., 1985]. Clofibric acid, mono(2-ethylhexyl)phthalate
(MEHP) and benzbromarone were peroxisome proliferators in primary cultures of rat hepatocytes, but not in primary cultures of human hepatocytes [Bichet et al., 1990]. In agreement, beclobric acid and clofibric acid showed no increase in peroxisomal 6-oxidation and number of peroxisomes in primary cultures of monkey and human hepatocytes whereas both compounds gave a 10-fold increase in peroxisomal 6-oxidation and a 3-fold increase in the relative number of peroxisomes in rat cultured hepatocytes [Blaauboer et al., 1990].
Therefore, at clinical doses of hypolipidemic drugs, the peroxisomal response seen in rodents may not predict the response in humans.
7. Methods in hepatic peroxisome proliferation studies
A. In vivo procedures
Animals can be fed with peroxisome proliferators followed by the morphological and biochemical examinations for pleiotropic responses of peroxisome proliferators including hepatomegaly and enzyme inductions. In vivo transplantation system was introduced by Reddy and coworkers [Reddy et al., 1984] for screening potential peroxisome proliferators and 27
studying the responses of different species.
B. In vitro procedures
In 1982 Lake and co-workers introduced the use of primary hepatocyte cultures from
rats and mice to provide an excellent in vitro system to assess the effects of peroxisome
proliferators in inducing peroxisome proliferation [Gray et al., 1982; Gray et a l , 1983; Lake
et a l, 1983], A wide variety of compounds, including hypolipidemic agents, herbicides,
plasticizers, and other industrial chemicals, have been shown to produce their peroxisome
proliferative effects in this in vitro system by Lake’s as well as other groups, including Bieri
et a l (1984), Mitchell et al (1984), Feller et al (1985), Elcombe and Mitchell (1986), and
Foxworthy and Eacho (1986).
Originally, the isolation of hepatocytes by in situ collagenase perfusion was introduced
by Berry and Friend (1969) and modified by Seglen (1976). Many of the characteristics of
peroxisome proliferation in vivo can be observed in rat hepatocytes. These characteristic
responses include the stimulation of replicative DNA synthesis, increased peroxisome
numbers, and inductions of peroxisomal and non-peroxisomal enzyme activities-e.g., fatty acyl-CoA oxidase, microsomal laurate hydroxylase, bifunctional enzymes, carnitine acetyltransferase and carnitine palmitoyltransferase [Moody et a l, 1991]. However, maintaining the cultured hepatocytes was difficult due to the variety of seeding and culture conditions used. The monolayers lose many of their hepatic properties, including some cytochrome P-450 isoenzymes. Nevertheless, the addition of 2 % dimethyl sulfoxide (DMSO)
[Muakkassah-Keller, et a l, 1988], 10'5 M hydrocortisone, or other naturally occurring or synthetic corticosteroids [Mitchell et a l, 1984], phenobarbital [Miyazaki et a l, 1985], and the use of supplemented Williams E media together with an addition of 6-aminolevulinic acid
[Feller et a l, 1985], were reported to improve the survival of the hepatocytes and maintain 28 considerably amount of cytochrome P-450 isoenzymes. Thus, like the in vivo studies, primary cultures of rat hepatocytes in an appropriate condition can be employed for a rapid screening for a peroxisome proliferative effect of chemicals, for screening compound metabolites as potential peroxisome proliferators [Mitchell et al. , 1985; Kocarek and Feller, 1987], as well as for studying structure-activity relationships [Lake et al., 1987; Esbenshade et al., 1990].
Recently, the use of rat hepatoma cells (H4IIEC3) has been developed and successfully used for the in vitro studies of peroxisome proliferation [Osumi et al., 1990]. The 7800 Cl
Morris hepatoma cell line has also been proven for its ability to maintain the enzyme activities necessary for peroxisome proliferation studies [Spydevold and Bremer, 1989]. For higher species, the use of primate primary cultures of hepatocytes [Foxworthy et al., 1990a] including human cultures [Butterworth et al., 1989; Bichet et al., 1990; Blaauboer et al.,
1990] gave a clear answer to the species differences in peroxisome proliferation. None of the human hepatocyte cultures were responsive to the effects of peroxisomal proliferators as reported in rodent cultures.
Recently, Issemann and Green (1990) have reported on the discovery of mouse peroxisome proliferator activated receptor (PPAR). In the studies, they constructed the chimeric expression plasmids in which the DNA encoding the probable ligand-binding domain of the receptor is ligated downstream of the cDNA encoding the DNA-binding domains of either the estrogen receptor gene or glucocorticoid receptor gene. Upon activation with peroxisome proliferator, the chimeric protein receptors bind to a second plasmid containing either an estrogen response element or a glucocorticoid response element, either of which drives the reporter. They demonstrated that the level of reporter enzyme (chloramphenicol acetyltransferase) activity caused by the binding of peroxisome proliferators to the chimeric receptor correlated well with the potency of the peroxisome proliferative effect and 29 hepatocarcinogenicity produced by the compounds. This method which becomes the first technique for studying the peroxisome proliferative effect of chemicals at a molecular level may be very useful in the evaluation of the peroxisome proliferative effect of xenobiotics as well as endogenous compounds. Moreover, it may be one of the most meticulous techniques for elucidating the mechanism of peroxisome proliferation caused by these compounds.
8. Significance of peroxisome proliferation studies
1. It is well established that a sustained increase in the number of peroxisomes in the liver after feeding peroxisome proliferators for extended periods of time leads to the development for hepatocarcinomas. Reddy and co-workers (1983) postulated that the mechanism for hepatocarcinogenicity of the compounds may arise from an imbalance in the production and degradation of H202 in the cells. Because of the direct involvement in cancer, it is very important to determine what causes the peroxisomes to proliferate and how can this happen.
2. Patients with peroxisomal disorders such as Zellweger disease have severe abnormalities in many organs. In these patients, peroxisomes are lacking or do not function properly. Study for the mechanism of peroxisome proliferators in increasing the number of peroxisomes may, somehow, be beneficial for the treatment of these diseases.
3. For scientists, peroxisome proliferation can be an excellent model whereby to study the growth of subcellular organelles. Nebert (1990) recently commented that the variety of biological phenomena related to growth and differentiation may be mediated by members of nuclear hormone receptor superfamily and genes encoding drug-metabolizing enzymes sharing center stage. One of these nuclear hormone receptor members is believed to be the peroxisome proliferator activated receptor (PPAR) freshly discovered by Issemann and Green 30
(1990).
E. Proposed Mechanisms of Peroxisome Proliferation
Two hypotheses have been proposed for the mechanism of peroxisome proliferation by drugs, chemicals and environmental agents. They are (a) activation of specific genes by the chemical or its metabolite, either directly or mediated by a specific receptor and (b) fatty acid overload, either as a consequence of lipolysis occurring outside the liver and causing an influx of fatty acids into the liver, or as an effect of peroxisome proliferators or their metabolites perturbing lipid metabolism [Reddy and Lalwani, 1983; Lock et al., 1989].
1. Receptor-mediated mechanism
Reddy and co-workers have proposed that peroxisome proliferators possess their effects by a ligand-receptor mediated mechanism [Reddy and Lalwani, 1983; Lalwani et al. ,
1983; Reddy and Rao, 1986; Lalwani et a l, 1987]. Reddy and co-workers suggested that these peroxisome proliferators exert their effect in the same way as does hormone-receptor interaction (Fig. 8). The peroxisome proliferators enter cells by diffusion through the plasma membrane and then bind with specific receptor proteins inside the cell. The binding of ligand activates the receptor, and this ligand-receptor complex binds with increased affinity to select site(s) in cellular DNA which then trigger the transcription of a peroxisome proliferator domain [Watanabe et al., 1985; Reddy and Rao, 1986]. These gene products defined as the specific peroxisome proliferator domain are responsible for the inductions of peroxisomal enzymes (e.g., enzymes in peroxisomal fatty acid B-oxidation system) and non-peroxisomal enzymes (e.g., cytochrome P450 IVA1 dependent lauric acid hydroxylation). The proposal of a receptor for foreign chemicals is not uncommon since aryl hydrocarbons such as dioxin 31 derivatives are also believed to exert their cytochrome P450 IA1 inductive effect through a cytosolic receptor protein, the Ah receptor [Poland and Knutson, 1982].
Some evidence is provided to support the receptor-mediated hypothesis. This evidence includes the following:
1. The tissue specificity in the induction of peroxisome proliferation. Peroxisome proliferation is inducible in hepatic parenchymal cells and to a limited extent in the proximal tubular epithelium of the kidney [Reddy and Lalwani, 1983]. Hepatocytes transplanted into the anterior chamber of the eye or into fatty pads of syngeneic or xenogeneic hosts are capable of recognizing and responding to peroxisome proliferators contained in the diet [Reddy et al.,
1984].
2. The rapid and significant increase in the rate of synthesis of mRNAs for peroxisomal B-oxidation and microsomal co-oxidation enzymes in liver and the rapidity of the transcriptional response of the gene [Reddy et al., 1986; Hardwick et al., 1987].
3. The demonstration of a reversible and specific binding of nafenopin to a cytosolic
70 kD protein in the liver [Lalwani et al., 1983; Lalwani et al. , 1987]. Recently, Alvares et al. (1990) isolated a protein that binds to clofibric acid and identified the protein as a member of the heat shock protein HSP70 family.
4. Structure-activity relationship supports the existence of a specific target site for peroxisome proliferators. Subtle structural differences between tetrazole-containing compounds related to LY-171,883 result in major differences in the induction of peroxisomal
B-oxidation [Eacho et al. , 1989]. Using a branched nonane as peroxisome proliferator, Ikeda et al. (1988) suggested that the receptor may contain a cationic domain which would recognize the negatively charged group (e.g., ionized carboxylic acid group) of the peroxisome proliferators, and a lipophilic domain which would have low structural specificity. 32
5. Issemann and Green (1990) recently reported on the cloning and characterization of the mouse peroxisome proliferator-activated receptor (PPAR) [Issemann and Green, 1990].
They presented a PPAR as a novel member of the nuclear hormone superfamily and demonstrated that the ligands for this receptor are peroxisome proliferators. This finding adds a step towards the development of a theory binding the receptor-mediated growth transduction pathway and the mechanism by which foreign chemicals or endogenous substances act as ligands [Nebert, 1990].
2. Substrate overload mechanism
Although there is evidence to support the existence of a receptor in peroxisome proliferation, direct pharmacological studies of the receptor have never been performed.
Other laboratories than Reddy’s have failed to detect the binding of either pHJ-nafenopin or
[3H]-ciprofibrate to the cytosolic protein. Milton et al. (1988) suggested that albumin is responsible for the binding activity in the original report. Moreover, because of the ability of structurally diverse chemicals to elicit peroxisome proliferation and the observation that a high-fat diet [Ishiiet al., 1980; Neat et al., 1980] and some physiological conditions cause peroxisome proliferation, Lock et al. (1989) proposed an alternative hypothesis to the receptor mechanism.
The enzyme induction during peroxisome proliferation is relatively specific to lipid metabolism; for example, peroxisomal fatty acid B-oxidase, microsomal fatty acid co-oxidase and carnitine acyltransferase activities are increased markedly after treatment with peroxisome proliferators. Excess influx of fatty acids into the liver has been reported to cause small increases in peroxisomal B-oxidation [Horie et al., 1981]. Because of these observations,
Lock et al. (1989) suggested that the peroxisome proliferators perturb lipid metabolism, which 33 results in a transient accumulation of lipid. However, Reddy et al. (1987) argued that
peroxisome proliferation has not been observed in fatty liver induced by ethanol, a choline-
deficient diet, or carbon tetrachloride.
Elcombe and Mitchell (1986) reported that administration of DEHP results in a
transient accumulation of small droplets of neutral lipid in the liver. They suggested that the
active peroxisome proliferator derived from DEHP, metabolite VI, caused a concentration-
dependent decrease in fatty acid oxidation in isolated cells and selectively inhibits medium-
chain fatty acid in isolated mitochondria. This could lead to an accumulation of medium-chain
fatty acids as their Co A or carnitine esters. Due to the depletion of CoA, all fatty acid 8-
oxidation reactions are inhibited. Lock et al. (1989) suggested that the medium-chain fatty
acids are responsible for the induction of microsomal cytochrome P-450 IVA1, the isozyme which catalyzes the co-oxidation of long-chain fatty acids to dicarboxylic acids, and that long- chain dicarboxylic acids are proximal stimuli for the induction of peroxisomal 8-oxidation (see
Fig. 9). This role of dicarboxylic acids in peroxisome proliferation was suggested earlier
[Sharmaet al. , 1988b; Sharma et al. , 1989].
Other inhibitors of mitochondrial fatty acid oxidation, including valproic acid [Horie and Suga, 1985] and 2-[5-(4-chlorophenyl)-pentyl]-oxiran-2-carboxylate(POCA) [Bone et al. ,
1982] induce peroxisomal 8-oxidation. In studies on the peroxisome proliferative effects of
LY-171,883, bezafibrate and bezafibroyl CoA, and 2-hydroxy-3-propyl-4-[6-(tetrazol-5- yl)hexyloxy]acetophenone (4-THA), Eacho and co-workers suggested that an inhibition of hepatic fatty acid oxidation at carnitine palmitoyltransferase I may be relevant to the mechanism of peroxisome proliferation [Eacho and Foxworthy, 1988; Foxworthy and Eacho,
1988; Foxworthy et al., 1990b].
Peroxisomes contain a fatty acid 8-oxidation system, which preferentially oxidize long- 34 chain fatty acids (C8-CM). The physicochemical properties of peroxisome proliferators, such as clofibric acid or non-metabolizable fatty acids have the hydrophobic carbon backbone that is closely similar to those of naturally occurring fatty acids. Reddy and Lalwani (1983) also suggest the possibility that these chemicals and/or their metabolites may serve as substrates for the peroxisome fatty acid B-oxidation, which leads to an induction of the enzymes by the substrate overload mechanism.
3. Proposed regulatory components in peroxisome proliferation
In addition to these two major hypotheses, participation of the cofactor coenzyme A
(CoA), calcium, fatty acid binding protein (FABP), and protein kinase C may participate as regulatory components in the mechanism of peroxisome proliferation.
3.1 CoA and the level of lone-chain acvl-CoA
Berge and Aarsland proposed that the induction of peroxisomal fi-oxidation by peroxisome proliferators and physiological conditions such as high-fat diets is exerted through an increased cellular level of long-chain acyl-CoA [Berge et al., 1984; Berge and Aarsland,
1985; Berge et al., 1987]. They suggested that peroxisome proliferators are generally activated to acyl-CoA thioesters and these derivatives play a causative role in the enzyme induction process [Hertz et al., 1985; Bronfman et al., 1986; Aarsland et al., 1990].
Recently, Aarsland and Berge (1991) added that the activation to the corresponding xenobiotic-CoA is a prerequisite for the proliferating effect of peroxisome proliferators; however, the rate of activation does not determine the potency of the peroxisome proliferators
[Aarsland and Berge, 1991].
Bronfman et al. (1989) have shown that acyl-CoA thioesters of peroxisome 35 proliferators greatly increase the activity of rat brain protein kinase C. Several enzymes in lipid metabolism are known to be regulated by the protein kinase [Hardie et al., 1989]. Thus, protein kinase C, a common regulatory protein, may account for the induction of peroxisome proliferation.
3.2 Fattv acid binding protein
As mention earlier, fatty acid binding proteins (FABP) were found to be induced during peroxisome proliferators treatment [Kawashima et al., 1983; Brandes et al., 1990].
Long-chain fatty acids are bound to FABP, and displacement of these fatty acids from FABP by peroxisome proliferators could contribute to a mechanism of induction mediated by substrate overload [Brandes et al., 1990].
3.3 The role of calcium
Recently, nicardipine, the calcium antagonist, has been reported to suppress the clofibrate-induced peroxisome proliferation in rat liver [Watanabe and Suga, 1988]; thus, a calcium-dependent mechanism may be responsible for the peroxisome proliferative effects.
Moreover, Itoga et al. (1990) also demonstrated that there might be a difference in the mechanism of peroxisome proliferation induced by xenobiotics and physiological condition- i.e., a high fat diet [Itoga et al., 1990].
F. Statement of the Problem
1. Objective and rationale
The discovery that peroxisomes contain several enzymes involved in the 6-oxidation of long-chain fatty acids [Lazarow, 1978, Lazarow and DeDuve, 1976], the proposal that 36
peroxisome proliferation is related to lipid metabolism and hypotriglyceridemia [Reddy and
Krishnakantha, 1975], and the reports on the peroxisome proliferation induced by high-fat
diets [Ishii et al. 1980; Neat et al., 1980] suggest the role of fatty acids in this phenomenon.
Although up until now a substantial amount of studies on hypolipidemic peroxisome
proliferators and other peroxisome proliferators have already been reported, studies concerning
the structural requirement for fatty acids in mediating peroxisome proliferation are limited.
Only a few studies have been reported such as the studies of the structural requirements of 2-
ethylhexanoic acid analogs [Lundgren et al., 1987b], of non-metabolizable 3-thia fatty acid
analogs [Berge et al., 1989a; Berge et al., 1989b; Aarsland et al., 1989], and of
perfluorinated fatty acid analogs [Ikeda et al., 1985; Kozuka et at., 1991]. Also, all of the
studies have been performed in vivo.
Primary cultures of rat hepatocytes have been demonstrated to be excellent models in
the investigation of peroxisome proliferation [Gray et al., 1982; Feller et al., 1985; Bieri et
al., 1987]. The advantages of using this in vitro system are the following:
(1) The ability to evaluate a large number of compounds at various concentrations for their peroxisome proliferative effects within the same animal used, thus, minimizing the
number of animals used.
(2) The elimination of whole animal variables such as gastrointestinal absorption and metabolism, plasma protein binding, and distribution in the body, as well as other extrahepatic parameters, such as thyroid hormones, estrogen, epinephrine, adrenocorticotropin, and glucagon. These hormones have been reported to affect peroxisomal function and biogenesis
[Russo and Black, 1982; Bohnet et al., 1991; Hertz et al., 1991].
(3) Since naturally occurring fatty acids are generally widely distributed in animal tissues including blood and adipose tissue, it is difficult to study the structural requirements 37 and the peroxisome proliferative activities of these natural occurring compounds in the intact animal. Also, one is able to use the fatty acid-free media to limit the amount of fatty acid exposed to the hepatocytes.
For these reasons, the study of the effects of fatty acids and fatty acid analogs in mediating peroxisome proliferation in primary cultures of rat hepatocytes was undertaken.
Chapter II highlights the chemical structural constraints of the fatty acid analogs for the peroxisome proliferative effects. The studies emphasize the comparison between the naturally occurring short- to medium-chain fatty acids as compared to their non-metabolizable perfluorinated fatty acid analogs of the same chain length. Moreover, the use of stereoisomers of selected compounds was also employed in order to gain more insight into the structural requirements of fatty acids analogs.
Although quite a number of reports exist on peroxisome proliferation in rats fed with high-fat diets, the active fatty acid components in the diets responsible for this phenomenon is still unclear. Partially hydrogenated fish oil and rapeseed oil have been shown to stimulate peroxisomal 6-oxidation in rat liver [Thomassen et al., 1982; Nilsson et al., 1987]. Since these dietary oils are rich in C22:, fatty acid, it has been suspected that the content of trans
C22:1 is responsible for the stimulation of peroxisomal 6-oxidation [Thomassen et al., 1982].
Christiansen et al (1985) demonstrated small increases in palmitoyl-CoA oxidation and carnitine acetyltransferase activities in hepatocytes cultures treated with 0.1 mM brassidic acid
(itrans C22:l) but not erucic acid (cis C22:1). However, the addition of cis and trans C22:1 acids to a diet high in soybean oil failed to induce peroxisomal activity [Flatmark et al., 1988].
Flatmark et al. (1988) concluded that other components in partially hydrogenated fish oil other than C22 are responsible for the peroxisome proliferation. In another report, Spydevold and
Bremer (1989) suggested that the inductive property of high fat diets may be due to their 38 content of shorter fatty acids such as C12 and C14.
Due to these controversial findings, Chapter III attempts to find the explanation for the induction of peroxisomal fatty acid 8-oxidation and peroxisome proliferation by natural fatty acids. The evaluations are on the carbon-chain lengths as well as the functional groups of the natural fatty acids, including unsaturated double bonds, dicarboxylic acid functions, and fatty acid alcohol. The studies on the effects of various chain-lengths of dicarboxylic acids are important since it has been proposed that dicarboxylic acids may represent the proximal stimuli for peroxisomal 8-oxidation [Sharma et a l, 1988b; Lock et a l, 1989].
Perfluorinated fatty acids are very interesting non-metabolizable analogs. They provoke the in vivo peroxisome proliferative effects which are as great as known peroxisome proliferators such as clofibric acid [Ikeda et a l, 1985]. Using cultured rat hepatocytes, the mechanism of peroxisome proliferation by perfluoro-n-octanoic acid (PFOA), the most potent fatty acid among all perfluorinated fatty acids tested [see Chapter II] was evaluated. To date, the peroxisome proliferative activity of perfluorinated fatty acids has not been examined in any in vitro experiment. Concerning the characterization of PFOA, Chapter IV addresses the following questions:
(1) Is the mechanism of peroxisome proliferation by PFOA similar to or distinct from those of xenobiotics such as ciprofibrate? The pharmacological approaches such as the concentration-response studies, time-course studies and the combination studies of the two compounds are used to answer the question.
(2) The characterization of the effect of non-metabolizable fatty acid are extended to the level of molecular mechanism. Since xenobiotic peroxisome proliferators have been reported to increase mRNA of the induced enzymes during peroxisome proliferation [Reddy et a l, 1986], however, at present, no reports have proposed on the mRNA induction by 39 PFOA eitherin vivo or in vitro. The effects of the inhibition of transcription and translation
on the peroxisome proliferative activity of PFOA are determined as compared to ciprofibrate
and a natural fatty acid.
(3) To date, the morphological changes in cultured rat hepatocytes exposed to non-
metabolizable perfluorinated fatty acid have not been performed. Our objective is, thus, to
reveal the ultrastructural changes in the cultured hepatocytes caused by this fatty acid and
related these changes to the increase in peroxisomal fatty acyl-CoA oxidase (FACO) activity
and in the level of the mRNA encoding FACO.
(4) The inhibition of mitochondrial fatty acid 6-oxidation may be an important
contributor to peroxisome proliferation as described by Lock et al., 1989, and by Elcombe
and Mitchell, 1986 [see section E.2, Substrate overload mechanisml. To investigate the role
of mitochondrial 6-oxidation in the mechanism of peroxisome proliferation by PFOA, various
mitochondrial blockers, i.e., the ketothiolase inhibitor, 2-bromooctanoic acid [Raaka and
Lowenstein, 1979]; the fatty acyl-CoA dehydrogenase inhibitor, 3-mercaptopropionic acid
[Sabbagh et al., 1985] and the carnitine palmitoyl transferase-I (CPT-I) inhibitor, 2- bromopalmitic acid [Mahadevan and Sauer, 1971] were applied to the studies. The enzymatic
functions of each enzyme in mitochondrial 6-oxidation are shown in Figure 1.
(5) Eacho and co-workers suggested that CPT-I is the site of action in the inhibition of mitochondrial oxidation by the peroxisome proliferators 4-THA and bezafibrate, since these compounds possessed CPT-I inhibitory action and cause peroxisome proliferation [Eacho and
Foxworthy, 1988; Foxworthy and Eacho, 1988; Foxworthy et al., 1990b]. POCA (2-[5-(4- chlorophenyl)pentyl]- oxiran-2-carboxylate) is another CPT-I inhibitor that induced peroxisomal 6-oxidation [Bone et al. , 1982]. According to this postulate PFOA as well as ciprofibrate and oleic acid are tested for their CPT I inhibitory effects in isolated 40 mitochondria.
(6) Calcium is reported to have a role in the proliferative effect of hypolipidemic peroxisome proliferators [Watanabe and Suga, 1988; Itoga et al., 1990]. It was also proposed that nicardipine can differentiate between the peroxisome proliferation by xenobiotic peroxisome proliferators and a physiological condition since nicardipine blocked only the effects of clofibrate but not high-fat diets in vivo [Itoga et al. , 1990]. Thus, it is interesting to determine whether the non-metabolizable fatty acid, PFOA, behaves more like xenobiotics or natural fatty acids in vitro. Moreover, the role of calcium in peroxisome proliferation induced by xenobiotics and fatty acid has never been investigated in vitro at present.
2. Significance
High-fat diets cause peroxisome proliferation, as reported by Neat et al. (1980) and
Ishii et al. (1980). The active composition of fatty acids in diets is not known. Since peroxisome function is expected to be regulated in part by fatty acids, understanding the chemical structures of the naturally occurring dietary fatty acids which possess peroxisome proliferative activities is quite significant. Our hypothesis proposes that fatty acids are important endogenous mediators of peroxisome proliferation. Using primary cultures of rat hepatocytes, the structural requirement of fatty acid analogs as active peroxisome proliferators can be revealed.
Perfluorinated fatty acids such as PFOA and PFDA are very potent peroxisome proliferators. These carbon-fluorine compounds are used commercially as surfactants, hydraulic fluids, heat exchangers, and firm-forming foams for fire extinguishers; thus, their mechanism of peroxisome proliferation and the relevance to humans is also of concern.
Finally, peroxisome proliferation represents a phenomenon that links to a growth of 41 subcellular organelles and hepatocarcinoma. Peroxisome proliferation once was thought to be only a toxicity of compounds and then should be avoided. Recently, however, the research on peroxisome proliferation has gained more attention due to a role of peroxisome proliferation in regulating many enzyme activities particularly those involved in fatty acid- metabolizing system and a proposed peroxisome proliferator-activated receptor [Issemann and
Green, 1990]. This phenomenon, thus, might be a natural event that occurs spontaneously so that cells can maintain their homeostasis. In this regards, it is necessary to clarify the role of fatty acids which may act as endogenous components in mediating this phenomenon. The studies should provide us more insights into the mechanism underlying peroxisome proliferation. 42 Table 1. Enzymes Associated with hepatic peroxisomes (Lazarow and DeDuve, 1976; Reddy et a l, 1980; Tolbert, 1981; Kindi and Lazarow, 1982; Goldfischer and Reddy, 1984; Hashimoto, 1987; Hawkins et a l, 1987].
Catalase H202-Generating oxidases Fatty acyl-CoA D-amino acid Urate Glyoxylate Polyamine Dehydrogenases 3-Hydroxyacyl-CoA NAD:gIyceroI phosphate NADP: Isocitrate Xanthine Glucose 6-phosphate Aldehyde 6-Phosphogluconate Other enzymes of fatty acid B-oxidation Fatty acyl-CoA synthetase Enoyl-CoA hydratase Thiolase 3-Hydroxyacyl-CoA epimerase 2,4-dienoyl-CoA reductase Acyl transferases Carnitine acety (transferase Carnitine octanoyltransferase Dihydroxyacetone phosphate acyltransferase Aminotransferases alanine-glyoxylate serine-pyruvate leucine-glyoxylate histidine-glyoxylate Other enzymes NADP:cytochrome c reductase Allantoinase Allantoicase HMG-CoA reductase Hydroxylase and cleavage enzymes of bile acid synthesis Epoxide hydrolase Alkyl dihydroxyacetone phosphate synthetase Acyl/alkyl dihydroxyacetone phosphate: NADPH oxidoreductase 43 Table 2. Classification of peroxisomal disorders (adapted from Naidu and Moser, 1990).
Class Pathogenesis Diseases
1 Reduced or absent Zellweger syndrome of peroxisome Neonatal adrenoleukodystrophy multiple enzyme defects (ALD) Infantile Refsum disease Hyperpipercolic acidemia
2 Normal number of single X-linked ALD peroxisomes; single enzyme Hyperoxaluria Type I defect Acatlasemia Psuedo-Zellweger syndrome- thiolase deficiency Acyl-CoA oxidase deficiency Bifunctional enzyme deficiency
3 Peroxisome present, Rhizomelic chondrodysplasia multiple enzyme defects punctata 44
R “ CH2“ fc - OH Peroxisome Mitochondrion ATP + CoASH I Tatty acyl-CoA synthalas* CamHirM-fadopondwrt CamlffM-dopondont AMPIP + PPI v l pathway pathway
R ” CH2 ~ i - SCoA
02 Fatty acyl-CoA oxldaso H202 u R “ CH = CH - fc “ SCoA
j ^ - wo Enoyl-CoA hydratai* Enoyl-CoA hydratai*
PH 9 R - CH - CH ~ C - SCoA
U- 3-Hydroxyoeyt-CoA 3-Hydroxyocyl-CaA J V NAW + H+ dohydrogonato d«hydrog*nas«
R “ fc “ CH.”" S - SCoA 2
CoASH Ir Bota-kotothlola** B*ta-k«tothlok>*«
R - 2 “ SCoA + CH3“ 2 “ SCoA
Figure 1. Fatty acid B-oxidation pathway in peroxisomes and mitochondria (Adapted from Moser, 1987). 45 V-
Cl Cl Clofibrate Ciprofibrate
.OH
Nafenopin Gemfibrozil
OCHi
Methylclofenapate Fenofibrate
Beclobrate Bezafibrate
Figure 2. Chemical structures of hypolipidemic (fibric acid) peroxisome proliferators. 46
V i V" 0 ,0H IsAHN
B M -15,766 Wy-14,643
■HNT XX .OH
DL-040 Cetaben
co2h
Tiadenol Tibric acid
Figure 3. Chemical structures of non-fibric acid hypolipidemic peroxisome proliferators. 2-Ethylhexanoic acid 2-EthyIhexaldehyde
.OH
2-Ethylhexanol Di(2-ethylhexyl)phthalate (DEHP)
Di(2-ethylhexyl)sebacate (DEHS) Di(2-ethylhexyl)adipate (DEHA)
Figure 4. Chemical structures of plasticizers and related compounds 48
Cl Cl •A Cl Cl
2,4-Dichlorophenoxyacetic acid (2,4-D) 2,4,5-Trichlorophenoxyacetic acid (2,4,5-T)
2-Methyl-4-chloropheoxyacetic acid (MCPA) Lactofen
Figure 5. Chemical structures of phenoxy acid herbicides 49
Valproic acid LY-171,883
OH
OCX ■CH, -C H
Acetylsalicylic acid Benzbromarone
HO'
Dehydroepiandrosterone Chlorpromazine
Figure 6. Chemical structures of miscellaneous drugs that cause peroxisome proliferation 50
,COz H ^ C 0 2h
"CC^H
Tetradecylthiopropionic acid or alky 1thioacetic acid 1, lO-Bis(carboxymethylthiodecane)
or 3-thiadicarboxylic acid
MEDICA-16 2,2,4,4,6,8,8,-heptamethylnonane
CF3(CF2)2C 0 2H CF3(CF2)6C 02H
Perfluoro-n-butanoic acid (PFBA) Perfluoro-n-octanoic acid (PFOA)
CF3(CF2)gC 0 2H CF3(CF2)7S 0 3H
Perfluoro-n-decanoic acid (PFDA) Perfluorinated octane sulfonic acid
Figure 7. Chemical structures of fatty acid analogs with peroxisome proliferative activity 51
PP + R
I PP.R
I
Binds To PP domain Nucleus
1 mRNA translation if Peroxisome proliferation
Figure 8. A receptor-mediated mechanism for peroxisome proliferation (adapted from Rao and Reddy, 1986). Peroxisome proliferators (PP) were proposed to enter cells by diffusion through the plasma membrane and bind to a specific receptor protein (R). The ligand-receptor complex (PP.R) then binds with increased affinity to select site(s) in cellular DNA and triggers the transcription of peroxisome proliferator domain (PP Domain). The increased levels of specific mRNAs lead to increased synthesis of specific proteins which accumulate within smooth membrane channels resulting in the formation of peroxisomes. 52
Peroxisome proliferator ‘(e.g., Metabolite VI of DEHP)
Inhibition of medium-chain Inhibition of long-chain fatty acid oxidation - -► CoA depletion fatty acid oxidation in mitochondria in mitochondria
Accumulation of medium-chain Accumulation of long-chain fatty acids fatty acids
-► Induction of cytochrome P450 IVA1<-
Production of long-chain dicarboxylic acids
Peroxisome proliferation
Figure 9. Proposed mechanism of peroxisome proliferation by DEHP; a substrate overload mechanism (adapted from Lock et al., 1989). CHAPTER II
STRUCTURAL REQUIREMENTS OF FATTY ACIDS ANALOGS:
PERFLUORINATED FATTY ACIDS AND ASYMMETRIC PHENOXYCARBOXYLIC .
ACIDS ON PEROXISOME PROLIFERATION IN
PRIMARY CULTURES OF RAT HEPATOCYTES
A. Introduction
Administration of hypolipidemic agents and phthalate ester plasticizers to rodents increases hepatic peroxisome numbers and peroxisome-associated enzyme activities [Paget
1963; Reddy and Krishnakantha, 1975; Reddy and Lalwani, 1983]. Certain nutritional states such as high fat diets [Nilsson et al., 1987] also cause peroxisome proliferation with an induction of the peroxisomal fatty acid 6-oxidation enzyme although the extent of the induction compared to xenobiotics is low. This may be explained by the rapid degradation of fatty acids in liver. In contrast, several metabolically stable perfluorinated carboxylic acids [Ikeda et al.,
1985], perfluorinated sulphonic acid [Ikeda et al., 1987] and non-6-oxidizable sulfur- containing fatty acid analogues, alkylthioacetic acids [Berge et al., 1989a; Berge et al.,
1989b], are potent peroxisome proliferators in vivo. Thus, the use of these metabolically stable analogs of fatty acids can be a valuable tool for evaluating the role of fatty acids in mediating peroxisome proliferation.
53 54
To further our understanding of structural requirements for fatty acids in mediating peroxisome proliferation, we have evaluated a series of metabolically stable perfluorinated fatty acids of chain length of 4,8 and 10 carbons (C4, C8 and C10, respectively) for their peroxisomal proliferating effects in cultured adult rat hepatocytes. Perfluorinated octanol was also examined to determine whether a carboxylic acid function is required for peroxisome proliferating action. The effects of these perfluorinated fatty acids were compared to the corresponding monocarboxylic acids of the same chain length (C4, C8 and C10).
To date, little information is available which has assessed the role of chirality among peroxisome proliferative agents. Enantiomers of clofibric acid analogs [R(+)- and S(-)-2-(4- chlorophenoxy)acetic acids] were reported to exhibit different potencies for biological activities in several systems [Feller et al., 1987a; Conte-Camerino et al. , 1988; Heiny et al.
1990]. The S(-)-isomers of asymmetric clofibrate analogs possessed more potent actions as inhibitors of chloride conductance in rat skeletal muscle fiber [Conte-Camerino et al., 1988] and as inducers of hepatic peroxisomal fatty acyl-CoA oxidase in cultured rat hepatocytes
[Esbenshade et al. , 1990], On the other hand, the R(+)-isomers showed greater antiplatelet activity in human platelets [Feller et al., 1987a]. Recently, Chinje and Gibson (1991) also demonstrated that there was a stereochemical specificity in the induction of microsomal cytochrome P450 IVA1 and of peroxisome proliferation. In this study, short(/i-propyl)- and medium(n-hexyl)-chain 2-(4-chlorophenoxy)acetic acids were investigated for the role of stereospecificity and carbon chain-length specificity for peroxisome proliferative effects.
B. Specific Aims
The specific aim of these experiments was to determine the structural requirements of fatty acids analogs in mediating hepatic peroxisome proliferation. The concentration- 55
dependent effects of a series of perfluorinated and asymmetric phenoxycarboxylic acids in
inducing the activities of peroxisome proliferation-associated enzymes: peroxisomal fatty acyl-
CoA oxidase (FACO) and microsomal laurate hydroxylase (LH) in primary cultures of rat hepatocytes were measured. In these experiments, clofibric acid was used as a standard agent
of peroxisome proliferation in cultured hepatocytes [Feller et al., 1987b], and the
corresponding natural monocarboxylic acids were included for comparison.
The chemical structures of the fatty acids analogs that were used in these experiments
are shown in Figure 10.
C. Methods
1. Materials
Biochemicals and their sources were the following: 6-aminolevulinic acid, antibiotic/antimycotic solution (100X), bovine serum albumin (fraction V and fatty acid free), clofibric acid, deoxyribonuclease I, dexamethasone, FAD, gentamicin sulfate, glucose 6- phosphate, glucose 6-phosphate dehydrogenase, hydrocortisone 21-hemisuccinate, 12- hydroxydodecanoic acid, insulin, lauric acid, NADH, NADP, palmitoyl-CoA, peroxidase, scopoletin and trypan blue (Sigma Chemical Company, St. Louis, MO): octanoic acid, decanoic acid, heptafluorobutyricacid, pentadecafluorooctanoicacid, nonadecafluorodecanoic acid and 2,2,3,3,4,4,5,5,6,6,7,7,8,8,8-pentadecafluoro-l-octanol(Aldrich Chemical Co.,
Milwaukee, WI), butyric acid and ScintiVerse M scintillation fluid (Fisher Scientific,
Cincinnati, OH), the optically pure R(+)- and S(-)-2-(4-chlorophenoxy)pentanoic acids and
R(+)- and S(-)-2-(4-chlorophenoxy)octanoic acids (gifts from Dr. V. Torterella, Department of Medicinal Chemistry, University of Bari, Italy), collagenase type IV (Cooper Biochemical,
Malvern, PA), [l-14C]lauric acid (58 mCi/mmole) (Amersham, Arlington Heights, IL), Nu- 56
Serum (Collaborative Research Inc., Lexington, MA), Vitrogen (The Collagen Corp., Palo
Alto, CA) and Williams Medium E (Gibco, Grand Island, NY). Other chemicals and organic solvents were of the highest purity available. Sources of other materials were the following:
Anasil G-250thin layer chromatography plates (Analab, Norwalk, CT), Corning tissue culture dishes (100 x 20 mm and 60 x 15 mm) and spectra Mesh (111 and 202 fim) (Fisher Scientific,
Cincinnati, OH), Syringe Filter 0.2 fim (Nalge Company, Rochester, NY) and Sterivex GA
Filters 0.22 fim (Millipore Corp., Medford, MA).
2. Animals
Male Sprague-Dawley rats (225-325 g) were obtained from Harlan Sprague-Dawley,
Inc. (Indianapolis, IN). The animals were housed in an animal facility accredited by the
American Association for the Advancement of Laboratory Animal Care and given food and water ad lib.
3. Isolation and preparation of primary cultures of rat hepatocytes
Hepatocytes were isolated from adult rats by the recirculating collagenase perfusion method of Seglen (1976) with minor modifications. After a 10-15 min perfusion, the cells were collected into a sterile crystallizing dish filled with approximately 25 ml of Williams
Medium E containing 1 % bovine serum albumin and 1 % of antibiotic/antimycotic solution.
The cell suspension was filtered through sterile 202 fim and 111 fim polyethylene
Spectra/Mesh. The crude suspension was purified by centrifuging 3 times for 5 min at 50 x g (the first 2 spins containing a few mg deoxyribonuclease I). Cell viability, determined by trypan blue exclusion, was in the range 75-95%. The hepatocyte suspensions were diluted with a Williams Medium E supplemented with 10 jiM hydrocortisone 21-hemisuccinate, 5 fiM. 57
dexamethasone, 20 mlU/ml insulin, 100 #tM 5-aminolevulinic acid, 50 U/ml penicillin, 50 tig!
streptomycin, 0.125 /ig/ml amphotericin B, 50 ng/ml gentamicin and 10% Nu-Serum.
Hepatocytes were then seeded at 3.5 x 10s cells onto collagen-coated petri dishes (100 mm
diameter) and placed into a humidified 37°, 95% air/5 % C02 incubator for 3 hr. After the
attachment period, freshly prepared medium containing compounds was added.
Compounds were dissolved into dimethylsulfoxide (DMSO) and added directly to
Williams E supplemented media. After the attachment period, media and unattached cells
were aspirated, and fresh media containing drugs in DMSO were added. The final
concentration of DMSO in the culture medium was 0.4% (v/v) in all cases including the
control (absence of drug). Culture medium with compounds was subsequently replaced every
24 hr.
After the desired time of incubation (generally, after 72 hr), hepatocytes were washed
twice with 0.154 M KC1/50 mM Tris-HCl buffer (pH 7.4) and harvested by scraping them
into 1.25 ml (for 100 mm dishes) or 0.5 ml (for 60 mm dishes) Tris-KCl. The cells were
homogenized by sonication for 5 sec at 60-70% output using a Sonic 300 dismembrator
(Fisher Scientific, Cincinnati, OH). The aliquots of each homogenate were taken for the assay
of protein and enzyme activities.
4. Biochemical assays
Protein concentrations of cell homogenates were determined by the method of Lowry
et al. (1951) utilizing bovine serum albumin as the standard. Fatty acyl CoA oxidase (FACO)
activity in cell homogenates was assessed by the fluorometric measurement of hydrogen peroxide (H202) production from palmitoyl-CoA as described by Walusimbi-Kisitu and
Harrison (1983). In the assay, H202 was coupled in a peroxidase-catalyzed reaction to the oxidation of scopoletin, a highly fluorescent compound, to a nonfluorescent product. The preincubation mixture contained the following: cellular protein (2-30 y,g), bovine serum
albumin (0.6 mg), FAD (50 jiM), peroxidase (3 units), scopoletin (1 jiM), Tris-HCl pH 8.3
(60 mM) and Triton X-100 (0.01%). The mixture was preincubated for 5 min at 37°C in a
shaking Dubinoff water bath and the reaction was initiated by the addition of 35 nM palmitoyl-CoA. After 20 min, incubation with palmitoyl-CoA, the reaction was terminated by adding 4 ml of 0.1 M sodium borate buffer (pH 10). The standards and samples were then read for fluorescence from a Aminco-Bowman spectrofluorometer using excitation and emission wavelengths of 395 nm and 470 nm, respectively.
Laurate hydroxylase (LH) activity was determined by measuring the amount of conversion of [l-14C]-lauric acid to the combination of 11-and 12-hydroxylauric acids according to the thin-layer chromatographic method described by Parker and Orton (1980) as modified for cultured rat hepatocytes by Lake et al. (1983) with some minor changes. The incubation mixture contained hepatocyte homogenates (1-2 mg protein), NADPH generating system (10 mM glucose 6-phosphate, 0.5 U/ml glucose 6-phosphate dehydrogenase, 6.25 mM
MgCl2 and 0.5 mM NADP) and 60 ixM (250,000 dpm) [l-,4C]-lauric acid in 50 mM Tris-HCI pH 7.5 for a total volume of 2 ml. The incubation mixtures (without lauric acid) were preincubated for 5 min in a 37° C Dubnoff shaking water bath and the reactions were started by the addition of lauric acid in 2 ptl 95 % ethanol. After a 10 min incubation with lauric acid, the reactions were terminated by adding 0.5 ml 4 N HC1. The lipids were extracted with 5 ml ether overnight and 1.5 ml of the ether layer was removed and evaporated. The resulting residues were dissolved in 50 /il of acetone which contained 1 mM of unlabelled 12- hydroxylauric acid. Twenty fi\ of the acetone dissolved residues were spotted onto Anasil G-
250 thin layer chromatography plates and chromatographed twice in a hexane/ether/glacial 59 acetic acid (90/28.5/1.5) solvent system. They were then sprayed with 1% potassium permanganate/6 % sodium carbonate and the spots corresponding to lauric acid and 11- and
12-hydroxylauric acids were identified by comparing the migrations with the standard sample spots of lauric acid and 12-hydroxylauric acid. The spots were scraped into 20 ml plastic liquid scintillation vials containing 2 ml 95 % ethanol. The silica gel was then dissolved by adding 0.25 ml of hydrofluoric acid and allowed to stand for approximately 1 hr. Ten ml of
ScintiVerse was then added to the samples and the radioactivities (CPM) were measured in a Beckman liquid scintillation spectrometer (model 6800, Palo Alto, CA). The data were expressed as nmoles of 11- and 12-hydroxy [14CJ-lauric acids being produced from
[14C]-lauric acid in 1 hr per 1 mg protein.
5. Determination of cytotoxicity produced bv the compounds
5.1 Morphological determination via light microscopy
The culture dishes containing hepatocytes were observed everyday for the morphological changes. The normal hepatocytes formed a monolayer of well-attached cells on the pre-collagen coated dishes. Cytotoxicity might be observed if the concentration of the test compound was too high. If cytotoxicity occurred, the monolayer of hepatocytes detached from the dishes. The cells became round and lost their nuclei. Consistent with the LDH measurement (see 5.2), morphological observations confirmed that at these concentrations, the compounds produced disruption of the hepatocyte monolayer and loss of cells.
5.2 Lactate dehydrogenase measurement
Lactate dehydrogenase (LDH) activities in media and cell homogenates were determined by following the pyruvate-dependent oxidation of NADH at 340 nm for 1 min at 60
30°C [Bergmeyer, 1974]. Enzyme reaction mixtures contained 0.6 mM pyruvate and 0.18
mM NADH in HEPES buffer (pH 7.4). The solution was added into sampling culture media
at desired time of incubation or hepatocyte homogenate and mixed immediately. Absorbances
were read every 10 sec for a period of 1 min with spectrophotometer (Gilford Instrument,
Model 240, Oberlin, OH). The activity of LDH that was leaked into the medium after 24 hr
or 72 hr incubation periods were expressed as nmole NADH oxidized/min/dish of cells.
Cellular LDH activity was expressed as nmole NADH oxidized/min/mg cellular protein.
Cytotoxicity of compounds was assessed in cell homogenates and media after 72 hr. Since
the compounds may cause an induction of LDH activity like some clofibric analogs that have
been reported earlier [Kocarek et al., 1987], cellular LDH activity was taken into account.
Therefore, percent of viability was estimated by dividing LDH activities in cell homogenates
by total LDH activities which were the activities in homogenates plus LDH that leaked into
culture media.
As shown in Table 3, at high concentrations, the perfluorinated analogues of fatty acid
were toxic to cultured hepatocytes. Treatment with PFBA (> 1 mM), PFOA (> 0.3 mM),
PFOL (> 0.3 mM) and PFDA (5: 0.1 mM) gave percent viabilities that were significantly
less than control. CPIB and all of the natural monocarboxylic acids, on the other hand, did
not provoke any cytotoxic effect even at a concentration up to 1 mM. Interestingly, cytotoxicity caused by these perfluorinated compounds seemed to increase in proportion to the carbon-chain length of the perfluorinated fatty acids i.e. PFBA < PFOA = PFOL < PFDA.
Treatment with PFBA (> 1000 fiM), PFOA (> 300 /*M), PFOL (> 300 fiM) and PFDA
(> 100 ixM) gave percent viabilities that were significantly (P < 0.05) less than control. On the other hand, CPIB and the other monocarboxylic acids did not produce any significant differences in cell viabilities from control even at concentrations up to 1 mM (data not 61 shown).
6. Analysis of data
All culture dishes that received the same compound and its respective concentration
were grouped together over all the experiments for the calculation of the results. In each
experiment, the number of samples for control or drug concentration was 3-4 dishes. Data
were presented in either in the absolute values of the enzyme activities or in the percent of the
corresponding control values within the same experiment. For the absolute values, FACO and
LH activities were expressed as nmole H202 produced/min/mg cellular protein and nmole
lauric acid hydroxylated/hr/mg cellular protein, respectively. Comparisons among the means
were made at P < 0.05 using a Student unpaired t-test (for direct comparisons of means) or
a Dunnett’s test (for multiple comparisons with a control) [Dunnett, 1955].
D. Results
1. Comparison of the effects of natural and metabolicallv stable analogs of
monocarboxylic acids on protein content. FACO and LH activities
As shown on Table 4, natural fatty acid (BA, OA and DA) at concentrations of 0.003 to 0.3 mM did not cause any significant increase in cellular protein content. On the other hand, perfluorinated fatty acids (PFBA, PFOA and PFDA) at concentrations up to 0.3, 0.1 and 0.01 mM respectively, significantly increase total cellular protein content (Table 5).
Corresponding to the LDH measurement, concentrations of perfluorinated fatty acids that produced cytotoxicity (PFBA > 1000 /iM, PFOA > 300 /*M and PFDA > 100 fiM) significantly reduced total protein content of the treated cultured hepatocytes due to the increase in cell death. 62
Along with the increase in cellular protein content, biochemical evaluation of the
activities of the peroxisome-proliferation associated enzymes, FACO and LH, revealed that
only the perfluorinated monocarboxylic acids (C4-CI0), but not their corresponding natural
monocarboxylic acids, elevated FACO and LH activities (Table 5 and Figs. 11-13).
Maximum inductions of FACO and LH activities by PFOA, PFDA and PFBA, compared to
the normalized control (as 100%), were 1372,333,390% and 450, 324,584%, respectively.
Enzyme activities in the presence of the monocarboxylic acids (BA, OA, and DA) at various
concentrations (30 to 1000 jiM) were not significantly different from control values (Figs. 11-
13).
2. Requirement of carboxvlic acid function in the structure of perfluorinated fattv acid
Perfluorinated octanol (PFOL) has the same structure as PFOA except that it has an alcohol moiety instead of a carboxy function. Whereas PFOA at concentrations up to 0.1 mM significantly increased total protein content, PFOL did not produce such significant increases at all of the concentrations tested (Table 6). However, like PFOA, PFOL (> 0.3 mM) significantly reduced cellular protein content at concentrations which caused cytotoxicity
(Table 6).
At the concentrations tested (0.001 mM to 1 mM), PFOL did not elevate FACO and
LH activities whereas PFOA, at 0.1 mM, increased FACO and LH activities 1372% and
450% over the control, respectively (Table 6 and Fig. 14).
3. Relative potencies of perfluorinated fattv acid analogs of different carbon chain-
leneth
PFBA, PFOA, PFDA and CPIB produced concentration-dependent increases in 63 peroxisomal FACO and microsomal LH activities (Figs. 15-16). PFOA (100 iiM) produced
a similar maximal FACO inductive response to that of CPIB (1000 /aM); FACO responses to
PFBA (300 fiM) and PFDA (60 nM) were only 46.2% and 33.5% of that of CPIB, respectively (Table 7). The FACO inductive responses of cultured hepatocytes to PFBA,
PFOA and PFDA decreased markedly at higher concentrations, and the effects were correlated to a reduction in cell viability. PFBA, PFOA and PFDA produced concentration-dependent
increases in LH activity giving maximal elevations at 1000 /aM, 30 /aM and 30 /aM which were 90.4%, 60.5% and 40.8%, respectively, of that exhibited by 1000 fiM CPIB. The
concentrations that produced 2.5-fold inductions and the relative potency for inductions of
FACO and LH by these perfluorinated fatty acids are shown in Table 7. The rank order of potency of the compounds for the inductions of FACO and LH was the same—i.e., PFOA >
PFDA > PFBA > CPIB. Interestingly, PFOA and PFDA were 12- and 5-fold, respectively, more potent than the prototypical peroxisome proliferator, CPIB (Table 7).
4. Effects of the enantiomers of two phenoxvcarboxvlic acid analogs on protein
content and FACO activity in primary cultured rat hepatocytes: comparison to
clofibric acid
The effect of the enantiomers and racemic mixture of 2-(4-chlorophenoxy)pentanoic acid (CPPA) as well as the enantiomers of 2-(4-chlorophenoxy)octanoic acid (CPOA) on cellular protein content and peroxisomal FACO activity are presented in Tables 8 and 9, respectively. Cytotoxicity was produced by the compounds when CPPA and CPOA were added in excess of 0.3 and 0.1 mM, respectively.
The enantiomers of CPPA and CPOA as well as achiral CPIB produced concentration- dependent increases in FACO activity (Figs. 17-18). S(-)-CPPA, at 0.3 mM, produced a 64
maximal increase in FACO activity which was 388% over the control activity. Whereas R(-)-
CPPA did not show any inductive effect at all concentration tested. In agreement with the
chiral 2-(4-chlorophenoxy)pentanoic acid analogs, studies of the enantiomers of 2-(4-
chlorophenoxy)octanoic acid also revealed that the S(-)-isomer was more active than the R(+)-
isomer in inducing FACO (Fig. 18). The maximal FACO induction by S(-)-CPOA was 376%
at the concentration of 0.1 mM whereas R(+)-isomer, at the same concentration, produced
only 118% of the control. Compared to CPIB, the rank order of stimulatory potency was
S(-)-CPOA > S(-)-CPPA > CPIB.
E. Discussion
Primary cultured rat hepatocytes showed demonstrable peroxisome proliferation after exposure to several xenobiotics [Gray et a l, 1983; Lake et a l, 1984; Feller et al., 1987].
Some problems such as loss of function and degenerative changes associated with culture may occur; however, the use of collagen and addition of 2% DMSO in the culture medium were reported to improve the survival of the hepatocytes [Bieri et a l, 1987]. Though in our experiment only 0.4% DMSO was used, the percent of viability and the enzyme activities were maintained after 72 hr treatment with the compounds.
Several studies have shown that fatty acids, including those present in fish oil [Nilsson et a l, 1987] and trans fatty acids [Christiansen et a l, 1985], which are poorly oxidized by the mitochondrial B-oxidation system are inducers of peroxisomal 6-oxidation enzymes. More recently, it has been shown that non-B-oxidizable fatty acids such as alkylthio acetic acids (3- thia fatty acids) [Berge et a l, 1989a; Berge et a l, 1989b] and B,B’-methyl-substituted hexadecanedioic acid (MEDICA 16) [Hertz et a l , 1988] are potent peroxisome proliferators.
Our results also reveal that only the non-oxidizable perfluorinated fatty acids but not natural 65 monocarboxylic acids induced peroxisomal B-oxidation enzymes. Thus, the ability of compounds to resist the mitochondria B-oxidation may be an important factor which determines their hepatic peroxisome proliferative activities [Aarsland et a l , 1989; Berge et al., 1989a; Berge et a l, 1989b].
Perfluorinated fatty acids are metabolically stable analogs of fatty acids due to their strong covalent bonds between carbon and fluorine atoms [Ophaug and Singer, 1980; Ikeda et a l, 1985]. Our experiments show that perfluorinated fatty acids were equally or more potent as peroxisome proliferators than the hypolipidemic phenoxycarboxylic acid, CPIB, in cultured rat hepatocytes. To compare the potencies of perfluorinated fatty analogues which possess differing maximal effects, we determined the concentration of each compound which gave a 2.5-fold induction of FACO and LH activities. In this regard, the rank order of potency for FACO and LH induction was identical (PFOA > PFDA > PFBA > CPIB).
Using hepatic catalase activity as a marker of in vivo peroxisome proliferation, Ikeda et al
(1985) observed the same pattern of rank order potency (PFOA > PFDA > PFBA).
Recently, Kozuka et al (1991) also reported on the same order of perfluorinated compounds in the ability to induce in vivo hepatic peroxisomal and mitochondrial enzymes. In addition, similar to other peroxisome proliferators [Lake et a l, 1984; Kocarek and Feller, 1987;
Sharma et a l, 1988a; Gibson et a l, 1990], the perfluorinated fatty acids also produced a close concentration-dependent relationship for inductions of peroxisomal FACO and the microsomal P450 IVA1-dependent laurate hydroxylase activities.
Ikeda et al (1985) found that a single injection of PFOL to rats caused peroxisome proliferation and suggested that PFOL, which has two hydrogen atoms around the hydroxylated carbon atom, is metabolized to an active compound, PFOA. In cultured hepatocytes, PFOL could not induce either FACO or LH activities whereas PFOA was a 66
potent inducer. Thus, the results indicate that either the oxidation of PFOL occurs to a
significant degree only in vivo or PFOL acts through an indirect mechanism independent of
the liver (e.g. hormone-mediated action). Nevertheless, the present studies strongly suggest
that a carboxylic group is an important structural requirement of perfluorinated fatty acid-
mediated peroxisome proliferation. The carboxylic acid group of fatty acids, in turn, may be
converted to an acyl-CoA thioester which either directly or indirectly perturbs lipid
metabolism leading to peroxisome proliferation [Berge and Aarsland, 1985].
An enantiomeric selectivity of fatty acid analogs was demonstrated for the induction
of peroxisomal FACO in primary cultured rat hepatocytes by the use of asymmetric clofibric
acid analogs. Earlier studies indicated that asymmetric clofibric acid analogs show a
stereoselective response [S(-) > R(+)] [Esbenshade et al. , 1990]. The present studies used
chiral analogs possessing a longer carbon chain-length (5 and 8 carbons) than the initial report and demonstrated that the isomers of CPPA and CPOA were stereospecific inducers of FACO activity, and the active S(-)-isomers possessed an activity which was much greater than CPIB or shorter-chained chiral analogs. Thus, stereospecificity at the a-carbon of the hydrophobic backbone of these chiral fatty acid analogs may provide important evidence for the involvement of peroxisome proliferation activated receptor (PPAR) [Issemann and Green,
1990] in the mechanism of action of these non-metabolized fatty acid analogs.
Chain-length of the carbon backbone may also play an important role in determining the potency of these fatty acid analogs. From the studies of a series of perfluorinated fatty acids (4, 8 and 10 carbon chain length) and a series of phenoxycarboxylic acid enantiomers
(5 and 8 carbon chain length), the stimulatory potency increased in proportion to the chain length with the maximum inductions of FACO activity occurring at a chain-length of 8 carbons. It is possible that longer chain-lengths (e.g. PFDA) were less soluble or more toxic to the cells; thus, it could not induce FACO and LH activities as much as PFOA.
Nevertheless, the present studies demonstrate that there is a dependency on the chain-length
of these fatty acid analogs to induce peroxisome proliferation (Fig. 19). Studies of
asymmetric clofibric acid analogs suggest that the potency for the induction of peroxisomal
enzymes is also dependent upon carbon chain-length, and this activity may be related to their
structural resemblance to stable fatty acids in addition to an intrinsic property associated with
the presence of a 4-chlorophenoxy group. It may be implied that the greater potency with an
increasing carbon-chain length of hydrophobic backbone is either, the more effective in
binding with the peroxisome proliferator activated receptor (PPAR) [Issemann and Green,
1990] or, the more effective in resisting fatty acid metabolism. Ikeda et al. (1988) and
Kozuka et al. (1991) suggested that a lipophilicity of a compound, which is directly related to the length of a hydrophobic carbon backbone of fatty acid analogs, is an important factor
for peroxisome proliferative activity because the greater lipophilicity of the compound, the better the compound can accumulate in the body. However, the results demonstrate that alipophilicity of compounds may not play only a pharmacokinetic role but also a pharmacodynamic role in the activity of peroxisome proliferators since differences in the proliferative activities of perfluorinated fatty acids of different chain-lengths were observed in the in vitro cultured hepatocyte system.
In conclusion, the results of this study have demonstrated that only metabolically stable analogues of fatty acids--i.e. perfluorinated fatty acids (C4, C8 and C,„) chain lengths-cause hepatic peroxisome proliferation as indicated by the increases in activities of peroxisomal
FACO and microsomal LH, a peroxisome proliferation-associated enzyme [Gibson et al.,
1990]. Whereas inductions of FACO and LH by the perfluorinated fatty acids (PFOA,
PFDA, PFBA) were either equal or more potent than CPIB for induction of FACO and LH 68 activities, corresponding monocarboxylic acids were unable to induce peroxisome-associated enzyme activities in primary cultures of rat hepatocytes. PFOL, which lacked a carboxylic group, did not induce peroxisome proliferation in culture hepatocytes. Thus, our data indicate that important requirements for fatty acid analogs as inducers of peroxisome proliferation include the presence of a carboxylic function linked to a hydrophobic backbone and an ability to resist mitochondrial fatty acid 6-oxidation. The stereoselectivity at the a-carbon may also play a role in this phenomenon. 69
CH3-CH2-CH2-COOH
Butanoic acid
CH3-CH2-CH2-CH2-CH2-CH2-CH2-COOH
Octanoic acid
CH3-CH2-CH2-CHr CH2-CH2-CH2-CH2-CH2-COOH
Decanoic acid
CF3-CF2-CF2-COOH
Perfluorobutanoic acid (PFBA)
CF3-CF2-CF2-CF2-CF2-CF2-CF2-COOH
Perfluorooctanoic acid (PFOA)
CF3-CF2-CF2-CF2-CF2-CF2-CF2-CF2-CF2-COOH
Perfluorodecanoic acid (PFDA)
Figure 10. Chemical structures, names and abbreviations of fatty acid analogs used for the studies of structural requirements of fatty acids as mediators for peroxisome proliferation. 70
Figure 10 (continued)
CF3-CF2-CF2-CF2-CF2-CF2-CF2-CH2OH
Perfluorooctanol (PFOL)
CH CH3-C-COOH CH3-CH2-CH2-CH-COOH I o o o o Cl Cl Clofibric acid (CPIB) 2-(4-Chlorophenoxy)pentanoicacid (CPPA)
* CH3-CH2-CH2-CH2-CH2-CH2-CH-COOH 6 o
2-(4-Chlorophenoxy)octanoic acid (CPOA) 71 Table 3. Effects of selected CPIB, mono- and perfluorinated fatty acids on cell viability assessed by LDH activity after 72 hr treatment.
% Viability1* after treatment with compound Comp at the concentration (mM) of ound8
0.01 0.03 0.1 0.3 1
CPIB 82.6+7.4 mmmQ 88.4±2.9 ____« 93.7±0.9
BA 85.4±7.4 88.2±0.7 90.8±2.0 89.4±2.9 97.1 ±1.7
OA — c 86.5 ±1.2 88.5± 1.5 87.6±1.7 80.4±6.9
DA 88.7±0.3 90.0±2.7 81.7±2.4 88.3±1.8 92.7±2.5
PFOA 93.4±1.0 93.3 ±0.7 92.6±1.1 41.1 ±7.2d 46.5±12.01
PFDA 94.3 ±0.9 84.4 ±3.4 68.0±9.3d 52.8±9.2d ___c
PFOL 91.3+0.6 85.9±3.8 88.4±0.7 40.2±10.6d 12.2±5.8d
8 Percent viability of control (no drug) was 90.7 ± 1.0%; n = 15. b Percent viability after 72 hr treatment were determined by (LDH activities of homogenates/total LDH activities) x 100. Values represent the mean ± S.E.M.; n = 3-15. c Not determined. d Means were significantly different from control (P < 0.05). 72
Table 4. Effects of natural monocarboxylic acids on cellular protein content in 72 hr primary cultured rat hepatocytes
Treatment Concentration Cellular protein content® (mM) (mg/dish)
Control 0 3.01 ± 0.06
BA 0.003 2.79 ±0.11 0.01 2.84 ± 0.15 0.03 2.75 ± 0.09 0.1 2.78 ±0.11 0.3 2.86 ± 0.09
OA 0.003 2.74 ± 0.07 0.01 2.88 ± 0.10 0.03 2.78 ± 0.04 0.1 3.11 ± 0.12 0.3 2.85 ± 0.17
DA 0.003 2.73 ± 0.08 0.01 2.84 ± 0.15 0.03 2.81 ± 0.09 0.1 2.74 ± 0.08 0.3 2.89 ±0.11 a Values are mean ± S.E.M., n = 6-13 73 Table 5. Effects of perfluorinated fatty acids and clofibric acid (CPIB) on cellular protein content, peroxisomal FACO and microsomal LH activities in 72 hr primary cultured rat hepatocytes
Treatment Cone. Cellular protein FACO LH
(mM) (mg/dish)a (nmole H202/min/mg)a (nmole/hr/mg)a
Control 0 3.01 ± 0.06 1.00 ± 0.20 2.40 ± 0.30
CPIB 1 3.34 ± 0.13b 9.07 ± 0.96b 17.39 ± 1.3?
PFBA 0.01 3.11 ± 0.16 0.84 + 0.32 1.76 ± 0.24 0.1 3.12 ± 0.13 2.52 ± 0.38” 6.38 ± 1.28b 1 3.22 ± 0.20 4.20 ± 0.94b 15.72 ± 2.90>
PFOA 0.003 3.28 ± 0.1 lb 0.84 + 0.12 2.98 ± 0.46 0.01 3.43 ± 0.13b 2.19 ± 0.17b 8.81 ± 0.63b 0.03 3.55 ± 0.08b 6.24 + 0.61b 10.53 ± 0.77b 0.1 3.26 ± 0.07b 9.41 + 0.7
PFDA 0.003 3.09 ± 0.20 0.76 ± 0.28 1.17 ± 0.23 0.01 3.58 ± 0.09b 0.97 ± 0.15 3.89 ± 1.37 0.03 3.18 + 0.16 2.97 + 0.25b 7.10 + 2.70b
0 Values are mean ± S.E.M., n = 6-12. b Means are significantly different than control (P < 0.05). c Data were discarded due to the cytotoxicity caused by the compounds at high concentrations. 74
O O Butanoic acid 400 PFBA
.1•*-> 300 u D
io 200
« 100
-5.0-5.5 -4.5 -4.0 -3 .5 -3 .0 -2 .5
800
600 c o u -a 400 c X _l 200
6.0 5.5 5.0 4.5 4.0 3.5 -3 .0 -2 .5 Log Molar concentrations
Figure 11. Comparison of monocarboxylic acids (O) and perfluorinated fatty acids (■ ) of the C4 carbon chain length on the induction of peroxisomal fatty acyl-CoA oxidase (FACO) (Top) and microsomal laurate hydroxylase (LH) (Bottom). Cultured hepatocytes were incubated for 72 hr in the presence of compound. The activities are expressed as a percent of the control (••••)• The control activity for FACO and LH were 0.79 ± 0.10 nmole H202/min/mg prot and 2.35 ± 0.34 nmole/hr/mg prot, respectively. Each point represents the mean ± S.E.M. of determination from 3-10 dishes of cells. Asterisk (*) indicates that the value is significantly different (P < 0.05) from the control. 75
1500 O O Octanoic acid ■ — ■ PFOA
£ 1000
u_ 500
0 •— - 6.0 -5 .5 -5 .0 4.5 -4 .0 -3 .5 -3 .0
600
500
.2 400 •4->o =J .1 300 _ix X 200
100
6.5 6.0 5.5 4.55.0 4.0 3.5 3.0 Log Molar concentrations
Figure 12. Comparison of monocarboxylic acids (O) and perfluorinated fatty acids (■ ) of the Cg carbon chain length on the induction of fatty acyl-CoA oxidase (FACO)(Top) and laurate hydroxylase (LH)(Bottom). Cultured hepatocytes were incubated for 72 hr in the presence of compound. The activities are expressed as a percent of the control (••••)• The control activity for FACO and LH were 0.79 ± 0.10 nmole H202/min/mg prot and 2.35 + 0.34 nmole/hr/mg prot, respectively. Each point represents the mean + S.E.M. of determination from 3-10 dishes of cells. Asterisk (*) indicates that the value is significantly different (P < 0.05) from the control. 76
400 O — O Decanoic acid ■ — ■ PFDA c o -+->o 300 Z3 TJ C O Q 200 if 100
-6 .5 - 6.0 -5 .5 -5 .0 -4 .5 -4 .0 -3.5
400
c o 300 o Z3 •O c 200 x
100
- 6.0 -5.5 -5.0 -4.5 4.0 -3 .5 -3.0 Log Molar concentrations
Figure 13. Comparison of monocarboxylic acids (O) and perfluorinated fatty acids (■ ) of the C10 carbon chain length on the induction of fatty acyl-CoA oxidase (FACO)(Top) and laurate hydroxylase (LH)(Bottom). Cultured hepatocytes were incubated for 72 hr in the presence of compound. TTie activities are expressed as a percent of the control (••••). The control activity for FACO and LH were 0.79 + 0.10 nmole H202/min/mg prot and 2.35 ± 0.34 nmole/hr/mg prot, respectively. Each point represents the mean + S.E.M. of determination from 3-10 dishes of cells. Asterisk (*) indicates that the value is significantly different (P < 0.05) from the control. 77 Table 6. Effects of perfluorinated octanoic acid (PFOA) and perfluorinated octanol (PFOL) on cellular protein content, peroxisomal FACO and microsomal LH activities in 72 hr primary cultured rat hepatocytes
Treatment Cone. Cellular protein FACO LH (mM) (mg/dish)B (nmole H202/min/mg)a (nmole/hr/mg)“
Control 0 3.01 ± 0.06 1.00 ± 0.20 2.40 ± 0.30
PFOA 0.01 3.43 ± 0.13b 2.19 + 0.17b 8.81 + 0.63b 0.03 3.55 ± 0.08b 6.24 ± 0.61b 10.53 ± 0.77b 0.1 3.26 ± 0.07b 9.41 + 0.7Qb 9.87 ± 1.28b
PFOL 0.03 3.02 ± 0.13 1.31 + 0.29 0.69 ± 0.22 0.1 2.92 ± 0.13 1.92 ± 0.63 1.68 ± 0.65 0.3 2.14 ± 0.14b 1.67 ± 0.40 2.00 ± 0.88 a Values are mean ± S.E.M., n = 6-12. b Means are significantly different than control (P < 0.05). 78
12 c LU '5 ■ PFOA 10 •*->o g OL 9 □ PFOL X cn o £ < \ o c o £ 6 *s CN O IN $X i = - g 3 if
0 -6 .0 -5 .5 -5.0 -4 .5 -4.0 -3 .5 -3 .0 -2.5
15
LU c 0 ■*->o £ Q. 10 § O l S E x > {±! \ < .22 5 DC O i E 3 3
0 •— - 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 LOG MOLAR CONCENTRATION
Figure 14. Comparison of the effect of perfluorinated octanoic acid (■ ) and perfluorinated octanol (□ ) on FACO (Top) and LH (Bottom) activities. The data were presented as absolute FACO activity (nmole H202/min/mg protein) and absolute LH activity (nmole/hr/mg protein). FACO and LH activities of the control were 1.0 ± 0.2 nmole H202/min/mg protein and 2.4 ± 0.3 nmole/hr/mg protein, respectively. Each point represents the mean ± S.E.M. of determination from 9-12 dishes of cells. Asterisk (*) indicates that the value is significantly different (P < 0.05) from the control. 79
Effects of Perfluorinated Fatty acids and CPIB on FACO induction
- O CPIB LlJ cn s A PFOA oX < ▲ PFDA o o
>- o < i = - 2 2 i£
-6.0 -5.5 -5.0 -4.5 -4.0 -3.5 -3.0 -2.5 LOG MOLAR CONCENTRATION
Figure 15. Concentration-response curves for the induction of peroxisomal fatty acyl-CoA oxidase (FACO) activity by perfluorobutanoic acid (PFBA), perfluorooctanoic acid (PFOA), perfluorodecanoic acid (PFDA) and clofibric acid (CPIB). Cultured hepatocytes were incubated for 72 hr in the presence of compound. Results are expressed as nmole H202 produced/min/mg protein and each point represents the mean ± S.E.M. of determinations from 6 to 9 dishes of cells. The control activity (FACO activity in the absence of compound) was 0.79 ± 0.10 nmole H202/min/mg protein. Inductions of enzyme activity which is greater than control activity (P < 0.05) are indicated by asterisks. 80
Effects of Perfluorinated Fatty Acids and CPIB on LH Induction
20 0 - - - 0 CPIB
Ll I CO - ▲ PFDA Xo O' Dl Q E >- 10 - X Ll I < O' O 5 - 3 i
0 -6.0 -5.5 -5.0 -4.5 -4.0 -3.5 -3.0 -2.5 LOG MOLAR CONCENTRATION
Figure 16. Concentration-response curves for the induction of microsomal laurate hydroxylase (LH) activity by perfluorobutanoic acid (PFBA), perfluorooctanoic acid (PFOA), Perfluorodecanoic acid (PFDA) and clofibric acid (CPIB). Cultured hepatocytes were incubated for 72 hr in the presence of compound. Results are expressed as nmole/hr/mg protein and each point represents the mean ± S.E.M. of determinations from 6 to 9 dishes of cells. The control activity (LH activity in the absence of compound) was 2.35 ± 0.34 nmole/hr/mg protein. Inductions of enzyme activity greater than control activity (P < 0.05) are indicated by asterisks. 81 Table 7. Relative potencies and maximal effects (E^t) for induction of fatty acyl-CoA oxidase (FACO) and laurate hydroxylase (LH) in primary cultured hepatocytes by CPIB and perfluorinated fatty acids.
FACO Activity LH Activity Compound
Concentration* Potencyb cE m« c Concentration* Potency1* “E mx d
CPIB 111.1 ± 5.1 1.00 9.1 ± 1.0f 112.8 ± 6.8 1.00 17.4 ± 1.3f
PFBA 83.0 ± 11.0 1.34 4.7 ± 0.8f 83.5 ± 31.0 1.35 15.7 ± 2.9f
PFOA 9.2 ± 2.7 12.07 9.4 ± 0.7f 3.2 ± 1.4 35.25 10.5 ± 0.8f
PFDA 21.4 ± 10.9 5.19 3.4 ± 0.2f 7.6 ± 1.9 14.84 7.1 ± 2.7f
PFOL mmj 6 1.4 ± 0.2* rmme _____C 2.0 ± 0.9* a Cells were cultured for 72 hr with varying concentrations of test compounds and the concentration (pM) of each compound required for a 2.5-fold induction of each enzyme activity was determined (n = 2-3 separate experiments). b Relative compound potencies were calculated as the ratio of potency of clofibric acid (pM) to potency of test compound G*M). c EmK values for FACO (nmole H202/min/mg prot) were determined using 1000, 300, 100 and 60 pM for CPIB, PFBA, PFOA and PFDA, respectively (Control effect = 1.0 ± 0.2 nmole H202/min/mg prot). d E ,^ values for LH (nmole/hr/mg prot) were determined using 1000, 1000, 30 and 30 pM for CPIB, PFBA, PFOA and PFDA, respectively (Control effect = 2.4 ± 0.3 nmole /hr/mg. e PFOL was not active in the concentration range of 10 to 1000 pM. f values were significantly different (P < 0.05) from corresponding control values. 8 E ^ value of PFOL (at 300 pM) were not significantly different (P > 0.05) from corresponding control values. 82
Table 8. Effects of enantiomers of 2-(4-chlorophenoxy)pentanoic acids (CPPA) and clofibric acid (CPIB) on cellular protein content and peroxisomal FACO activity in 72 hour primary cultured rat hepatocytes.
Treatment Concentration Cellular protein FACO (mM) (mg/dish)a (nmole H202/min/mg)'
Control 0 1.44 ± 0.22 0.59 ± 0.25
R(+)-CPPA 0.1 1.75 ± 0.48 0.66 ± 0.34 0.3 1.39 + 0.17 0.24 + 0.09
S(-)-CPPA 0.1 1.45 ± 0.14 0.77 ± 0.23 0.3 1.59 ± 0.13 1.82 ± 0.42b
(±)-CPPA 0.1 2.81 ± 0.69 0.92 ± 0.23 0.3 1.84 ± 0.17 1.89 ± 0.98b
CPIB 1 1.32 ± 0.32 2.26 ± 0.25b a Values are mean ± S.E.M., n = 3-6. b Means are significantly different than control (P < 0.05) 83 Table 9. Effects of enantiomers of 2-(4-chlorophenoxy)octanoic acids (CPOA) and clofibric acid (CPIB) on cellular protein content and peroxisomal FACO activity in 72 hour primary cultured rat hepatocytes
Treatment Concentration Cellular protein FACO (mM) (mg/dish)a (nmole H202/min/mg)a
Control 0 2.17 ± 0.14 0.65 ± 0.04
R(+)-CPOA 0.03 2.76 ± 0.27 0.78 ± 0.16 0.1 3.00 + 0.26b 0.76 ± 0.02
S(-)-CPOA 0.03 2.63 + 0.10 1.40 ± 0.25b 0.1 2.88 ± 0.15b 1.57 ± 0.13b
CPIB 1 2.45 ± 0.35b 2.36 ± 0.49b a Values are mean ± S.E.M., n = 3-6 b Means are significantly different than control (P < 0.05) 84
Effects of Enantiomers of 2—(4-Chlorophenoxy)pentanoic acid on FACO Induction
500 + O — O R (+)—CPPA • ----- # S(—)—CPPA T #oc 400 ■ o CPIB 1 3 TJ C 300 O g 200
100 -
0 +■ + 1------1------5 .5 -5 .0 -4.5 -4.0 -3.5 -3 .0 -2 .5 Log Molar concentrations
Figure 17. Concentration-response curves for the induction of fatty acyl-CoA oxidase (FACO) activity by the enantiomers of 2-(4-chlorophenoxy)pentanoic acid [R(+)-CPPA and S(-)-CPPA] as compared to clofibric acid (CPIB). Cultured hepatocytes were incubated for 72 hr in the presence or absence of compound. The results are expressed as the percent of FACO induction as compared to the control activity (0.59 ± 0.25 nmole H202/min/mg protein). Each point represents the mean + S.E.M. of determinations from 6 dishes of cells. 85
Effects of Enantiomers of 2—(4—Chlorophenoxy)octanoic acid on FACO induction
500 A — A R (+ )- cpo a CPIB
A — A s (-)- cpo a § 400 T ’-P A o | 300 o § 200 X
1 0 0 ■A-
o -5.5 -5.0 -4.5 -4.0 -3.5 -3.0 -2.5 Log Molar concentrations
Figure 18. Concentration-response curves for the induction of fatty acyl-CoA oxidase (FACO) activity by the enantiomers of 2-(4-chlorophenoxy)octanoic acid [R(+)-CPOA and S(-)-CPOA] as compared to clofibric acid (CPIB). Cultured hepatocytes were incubated for 72 hr in the presence or absence of compound. The results are expressed as the percent of FACO induction as compared to the control activity (0.65 ± 0.04 nmole H202/min/mg protein). Each point represents the mean ± S.E.M. of determinations from 6 dishes of cells. 86
Effects of a series of perfluorinated fatty acids and 2 -(4 —chlorophenoxy)carboxylic acids on FACO induction
A ----A PFBA O — O S(—)—CPPA □ ----□ S(—)—CPOA c 600-- ■ ----■ PFOA
T)
Li-
200 ~
- 6.0 -5 .5 -5 .0 -4 .5 -4 .0 -3 .5 -3 .0 Log Molar concentrations
Figure 19. Concentration-response curves for the induction of fatty acyl-CoA oxidase (FACO) activity by perfluorobutanoicacid (PFBA), perfluorooctanoic acid (PFOA), S(-)-2-(4- chlorophenoxy)pentanoic acid (CPPA) and S(-)-(4-chIorophenoxy)octanoic acid (CPOA). Cultured hepatocytes were incubated for 72 hr in the presence or absence of compound. The results are expressed as the percent of FACO induction as compared to the control activity (0.7 ±0.1 nmole H202/min/mg protein). Each point represents the mean ± S.E.M. of determinations from 6-12 dishes of cells. CHAPTER III
NATURALLY OCCURRING FATTY ACIDS AS MEDIATORS FOR PEROXISOME
PROLIFERATION: THE SIGNIFICANCE OF FUNCTIONAL GROUPS
IN AN ACTIVE PROLIFERATOR STRUCTURE
AND THE ROLE OF DICARBOXYLIC ACIDS
A. Introduction
High-fat diets as well as conditions that increase lipid influx to the liver such as diabetes, starvation, and vitamin E deficiency, induce peroxisomal fatty acid 8-oxidation and peroxisome proliferation [Ishii et ah, 1980; Neat et al., 1980; Horie et ah, 1981; Reddy et al, 1981; Thomassen et ah, 1982; Osmundsen et ah, 1987]. However, in contrast to effects observed with administration of xenobiotics, the increase in peroxisomal 8-oxidation caused by these dietary conditions are relatively small (2-fold at maximum) [Ishii et ah, 1980; Neat et ah, 1980; Thomassen et ah, 1982; Reddy and Lalwani, 1983; Moody et ah, 1991].
Dietary conditions that produce maximal peroxisome proliferation occur when rodents are fed partial hydrogenated fish oil or rapeseed oil in vivo [Thomassen, 1981].
The optimal structure of naturally occurring fatty acids as peroxisome proliferators is not known. Partial hydrogenated fish oil and rapeseed oil are rich in C22:i fatty acids such as erucic acid or brassidic acid [Thomassen et ah, 1982]. It was suggested that the C22:I, and in particular trans fatty acid (brassidic acid) which are poorly oxidized by mitochondria, may be responsible for the peroxisomal proliferative effect of these dietary oils. More recently;
87 88 however, Flatmark et al. (1988) did not find increases in peroxisomal fatty acid B-oxidation enzymes in rats fed with C22;1 added soybean oil. Spydevold and Bremer (1989) suggested that the inductive property of high fat diets may be due to its content of shorter-chain fatty acids such as C12 and C,4 rather than the content of long-chain fatty acids. Since only a few structure-activity relationship works have been performed with these natural fatty acids
[Christiansen et al. , 1985; Hertz et al. , 1985; Lundgren et al. , 1987b; Spydevold and Bremer,
1989], the role of naturally occurring fatty acids involved in peroxisome proliferation remains unclear.
The substrate overload mechanism of peroxisome proliferation (Fig. 9) suggests that the accumulation of fatty acids caused by either the high influx of fatty acid into the liver or the inhibition of mitochondrial fatty acid B-oxidation by peroxisome proliferators leads initially to an induction of cytochrome P450IVA1 (P452), now classified as CYP4A1 [Nebert et al.,
1991] and subsequently to an induction of peroxisomal fatty acyl-CoA oxidase (FACO) [Lock et al., 1989]. A close association has always been observed between the induction of this cytochrome and peroxisomal B-oxidation enzymes by peroxisome proliferators [Sharma et al. ,
1988a; Lock et al., 1989; Gibson et al. , 1990]. Induction of cytochrome P450IVA1 which exhibits a narrow substrate specificity for the to- or gj-1-hydroxy lation of fatty acid [Tamburini et al. , 1984] causes an accumulation of cellular &>-hydroxy fatty acid which is then further oxidized to the corresponding dicarboxylic acids. Dicarboxylic acids, in particular of the long-chain length, are proposed to act as proximal stimuli for the observed hepatic peroxisome proliferation [Sharma et al. , 1988b; Lock et al. , 1989].
B. Specific Aims
The substrate overload hypothesis suggested that peroxisome proliferators exert their 89 effects by inhibiting fatty acid oxidation and causing fatty acid accumulation. To validate this hypothesis, a new scheme emphasizing only the role of fatty acids was drawn (Fig. 20).
According to Fig. 20, various structures of natural fatty acids (1) were applied to cultured hepatocytes to represent the situation where fatty acids are overloaded. The effects of these natural fatty acids were evaluated for their peroxisome proliferative activity in hepatocytes.
These selected fatty acids are fatty acids of short-, medium- and long-chain monocarboxylic acids (C4-C18) as well as a series of long-chain saturated and unsaturated fatty acids (C18:0,
Cjg;1, C18:2 and Ci8:3). Long-chain polyunsaturated fatty acids (C20;5 and C22:6) which are commonly found in fish oil are also investigated since they are substrates for peroxisomal but not mitochondrial fatty acid 8-oxidation. These findings should provide a better understanding on the active structures of naturally occurring fatty acids such as those that are present in high fat diets.
Another important objective of this chapter is to determine if dicarboxylic acids (3) act as a proximal stimulus for peroxisome proliferation as described in the substrate overload hypothesis [Sharmaet a l, 1988b; Lock et al., 1989]. According to this hypothesis, dicarboxylic acids should induce hepatic peroxisomal fatty acid 8-oxidation enzymes (i.e.,
FACO) without an accompanied increase in microsomal cytochrome P450 IVA1 as assessed by the increase in the activity of laurate hydroxylase (LH). Dicarboxylic acids of short-, medium- and long-chain length (C4-C16) were included in this investigation. The studies also extend to the w-hydroxy fatty acids (2), which are the products of cytochrome P450 IVA1 hydroxylation and are further oxidized to corresponding dicarboxylic acids.
The chemical structures, names and abbreviations of the fatty acids and analogs that were used in the studies are shown in Table 10. 90
C. Methods
1. Materials
The biochemicals used and their sources were the following: butyric acid (Fisher
Scientific, Cincinnati, OH), octanoic acid, decanoic acid, 2-bromohexadecanoic acid, (±)-2- bromooctanoic acid (Aldrich Chemical Co., Milwaukee, WI), 3-mercaptopropionic acid, myristic acid, lauric acid, stearic acid, oleic acid, linoleic acid, linolenic acid, sebacic acid, suberic acid, succinic acid, 1,12-dodecanedioic acid, hexadecanedioic acid, 12- hydroxydodecanoic acid, 16-hydroxyhexadecanoic acid, cycloheximide, 5,5’-dithiobis-(2- nitrobenzoic acid) [DTNB], EDTA and DL-camitine (Sigma Chemical Company, St. Louis,
MO). Other materials used were the same as those described in Chapter II, section C .l,
Materials.
2. Isolation and preparation of primary cultures of rat hepatocytes
The animals and hepatocyte isolation procedures used were the same as those described in Chapter II, section C.2, and C.3, Animals and Isolation and preparation of primary cultures of rat hepatocytes. To determine the inductive effects of naturally occurring fatty acids on
FACO and LH activities, hepatocyte cultures were treated with the fatty acids in the same manner as that described in Chapter II. Media and tested compounds were changed daily and cells maintained for a maximum of 5 days. Unless noted otherwise, fatty acid analogs were tested at concentration ranges from 0.1-1 mM. Standard peroxisome proliferators were used at 0.1 mM and 1.0 mM for PFOA and CPIB, respectively. For most studies, cultures were harvested after 3 days. For experiments on the inhibition of mitochondrial fatty acid 6- oxidation, both the mitochondrial 6-oxidation blockers (2-bromopalmitic acid, 2- bromooctanoic acid and 3-mercaptopropionic acid) and the tested fatty acids were dissolved 91 in DMSO before being added together at the desired concentrations into the culture media.
The final concentration of DMSO in the media was 0.4%(v/v) in all cases including the control. To study the cycloheximide effect, cycloheximide (1 jig/ml) was added with fatty acid (oleic acid) in the last two days of incubation. For the time-course study, the treated cultures were harvested every 24 hr and measured for enzyme activities which include FACO and carnitine acetyltransferase (CAT) throughout a 5 day time period. The harvesting procedures are the same as those described in Chapter II, section C.3, Isolation and preparation of primary cultures of rat hepatocytes.
3. Biochemical assays
The assays for total cellular protein, FACO and LH activities are the same as those described in Chapter II, section C.4 Biochemical assays. In addition, carnitine acetyltransferase (CAT) activity was included as another biochemical marker for peroxisome proliferation [Gray et al., 1982; Feller et al., 1987b]. CAT activity was assessed by measuring the carnitine-stimulated formation of coenzyme A from acetyl CoA as described by Gray et al. (1982) with some modification. Reagent mixtures contained 0.25 mM acetyl
CoA, 0.156 mM 5,5’-dithiobis-(2-nitrobenzoicacid), 1.25 mM EDTA, 100-200 fig protein of cultured homogenates, 3.125 mM DL-camitine (experimental tube only) and 50 mM Tris-
HC1 buffer pH 8.0 in a total volume of 1 ml. After a 25 min-incubation period at room temperature, CAT activity was measured by using a Gilford model 240 spectrophotometer with an autosampler at 412 mm, and enzyme activity calculated using an extinction coefficient of 13.6 cm'1 mM'1 [Gray et al., 1982]. 92 4. Determination of cvtotoxicitv produced by the compounds
Cytotoxicity caused by tested fatty acid was determined by morphological determination as well as by LDH activity released into the media expressed as a ratio to LDH activity in the cell homogenates as described in Chapter II, section 5. None of the fatty acid concentrations presented in this chapter caused any cytotoxicity. However, when concentrations of these fatty acids exceeded 1 mM, increased LDH concentrations were observed in the culture medium. Moreover, some cytotoxicity was observed through morphological determinations; the hepatocyte monolayer started to detach and the cells appeared in a circular shape instead of a flat elongated shape which was seen in the normal cultured hepatocytes.
5. Analysis of data
Data were statistically analyzed by comparisons among the means using Student’s unpaired t-test for direct comparisons of means. In case of multiple comparisons between means of different treatments (i.e., no compound, one compound, and combination of compounds), one-way analysis of variance followed by a Student-Newman-Keuls test was used
[Tallarida and Murray, 1987].
D. Results
1. Comparison of various chain length of naturally occurring monocarboxvlic fatty
acids
Naturally occurring saturated monocarboxylic acids of various chain length (C4:0, C8:0,
Cio:o> C18:0 and C22:0) were investigated for their abilities to increase the activity of peroxisomal fatty acyl-CoA oxidase (FACO) and microsomal laurate hydroxylase (LH). AH of these fatty 93 acids at the concentration tested (0.1 and 1 mM) did not produce any significant increase in
FACO activity as compared to the control (as 100%) (Fig. 21, top). Similarly, these natural fatty acids did not increase the activity of microsomal LH; therefore, they did not induce cytochrome P450IVA1 (Fig. 21, bottom).
The positive control, 0.1 and 1 mM CPIB, increased both FACO and LH activities.
The maximum induction of FACO and LH, occurred at 1 mM CPIB, were 758% and 597% of the control, respectively. PFOA, the non-metabolizable fatty acid, also revealed inductions of both FACO and LH activities with the maximum (at 0.1 mM) of 1100% and 373% of control, respectively (Fig. 21).
2. Comparison between saturated and unsaturated fattv acids of the same chain length
Fatty acids of the same chain length but different in degree of unsaturation (C18:0, C18:1,
C18:2 and C18;3) were compared. Concentration-dependent studies reveal that only oleic acid
(C18:1) and linoleic acid (C18:2) produced a concentration-dependent increases in FACO and LH activities (Fig. 22, Table 11). Stearic acid (C18:0) did not increase FACO or LH activities
(Fig. 22, Table 11). Linolenic acid (C18:3), at a concentration up to 0.1 mM, did not increase
FACO or LH activities and at a high concentration (1 mM) caused cytotoxicity to hepatocytes
(Table 11). The most potent C18 fatty acid in increasing peroxisomal FACO activity was oleic acid (1 mM) (Figs. 22-23). Linoleic acid, having 2 double bonds in its structure, at the concentration of 1 mM increased microsomal cytochrome P450IVA1 (LH) greater than oleic acid at 1 mM (Figs. 22-23).
Table 12 shows the effects of long-chain polyunsaturated fatty acids ( C ^ and C22:6) on the peroxisomal enzyme activity. At a low concentration (0.01 mM), the long-chain polyunsaturated fatty acids slightly though not significantly increased FACO activity. 94
However, at higher concentration (0.1 mM), these fatty acids produced cytotoxicity (Table
12).
3. Characterization of oleic acid-increased enzvme activities: time-course study and
effect of cycloheximide
Among all naturally occurring fatty acid tested, oleic acid (1 mM) showed the greatest effect on the peroxisomal FACO activity (approximately 2-fold increase over the control).
Therefore, studies were designed in order to further characterize the time-dependent effect of this fatty acid on peroxisomal FACO activity. Time-course study revealed that the increase in FACO activity appeared after the cultures were exposed to oleic acid for 3 days (Fig. 24).
Oleic acid also produced a time-dependent increase in CAT activity: another enzyme which is used to assess the effects of peroxisome proliferators [Feller et al., 1987](Fig. 25). The increase in CAT activity was also seen after 3 days of fatty acid treatment and continued to increase throughout a 5 day-incubation with oleic acid (1 mM).
Cycloheximide, an inhibitor of protein synthesis (translation process), did not inhibit the induction of peroxisomal FACO by oleic acid as shown in Table 13. In contrast, cycloheximide blocked the ciprofibrate-induced FACO activity by 46% (Table 13).
4. Role of dicarboxylic acids: effects of short-, medium-, and long-chain dicarboxylic
acids and qj-hvdroxv fattv acids
The effects of various chain lengths of dicarboxylic acids in peroxisomal FACO and microsomal LH activities are given in Table 14. Short- and medium-chain dicarboxylic acids did not increase FACO and LH activities; moreover, they appeared to significantly attenuate both enzyme activities (Table 14). Long-chain dicarboxylic acids (C12 and Ct6) at 0.01 mM 95 slightly but significantly increased FA activity without an effect on LH activity (Table 14).
Higher concentrations of these fatty acids had no effect on these enzyme activities. At 1 mM, the C12 and Ci6 dicarboxylic acids exhibited cytotoxicity.
The effects of co-hydroxy fatty acids are shown in comparison to their corresponding dicarboxylic acids (Table 15). Both types of the fatty acids significantly induced FACO at
0.01 mM, and the maximum induction was approximately 2-2.5 fold of the control.
5. Effects of various mitochondrial fatty acid B-oxidation blockers on the effects of
short- and medium-chain mono- and di-carboxvlic acids
2-Bromooctanoic acid (BOA), mercaptopropionic acid (MPA) and 2-bromopalmitic acid (BPA) were used in these experiments as inhibitors of mitochondrial fatty acid B- oxidation. By comparison to their own control (vehicle with mitochondrial blocker and without fatty acid), it is seen that the FACO activity did not increase or decrease by mono- or di-carboxylic acid C4-Cio in the presence of these mitochondrial B-oxidation blockers (Table
16). Thus, by blocking mitochondria B-oxidation, short- and medium chain mono- and di carboxylic acids did not produced any significant changes in their FACO profile. It is noteworthy to notice that BPA, the carnitine palmitoyltransferase I inhibitor, alone, produced a slight but significant increase in FACO activity (Table 16). This interesting finding will be discussed more in Chapter IV.
E. Discussion
As shown in Figure 21, saturated monocarboxylic acids of C4,Cg, C10, C18 and C22 did not produce any significantly increase in either FACO or LH activity, whereas the xenobiotics, CPIB and perfluorooctanoic acid, markedly induced both FACO and LH 96 activities. Our results, thus, indicate that saturated monocarboxylic acids of short- (C4 to C10) or long-carbon chain length (C18 to C22) did not play a role in mediating peroxisome proliferation.
Hertz and Bar-Tana (1985) showed that dodecanoic acid (C12) increased enoyl-CoA hydratase and palmitoyl-CoA oxidase in cultured rat hepatocytes. In this regard, Spydevold and Bremer (1989) suggested that medium-chain fatty acids (C12-C,4) presented in high fat diets are responsible for peroxisome proliferation. They reported that myristic acid, C14 saturated monocarboxylic acid, induced the peroxisomal palmitoyl-CoA oxidation in 7800 Cl
Morris hepatoma cells. The induction was approximately 8-fold over the control by 1 mM myristic acid, and there was a rapid drop in the effect with longer chain length. In our preliminary experiment, myristic acid, at the concentrations of 0.1 and 1 mM, increased peroxisomal FACO activity in cultured rat hepatocytes (data not presented). Thus, there is a possibility that medium-chain (C,2 to C,4) monocarboxylic acids may contain suitable lengths as mediators for peroxisome proliferation. Since these fatty acids are good substrates for laurate hydroxylase, which specifically co-hydroxylates medium-chain monocarboxylic acids, their proliferative activities might occur through the action of their corresponding oo- hydroxylated fatty acids and dicarboxylic acids. The effects of medium-chain dicarboxylic acids and hydroxylated fatty acids are discussed below.
Although a saturated C,8 fatty acid (stearic acid) did not increase FACO activity, selected unsaturated fatty acids of C18 chain length (oleic and linoleic acids) significantly increased FACO activity in cultured rat hepatocytes. These findings indicate that an unsaturated function in the structure of fatty acids is important for their peroxisome proliferative effects. Unsaturated fatty acids are relatively more resistant to mitochondrial fatty acid 6-oxidation than their saturated acids [Osmundsen et al., 1987]. At least 2 more 97 enzymes-i.e., cis-3, trans-2-enoyl CoA isomerase, and 3-hydroxyacyl-CoA epimerase-are required for the degradation of unsaturated fatty acids in mitochondria. These enzyme reactions also occur during the degradation of unsaturated fatty acids in peroxisomes; however, there is no direct proof of these additional enzymes in this organelle [Mizugaki et al., 1982]. Kunau and Dommes (1978) demonstrated that 2,4-dienoyl-CoA reductase is an alternative pathway for the degradation of unsaturated fatty acids in mitochondria, and peroxisomes and this reductase was significantly induced by peroxisome proliferators
[Mizugaki et al., 1982]. Polyunsaturated fatty acids (C ^ and C22:6) are fatty acids that are present in fish oils [Stansby, 1990]. These fatty acids are poorly metabolized by mitochondria
[Osmundsen, 1982; Mannaerts and VanVeldhoven, 1990], and this may explain the increase in FACO activity by these very long-chained fatty acids. Collectively, the results suggest that the fatty acids that increase FACO activity are also relatively resistant to fatty acid B-oxidation mainly due to their poor susceptibility to mitochondrial oxidation. Therefore, ability to resist mitochondrial fatty acid B-oxidation is an important factor for the natural fatty acids to possess the peroxisome proliferative effect.
In vivo studies by Nilsson et al. (1987) showed a parallel stimulation of peroxisomal
B-oxidation and microsomal co-oxidation by high-fat diets. Similarly, in these present studies, unsaturated fatty acids, oleic acid (C18:,)and linoleic acid (C18:2), increased both FACO and LH activities in primary cultures of rat hepatocytes. Linolenic acid (C18:3), however, did not increase the enzyme activity which may be explained by its cytotoxic effect to the hepatocytes.
Oleic acid, the C18 fatty acid having only one double bond, was a more potent stimulator of
FACO activity than linoleic acid which possesses two double bonds (Fig. 23). Therefore, the ability of unsaturated fatty acids to increase peroxisomal FACO activity was not correlated with an increase in the number of unsaturated bonds. In contrast, the activity of microsomal 98
LH was elevated in proportion to the degree of unsaturation; linoleic acid was much more
potent than oleic acid in stimulating LH activity (Fig. 23).
Characterization of the enzyme induction by oleic acid revealed that the stimulatory
effect occurred after 72 hr of exposure. The time-course and magnitude of FACO activity
changes indicated that the increase in enzyme activity may not be due to the true induction of
enzyme synthesis. The maximal increase in FACO activity that appeared after 120 hr of
exposure was not greater than the cellular FACO activity assayed immediately after cell were
isolated (Fig. 24, top). Further, cycloheximide, the inhibitor of protein synthesis, did not
inhibit the increase in FACO activity by oleic acid whereas it inhibited the effect of
ciprofibrate (Table 13). Thus, the increase in FACO activity by oleic acid is not related to de novo enzyme synthesis. This suggests that oleic acid’s effect on FACO may be associated
with a decrease in enzyme degradation. Horie and Suga (1989) reported on the change in turnover rate of peroxisomal FACO in rats fed a partially hydrogenated fish oil diet. They
indicated that the observed increase in FACO activity was due to a reduced rate of FACO
degradation.
According to the hypothesis of Lock et al. (1989), dicarboxylic acids are proximal
stimuli for hepatic peroxisome proliferation. Dicarboxylic acids of C4 to C10 did not reveal any peroxisome proliferative effects in rat hepatocyte cultures (Table 14).* These may be because C4 to C10 dicarboxylic acids are not substrates for peroxisomal fatty acid B-oxidation and/or they are readily degraded by mitochondrial B-oxidation [Mannaerts and VanVeldhoven,
1990]. The effect of dicarboxylic acid as well as its corresponding monocarboxylic acid of
C8 chain length was examined in the presence of various inhibitors of mitochondrial fatty acid
B-oxidation. 2-Bromopalmitic acid (BPA) inhibits mitochondrial B-oxidation by preventing the transportation of fatty acid into the mitochondrial compartment due to the inhibition of 99 carnitine palmitoyl transferase-I [Mahadevan and Sauer, 1971; Schulz, 1987].
Mercaptopropionic acid (MPA) [Sabbagh et a l, 1985] and 2-bromooctanoic acid (BOA)
[Raaka and Lowenstein; 1979; Schulz 1987] block mitochondrial B-oxidation by inhibiting fatty acid dehydrogenase (the first enzyme in mitochondrial B-oxidation) and 3-ketothiolase, respectively. In the presence of these inhibitors, octanoic acid and octanedioic acid still did not increase FACO activity (Table 16). Therefore, it is obvious that although mitochondrial
B-oxidation was blocked, these short-chain mono- and di-carboxylic acids were still not substrates for the peroxisomal fatty acid B-oxidation system. In fact, the data showed that short-chain dicarboxylic acids, in particular, those of C8 and C10 carbon chain length, not only exhibited no FACO inductive effects, they produced a concentration-dependent inhibition of peroxisomal FACO and microsomal LH (Table 14). In agreement, Poosch and Yamazaki
(1989) also demonstrated the substrate inhibition of peroxisomal FACO activity by mono-CoA esters of hexanedioic, octanedioic and decanedioic acids. These shorter chain dicarboxylic acids accumulated in hepatocytes and slowed the peroxisomal B-oxidation cycle [Poosch and
Yamazaki, 1989].
The present experiments demonstrated that peroxisomal FACO activity, and to a lesser extent, microsomal LH activity are increased only by long-chain (C12- and C16) dicarboxylic acids and their structurally related w-hydroxylated fatty acids as well as unsaturated fatty acids of C18. Thus, the chain-length of carbon backbone and an unsaturated function of fatty acids play an important role in designating fatty acids as substrates for mitochondrial or peroxisomal oxidation. The present experiments indicate that only the appropriate substrates for peroxisomal fatty acid oxidation can increase FACO activity.
Taken collectively, our results reveal that C12 and C16 dicarboxylic acids as well as their corresponding w-hydroxyfatty acids, which are good substrates for the peroxisome fatty 100 acid B-oxidation pathway [Poosch and Yamazaki, 1989], were able to increase FACO activity.
These findings are in agreement with the substrate overload theory presented by Lock et al.
(1989) and Sharma et al. (1988b). Similarly, Nilsson et al. (1987) previously reported a slight induction of peroxisomal fi-oxidation in the liver of rat fed with high hexadecanedioic acid diet. However, since only small increases in FACO activities are observed by these dicarboxylic acids makes it not likely to conclude that long-chain dicarboxylic acids are proximal stimuli for peroxisome proliferation induced by other peroxisome proliferators. By contrast, the increases in FACO activity by xenobiotics are as great as 10- to 30-fold over the control. Accordingly, the substrate overload theory by Lock et al. (1989) and Sharma et al.
(1988b) may be applied for the case of the inductive effect by naturally occurring fatty acids that are either relatively resistant to the mitochondrial fatty acid B-oxidation (i.e., unsaturated long-chain fatty acids), and/or are good substrates for the peroxisomal fatty acid B-oxidation
(i.e. medium- and long-chain dicarboxylic acids). The substrate overload theory; however, may not be responsible for the mechanism by which exogenous compounds exert their powerful peroxisome proliferative effect. In fact, although diethyhexyl phthalate (DEHP) treatments in vivo induced w-hydroxylation of fatty acids, increases in the levels of w-hydroxy fatty acids and dicarboxylic acids have not been observed in liver or plasma of rats fed DEHP
[Okita and Okita, 1990]. Thus, the mechanism underlying a potent FACO inductive effect by the non-metabolizable perfluorinated fatty acid is addressed further in Chapter IV. 101
Fatty acids (1) if Induction of cytochrome P450 IVA1 (LH) if w-OH fatty acids (2) if Dicarboxylic acids (3) if Substrates for peroxisome fatty acid B-oxidation system if t Peroxisome B-oxidation enzymes (FACO) and Peroxisome proliferation
Figure 20. Schematic illustration of the hypothesis presented in Chapter III. Some naturally occurring fatty acids (1) which meet the structural requirement as peroxisome proliferators are suggested to initially increase cytochrome P450IVA1 activities. This induction results in an increase in the level of w-hydroxyfatty acid (2) and subsequently dicarboxylic acids (3). Dicarboxylic acids, good substrates for peroxisomal fatty acid B-oxidation, are proposed to be the proximal stimuli for induction of peroxisomal enzymes (e.g., FACO) and peroxisome proliferation. To examine the hypothesis, each selected fatty acid (i.e., 1,2 and 3) was tested for its ability to increase activities of FACO and, in some cases, LH. The results from this study should help validating the substrate overload mechanism by fatty acids. 102 Table 10. Chemical structures and names of fatty acids and analogs used in the effect of naturally occurring fatty acids on peroxisome proliferation.
Structures Names
Monocarboxvlic acids
CH jCCH^COOH Butanoic acid
CH3(CH2)6COOH Octanoic acid
CH^CH^COOH Decanoic acid
Lone-chain saturated and unsaturated fattv acids of C,„
CH^CH^COOH Stearic acid (18:0)
CH3(CH2)7CH=CH(CH2)7COOH Oleic acid (18:1)
CH3(CH2)4(CH=CHCH2)2(CH2)6COOH Linoleic acid (18:2)
CH3CH2(CH=CHCH2)3(CH2)6COOH Linolenic acid (18:3)
Dicarboxvlic acids and co-hvdroxvfattv acids
HOOQCH^COOH Butanedioic acid
HOOC(CH2)6COOH Octanedioic acid
HOOC(CH2)8COOH Decanedioic acid
HOOC(CH2)10COOH Dodecanedioic acid
HOOC(CH2)14COOH Hexadecanedioic acid
HOH2C(CH2)10COOH 12-Hydroxydodecanoic acid
HOH2C(CH2)14COOH 16-Hydroxyhexadecanoic acid Effects of monocarboxylic acids on FACO activity
1250--
oc 1000-- u-« •s. 3o TJC 750-- o g 500--
250-- n^"T1rt""rTft"--TTrl""rii3i'"' 0.1 1 0.1 1 0.1 1 0.1 1 0.1 1 0.01 0.1 m w C4 C8 PFOA Effects of monocarboxylic acids on LH activity
600 c o -g 400 c 1 1 X * JL * 200
0 l a Tiii. fin.. lift 0.1 1 0 .1 1 0 .1 1 0 .1 1 0.1 1 o.oi o.i mM C4 C8 C10 C22 CPIB PFOA
Figure 21 Effects of various chain lengths of monocarboxylic acids (C4, C8, C10> C18 and as compared to clofibric acid (CPIB) and perfluorooctanoic acid (PFOA) on peroxisomal fatty acyl-CoA oxidase (Top) and microsomal laurate hydroxylase (Bottom) activities in cultured rat hepatocytes. Each bar represent mean ± S.E.M of determinations from 6-9 dishes of cells. Asterisks indicate the values that are significantly different from control (P < 0.05). 104
Long-chain Fatty Acids and FACO activity
% increase In FACO activity 250
200
150
100
50
0 0.001 0.01 0.1 1 Concentrations (mM)
Long-chain Fatty Acids and LH Activity
% increase in LH activity 200
160
100
50
0 0.001 0.01 0.1 Concentrations (mM)
Stearic Acid (18:0) Oleic Acid (18:1) IS&i Linoleic Acid (18:2) C o n tro l
Figure 22. Concentration-dependent studies of the effects of long-chain saturated and unsaturated C18 fatty acids in increasing peroxisomal fatty acyl-CoA oxidase (FACO) (Top) and microsomal laurate hydroxylase (LH)(Bottom). Cultured hepatocytes were incubated for 72 hr in the presence or absence of compound. The results are expressed as the percent of FACO and LH induction as compared to the control FACO and LH activities (0.42 + 0.05 nmoles H202/min/mg protein and 1.07 + 0.18 nmole/hr/mg protein, respectively). Each point represents the mean + S.E.M. of determinations from 6-9 dishes of cells. Asterisks indicate values that are significantly different from control (*, P < 0.05 **, P < 0.01). 105 Table 11. Comparison of the effects of Clg fatty acids of various degree of unsaturation on peroxisomal fatty acyl-CoA oxidase (FACO) and microsomal laurate hydroxylase (LH) activities.
FACO LH Treatment?
activityb %c activityd %b
Control 0.420 ± 0.048 100 1.073 ±0.184 100
18:0 (ImM) 0.435 ± 0.085 109.5 1.029 ± 0.312 95.8
18:1 (ImM) 0.883 ± 0.109® 208.6° 1.384 ± 0.12° 128.9°
18:2 (ImM) 0.710 ± 0.095° 175.7° 3.065 ±1.12° 285.6°
18:3 (O.lmM) 0.358 ± 0.18 60.4° 0.763 ± 0.216 71.1
(lmM)f 0 ± 0 0° 0.515 ± 0.17 48° a Fatty acids were added each day for 3 days. b Values are mean ± S.E.M. presented as absolute peroxisomal FACO activity (nmole H202/min/mg protein) n= 6-9 dishes of cells. c Values are mean ± S.E.M. presented as the percent of increases in enzymes activity over the control values. d Values are mean ± S.E.M. presented as absolute microsomal LH activity (nmole/hr/mg protein) n= 6-9 dishes of cells. * Significantly different from control (P < 0.05). f At the concentration of 1 mM, Ci8:3 caused toxicity to the hepatocytes. 106
Effects of degrees of unsaturation on FACO and LH activities
C ontrol CK2 FACO □ LH > a o 200 -- o ■+-> c o o >4— o 100 -
18:0 18:1 18:2 18:3 Degrees of unsaturation
Figure 23. Effects of degrees of unsaturation on fatty acyl-CoA oxidase (FACO) and laurate hydroxylase (LH) activities in primary cultures of rat hepatocytes. A series of C18 fatty acids (C18.0, C18:1 and C18:2) was applied to the cultures for 3 days, and FACO and LH activities were determined. Results are expressed as the percent of the control activities determined from 6-9 dishes of cells. The control FACO and LH activities were 0.42 ± 0.05 nmole H202/min/mg protein and 1.07 ± 0.18 nmole/hr/mg protein, respectively. Asterisks indicate that the values are significantly different from the control values (P < 0.05). 107 Table 12. The effect of long-chain polyunsaturated fatty acids on fatty acyl-CoA oxidase (FACO) activity in primary cultures of rat hepatocytes.
Treatment3 % of FACO activityb
22:5 (0.01 mM) 130.0 ± 9.5
(0.1 mM) 31.9 ± 22.0
22:6 (0.01 mM) 140.4 ± 10.3
(0.1 mM) 50.7 ± 22.3 a Concentrations of fatty acids were added throughout 3 days of incubation; n = 3 b Expressed as a percent of control activity. 108
2.0 C* O--" O Control o L. ♦ -----♦ Oleic acid 1 mM
c I o ^ o o < CM u: x a> o 0.5 E c
0.0 L 1 2 3 4 5 1000 r Control 800 ♦ Oleic acid 1 mM c o I 600 c o ^ 400 Lu X 200
0 1 2 3 4 5 Time (day)
Figure 24. Time courses for the induction of peroxisomal fatty acyl-CoA oxidase (FACO) activity by oleic acid (1 mM) and by control (no drug). Hepatocytes were incubated for 0 to 5 days. Each point represents the mean ± S.E.M. of determinations from 6 dishes of cells. Top: Results are expressed as absolute activity of the enzyme in nmole/hr/mg protein. Bottom: Results are expressed as the percent FACO induction as compared to the corresponding control activity examined at the same incubation period. 109
O control oleic acid 1 mM ♦
1000 Control
♦ Oleic acid 800
600
400
200
0 1 2 3 4 5 Time (day)
Figure 25. Time courses for the induction of carnitine acetyltransferase (CAT) activity by oleic acid (1 mM) and by control (no drug). Hepatocytes were incubated for 0 to 5 days. Each point represents the mean + S.E.M. of determinations from 6 dishes of cells. Top: Results are expressed as absolute activity of the enzyme in nmole/min/mg protein. Bottom: Results are expressed as the percent CAT induction as compared to the corresponding control activity examined at the same incubation period. 110 Table 13. Effect of cycloheximide on oleic acid- and ciprofibrate-induced FACO activity in primary cultures of rat hepatocytes.
Treatment FACO activity % FACO induction
(nmole H202/min/mg)
Control 0.38 ± 0.09 100
+ Cycloheximide (1 /ig/ml) 0.36 ± 0.04 100
Oleic acid (1 mM)a 0.93 ± 0.08° 241
+ Cycloheximide (1 jig/ml) 1.23 ± 0.05c 338
Ciprofibrate (0.08 mM)b 1.39 ± 0.17c 361
+ Cycloheximide (1 /ttg/ml) 0.75 ± 0.08* 207 a Oleic acid (1 mM) was added each day for 3 days. Cycloheximide was added for the last 2 days of incubation to the control- and the oleic acid-treated cultures. Cells were washed and removed by scraping, and homogenates prepared for assay of protein and FACO activity. b Ciprofibrate (0.08 mM) was added each day for 2 days. Cycloheximide was added for the last day of incubation to the control- and the ciprofibrate acid-treated cultures. Cells were washed and removed by scraping, and homogenates prepared for assay of protein and FACO activity. c Significantly different from control (P < 0.05). d Significantly different from ciprofibrate-induced FACO activity. Ill Table 14. Effects of dicarboxylic acids of various chain length on FACO and LH activities in primary cultures of rat hepatocytes.
Enzyme activityb
Fatty acid* Concentration
(mM) % FACO % LH
c 4 0.01 99.4 ± 39.8 79.0 ± 5.5 0.1 92.3 ± 32.2 58.2 ± 8.7' 0.3 20 ± 10° 50.7 ± 9.9'
C8 0.01 59.4 ± 9.3C 42.3 ± 9.3' 0.1 40.1 ± 19' 39.4 ± 4.9° 0.3 17.7 ± 11.8' 50.5 ± 4.7'
C-10 0.01 75.9 ± 3.4 26.4 ± 8.4' 0.1 37 ± 15.5' 65.7 ± 10.2 0.3 26.8 ±11.7' 54.1 ± 10.1'
CJ2 0.001 108.5 ± 2.3 _ 0.01 139.5 ± 2.9* 83.0 ± 16.7 0.1 111.2 ± 17.5 91.7 ± 24.1
C]6 0.001 102.7 ± 18.9 40.4 ± 14.1 0.01 143.6 ± 19* 65.7 ± 7.7 0.1 122.0 ± 21 156.2 ± 18.1
8 Dicarboxylic acids were added throughout a 3 day incubation period. Cells were washed and removed by scraping, and homogenates prepared for assay of protein, FACO and LH activities. b Expressed as a percent of the corresponding control activity. 0 values are significantly (P < 0.05) less than control. d values are significantly (P < 0.05) greater than control. 112 Table 15. Effects of dicarboxylic and hydroxycarboxylic acids of C12 and C16 on FACO activity in primary cultures of rat hepatocytes.
FACO activity*1 Fatty acid8 at the concentration (mM) of
0.001 0.01 0.1
C12-dicarboxylic 0.76 ± 0.18 1.30 ± 0.28° 1.04 ± 0.1
C,2-hydroxycarboxylic 0.85 ± 0.27 1.31 ± 0.22° 1.19 ± 0.21
C16-dicarboxylic 1.05 ± 0.35 1.41 ± 0.42c 0.93 ± 0.13
C16-hydroxycarboxylic 1.04 + 0.22 1.80 ± 0.04c 1.27 ± 0.58 a Fatty acids were added throughout a 3 day incubation period. b FACO activity were expressed as nmole H202/min/mg protein. The control FACO activity was 0.75 ± 0.21 nmole H202/min/mg protein. c Values are significantly different from control (P < 0.05). 113 Table 16. Effects of the inhibition of mitochondrial fatty acid B-oxidation on natural mono- and di-carboxylic acids in primary cultures of rat hepatocytes.
Treatment (mM)* FACO activity % of Control (nmoles/min/mg prot)
Control (0) 0.96 ± 0.10 100 +BPA (0.03 mM) 1.53 ± 0.56* 100 +MPA (0.03 mM) 0.83 ± 0.23 100 4-BOA (0.03 mM) 0.73 ± 0.25 100
Octanoic acid (0.3 mM) 0.96 + 0.19 98.6 +BPA 1.26 ± 0.41b 93.9 +MPA 0.65 + 0.14 105.7 +BOA 0.77 + 0.29 84.9
Octanedioic acid (0.3 mM) 0.43 ± 0.16 65.1 +BPA 1.34 ± 0.55b 80.0 +MPA 0.45 ± 0.10 82.8 +BOA 0.43 ± 0.16 48.1
0 Cells were incubated with mono- (octanoic acid) and di- (octanedioic acid) carboxylic acids for 3 days. Mitochondrial inhibitors were 2-bromopalmitic acid (BPA), mercaptopropionic acid (MPA) and 2-bromooctanoic acid (BOA). All inhibitors were applied at the concentration of 0.03 mM throughout the 3 day-incubation period. b Significantly different from control (P < 0.05). CHAPTER IV
CHARACTERIZATION OF PERFLUORINATED OCTANOIC ACID (PFOA)
AS NON-METABOLIZABLE FATTY ACID PEROXISOME PROLIFERATOR
IN PRIMARY CULTURES OF RAT HEPATOCYTES
A. Introduction
A variety of structurally diverse chemicals and dietary treatments have been shown both in vivo and in vitro to cause hepatic morphological and biochemical changes in rodents
[Lake etal, 1983; Reddy and Lalwani, 1983; Feller etal., 1989; Lock etal. , 1989; Moody et at., 1991]. The hepatic morphological changes are hepatomegaly and proliferation of hepatic endoplasmic reticulum, mitochondria and peroxisomes. Biochemical changes during peroxisome proliferation include inductions of peroxisomal and non-peroxisomal enzymes that are involved in lipid metabolism such as fatty acyl CoA oxidase [Lazarow, 1977], carnitine aceyltransferase [Moody et al., 1974], the bifunctional polypeptide of 80 kD [Feller et al.,
1987b] and microsomal cytochrome P450 IVA1 [Lake et al., 1984; Gibson et al. , 1990].
Among all peroxisome proliferators that have been investigated for their effects, perfluorinated fatty acids have structures that are closer to naturally occurring fatty acids.
These fatty acids are non-metabolizable due to the strength of the covalent bond between carbon and fluorine atoms. Indeed, the stability of C-F bond and its steric similarity to C-H bond has led to the development of fluorinated drugs such as 5-fluorouracil [Duschinsky and
114 115 Pleven, 1957] and fluorinated imidazoles [Kirk and Cohen, 1976]. Administration of perfluorinated octanoic acid (PFOA) by stomach intubation to rats revealed that PFOA was not biologically oxidized or defluorinated [Ophang and Singer, 1980], Thus, the use of these non-metabolizable fatty acids may be an important tool for investigation of the role of fatty acids and structurally related analogs in mediating peroxisome proliferation.
Taves (1968) found that fluoride in human serum also exists in an non-ionic form which was subsequently identified as perfluorinated fatty acids of the C„ chain length (PFOA)
[Guy et al., 1976]. In industry, perfluorinated fatty acids are widely used as surfactants, lubricants, plasticizers, wetting agents and corrosion inhibitors [Guenthner and Vietord, 1962].
Some perfluorinated compounds have also been proposed to be used as vascular replacement fluids and as contrast media in computer-assisted tomography [Olson and Andersen, 1983].
In 1981, toxicity of these perfluorinated fatty acids in laboratory animals was revealed
[Andersen et al., 1981]. Evidence indicated that these analogs caused hepatomegaly
[VanRafelghem et a l, 1987], decreases in body weight and food consumption [Olson and
Anderson, 1983], and androgenic deficiency which may result in testicular atrophy [Bookstaff et al. , 1990]. Peroxisome proliferative activity of perfluorinated fatty acids in rat was initially reported by Ikeda et al. in 1985.
Among these perfluorinated fatty acids, PFOA was the most potent peroxisome proliferation in cultured rat hepatocytes [Intrasuksri and Feller, 1991; see also Chapter II].
In vivo experiments support this finding [Ikeda et al., 1985; Kozuka et al., 1991]. PFOA, unlike non-perfluorinated octanoic acid, produced large peroxisome proliferation responses which were easily quantified in cultured rat hepatocytes. The characterization of the effects of PFOA may provide insights into the mechanism of peroxisome proliferation which is mediated by endogenous fatty acids and closely related analogs. The use of inhibitors of peroxisome proliferation represents another meaningful tool to examine the mechanism of peroxisome proliferation by fatty acid analogs. Thioridazine,
a phenothiazine drug, selectively inhibited peroxisomal B-oxidation in vivo [Van den Branden
and Roels, 1985]; however, there was no report of its effect on peroxisome proliferation.
Carnitine palmitoyltransferase I (CPT-I) inhibitors prevented the induction of peroxisome proliferation by clofibrate and hypolipidemic analogs. These CPT-I inhibitors include 2- bromopalmitic acid (BPA), 2-[5-(4-chlorophenyl)penty 1]oxirane-2-carboxylate (POCA), 2- tetradecylglycidic acid and etomoxir which were shown to inhibit the induction of peroxisome
B-oxidation enzymes by bezafibrate in vivo and in vivo [Hertz and Bar-Tana, 1987; Gerondaes
et a l, 1988]. Calcium may also play a role in peroxisome proliferation. In vivo administration of calcium antagonists, nicardipine, nifedipine and diltiazem was also reported to suppress clofibrate-evoked peroxisome proliferation in rat liver [Watanabe and Suga, 1988;
Itoga et a l, 1990]. In this chapter, the effects of a CPT-I inhibitor, BPA, and a calcium antagonist, nicardipine, on FACO induction by PFOA were investigated in primary cultures of rat hepatocytes.
Peroxisome proliferators such as di-(2-ethylhexyl)phthalate (DEHP) and LY-171883 caused transient lipid accumulation in liver within 24 hr upon administration [Elcombe and
Mitchell, 1986; Lock et a l , 1989; Foxworthy et a l , 1990b]. Thus, the researchers suggest that the lipid accumulation may be the result of the inhibition of mitochondrial fatty acid oxidation by DEHP and LY-171,883. The lipid accumulation generated fatty acid overload which then induced peroxisome proliferation. It was hypothesized that mitochondrial fatty acid oxidation was inhibited at the step of transferring long-chain fatty acyl CoA into the mitochondrial compartment through the action of CPT-I (Fig. 26), the rate-determining enzyme for mitochondrial B-oxidation [Eacho and Foxworthy, 1988; Brady et a l, 1989]. In 117
this regard, LY-171883 and bezafibrate were inhibitors of CPT-I activity [Eacho and
Foxworthy, 1988; Foxworthy et a l , 1990b]. Therefore, it is important to determine whether
or not the peroxisome proliferative effect of PFOA is linked to an inhibition of liver CPT-I
activity.
B. Specific Aims
The objective of this study is to extensively characterize the effect of PFOA on the
morphology and biochemistry of peroxisomes in primary cultures of adult rat hepatocytes as
well as to identify the nature of peroxisomal fatty acid 6-oxidation enzyme induction by PFOA
by using pharmacological, biochemical and molecular approaches. The specific aims are as
follows:
1. To determine the relative potency of PFOA as compared to clofibric acid (CPIB),
a prototypical xenobiotic peroxisome proliferator, and its more potent analog, ciprofibrate
(CIPRO). The pharmacological approaches used included concentration-response and time-
course profile studies, and drug combination studies of structurally distinct or similar compounds.
2. To investigate the effect of nicardipine [Watanabe and Suga, 1988] on FACO induction by PFOA and to compare this effect to the induction by xenobiotic peroxisome proliferator, CIPRO, as well as to the naturally occurring fatty acid, oleic acid.
3. To examine the prevention of PFOA-induced peroxisome proliferation by CPT-I inhibitor, BPA. In addition to the CPT-I inhibition, the effects of mitochondrial oxidation inhibitors of 3-ketothiolase (2-bromooctanoic acid, BOA), and fatty acyl-CoA dehydrogenase
(mercaptopropionic acid, MPA) were also applied to the study to evaluate the effect of general inhibition of mitochondrial fatty acid oxidation on peroxisome proliferation as compared to 118
the specific inhibition at the carnitine palmitoyltransferase step.
4. To determine whether PFOA possesses CPT-I inhibitory activity in rat liver
mitochondrial fractions. The studies also included the use of CIPRO and oleic acid as
comparative inducers of peroxisomal proliferators. BPA was used as a standard inhibitor of
CPT-I.
5. To determine the morphology of peroxisomes induced by PFOA in cultured rat
hepatocytes as compared to those of CIPRO and control. The morphometric relationships
between the ultrastructural changes in peroxisomes and induction of peroxisomal enzymes in
cultured hepatocytes were investigated.
6. To assess the extent of transcriptional and translational regulation of peroxisome
proliferation by PFOA, CIPRO and oleic acid in rat hepatocyte cultures. The induction of
mRNA encoding FACO and the effect of the protein synthesis inhibitor, cycloheximide, were
examined in cultured hepatocytes.
C. Methods
1. Materials
The biochemicals used and their sources were: pentadecafluorooctanoic acid,
nonadecafluorodecanoic acid, octanoic acid, 2-bromopalmitic acid and (±)-2-bromooctanoic
acid (Aldrich Chemical Co., Milwaukee, WI); clofibric acid, 3-mercaptopropionic acid,
suberic acid, cycloheximide, 5,5’-dithiobis-(2-nitrobenzoicacid), EDTA, DL-carnitine, 3,3’-
diaminobenzidine and sodium cacodylate (Sigma Chemical Company, St. Louis, MO);
ciprofibrate (Sterling-Winthrop Research Institute, Rensselaer, NY); osmium tetroxide
(Jennelle Chemical Co., Cincinnati, OH); and LR White resin (London resin company limited,
Basingstoke, England). Chemicals used for the RNA isolation and Northern blotting were 119 provided by Dr. P.E. Kolattukudy (Biotechnology Center, The Ohio State University,
Columbus, OH). Other materials used were obtained form the sources described in Chapter
II and Chapter III, section C .l.
2. Isolation and preparation of primary cultures of rat hepatocytes
The animals and procedures used were the same as those described in Chapter II,
section C.2 and C.3. To compare the inductive effects of PFOA, ciprofibrate and clofibric
acid on FACO and LH, hepatocyte cultures were treated with selected concentrations of the
fatty acids as that described in Chapter II. Nicardipine was dissolved in DMSO and added
with PFOA, CIPRO or oleic acid into the William’s E supplemented media. The cultures
were harvested 2 days and 4 days after exposure to nicardipine with PFOA or CIPRO and
oleic acid, respectively.
Various inhibitors of mitochondrial fatty acid B-oxidation (2-bromohexadecanoicacid,
2-bromooctanoic acid and 3-mercaptopropionic acid) were dissolved in DMSO and applied at
the desired concentration (0.03 mM) to the media containing PFOA and cultured for 3 days.
The effects of mono- and di-carboxylic acids on the peroxisome proliferative effects of PFOA were also investigated by adding the combination of either octanoic acid or octanedioic acid and PFOA. In the studies of additive or inhibitory effects of drug combinations (i.e., PFOA
+ CIPRO; CPIB + CIPRO), all compounds were dissolved directly in DMSO and added at the desired concentration. In all cases, the final concentration of DMSO in culture dishes was
0.4% (v/v).
Cycloheximide (1 £ig/ml) was added at 24-48 hr of incubation to assess the role of protein synthesis. The cultures were harvested after 2 days exposure to the peroxisome proliferators. FACO and CAT activities were determined as described in Chapter II and III, 120 section C.4.
For time-course studies, treated cultures were harvested every 24 hr by the same method described in Chapter II, section C.3. Homogenates of hepatocytes prepared by sonication were assayed for enzyme activities which include FACO, LH and CAT as described in Chapter II and Chapter III.
3. Biochemical assays
The assays for total cellular protein, FACO and LH activities were the same as those described in Chapter II, section C.4. CAT activity was assayed according to the method indicated in Chapter III, section C.3.
4. Assay of carnitine palmitoyltransferase activity in isolated rat liver mitochondria
4.1 Isolation of rat liver mitochondria
Mitochondria were isolated from the liver of adult Sprague-Dawley rats (250-300 g) as follows: animals were terminated by exposure over dry ice in a closed chamber, the abdomen opened and the livers removed. Livers were minced with a scissors and homogenized with 10 vol (1:10; w/v) of ice-cold Tris buffer (pH 7.5) containing 0.25 M sucrose/0.2 mM EDTA using a power driver teflon-glass homogenizer [Bieber et al., 1972].
Tissue homogenates were centrifuged for 12 min at 750g, and the recovered 750# supernatant was collected and centrifuged at 6700g for 20 min. The mitochondrial pellets were resuspended and the 6700g centrifugation step was repeated. The final mitochondrial pellet was suspended in 5-10 ml of Tris buffer pH 8.0 and was sonicated. The protein concentration of the mitochondrial suspension was determined by the method of Lowry et al. (1951) using bovine serum albumin as the standard. 121 4.2 Carnitine palmitoyltransferase I assay
Carnitine palmitoyltransferase I (CPT-I) activity was determined in isolated mitochondria. The effect of 2-bromopalmitic acid (BPA) as a known inhibitor of CPT-I
[Schulz, 1987] was determined. CPT-I activity was assayed by measurement of palmitoyl- camitine and CoASH formation from palmitoyl CoA by mitochondria in the presence of DL- carnitine as described by Bieber et al. (1972) with some modification [Gray et al., 1982;
Feller et al., 1987b]. The determination is based on the initial rates of CoASH release and reaction with DTNB [Bieber et a l, 1972]. The optimal conditions for the assay of CPT-I activity as follows: CoASH formation was linear through 10 min in the presence of mitochondrial protein concentrations ranging from 100 to 750 /tg/1.5 ml (data not presented).
Incubation mixtures contained 75 fig palmitoyl CoA, 92.8 ng DTNB, 698 fig EDTA, 500 fig sonicated mitochondrial protein, 928 fig DL-carnitine (experimental tube only), various concentrations (0.001-1000 fiM) of the compounds (PFOA, CIPRO, oleic acid and BPA) and
50 mM Tris-HCl (pH 8.0) in a total volume of 1.5 ml. After 5 min of incubation period,
CPT-I activity was measured at 412 nm on a Gilford model 240 spectrophotometer using an extinction coefficient of 13.6 cnr’mM'1.
5. Total RNA isolation and Northern blotting
5.1 Isolation of total mRNA
After the hepatocyte cultures were exposed to the compounds (i.e., 0.1 mM PFOA,
0.1 mM CIPRO and 1 mM oleic acid) for certain periods of time (i.e., 0, 2, 6 , and 20 hr), cultures were lysed to obtain total RNA by the following procedure. Media were aspirated and cells were washed with Dulbecco’s phosphate buffer 3 times. Two and one half ml of lysate buffer (4 M guanidine isothiocyanate, 5 mM sodium citrate, 0.5 % sodium sarcosinate 122 and 0.1 M B-mercaptoethanol) were then applied to the washed cultures. Cesium chloride
(0.5 g/2.5 ml) lysate buffer was added directly to culture dish. Using a SW41 tube, cell
lysates were transferred to the top of 3.3 ml of 5.7 M CsCl in 0.1 M EDTA (pH 7.4) and
centrifuged for 24 hr at the speed of 32000 rpm in Beckman Ultracentrifuge Model L5-75 or
L7-65 (Palo Alto, CA). The pellets were collected and resuspended in 10 mM Tris buffer
(pH 7.4) containing 5 mM EDTA and 1% sodium dodecyl sulfate (SDS) and were then
extracted with chloroform and butanol (4:1). RNA is recovered by ethanol precipitation
(100% ethanol was added to the supernatant and the samples were frozen at -80°C overnight).
Total RNA concentration in each sample was quantified by spectrophotometry (260 nm) as described by Maniatis et al. (1982).
5.2 Northern blotting and RNA transfer
Total RNA was denatured and electrophoresed through formaldehyde agarose gels as described by Foumey et al. (1987). Three kinds of buffers were used. The gel preparation buffer (10X MOPS/EDTA buffer) was composed of 0.2 M 3-(N-morpholino)propanesulfonic acid (MOPS), 50 mM sodium acetate, 10 mM EDTA (pH 7.0). The electrophoresis sample buffer was freshly prepared by adding 0.75 ml deionized formamide, 0.24 ml formaldehyde,
0.1 ml deionized RNase-free water, 0.1 ml glycerol and 0.08% 10% (v/v) bromophenol blue to 0.15 ml lOx MOPS buffer. The last buffer used was the electrophoresis (running) buffer or IX MOPS/EDTA. Briefly, samples were prepared by adding RNA (20 fig) to electrophoresis sample buffer and heated at 65°C for 15 min. Ethidium bromide was added into each sample for RNA visualization and the sample were then loaded onto the agarose- formaldehyde gel (11x14 cm). The gel was electrophoresed at 30 V (constant voltage) at room temperature for 18 hr. Bromophenol blue migrated about 10 cm into the gel. 123
Following electrophoresis, RNA bands were directly visualized and photographed by a short wave transilluminator (254 nm). RNA on the gel was then transferred to a nylon membrane by capillary action using a sponge to enhance the action [Fourney et al., 1987].
RNA was fixed to the membrane by baking at 80 °C for 2 hr.
The cDNA probe for rat peroxisomal FACO was contributed by Dr. P.E. Kolattukudy
(provided by Dr. T. Hashimoto, Depart of Biochemical, Shinshu U., Nagano, Japan). The probe was obtained from the recombinant plasmid pMJ115 which was cleaved by restriction enzyme PstI [Miyazawa et a l , 1987]. It was then labelled by random priming with p 2P] ATP using Boehringer Mannheim random primed DNA labeling kit and purified through high and low salt column (Elutip-d column). Hybridization was performed with the methods described by Maniatis et al. (1982). Following hybridization, the membranes were washed and the
[32P]cDNA/mRNA hybrids were visualized by autoradiography. The mRNA levels were quantified by scanning densitometry using a densitometer Model 1650 and a recorder Model
1321 (Biorad, Richmond, Ca).
6. Electron microscopy
Hepatocyte cultures after treatment with 0.4% DMSO (control), 0.1 mM PFOA or 0.1 mM CIPRO, were prepared for electron microscopy as outlined by Furukawa et al. (1984).
After 72 hr, cultures were washed 2 times with 10 mM HEPES buffer (pH 7.4). The cultured cells were fixed directly on the dishes with 2.5% glutaraldehyde-0.1 M sodium cacodylate buffer (pH 7.4) at 4°C for 6 hr. 3,3’-Diaminobenzidine (DAB) in alkaline medium (pH 9.7) containing KCN and hydrogen peroxide (HjOa) were applied to the cultures for 2 hr (the first hr without HjOz) for peroxisome staining [Novikoff et a l, 1972]. The cells were postfixed in 2% osmium tetroxide in 0.1 M sodium cacodylate buffer (pH 7.4), dehydrated and then 124
embedded in LR White resin. Sections were prepared parallel to the surface of the Petri dish
and stained with uranyl acetate and lead citrate. The electron micrographs were taken at
x3000 and x3500 from Hitachi TM H600 (75 KV) and the negatives were printed and
magnified by 3 times on paper. Peroxisomes in a cytoplasmic area of 100 fim2 were manually
counted, and peroxisome diameters were measured from 9-11 micrographs for each treatment.
7. Data analysis
The data were statistically analyzed by comparisons among the means using the
Student’s unpaired t-test for direct comparison of means. In case of multiple comparisons between means of different treatments (i.e., no compound, one compound, and combination of compounds), one-way analysis of variance followed by a Student-Newman-Keuls test are used [Tallarida and Murray, 1987].
For the morphology analysis of electron micrographs, the results are expressed as number and diameter of peroxisomes per 1 0 0 f i m 2, and percent of cytoplasmic area occupied by peroxisomes which were calculated from the number and diameter of peroxisomes in each
100 nm2. The Student’s unpaired t-test was used to test for significance (P < 0.05) between data of control and drug treated cells (PFOA or CIPRO).
D. Results
1. Biochemical characteristic of peroxisome proliferation bv PFOA
1.1 Concentration-response studies
Concentration-response curves of PFOA for the inductions of FACO and LH activities were constructed and compared to those of CIPRO and CPIB (Figs. 27-28). The cultured hepatocytes were exposed to the compounds for 72 hr. Control FACO and LH activities were 125
1.0 ± 0.2 nmole H 20 2/min/mg protein and 2.4 ± 0.3 nmole/hr/mg protein. EQo values of
each compound estimated from the concentration-response curves are shown in Table 17. The
rank order of potency determined by their EC^’s was CIPRO > PFOA > CPIB, for FACO
induction and PFOA > CPIB, for LH induction.
1.2 Time-courses for induction of FACO. LH and CAT activities by PFOA as
compared to CPIB
PFOA (0.1 mM) produced time-dependent increases in FACO, LH and CAT (Figs.
29-31) similar to those produced by CPIB (1 mM) (Figs. 29, 31). Control FACO activity
decreased throughout the incubation period (0-4 days) (Fig. 29, top). PFOA increased FACO
activity throughout a 4 day-incubation. CPIB also increased FACO activity with time, but the
activity reduced after 3 days of incubation. Fig. 29 shows percent of FACO induction as
compared to the corresponding control measured at the same time. Percent of FACO
inductions by both PFOA (0.1 mM) and CPIB (1 mM) were significant (P < 0.05) at the first
day of incubation and the maximum inductions were observed after 3 days of incubation.
Figure 30 displays the induction of LH by 0.1 mM PFOA. The control LH activity
decreased after 1 day incubation and remained relatively stable up to 5 days of incubation.
PFOA increased LH activity throughout a 5 day-period. A significant induction of LH was
observed after 2 days of incubation, and the maximum induction was found after 5 days of
incubation with PFOA 0.1 mM (Fig. 30).
PFOA (0.1 mM), like CPIB (1 mM), also increased CAT activity in a time-dependent
manner (Fig. 31). In contrast to FACO and LH of the control hepatocytes, the control CAT
activity was relatively stable from the first day and maintained for 4 days (Fig. 31, top). Both
PFOA and CPIB caused CAT induction that was observed within one day after the hepatocyte 126 cultures were exposed to the compounds. The induction by both compounds reached a maximum after a 4-day incubation (Fig. 31).
2. Drue combination studies
2.1 Effects of drug combination on FACO activity
The effects of PFOA (0.001, 0.01 and 0.1 mM) and CPIB (0.1 and 1 mM) on the concentration-response curves of CIPRO were constructed for any possible inhibitory or potentiating effects (Figs. 32-33). None of the combinations were toxic to the cells as assessed by morphology and LDH leakage determination.
Maximum FACO induction was found when the cultures were exposed to CIPRO (0.1 mM) and PFOA (0.1 mM). PFOA at the concentrations of 0.001, 0.01 and 0.1 mM increased FACO 15.1 %, 33.3 % and 120.4 % of the maximum response, respectively (Fig.
32). At a low concentration (0.001 mM), PFOA significantly reduced the effect o f0.003 and
0.01 mM CIPRO in increaseing FACO activity (Fig. 32). At 0.01 mM of PFOA, the curve was not significantly different from the CIPRO control curve (CIPRO alone). With 0.1 mM
PFOA, the CIPRO curve was obviously altered by shifting to the left. The percent of FACO induction of CIPRO concentrations (0.003-0.1 mM) in the presence of 0.1 mM PFOA became / similar to the FACO induction observed when 0.1 mM PFOA was present alone (Fig. 32).
No additive effects were found by the combination of these two structurally different peroxisome proliferators.
Addition of CPIB to hepatocyte cultures produced a maximum FACO induction at 1 mM. When added with CIPRO (0.003-0.1 mM), the low concentration (0.1 mM) of CPIB significantly inhibited the effects of 0.01 mM CIPRO (Fig. 33). Similar to PFOA (Fig. 32), a high concentration of CPIB (1 mM) significantly shifted the curve of CIPRO to the left with 127 the same percent of FACO induction which was similar to that produced by CPIB (1 mM) alone (Fig. 33). The combination of CIPRO (0.1 mM) and CPIB (1 mM) caused a reduction of FACO activity as compared to that of each drug alone, suggesting additive toxicity (Fig.
33).
2.2 Effect of PFDA on FACO induction bv PFOA
The combination study of PFOA and its longer-chain fatty acid analog, perfluorodecanoic acid (PFDA) was studied. PFDA (0.01 mM) induced FACO activity to
193% of control. Addition of PFDA (0.01 mM) to either 0.01 or 0.03 mM PFOA did not show any additive or inhibitory effect since the FACO induction in the presence of both compounds was not significantly different from that of PFOA alone (Fig. 34).
2.3 Effects of octanoic and octanedioic acids on PFOA response
Octanoic acid and octanedioic acid are naturally occurring mono- and di-carboxylic acids of an 8 -carbon chain length. These fatty acids did not increase FACO activity in cultured hepatocytes (see Fig. 12, Chapter II). Moreover, the dicarboxylic acid produced a significant reduction of FACO activity (see Table 14, Chapter III). The effects of these two fatty acids on FACO induction by PFOA, the perfluorinated fatty acid of the same chain length, were investigated. Whereas the control FACO activity was 1.1 ± 0.2 nmole
H20 2/min/mg protein, PFOA 0.01 mM and 0.03 mM increased FACO activity to 2.1 ± 0.6 and 3.6 ± 0.2 nmole H 20 2/min/mg protein which are 174% and 325% of the control, respectively. Octanoic acid (0.3 mM) did not change any FACO inductive activity of PFOA since, upon addition of octanoic acid, the percent of FACO induction by 0.01 and 0.03 mM
PFOA were 244% and 309% and the values are not significantly different from FACO 128 induction by PFOA alone (Fig. 35). However, octanedioic acid (0.3 mM) which by itself reduced FACO activity to only 33% of the control, did not block the stimulatory effect of
PFOA. In contrast, it significantly potentiated the effect of 0.03 mM PFOA. The FACO induction by 0.03 mM PFOA increased to 459% of control in the presence of 0.3 mM octanedioic acid (Fig. 35).
3. Effect of nicardipine on FACO induction
The effect of an addition of the calcium antagonist, nicardipine (at 0.001 and 0.01 mM) to the hepatocyte cultures treated with PFOA (0.08 mM), CIPRO (0.08 mM) and oleic acid (1 mM) is shown in Fig. 36. At these concentrations, nicardipine did not cause any changes in FACO activity. Higher concentrations (> 0 .1 mM) of nicardipine were cytotoxic to hepatocytes. Nicardipine only slightly but not significantly reduced FACO induction produced by CIPRO and PFOA. FACO induction produced by a submaximal concentration
(0.08 mM) of CIPRO was 351% of the control value. The induction was reduced to 246% and 262% of control when the CIPRO-treated cultures were supplemented with nicardipine at 0.001 and 0.01 mM, respectively. FACO induction by a submaximal concentration (0.08 mM) of PFOA was 241 % of control. Nicardipine (0.001 and 0.01 mM) reduced the induction of FACO by PFOA to 199% and 188% of control, respectively. Oleic acid (1 mM) increased
FACO activity to 181% of the control, and in the presence of 0.001 and 0.01 mM nicardipine, the activities were 142% and 155% of the control, respectively (Fig. 36).
4. Mitochondrial fattv acid B-oxidation and PFOA induction
The effect of mitochondrial fatty acid 6 -oxidation inhibitors on FACO induction by
PFOA is shown in Figure 37. The mitochondrial inhibitors and concentrations used were 0.3 mM 2-bromooctanoic acid (BOA), 0.3 mM mercaptopropionic acid (MPA) and 0.3 mM 2-
bromopalmitic acid (BPA). The concentrations selected were those previously reported to
impair mitochondrial fatty acid 6 -oxidation in cultured hepatocytes [Hertz and Bar-Tana,
1987], The control FACO activity was 1.0 ± 0.2 nmole H 20 2/min/mg protein. PFOA 0.03
mM increased FACO activity to 2.4 ± 0.4 nmole H 20 2/min/mg protein which is 238% of the
control value. It was found that BOA and MPA, the inhibitors of 3-ketothiolase and fatty
acyl-CoA dehydrogenase, respectively, did not cause any significant changes in the percent
of FACO induction by PFOA (Fig. 37). The percent of FACO induction by PFOA in the
presence of BOA and MPA were 230% and 231%, respectively. In contrast, the carnitine
palmitoyltransferase-I (CPT-I) inhibitor, BPA, inhibited FACO induction by PFOA. FACO
induction by 0.1 mM PFOA was decreased to 43% of the control after the cultures exposed to 0.03 mM BPA (Fig. 37).
5. Effect of CPT-I inhibitor on FACO induction bv PFOA and CIPRO
BPA at 0.003 and 0.03 mM produced a slight but significant induction of FACO over the control value (184% and 250%, respectively) (Fig. 38). The control activity was 0.55 ±
0.1 nmole H 20 2/min/mg protein. When BPA (0.003 and 0.03 mM) was added into cultures with CIPRO (0.1 mM) or PFOA (0.1 mM), the percent FACO induction by the two peroxisome proliferators decreased significantly (Fig. 38). The inhibitions of FACO induction by BPA (0.003 and 0.03 mM) were concentration-dependent (Fig. 38). The maximal FACO response produced by CIPRO (0.1 mM) was 707%, and this induction was reduced to 321% and 328% of the control after addition of 0.003 mM and 0.03 mM BPA, respectively (Fig.
38). PFOA (0.1 mM) gave maximum response which was 538% of the control value.
Addition of 0.003 mM BPA to the cultures did not alter the FACO inductive response to 130 PFOA. However, at a higher concentration of BPA (0.03 mM), FACO induction by PFOA
was inhibited to 139% of the control value which was equivalent to 26% of the maximum
PFOA response.
6 . Effect of PFOA. oleic acid and CIPRO on CPT-I activity in isolated rat liver
mitochondria
As expected, BPA, the known inhibitor of CPT-I activity [Schulz, 1987], blocked
CPT-I activity of rat liver mitochondria in the concentration-dependent manner (Fig. 39). The
control CPT-I activity in freshly isolated mitochondria was 3.2 + 0.7 nmole/min/mg. The
maximum inhibition of CPT-I activity by BPA was 20% and the IC 50 value calculated from
the inhibition-response curve was 0.19 fiM.
In contrast to BPA, CIPRO did not show any significant inhibition of CPT-I activity
in the range of tested concentrations (0.001 to 1000 /zM) (Fig. 39). PFOA displayed a slight
inhibition of CPT-I activity in isolated mitochondria; however, the maximum inhibition was
only 60% of the control at the highest concentration (100 fiM) (Fig. 40). The unsaturated
fatty acid, oleic acid, slightly impaired CPT-I activity. The maximum reduction of CPT-I
activity by oleic acid was 61% of the control value at a concentration of 1 mM (Fig. 40).
7. Morphological analysis of peroxisome proliferation induced bv PFOA as compared
to CIPRO
Electron micrographs of cultured hepatocytes treated with 0.1 mM CIPRO and 0.1 mM PFOA for 72 hr were compared to the control (0.4% DMSO) (see Figs. 41, 42, 43, respectively). Peroxisomes are visualized as more darkly stained organelles containing a dense core or nucleoid within the structures. Increases in peroxisome numbers were observed from both CIPRO and PFOA treatments (Table 18). Peroxisome numbers increased from the average of 8.0 to 19.7 and 13.8 peroxisomes per 100 nm2 after a 3-day treatment of cultured rat hepatocytes with CIPRO (0.1 mM) and PFOA (0.1 mM), respectively. Diameter of induced peroxisomes by 0.1 mM CIPRO was significant larger than those of the control peroxisomes (P < 0.05) (Table 18). Peroxisomes induced by 0.1 mM PFOA were slightly though not significantly larger than the control peroxisomes (Table 18). Percent area of cytoplasm occupied by peroxisomes were significantly increased (P < 0.01) by CIPRO and
PFOA treatment. In the control cells, peroxisomes occupied 2.3% of the cytoplasmic area and altered to occupy 7.8% and 5.0% of the cytoplasmic area after CIPRO and PFOA treatments which represents 340% and 217% increases, respectively, as compared to the control hepatocytes (Table 18). Corresponding to the increase in volume occupied by peroxisomes, the activity of the peroxisomal enzyme, FACO, was also increased after CIPRO and PFOA treatment (Table 18).
8 . Molecular mechanisms of PFOA as compared to CIPRO and oleic acid
8.1 Induction of mRNA encoding peroxisomal FACO in cultured rat hepatocytes
Northern blot analysis of RNA isolated from hepatocyte cultures exposed to 0.1 mM
CIPRO, 0.1 mM PFOA and 1 mM oleic acids is shown in Figure 44. Hepatocyte RNA was isolated at 0, 2, 6 and 20 hr after incubation with CIPRO and PFOA and at 2 and 20 hr after exposure to oleic acid. Using the cDNA probe for peroxisomal FACO, it has been shown that the level of mRNA encoding FACO increased in the time-dependent manner after CIPRO and
PFOA treatment (Fig. 44).
PFOA increased mRNA of FACO within 6 hr and maximum levels were attained at
20 hr. Using the densitometer to estimate the amount of mRNA levels after hepatocytes were 132 incubated with PFOA, the level of mRNA encoding FACO increased to 1.2- and 2.6-fold of the control mRNA levels determined at 6 and 20 hr, respectively (Fig. 45).
Comparison of the mRNA levels of 20 hr incubation with no drug, 0.1 mM CIPRO,
0.1 mM PFOA and 1 mM oleic acid is shown in Fig. 46. PFOA and CIPRO produced significant increases in the mRNA of FACO. However, oleic acid did not cause any detectable increase in mRNA level as compared to the control level (Fig. 46).
8.2 Effect of cvcloheximide on FACO induction bv PFOA. CIPRO and oleic acid
Addition of 1 ptg/ml cycloheximide, an inhibitor of protein synthesis, to the cultured hepatocytes resulted in the reduction of FACO induction produced by CIPRO and PFOA.
Cycloheximide alone did not produce any significant change in FACO activity as compared to the control. FACO activity of the control and the cycloheximide-treated cultures were 0.38
± 0.09 and 0.26 ± 0.04 nmole H 20 2/min/mg protein, respectively.
CIPRO at the concentration of 0.08 mM caused a 346% FACO induction over that of the control activity. Upon addition of cycloheximide, the extent FACO induction by
CIPRO was significantly decreased to 196% of the control (Fig. 47). Similar to CIPRO,
FACO induction by 0.08 mM PFOA was reduced from 559% to 218% after exposure of cultures to cycloheximide (Fig. 47). However, cycloheximide did not inhibit the stimulation of FACO activity by 1 mM oleic acid. Oleic acid, the natural unsaturated fatty acid, caused a 274% increase in FACO activity, and in the presence of cycloheximide (1 ng/ml), the
FACO induction was significantly increased to 364% of the control values (Fig. 47).
E. Discussion
Primary cultured hepatocytes are a good model system for the study of a 133
concentration-response and a time-course profile of peroxisome proliferators. Similar to the
hypolipidemic peroxisome proliferators, PFOA produced concentration-dependent and time-
dependent increases in both FACO and LH activities (Figs. 27-30). Interestingly, PFOA, the
non-metabolizable fatty acid structurally unrelated to hypolipidemic peroxisome proliferators,
was 12- and 40-fold more potent than CPIB in the induction of FACO and LH activities
respectively (Table 17). Induction of FACO by CIPRO, one of the most potent peroxisome
proliferators, was only 7-fold greater than that of PFOA. In contrast, CIPRO was * 70-fold
more potent than CPIB as an inducer of FACO (Table 17). These results agree with the
findings of Feller et al. (1987) and Kocarek and Feller (1989) on the greater potency of
CIPRO than CPIB in the induction of CAT, FACO and LH in cultured hepatocytes.
Lake et al. (1984) reported on a high degree of correlation between the inductions of
peroxisomal fatty acid oxidation enzymes and microsomal laurate hydroxylase by
hypolipidemic peroxisome proliferators in vitro. Using two structurally distinct chemicals
(hypolipidemic peroxisome proliferator and non-metabolizable fatty acid peroxisome
proliferator), the results also show a correlation between the inductions of FACO and
microsomal LH since the rank order of potency (PFOA > CPIB) and the ECso’s of FACO
and LH inductions by PFOA and CPIB were relatively similar. Although PFOA possessed
high efficacy in FACO induction (as determined from E ^ value), its efficacy in increasing
LH was not as great as that produced by the hypolipidemic peroxisome proliferators, CIPRO
and CPIB (see Fig. 28). The maximum LH induction of PFOA was only 69% of that of
CPIB.
Time-course profiles of PFOA for the induction of peroxisomal FACO, microsomal
LH, and CAT were similar to those of CIPRO. All enzyme inductions were detected within the first or the second day and reached the maximum within 3 days after the cultures were 134 exposed to the compound. In contrast to the profiles of a non-metabolizable fatty acid
(PFOA) and a hypolipidemic peroxisome proliferator (CPIB), the time-course profile of
FACO activity for a naturally occurring fatty acid (oleic acid) was different (See Chapter III).
The increase in enzyme activity by oleic acid was detected after 3 days of treatment. Relative to the control activity, the potency and the maximal inductions of all enzymes produced by the natural fatty acid were very low (* 2-fold) as compared to PFOA (== 13-fold) and CPIB
(*= 10-fold). These differences in potency and time-course profile might indicate that differences exist in the mechanism of action between xenobiotics and naturally occurring fatty acids.
The combination studies of PFOA and CIPRO, and, CPIB and CIPRO demonstrate that PFOA, CPIB and CIPRO may have a common pathway in mediating peroxisome proliferation. In these experiments, a low concentration of PFOA (0.001 mM) blocked the effect of CIPRO (0.003 and 0.01 mM). At a high concentration (0.1 mM) of PFOA, the
CIPRO concentration-response curve (0.003-0.1 mM) was shifted to the left (Fig. 32). No additive effects were found at any concentration of the combinations of PFOA and CIPRO.
Similar effects of CPIB on the concentration-response curve of CIPRO were found (Fig. 33).
These results can be explained in two ways. One possibility is that PFOA acted distinctively at low and high concentrations. At a low concentration, PFOA behaved as an inhibitor of peroxisome proliferation, and blocked CIPRO responses whereas, at a high concentration of
PFOA, it became an inducer of peroxisome proliferation. The second possibility is that
PFOA may act through a common pathway as CIPRO but with a lower potency. As mention earlier (Chapter I), Issemann and Green (1990) recently discovered a nuclear receptor that can be activated solely by peroxisome proliferators and named this receptor a " peroxisome proliferator-activated receptor" or "PPAR". Assuming that PFOA interacts with a low 135 potency on the same receptor as CIPRO, PFOA at a low concentration may interfere with the
CIPRO-receptor interaction by occupying some receptors and producing less response than
CIPRO. At a high concentration, however, PFOA occupied most of the receptors and provoked its own effect.
The possibility that PFOA acts through a common pathway as CIPRO was substantiated when combinations of CIPRO and CPIB were investigated (Fig. 33). CIPRO and CPIB combination studies revealed results quite similar to those of PFOA and CIPRO combination studies. A low concentration of CPIB shifted the CIPRO-concentration response curve to the right and, at a high concentration of CPIB, the curve was shifted to the left (Fig.
33). The studies also revealed no additive effects on FACO induction (Fig. 33). Feller et al.
(1987) previously reported on the no inductive effects on CAT induction between the CIPRO-
CPIB combinations. CIPRO is much more potent than CPIB in producing peroxisome proliferation [Feller et al., 1987] and it should be implied that these two structurally similar peroxisome proliferators possess the same mechanism of action. Taken collectively, it is suggested that, similar to that of CPIB and CIPRO, PFOA and CIPRO may act through the same common pathway leading to peroxisome proliferation. PFOA and PFDA combinations also demonstrated no additivity suggesting a common mechanism (Fig. 34). Since the aim of this study is not to prove the existence of peroxisome proliferation-associated receptors, the common pathway leading to the similar mechanism of peroxisome proliferation by a non- metabolizable fatty acid and hypolipidemic drugs were not established.
Natural monocarboxylic acids (e.g., octanoic acid) and dicarboxylic acids (e.g., octanedioic acid) did not inhibit FACO inductive effect of PFOA (Fig. 35). Interestingly, although octanedioic exhibited inhibitory effect on FACO activity (see Chapter III, Table 14), the combinations of octanedioic acid and PFOA revealed an additive effect. The dicarboxylic 136 acid may pharmacokinetically facilitate PFOA-transportation to its site of action or pharmacodynamically stimulate the binding of PFOA to its effector site. Lock et al. (1989) suggested that long-chain dicarboxylic acids are the proximal stimulators of peroxisome proliferation. Although octanedioic acid, a medium chain dicarboxylic acid, did not cause peroxisome proliferation, in the presence of a potent inducer, it might become active and produce additive effect.
It is known that calcium signalling is involved in the molecular regulation of many proteins such as contractile protein, secretory protein, cell growth and protein phosphorylation
[Itoga et al., 1990]. In addition, it was suggested to function in the regulation of peroxisome proliferation [Watanabe and Suga, 1988; Itoga et a l, 1990]. Nicardipine, a calcium antagonist, was reported to inhibit peroxisome induction by clofibrate in vivo. The inductions of peroxisomal fatty acyl-CoA oxidizing enzymes and CAT activities by clofibrate were observed on the first day but the suppression of the enzyme inductions were found after 5 days of nicardipine treatment. Itoga et al. (1990) concluded that peroxisome proliferation produced by clofibrate in vivo is composed of 2 steps, i.e, a triggering step and an enhancing step, and nicardipine inhibited the later step with a maximum suppression of only 52%. According to the present experiments in cultured hepatocytes, nicardipine did not significantly suppress
FACO induction by CIPRO or PFOA after 2 day-incubation with the compounds (Fig. 36).
The results suggest that either nicardipine inhibitory effect needs a participation of other components such as hormones; thus, the inhibitory effect could not be demonstrated in vitro, or, in the in vitro model only a triggering step occurred. Although, in general, incubation of hepatocytes with PFOA or CIPRO for 2 days resulted in the increase in FACO activity that almost reached the maximum induction, the possibility remains that an enhancing step, if it exists in this in vitro system, may occur after hepatocyte cultures were exposed to the 137 compounds for more than 2 days.
Elcombe and Mitchell (1986) presented that metabolite VI, the active peroxisome proliferator derived from DEHP, inhibited fatty acid oxidation in isolated hepatocytes and selectively inhibited medium-chain fatly acid oxidation in isolated mitochondria. They suggested that the inhibition of mitochondrial B-oxidation by this metabolite led to an accumulation of lipids. In order to maintain cellular homeostasis from the fatty acid overloaded situation, cells increased the synthesis of organelles and enzymes involved in fatty acid oxidation. These organelles and enzymes include peroxisomes and endoplasmic reticulum, and, peroxisomal FACO and microsomal LH [Elcombe and Mitchell, 1986; Lock et al. , 1989]. In this regard, if an inhibition of mitochondrial B-oxidation was the mechanism of peroxisome proliferation by a non-metabolizable fatty acid (PFOA), additions of mitochondrial inhibitors should alter PFOA effects. However, an inhibitor of 3-ketothiolase
(2-bromooctanoic acid) and fatty acyl CoA dehydrogenase (mercaptopropionic acid) had no effect on the induction of FACO by PFOA (Fig. 37). Therefore, the results indicate that
PFOA did not exhibit its peroxisome proliferative effects through the inhibition of 3- ketothiolase or fatty acyl-CoA dehydrogenase, the two enzymes in the mitochondrial fatty acid
B-oxidation system (Fig. 1).
By contrast, carnitine palmitoyltransferase (CPT) inhibitor (2-bromopalmitic acid,
BPA), which inhibits the transport of long-chain fatty acyl-CoA into mitochondrial matrix by irreversibly binding to CPT-I (Fig. 26), inhibited the FACO induction by PFOA (Fig. 37-38).
Hertz and Bar-Tana (1987) reported on the prevention of bezafibrate induced-peroxisome proliferation in cultured hepatocytes by CPT inhibitors: 2-bromopalmitate, 2-[5-(4- chlorophenyl)pentyl]oxirane-2-carboxylate(POCA) and 2-tetradecylglycidic acid. Since other mitochondrial inhibitors had no effects on PFOA, an inhibition of PFOA-induced peroxisome 138
proliferation by BPA may not be attributed to a function of mitochondrial oxidation pathway.
CPT-I inhibitors share some common effects with hypolipidemic peroxisome
proliferators [Gerondaes et a l , 1988]. BPA as well as other CPT-I inhibitors such as POCA
and etomoxir increased peroxisomal fatty acid oxidizing enzymes both in vivo and in vitro
[Hertz and Bar-Tana, 1987; Osmundsen et a l, 1987; Gerondaes et al.t 1988]. The CPT-I
inhibitors, similar to clofibrate, increase hepatic carnitine and CoA levels after chronic
treatment [Koundakjian# al. , 1981]. The present experiments showed that incubation of 0.03
mM BPA with cultured hepatocytes increased FACO activity by 2.5-fold over the control
(Fig. 38). Further, BPA exhibited a concentration-dependent inhibition of FACO induction
by PFOA and CIPRO (Fig. 38). This evidence indicated that there might be a common or
a competing pathway between these peroxisome proliferators and CPT-I inhibitors.
Eacho and Foxworthy (1988) reported on the inhibition of CPT-I by bezafibrate and
bezafibroyl-CoA. They suggested that the CPT-I inhibition may be relevant to the mechanism
of peroxisome proliferation by xenobiotics. The effect of PFOA as well as CIPRO and oleic
acid on hepatic CPT-I was investigated to assess their direct interactions with this enzyme.
In the assay, BPA inhibited CPT-I with an IC^ of 0.19 fiM (Fig. 39). However, in our
experiment, CIPRO, a potent peroxisome proliferator, did not possess CPT-I inhibitory effect
at any concentration tested (Fig. 39). Moreover, PFOA, which exhibited FACO inductive
activity that was over 1 0 0 -fold greater than oleic acid, showed similar or less effect in
impairing CPT-I activity in isolated rat mitochondria (Fig. 40). Brandes et al. (1990) examined the effects of tetradecylglycidic acid (TDGA) on the inhibition of bezafibrate- dependent induction of FACO and liver fatty acid binding protein (L-FABP) and suggested that this FACO and L-FABP inhibitory effect of TDGA involved a process other than, or in addition to, inhibition of CPT-I. Taken together, the results with PFOA, CIPRO and oleic 139 acid do not support the proposal that peroxisome proliferators exert their proliferative effects
through an inhibition of CPT-I. Although BPA, which is a CPT-I inhibitor, increased FACO
activity and prevented FACO induction by CIPRO and PFOA, its CPT-I inhibitory effect may
not be related to peroxisome proliferation. Like other fatty acid analogs, BPA has a structure
that contains a long hydrophobic backbone and a carboxylic function and is relatively resistant
to fatty acid oxidation. This structure indeed meets the requirement of a fatty acid analog that
causes peroxisome proliferation (See Chapter II). Thus, BPA effect on the inhibition of
PFOA- or CIPRO-dependent FACO induction may due to its low potency as a peroxisome proliferator.
Morphological analysis revealed that PFOA increased the number and the cytosolic volume occupied by peroxisomes in cultured hepatocytes as compared to the control cultures
(Fig. 41, 43). In agreement with the increase in the number of peroxisomes by CIPRO in vivo [Reddy and Lalwani, 1983] and in vitro [Feller et al. , 1987b], significant increases in the number of peroxisome and diameter were observed in this cultured hepatocyte model (Fig.
42). The volume occupied by peroxisomes in the normal cells increased from 2.3% to 7.8% and 5.0% by CIPRO and PFOA, respectively (Table 18). Although not determined experimentally, mitochondria also increased in number upon PFOA treatment. The induction of peroxisomes by CIPRO and PFOA (Figs. 42-43) correlated well with the induction of peroxisomal enzyme FACO (Table 18) although Baumgart et al. (1990) has suggested that peroxisome proliferation and the induction of the fatty acid 6 -oxidation are regulated separately.
Molecular approaches revealed that the induction of FACO by PFOA and CIPRO were the result of an increase in mRNA synthesis. CIPRO has been shown to increase the mRNA levels of FACO, enoyl-Co A hydratase/3-hydroxyacyl-CoAdehydrogenase bifunctional protein 140 and thiolase both in vivo [Osumi et al., 1985; Reddy et al., 1986] and in vitro [Thangada et
al., 1989; Bieri et al., 1991]. However, to our knowledge, the transcriptional regulation of
FACO by PFOA has not been demonstrated before either in vivo or in vitro. The present
results reveal that PFOA, like other hypolipidemic peroxisome proliferators, increases the
mRNA levels encoding fatty acyl-CoA oxidase, the first and rate-limiting enzyme of the
peroxisomal fatty acid 8 -oxidation system (Figs. 44). The induction of mRNA of FACO was
time-dependent (Fig. 45). The present experiments also showed that oleic acid did not
significantly increase mRNA of FACO in cultured hepatocytes after a 20-hr exposure to the
fatty acid (Fig. 44, 46). Comparison between the mRNA induction of CIPRO and PFOA
revealed that CIPRO, although more potent than PFOA as a peroxisome proliferator and an
inducer of FACO activity, appeared similar or less active as an inducer of mRNA encoding
FACO (Fig. 46).
Cycloheximide, the inhibitor of translation process (inhibit protein synthesis), prevented the induction of FACO by CIPRO and PFOA (Fig. 47). The results, thus,
demonstrate that increases in FACO activity by CIPRO and PFOA are due to de novo enzyme
synthesis. Feller et al. (1987b) also demonstrated an inhibition of CIPRO-induced CAT activity by actinomycin D and cycloheximide in cultured rat hepatocytes. In addition,
Thangada et al. (1989) showed that addition of cycloheximide to CIPRO-treated cultures did not inhibit or superinduce the mRNA levels encoding various peroxisomal enzymes indicating that the induction of these enzymes represented the direct effect of CIPRO on the expression of the genes.
In contrast, cycloheximide did not inhibit oleic acid-dependent increase in FACO activity. Moreover, as mentioned above, oleic acid also did not significantly increase mRNA level. Taken collectively, an increase in FACO activity by oleic acid is not mediated by an 141 increase in de novo enzyme synthesis (transcription or translation) but may be related to a
decrease in enzyme degradation. Decreases in the degradation rate of hepatic acyl-CoA
oxidase in liver of rat fed with partially hydrogenated fish oil enriched with unsaturated fatty
acid were reported [Horie and Suga, 1989]. Therefore, peroxisome proliferation produced
by xenobiotics and natural fatty acids are regulated by two distinct mechanisms. Interestingly,
addition of cycloheximide significantly potentiated an increasing FACO activity by oleic acid
(Fig. 47). This experiment suggested that there might be a "repressor" protein involved in
the increase in FACO activity by oleic acid. Cycloheximide inhibits all protein synthesis
including this theoretical "repressor" protein. A decrease in this repressor protein may result
in the potentiation of FACO activity after cycloheximide addition.
Dissimilarities between the nature of peroxisome proliferation by xenobiotics and
natural compounds have been demonstrated by many researchers [Farrants et al., 1990; Horie
and Suga, 1990; Itoga et al., 1990]. Firstly, Horie and Suga (1990) demonstrated that feeding
rats with partially hydrogenated fish oil increased peroxisomal acyl-CoA oxidase activity
without producing an increase in the mRNA for this enzyme whereas Flatmark et al. (1988)
reported on the 12-fold induction of the mRNA encoding the bifimctional enzyme of
peroxisomal 6 -oxidation. In the present work, addition of oleic acid to cultured hepatocytes
increased FACO activity without any detectable increase in the mRNA encoding the enzyme
which is in agreement with Horie and Suga (1990). Secondly, in the increase of peroxisomal
6 -oxidation enzymes by natural compounds or physiological manipulation such as vitamin-E
deficiency and diabetes, the magnitudes of induction are very small as compared to those
produced by xenobiotic peroxisome proliferators [Moody et al., 1991]. Thirdly, Watanabe
and Suga (1988) and Itoga et al. (1990) reported on the in vivo inhibition of clofibrate-induced peroxisome proliferation but not high fat diet-induced peroxisome proliferation by calcium 142 antagonists. Finally, Farrants et al. (1990) demonstrated that oxidation of 3a,7a, 12a-
trihydroxy-SB cholestanoic acid to cholic acid which follows a similar reaction mechanism to
that of peroxisomal B-oxidation was induced in liver of rat fed with partial hydrogenated fish
oil but not clofibrate. Taken collectively, two separate mechanisms for the induction of
peroxisomal enzymes by xenobiotic and natural compounds may exist.
In addition, our experiments demonstrate that primary cultures of hepatocytes provide
a useful system for performing pharmacological, biochemical, morphological as well as
molecular characterization of peroxisome proliferation produced by a non-metabolizable fatty
acid analog, perfluorooctanoic acid (PFOA). The system maintains good conditions for the
study of concentration-response and time-course profile of the peroxisome proliferator. The
experiments on effects of drug combinations give a relatively simple and direct way to
investigate the effects of both drug with less pharmacokinetic interference when compared to
the in vivo methods. Moreover, comparable to in vivo studies in which thin liver slices of
treated-animals were stained and observed through electron microscope, sections of treated-
cultured hepatocytes can also be prepared and employed as excellent tissues to reveal
morphological changes during peroxisome proliferation. To date, this is the first report
verifying hepatocyte ultra structural changes in peroxisomes in cultured hepatocytes by a non-
metabolizable fatty acid. Similar to other approaches, molecular techniques can be applied
to the cultured hepatocyte system. Inductions of mRNA encoding specific enzymes can also
be determined in this in vitro system. In this present work, it is demonstrated, for the first
time, that similar to hypolipidemic peroxisome proliferator, a non-metabolizable fatty acid analog also regulates peroxisomal enzyme synthesis at a transcriptional and a translational level. 143
Mitochondrial inner membrane
Cytosol Matrix
. 0
O 'I O A Carnitine RC—CoA RC— CoA Carnitine
Carnitine C arnitine acyitransterase 1 acyitransterase
RC—Carnitine. ■ R C — Carnitine CoA CoA II II O O
Figure 26. Carnitine carries long-chain fatty acyl CoA into the mitochondrial matrix for fatty acid oxidation. Long-chain acyl CoA molecules are carried across the inner mitochondrial membrane by carnitine, a zwitterionic compound formed from lysine.. The acyl group is transferred from the sulfur atom of CoA to the hydroxyl group of carnitine to form acyl carnitine. This reaction is catalyzed by carnitine acyltransferase I which is located on the cytosolic face of the inner mitochondrial membrane. The entry of acyl carnitine into the mitochondrial matrix is mediated by translocase. The acetyl group is transferred back to CoA on the matrix side of the membrane by carnitine acyltransferase II. Carnitine palmitoyltransferase (CPT) is carnitine acyltransferase that preferentially transfers palmitoyl-CoA and other long-chain acyl CoA whereas carnitine acetyltransferase (CAT) and carnitine octanoyltransferase favor the transfer of short-chain fatty acyl-CoA such as acetyl-CoA and medium-chain acyl-CoA e.g. octanoyl-CoA, respectively. 144
Concentration—Response Curves of PFOA, CIPRO and CPIB on FACO Induction
* EC-o GliM) S 1500" PFOA =27.8 II CIPRO = 4.2 o CPIB = 319.1 o 1000-
-o 5 0 0 -
Ll_
0.0100.1000.001 1.000 Concentrations (mM)
Control ■A PFOA O O CIPRO □ ---- □ CPIB
Figure 27. Concentration-response curves for the induction of fatty acyl-CoA oxidase (FACO) activity by perfluorooctanoic acid (PFOA), ciprofibrate (CIPRO) and clofibric acid (CPIB). Hepatocytes were incubated for 72 hr in the presence or absence of the compound. The control FACO activity was 1.0 + 0.2 nmole H 20 2/min/mg protein. Results are expressed as a percent of the control activity. Each point represents the mean + S.E.M. of determination from 6-12 dishes of cells (2-3 separate experiments). The EC 50 values were determined from the curves using 2 -degree polynomial curve fit. 145
Concentration—Response Curves of PFOA and CPIB on LH Induction
7 5 0 - EC50 ^ M) PFOA = 7.4 CPIB =311.2 c o v 5 0 0 - o ■o3 c X « 2 5 0 -
0.001 0.01 0.1 1 Concentrations (mM) Control ▲— A PFOA □ ---- □ CPIB
Figure 28. Concentration-responsecurves for the induction of microsomal laurate hydroxylase (LH) activity by perfluorooctanoicacid (PFOA) and clofibric acid (CPIB). Hepatocytes were incubated for 72 hr in the presence or absence of the compound. The control LH activity was 2.4 + 0.3 nmole/hr/mg protein. Results are expressed as a percent of the control activity. Each point represents the mean ± S.E.M. of determination from 9-12 dishes of cells (3 separate experiments). The EC50 values were determined from the curves using 2-degree polynomial curve fit. 146
Table 17. EC 50 values for the induction of FACO and LH activities by PFOA, CPIB and CIPRO
ECjo“ 0*M) for induction of Compound FACO LH
PFOA 27.8 ± 4.3 7.4 ± 1.9
CPIB 319.1 ± 88.9 311.2 ± 35.8
CIPRO 4.2 ± 0.7 ___ b
a ECjo values were determined from corresponding concentration-response curves. The values are mean ± S.E.M. of 2-3 separate experiments b not determined 147
Tim e-course of FACO induction by PFOA and CPIB
^ O—O Control •| 6 " A A PFOA 0.1 mM 5 - - □ — □ CPIB 1 mM
Time (day)
2500-- .... Control (100%) A A PFOA 0.1 mM □ — □ CPIB 1 mM -a 1500--
^ 1000 -- Li -
500--
0 2 3 4 Time (day)
Figure 29. Time courses for the induction of peroxisomal fatty acyl-CoA oxidase (FACO) activity by 0.1 mM perfluorooctanoic acid (PFOA) and 1 mM clofibric acid (CPIB) and control (no drug). Hepatocytes were incubated for 0 to 4 days. Each point represents the mean ± S.E.M. of determinations from 6 dishes of cells. Top: Results are expressed as absolute activity of the enzyme in nmole H202/min/mg protein. Bottom: Results are expressed as the percent FACO induction as compared to the corresponding control activity examined at the same incubation period. 148
Time-course of LH induction by PFOA
O O control 15-- A— A PFOA 0.1 mM cn
10--
5--
0 2 3 4 5 Time (day)
.... Control (100%) 600-- A — A PFOA 0.1 mM
oc o 3 400-- •oc X_i * 2 0 0 --
0 1 2 3 4 5 Time (day)
Figure 30. Time courses for the induction of microsomal laurate hydroxylase (LH) activity by 0.1 mM perfluorooctanoic acid (PFOA) and control (no drug). Hepatocytes were incubated for 0 to 5 days. Each point represents the mean ± S.E.M. of determinations from 6 dishes of cells. Top: Results are expressed as absolute activity of the enzyme in nmole/hr/mg protein. Bottom: Results are expressed as the percent LH induction as compared to the corresponding control activity examined at the same incubation period. 149 Time—course of CAT induction by PFOA and CPIB
8 O ■O control o» E A ■A PFOA 0.1 mM T A \c □ CPIB 1 mM E \ _a> o E c £ ho-i < Time (day) 1000-- .... Control (100*) A— A PFOA 0.1 mM c 800 □ ----□ CPIB 1 mM ■o 6 0 0 -- o 4 0 0 -- 2 0 0-- 0 1 2 3 4 Time (day) Figure 31. Time courses for the induction of carnitine acetyltransferase (CAT) activity by 0.1 mM perfluorooctanoic acid (PFOA), 1 mM ciprofibrate (CPIB) and control (no drug). Hepatocytes were incubated for 0 to 4 days. Each point represents the mean + S.E.M. of determinations from 6 dishes of cells. Top: Results are expressed as absolute activity of the enzyme in nmole/min/mg protein. Bottom: Results are expressed as the percent CAT induction as compared to the corresponding control activity examined at the same incubation period. 150 Effect of various concentrations of PFOA on CIPRO concentration-response curve <1 O CIPRO (alone) • CIPRO + PFOA 0.001 mM A CIPRO + PFOA 0.01 mM 0.001 CIPRO + PFOA 0.1 mM 0.003 0.01 0.03 Concentrations of CIPRO (mM) Figure 32. Effect of various concentrations of perfluorooctanoic acid (PFOA) on ciprofibrate (CIPRO)-concentration-response curve. Concentration-response curve of CIPRO (0.003- 0.1 mM) was constructed in the absence or presence of three different concentrations of PFOA (0.001,0.01 and 0.1 mM). Both compounds at the desired concentration were added together to cultured hepatocytes and incubated for 2 days. Three stacked vertical bars on the left of the figure indicate the fatty acyl-CoA oxidase (FACO) inductive activity by PFOA (0.001, 0.01 and 0.1 mM) alone. All results are expressed as the percent maximum FACO induction produced by CIPRO 0.1 mM. Each point represents the mean ± S.E.M. of determination from 6 dishes of cells (2 separate experiments). Asterisks indicate significant different (P < 0.05, Student’s t test) from the control CIPRO concentration-response curve. 151 Effect of various concentrations fo CPIB on CIPRO concentration-response curve 150 125-- O 1 0 0 - 7 5 - CPIB 1 mM 50-- o — O CIPRO (alone) 2 5 - ■ — ■ CIPRO + CPIB 0.1 mM 0.1 □ — □ CIPRO + CPIB 1 mM 0.003 0.01 0.03 0.1 Concentrations of CIPRO (mM) Figure 33. Effect of various concentrations of clofibric acid (CPIB) on ciprofibrate (CIPRO)- concentration-response curve. Concentration-response curve of CIPRO (0.003- 0.1 mM) was constructed in the absence or presence of two different concentrations of CPIB (0.1 and 1 mM). Both compounds at the desired concentration were added together to cultured hepatocytes and incubated for 2 days. Two stacked vertical bars on the left of the figure indicate the fatty acyl-CoA oxidase (FACO) inductive activity by CPIB (0.1 and 1 mM) alone. All results are expressed as the percent maximum FACO induction produced by CIPRO 0.1 mM. Each point represents the mean ± S.E.M. of determination from 3 dishes of cells. Asterisks indicate significant different (P < 0.05, Student’s t test) from the control CIPRO concentration-response curve. 152 Effect of PFDA on PFOA response 6 -- E S PFOA 0.01 mM CX3 PFOA 0.03 mM o> E -P' = c 4.. E o CM o a O X JO 1 Q £o ----- E c I * 1 None PFDA 0.01 mM Figure 34. Effect of the combination of perfluorooctanoic acid (PFOA) and perfluorodecanoic acid (PFDA) concentrations on fatty acyl-CoA oxidase (FACO) activity in rat hepatocytes. Effect of addition of 0.01 mM PFDA into cultures treated with PFOA 0.01 and 0.03 mM were investigated. The cultures were incubated for 3 days. Results are expressed as absolute FACO activity (nmole H202/min/mg protein) determined from 6 dishes of cells. Points with letters indicate FACO activity that were significantly greater than control and points with different letters indicate that the values were significantly different from each other (P < 0.05, Student-Newman Keuls test). 153 Effects of octanoic and octanedioic acids on PFOA response E S PFOA 0.01 mM 6 -- □ □ PFOA 0.03 mM o> ll 4 - 8 ^ ° <•o (D u: o E 2- c I & None Octanoicacid Octanedioic acid 0.3 mM 0.3 mM Figure 35. Effect of octanoic acid and octanedioic acid on fatty acyl-CoA oxidase (FACO) induction by perfluorooctanoic acid (PFOA). Effect of addition of 0.3 mM octanoic acid and 0.3 mM octanedioic acid into cultures treated with PFOA 0.01 and 0.03 mM were investigated. The cultures were incubated for 3 days. Results are expressed as absolute FACO activity (nmole H202/min/mg protein) determined from 6 dishes of cells. Points with letters indicate FACO activity that were significantly greater than control and points with different letters indicate that the values were significantly different from each other (P < 0.05, Student-Newman Keuls test). 154 Effect of nicardipine on &FACO induction by CIPRO, PFOA and oleic acid EH] No nicardipine Hd Nicardipine 0.001 mM 4 0 0 ES Nicardipine 0.01 mM c 0 Control 1 3 0 0 "O C. o Q 200 £ 1 0 0 Control CIPRO PFOA Oleic acid 0.08 mM 0.08 mM 1 mM Figure 36. Effect of nicardipine on fatty acyl-Co A oxidase (FACO) induction by perfluorooctanoic acid (PFOA), ciprofibrate (CIPRO) and oleic acid in cultured rat hepatocytes. Nicardipine (0.001 and 0.01 mM) was added to hepatocyte cultures treated with 0.08 mM CIPRO, 0.08 mM PFOA or 1 mM oleic acid. Effects of the FACO induction by CIPRO and PFOA were determined after 2 days of incubation with and without nicardipine. In case of oleic acid, the enzyme activity was determined after 4 days of incubation: Results are expressed as percent of FACO induction over the control activity which was 0.83 ± 0.10 nmole H202/min/mg protein. Each point represents mean ± S.E.M. determined from 6-9 dishes of cells. Significant inhibition by nicardipine (0.001 and 0.01 mM) on FACO induction produced by CIPRO, PFOA and oleic acid was not observed by Student-Newman Keuls test. 155 Effect^of various mitochondrial |8-oxidation blockers on PFOA c o tj 200 — T 3 #C O O • £ 100 - Inducer (mM) PFOA 0.03 PFOA 0.03 PFOA 0.03 PF0A 0.03 Mitochondrial blocker - + boa 0.03 +MPA0.03 b + p a o .03 (mM) Figure 37. Effects of various inhibitors of mitochondrial fatty acid 6-oxidation on fatty acyl- CoA oxidase (FACO) induction by 0.03 mM perfluorooctanoic acid (PFOA). The inhibitors used were 2-bromooctanoic acid (BPA), the inhibitor of 3-ketothiolase, mercaptopropionic acid (MPA), the inhibitor of fatty acyl CoA dehydrogenase, and 2-bromopaImitic acid (BPA), the inhibitor of carnitine palmitoyltransferase I (CPT-I). All inhibitors were applied at 0.03 mM concentration together with 0.03 mM PFOA as an inducer to the cultured hepatocytes and incubated for 3 days. Results are expressed as percent of FACO induction over the control activity which was 1.0 ± 0.2 nmole H202/min/mg protein. Each point represents mean + S.E.M. determined from 6 dishes of cells. 156 Effect of CPT inhibitor (BPA) on FACO induction by CIPRO and PFOA 1000 Control I I no BPA -- G J BPA 0.003 mM | 7 5 0 U-j o ES9 BPA 0.03 mM ZJ TJ •£ 500 o o « 250 0 BPA CIPRO 0.1 mM PFOA 0.1 mM Figure 38. Effects of a carnitine palmitoyltransferase I inhibitor on fatty acyl-CoA oxidase (FACO) induction by 0.1 mM ciprofibrate (CIPRO) and 0.1 mM perfluorooctanoic acid (PFOA). Two concentrations of 2-bromopalmitic acid (BPA) (0.003 and 0.03 mM) were applied to the cultured hepatocytes treated either with CIPRO or PFOA and incubated for 3 days. Results are expressed as percent of FACO induction over the control activity which was 0.55 ± 0.10 nmole H202/min/mg protein. Each point represents mean ± S.E.M. determined from 6 dishes of cells. Asterisks indicate the concentrations of BPA that significantly inhibited the effects of CIPRO and PFOA in increasing FACO activity. 2 —BROMOPALMITIC ACID (BPA) 150 O* O £ 1 0 0 *- o c o _a_ ' £ > 5 0 - o a a. o * 0.001 0.01 0.11 3 BPA (jM ) CIPR0FIBRATE g 150 0 T“ II 1 100 o & tj 50 a c l o * 0 —I------1------1------1------1------1------t— 0.001 0.01 0.1 1 10 100 1000 Ciprofibrate (jiM) Figure 39. Effects of 2-bromopaImitic acid (top) and ciprofibrate (bottom) on the activity of carnitine palmitoyltransferase I (CPT-I) in isolated rat liver mitochondria. The enzyme activity was assayed as described in Methods. The incubation mixture contained 500 fig of mitochondrial protein as a source of enzyme, 75 fig palmitoyl CoA, 92.8 fig DTNB, 698 fig EDTA, 928 fig DL-carnitine (experimental tube only) and the presence or absence of desired concentrations of BPA (0.001-3 fiM) or CIPRO (0.001-1000 fiM) and q.s. to 1.5 ml with 50 mM Tris pH 8.0. The enzyme activity was determined 5 min after incubation at room temperature. Results are expressed as percent of CPT activity as compared to control which was the activity of CPT-I of mitochondrial protein in the absence of drug. The control activity was 3.2 + 0.7 nmole/min/mg mitochondrial protein. BPA inhibited CPT-I activity with the IC50 of 0.19fiM. No CPT-I inhibition was shown by- CIPRO. PFOA 150 o o 100 o D I- Q_ O 0.001 0.01 0.1 1 10 100 1000 PFOA (/zM) Oleic acid (18:1) 200 o o 7 1 5 0 - 2 •«->c o o 100 £ > o a i— 5 0 - Q . O 0.001 0.01 0.1 10 100 Oleic acid (//M) Figure' 40. Effects of perfluoroottanoic acid (top) and oleic acid (bottom) on the activity of carnitine palmitoyltransferase I (CPT-I) in isolated rat liver mitochondria. The enzyme activity was assayed as described in Methods. The incubation mixture contained 500 fig of mitochondrial protein as a source of enzyme, 75 fig palmitoyl CoA, 92.8 fig DTNB, 698 fig EDTA, 928 fig DL-carnitine (experimental tube only) and the presence or absence of desired concentrations of PFOA (0.001-1000 ptM) or oleic acid (0.001-100 fiM) and q.s. to 1.5 ml with 50 mM Tris pH 8.0. The enzyme activity was determined 5 min after incubation at room temperature. Results are expressed as percent of CPT activity as compared to control which was the activity of CPT-I of mitochondrial protein in the absence of drug. The control activity was 3.2 + 0.7 nmole/min/mg mitochondrial protein. Figure 41. Electron micrographs of the control cultured rat hepatocytes. Hepatocytes were incubated with 0.4% DMSO and no drug for 3 days. The media were removed and cells were fixed with glutaraldehyde and stained with alkaline 3,3’-diaminobenzidine (DAB) for peroxisome visualization as described in Methods. Note the normal mitochondria (M) and the few peroxisomes (P) were darkly stained and contained a nucleoid core. Magnification, x 5250. The bar represents 1 pm. 160 Figure 42. Electron micrographs of ciprofibrate-treated cultured rat hepatocytes. Hepatocytes were incubated with 0.1 mM ciprofibrate and 0.4% DMSO as vehicle for 3 days. ITie media were removed and cells were fixed with glutaraldehyde and stained with alkaline 3,3’- diaminobenzidine (DAB) for peroxisome visualization as described in Methods. Mitochondria (M) were swollen. Peroxisomes (P) were stained darker than the control culture and an obvious increase in numbers and diameter as compared to the control (Fig. 41). Magnification, x 5250. The bar represents 1 f i m . 161 Figure 43. Electron micrographs of PFOA-treated cultured rat hepatocytes. Hepatocytes were incubated with 0.1 mM PFOA and 0.4% DMSO as vehicle for 3 days. The media were removed and cells were fixed with glutaraldehyde and stained with alkaline 3,3’- diaminobenzidine (DAB) for peroxisome visualization as described in Methods. Note the increase in number of mitochondria (M) and peroxisomes (P) as compared to the control (Fig. 41). Magnification, x 5250. The bar represents 1 nm. 162 Table 18. Morphological changes of peroxisomes after the hepatocytes were treated with CIPRO (0.1 mM) and PFOA (0.1 mM) for 3 days. The peroxisome proliferation was shown as increases in number, diameter and percent of the area occupied as compare to control. Peroxisome Compound FACO-1 Number8 Diameterb % area occupied® Control 8.0 ± 0.4 0.60 ± 0.02 2.33 ± 0.22 1.09 ± 0.28 CIPRO 19.7 ± 1.8® 0.72 ± 0.04f 7.84 ± 0.84® 7.05 ± 0.96® PFOA 13.8 ± 1.1® 0.66 ± 0.03 5.05 ± 0.93® 6.39 ± 0.95® 0 Values are shown as mean ± S.E.M. determined from 9-11 electron micrographs of each treatment. The numbers of peroxisomes were expressed per the area of 100 fim2 of the cytoplasm. b Diameters in fim are shown as mean ± S.E.M. determined from each micrographs (n = 9- 11) c Percent of area occupied are shown as mean ± S.E.M. determined from each micrographs (n = 9-11). Values were calculated from numbers of peroxisomes x 7r(radius of peroxisome)2 divided by total cytoplasmic area measured which was 100 fim2 d FACO were expressed as nmole H202/min/mg protein 6 Values are significantly different from the control culture (P < 0.01) f Values are significantly different from the control culture (P < 0.05) 163 0 2 6 20 2 6 20 2 6 20 2 20 I------II------II------II------1 Control CIPRO PFOA Oleic A. 0.1 mM 0.1 mM 1 mM Figure 44. Northern blot analysis (20 f i g total RNA/lane) of time course changes in the mRNA level encoding fatty acyl-CoA oxidase (FACO) in hepatocyte cultured in the absence (control) or presence of 0.1 mM ciprofibrate (CIPRO), 0.1 mM perfluorooctanoic acid (PFOA) or 1 mM oleic acid for 0, 2, 6 and 20 hr. The mRNA of FACO is 3.8 kb in size. 164 ‘Cv.-V. p :|S -3.8kb 0 2 6 20 Control 0 2 6 20 Hr PFOA 0 2 6 20 Hr Figure 45. Northern blot analysis of time course changes in the mRNA level encoding fatty acyl-COA oxidase (FACO) in cultured hepatocytes treated with 0.1 mM perfluorooctanoic acid (PFOA) (Bottom) as compared to the corresponding control (top). Drawings on the left represent the density peaks determined from scanning densitometer. PFOA increased mRNA of FACO over the corresponding control by approximately 1.2- and 2.6-fold after 6 and 20 hr treatments. The mRNA of FACO is 3.8 kb in size. 165 W i i A B C D Figure 46. Northern blot analysis of the mRNA levels of FACO after 20 hr treatment with no drug (A), 0.1 mM ciprofibrate (B), 0.1 mM perfluorooctanoic acid (C), and 1 mM oleic acid (D). The size of mRNA of FACO is 3.8 kb. 166 Effect of cycloheximide on FACO induction by CIPRO, PFOA and oleic acid CZ1 no cycloheximide with cycloheximide c 6 0 0 - o 1 ^ g /m l o 3 TJ C O o CIPRO PFOA Oleic acid 0.08 mM 0.08 mM 1 mM Figure 47. Effect of cycloheximide on fatty acyl-CoA oxidase (FACO) induction produced by 0.08 mM ciprofibrate (CIPRO), 0.08 mM perfluorooctanoic acid (PFOA) and 1 mM oleic acid. Cycloheximide 1 fig/ml cultured media were added to the hepatocyte cultures after the cells were exposed to CIPRO, PFOA or oleic acid for 24 hr as described in Methods. The cells were harvested after 2 days of incubation with CIPRO and PFOA and after 4 days of incubation with oleic acid. Results are expressed as percent FACO induction over the control values. The control FACO activity was 0.38 ± 0.09 nmole H202/min/mg protein. Each point represents the mean + S.E.M. of determination from 3-6 dishes of cells. Asterisks indicate significant inhibition of FACO induction with cycloheximide (P < 0.05, Student’s t test). CHAPTER V SUMMARY AND CONCLUSIONS A. Summary Primary cultures of adult rat hepatocytes provide a useful system for investigating the role of fatty acids and fatty acid analogs in mediating peroxisome proliferation. As well as being a practical system for conducting pharmacological and biochemical evaluation of the selected fatty acids, morphological and molecular techniques can also be applied to this in vitro system. By using the cultured hepatocytes, a number of naturally occurring fatty acids and non-metabolizable fatty acid analogs were investigated in order to determine the structural requirements needed for their peroxisome proliferative roles (Chapter II). In addition, the effect of stereoisomerism was studied to gain more insight into the structural requirements of fatty acid analogs (Chapter II). The experiments attempting to find active structures of naturally occurring fatty acids in peroxisome proliferation and to validate the fatty acid overload theory by Lock et al. (1989) were also conducted (Chapter III). Moreover, the mechanism of a non-metabolizable fatty acid analog (perfluorinated octanoic acid) as a potent peroxisome proliferator was characterized as compared to a hypolipidemic peroxisome proliferator, ciprofibrate (Chapter IV). The major findings of these experiments are listed below. 1. Comparison of the effects of natural and metabolically stable analogs of 167 168 monocarboxylic acids reveal that only the non-metabolizable perfluorinated fatty acids but not its corresponding monocarboxylic acids (C4, C8 and C10) induced peroxisomal fatty acyl-CoA oxidase (FACO) and microsomal laurate hydroxylase (LH). Maximum inductions of FACO and LH activities by 0.1 mM perfluorooctonoic acid (PFOA, C8), 0.01 mM perfluorodecanoic acid (PFDA, C10) and 0.3 mM perfluorobutanoic acid (PFBA, C4), as compared to the normalized control (100%), were 1372, 333, and 390% and 450, 324, and 584%, respectively. 2. At the concentrations tested (0.001 mM to 1 mM), the perfluorinated alcohol, perfluorooctanol (PFOL), did not elevate FACO and LH activities whereas its fatty acid analogs, PFOA, at the concentration of 0.1 mM, increased FACO and LH activities 1372% and 450% over the control, respectively. 3. PFBA, PFOA and PFDA produced concentration-dependent increases in FACO and LH activities. The rank order of potency of these perfluorinated fatty acid analogs for the inductions of FACO and LH activities was similar—i.e., PFOA > PFDA > PFBA > CPIB. 4. The (-) enantiomers of 2-(4-chlorophenoxy)pentanoic acid (CPPA) and 2-(4- chlorophenoxy)octanoic acid (CPOA) produced concentration-dependent increases in FACO activity. S(-)-CPPA (0.3 mM) produced a maximal increase in FACO activity which was 388% over the control value whereas R(+)-CPPA did not show any in inductive effect at all concentrations tested (0.01-1 mM). Similar to the CPPA enantiomers, S(-)-CPOA was active as peroxisome proliferator while R(+)-CPOA was inactive. The maximum induction of FACO by S(-)-CPOA which occurred at the concentration of 0.1 mM, was 376% of the control. The rank order of FACO stimulatory potency was S(-)-CPOA > S(-)-CPPA > CPIB. 169 5. Naturally occurring saturated monocarboxylic acids of C4, C8, Ci„, C18, and C22 chain length did not produce any changes in peroxisomal FACO or microsomal LH activities. However, unsaturated long-chain fatty acids (C18) significantly increased FACO and LH activities. Among all natural fatty acid, oleic acid (C18:I) displayed the greatest effect in increasing FACO while linoleic acid (C18:2) revealed the greatest effect in increasing LH activity; linolenic acid (C18;3), like stearic acid (Ci8;0), was inactive. However, the maximum enzyme inductions by natural fatty acids were only 200% to 300% of the control values. 6. Oleic acid increased FACO and CAT activities in the concentration- and time- dependent manner. For FACO, the increase in enzyme activity appeared after the cultures were exposed to the fatty acid for 3 days and the maximum increase was lower than FACO activity of the freshly isolated hepatocytes (day 0). 7. Studies on the effects of dicarboxylic acids of C4, C8, C10, C12, and C16 chain length were performed. Dicarboxylic acids of C4, C8, and C10 chain length were inactive in increasing both FACO and LH activities. In fact, these short-chain dicarboxylic acids significantly attenuated both enzyme activities. On the other hand, longer-chain dicarboxylic acids (C12, C16) significantly increased FACO activity with the maximum of approximately 1.5- to 2-fold over the control. The 12-hydroxydodecanoicacid and 16-hydroxyhexadecanoic acids also showed a 2-fold increase in peroxisomal FACO activity. 8. BOA, MPA and BPA are inhibitors of mitochondrial fatty acid 6-oxidation that block 3-ketothiolase, fatty acyl-CoA dehydrogenase and carnitine palmitoyltransferase-I(CPT- I), respectively. In the presence of these inhibitors, octanoic acid and octanedioic acid, which are fatty acids that are rapidly degraded by mitochondrial B-oxidation, still did not exhibit any FACO inductive effects. 9. Among all fatty acid analogs investigated, PFOA was the most potent peroxisome 170 proliferator as determined by the ability to increase peroxisomal FACO activity. The EQo values of CIPRO, PFOA and CPIB determined from concentration-response curves were 4.2, 27.8 and 318.1 fiM, respectively. PFOA was much more potent than CPIB in inducing both FACO and LH activities. 10. PFOA produced time-dependent increases in FACO, LH and CAT. The time- course profiles were similar to that produced by CPIB--i.e., all enzyme activities were increased after the first day of incubation with PFOA and maximal increases were reached after 3-4 days of treatment. 11. Drug combination (PFOA, PFDA, CPIB, CIPRO) studies were performed to examine whether their effects on peroxisomal FACO are additive or synergistic. Addition of PFOA and CPIB provoked similar effects on the CIPRO-concentration response curve (0.003- 0.1 mM) for FACO. At a low concentration of PFOA or CPIB, CIPRO concentration- response curve of FACO was shifted to the right whereas at a high concentration of PFOA or CPIB, the curve was shifted to the left. No additive effects were detected in the combinations of either CIPRO and PFOA, CIPRO and CPIB or PFOA and PFDA. 12. The effects of natural mono-(octanoic acid) and di-(octanedioic acid) acids on the PFOA-mediated increases in FACO were examined. Octanoic acid did not produce any effect of the induction of FACO by PFOA. However, octanedioic acid potentiated the effect of PFOA. Addition of 0.3 mM octanedioic acid to 0.03 mM PFOA increased the FACO activity from 309% to 459% of the control value. 13. Nicardipine (at 0.001 mM and 0.01 mM) slightly though not significantly inhibited FACO induction by 0.08 mM CIPRO and 0.08 mM PFOA. Nicardipine, at both concentrations, was without effect on the increase in FACO activity by 1 mM oleic acid. 14. Whereas BOA and MPA did not produce any effect on FACO induction by 171 PFOA, the CPT-I inhibitor, BPA, significantly inhibited the effect of PFOA. BPA (0.03 mM) decreased the FACO induction by 0.1 mM PFOA from 538% to 139% of the control value. Moreover, BPA (0.003 and 0.03 mM) reduced 0.1 mM CIPRO response from 707 % to 321 % and 328% of the control, respectively. 15. BPA inhibited hepatic CPT-I activity in isolated mitochondria with the IC 50 of 0.19 /xM. CIPRO did not show any inhibitory effect on CPT-I activity. PFOA and oleic acid showed slight inhibitory effects on CPT-I activity at high concentrations. The maximum inhibitions by PFOA and oleic acid were 60% of the control CPT-I activity. 16. Morphometric analysis demonstrated that, similar to CIPRO, PFOA increased the peroxisome number, peroxisome diameter, and cytoplasmic area occupied by peroxisomes. As compared to the control, peroxisome numbers increased from 8 to 19.7 and 13.8 peroxisomes per 100 /xm 2 after a 3-day treatment with CIPRO (0.1 mM) and PFOA (0.1 mM), respectively. The area occupied by peroxisomes also increased from 2.3% to 7. 8 % and 5.1% after CIPRO and PFOA treatment. The increases in cytoplasmic area occupied by peroxisomes correlated with the increases in peroxisomal FACO activity produced by these two peroxisome proliferators. 17. PFOA increased the mRNA which encodes FACO in the time-dependent fashion. The induction of mRNA was 2.6-fold over the control after the cultures were exposed to 0.1 mM PFOA for 20 hr. Similarly, CIPRO increased the mRNA encoding FACO after a 20 hr incubation. However, oleic acid (1 mM) did not produce a detectable increase in the mRNA level after the cultures being exposed to the fatty acid for 20 hr. 18. Cycloheximide (1 /xg/ml), an inhibitor of protein synthesis, inhibited the induction of FACO by PFOA (0.08 mM) and by CIPRO (0.08 mM). FACO induction by PFOA and CIPRO decreased from 559% to 218% and 346% to 196%, respectively. In contrast, 172 cycloheximide (1 /tg/ml) did not inhibit but potentiate the increase in FACO activity produced by 1 mM oleic acid. Oleic acid increased FACO activity by 274%, and in the presence of cycloheximide, the activity was raised to 364% of the control activity. B. Conclusions In conclusion, these results reveal that: 1. Fatty acids and fatty acid analogs that are active as peroxisome proliferators exhibit these structure constraints: A) a hydrophobic backbone whose length determined the peroxisome proliferative potency. B) a carboxylic acid function. C) a poor susceptibility to fatty acid metabolism or a poor substrate specificity for mitochondrial fatty acid B-oxidation. 2. Naturally occurring fatty acids that increase peroxisomal FACO activity are fatty acids that are poorly metabolized by mitochondria and/or are good substrates for peroxisomal fatty acid B-oxidation. These fatty acids include unsaturated long-chain fatty acids, medium- and long-chain dicarboxylic acids and co-hydroxy carboxylic acids. The maximum FACO induction produced by these natural fatty acids is only 2- to 3-fold over the control. 3. The substrate overload hypothesis proposed by Lock et al. (1989) explains the effect of naturally occurring medium- to long-chain fatty acids that accumulate in cells, and are causally link to peroxisome proliferation. These fatty acids, which are substrates for peroxisomal fatty acid B-oxidation or are transformed (by the action of microsomal LH) to substrates for peroxisomal B-oxidation (e.g., medium- and long-chain dicarboxylic acids), overload the peroxisomal B-oxidation system and increase the activities of peroxisomal enzymes as well as other cellular fatty acid metabolizing enzymes. In the present studies; however, the dicarboxylic acids and related co-hydroxy fatty acid precursors increased FACO activity by up to only 2-fold while other peroxisome proliferators such as CIPRO and PFOA 173 increased FACO activity by 10- to 20-fold over the control. Thus, based upon our results, the proposed role of dicarboxylic acids as proximal stimuli for the peroxisome proliferative effect of potent peroxisome proliferators [Sharma et al. , 1987b; Lock et al., 1989] is questioned. More likely, the stimulatory effects of these fatty acids on FACO is related more to a regulation on other homeostatic mechanisms in the cell and is not the proximal stimulus for xenobiotic peroxisome proliferators. 4. PFOA, a non-metabolizable fatty acid analog, was a potent peroxisome proliferator, and its effect can be displayed morphologically in cultured rat hepatocytes. The results indicate that an inhibition of CPT-I activity is not directly associated with a proliferative effect of peroxisome proliferators as has been previously proposed by Eacho and Foxworthy (1986). The results also demonstrate that non-metabolizable fatty acid analogs (e.g., PFOA and PFDA) and xenobiotic peroxisome proliferators (e.g., CIPRO and CPIB) act through a common pathway leading to peroxisome proliferation. Although this common pathway is not known, these two structurally distinct peroxisome proliferators act directly on the regulation of gene transcription. An increase in the peroxisomal FACO activity by these peroxisome proliferators occurred through a de novo enzyme synthesis. In contrast, the increase in the peroxisomal enzyme activity by oleic acid was independent of de novo synthesis, and the effect of natural fatty acids follow another pathway. C. Synopsis of research work Collectively, these studies have provided new insights into the role of fatty acids and their analogs in mediating peroxisome proliferation and have further characterized this response pharmacologically, biochemically, morphologically and genetically. This new information increases our understanding of the phenomenon of peroxisome proliferation which 174 may be an important event leading to a regulation of the growth of subcellular organelles and a regulation of enzymes needed to maintain the cellular homeostasis. Although the mechanism of peroxisome proliferation and the direct involvement of fatty acids in this phenomenon are still unclear, our results indicate that mechanisms of peroxisome proliferation may consist of more than one pathway (Fig. 48). Xenobiotics as well as non-metabolizable fatty acids may interact with peroxisome proliferator activated receptors (PPAR)[Issemann and Green, 1990], either directly or by stimulation of the endogenous ligand of the receptors. The activated receptors, thus, bind to their DNA domains which regulate a transcription of the genes encoding fatty acid-metabolizing enzymes. On the other hand, the second mechanism may be operated when liver cells are faced with a fatty acid overloaded situation. In the presence of increased intracellular fatty acids, stimulation of cytochrome P450IVA1 occurs which increases production of co-hydroxy, and subsequently dicarboxylic acids that are excellent substrates for the peroxisomal 8-oxidation enzyme system. The substrates induce peroxisomal enzyme activity and cause peroxisome proliferation. Whereas the magnitude of enzyme inductions by the receptor-mediated mechanism can exceed 10-30 fold over the control, the substrate overload mechanism may cause only a 2-3 fold induction of the enzyme activity. The functions of a peroxisome proliferation-associated receptor (PPAR) which is activated by xenobiotics (foreign compounds) or other endogenous ligands (which may be naturally occurring compounds), should be an interesting issue to study in the future. 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